A screen of apoptosis and senescence regulatory genes for life span effects when over-expressed in Drosophila

Results

Characterization of Geneswitch drivers in adult flies using the UAS-GFP reporter

To facilitate the screen of apoptosis and senescence-regulatory genes for life span effects, several Geneswitch system drivers were characterized for their tissue-specificity of transgene activation using a UAS-GFP reporter, both in adult flies and during larval development. The UAS-GFP reporter employed was "UAS-ultraGFP" which contains multiple copies of a UAS-eGFP construct, and yields particularly high levels of GFP expression [18]. Three Geneswitch system drivers were characterized: The Act-GS-255B driver strain contains multiple inserts of a construct in which the promoter from the cytoplasmic actin gene Actin 5C drives Geneswitch, and is expected to yield tissue-general expression [16]. The Elav-GS driver contains Geneswitch under control of the Elav gene promoter and produces nervous-system-specific expression [19]. Finally the whole-body fat-body Geneswitch driver strain ("WB-FB-GS") contains both a head fat-body driver (S₁-32) and a body-fat-body driver (S₁-106) [20-22], and is expected to yield expression in the fat-body tissue throughout the animal. The three driver strains were crossed to the UAS-ultraGFP reporter strain to produce adult progeny containing both the driver and reporter constructs, and the flies were cultured in the presence and absence of drug for two weeks. GFP expression was scored in live adult flies as well as in several dissected tissues (Figure 1). The Act-GS-255B driver was found to yield tissue-general expression of the UAS-ultraGFP reporter in adult flies. In whole adults, GFP expression was observed throughout the body of both males and females, with greater expression levels observed in females relative to males. Similarly with heads dissected in half and bodies dissected in half, expression was observed in all tissues, including abundant expression in nervous system, muscle (including flight muscle), and fat-body tissue. Note that flight muscle in male has lower expression than flight muscle in female, however inspection of the GFP-only image for male flight muscle (inset) reveals expression throughout this tissue. Abundant expression was also observed throughout dissected gut tissue, ovary and testes. The expression level was greater in some regions of the gut than others, however all regions of the gut exhibited staining, as revealed by inspection of the GFP-only images (inset). All tissues observed showed significant GFP expression, and therefore we conclude that Act-GS-255B yields truly tissue-general expression in adult flies. The WB-FB-GS driver produced GFP expression in the head-fat-body and body-fat-body tissues, as expected, as well as in the gut and testes, and very faint expression in ovary; there was no detectable expression in nervous, muscle, or other tissues. Notably, the expression in adult male head fat body was much reduced relative to female head fat body, consistent with recent characterization of the fat body drivers using a LacZ reporter [17]. Finally, the Elav-GS driver produced abundant expression in the brain and ventral nerve cord, as expected, and there was no detectable expression in any other tissues; for example, the muscle, gut and gonads were clearly negative. Note the GFP-only image for the gut (inset) shows a lack of expression. The Elav-GS driver was found to produce similar levels of UAS-GFP reporter expression in male versus female in our experiments.

Figure 1. Expression pattern produced by Geneswitch drivers and UAS-GFP reporter in adult flies. The indicated GeneSwitch drivers Act-GS-255B ("255B"), Elav-GS ("Elav") and WB-FB-GS ("FB") were crossed to the UAS-ultraGFP reporter and adult progeny containing both constructs were scored for GFP expression in various tissues. Control flies were generated by crossing UAS-ultraGFP to white¹¹¹⁸strain flies to produce progeny containing only UAS-ultraGFP. Age-synchronized flies were cultured in the presence and absence of the drug RU486 for two weeks prior to assay, and GFP expression was scored in whole adult flies and dissected tissues, as indicated. Each image is the overlay of the visible light and GFP images. Insets show details of the regions boxed in white, GFP image only. M = male, F = female. Pictures were taken at the magnification of 20X, 50X, 32X, 20X, 50X, and 80X, for whole fly, head in half, body in half, gut, ovary, and testes, respectively. The white arrow indicates a region of 255B Female flight muscle that is obscured by a fragment of cuticle.

Characterization of Geneswitch drivers in larvae using the UAS-GFP reporter

The Geneswitch driver strains were also scored for expression patterns in 3^rd instar larvae and dissected tissues (Figure 2). The Act-GS-255B driver was found to yield tissue-general expression, including abundant expression throughout the body of whole 3^rd instar larvae, as well as in dissected brain, gut, salivary gland, imaginal discs and fat-body tissues; all tissues observed showed abundant GFP expression (Figure 2A). The inset for the Act-GS-255B 3^rd instar larval brain shows detail of the GFP-only image, and indicates that expression was present throughout the brain, with higher-level expression in a subset of cells. The WB-FB-GS driver was found to drive abundant expression in salivary gland and anterior midgut, but notably no expression in any other larval tissues including larval fat-body. Finally the Elav-GS driver produced abundant expression in larval nervous system and no detectable expression in any other larval tissues. The inset for the Elav-GS 3^rd instar larval brain shows detail of the GFP-only image, and shows that expression was present throughout the brain, with higher-level expression in a subset of cells. Notably this subset of cells was different from that observed above with Act-GS-255B. Each of the three drivers was found to produce similar patterns of expression in 1^st and 2^nd instar larvae as well (Figure 1B). When the Act-GS-255B driver was induced using dilutions of RU486 drug in the culture media, it produced a dose-response of GFP expression in 3^rd instar larvae (Figure 1D), as well as in adult flies (data not shown).

Figure 2. Expression pattern produced by GeneSwitch drivers and UAS-GFP reporter in larvae. The crosses are the same as Figure 1, but larvae were cultured in the presence and absence of drug in the food, from hatching to the indicated developmental stage. A. Expression patterns in 3^rd instar larvae and dissected tissues. For the Elav-GS driver ("Elav") a 1:10 dilution of drug was used because of the toxic effects of drug observed in larvae with this driver. Pictures were taken at the magnification of 25X, 100X, 20X, 50X, 100X, 80X, for whole larvae, brain, gut, salivary gland, imaginal discs, and fat body, respectively. B. Expression patterns in the three larval stages. For Elav-GS a 1:10 dilution of drug was used to avoid toxic effects. GFP pictures were taken at the magnification of 100X, 50X, 25X, for 1^st instar, 2^nd instar, and 3^rd instar, respectively. C. Expression in 3^rd instar larvae using Act-GS-255B and titrations of drug. ETOH indicates the ethanol solvent for the drug alone. Pictures were taken at the magnification of 25X.

Effect of apoptosis and senescence-regulatory gene over-expression on life span

Fourteen apoptosis and senescence regulatory genes were chosen for analysis based on their relevance to human apoptosis and senescence pathways and the availability of reagents for Drosophila. Ras85D is a Drosophila homolog of the human oncogene Ras that encodes a GTPase involved in signal transduction.

Ras85D activated form contains an amino acid substitution that causes Ras to be constitutively active [23], and Ras85D dominant negative (DN) form contains an amino acid substitution that causes it to inhibit the endogenous Ras protein [23,24]. Wingless is a Drosophila homolog of the human Wnt signaling protein involved in development and tumorigenesis [25]. Pk61C is a serine/threonine protein kinase related to human PDK-1 and involved in growth signaling [26]. DIAP1 is a Drosophila member of the inhibitor of apoptosis protein (IAP) family [27]. Baculovirus p35 is a caspase inhibitor protein also related to the IAPs. Nemo (nmo) is the Drosophila homolog of a human protein kinase regulatory subunit involved in NF-kappaB signaling pathway [28]. Egfr is the Drosophila homolog of the human epidermal growth factor receptor [29]. The Drosophilapointed (pnt) gene encodes a transcription factor homologous to human Ets1 that is involved in the Ras signaling pathway. The DrosophilaMatrix metalloproteinase 2 gene (Mmp2) is involved in tissue remodeling and tumor progression and is related to a family of human matrix metalloproteinases [30]. The DrosophilaStat92E gene encodes a homolog of the human Stat transcription factor, which is a target of the Jak-Stat growth-regulatory pathway [31]. The Drosophilapuckered (puc) gene encodes a phosphatase homologous to the human VH-1 family that antagonizes JNK signaling, and heterozygous puc mutant flies have been reported to have increased stress resistance and life span [32,33]. The DrosophilaSphingosine kinase 2 (Sk2) gene encodes a lipid kinase involved in activation of protein kinase C-family signaling, and the human homolog Sphk2 is implicated in regulation of apoptosis [34]. Finally the CG14544 gene encodes a predicted methyltransferase, and the Drosophilabantam (ban) gene encodes a micro-RNA that inhibits expression of pro-apoptotic genes [35]. Each of these genes of interest was over-expressed in adult flies or during larval development, and assayed for effects on adult fly life span.

To control for any possible effects of the Geneswitch system and the RU486 drug itself, life span was assayed in flies that were the progeny of Act-GS-2555B driver crossed to either Oregon-R (Or-R) wild-type strain or to the w¹¹¹⁸ control strain, to produce progeny containing only the driver. In these control flies, treatment with drug produced small, but statistically significant reductions in life span in both male and female adults: treatment during adulthood reduced mean life span by -4% to -10%, while treatment in larval stages reduced adult life span by -8% to -16% (Figure 3A, B; Figure 4A, B; Tables 2, 3). There were no significant increases in life span in control flies treated with RU486 in any of the replicate experiments. These data indicate that in these experiments, when the Act-GS-255B driver is present, the RU486 can cause small but significant reductions in adult life span, and this effect must be taken into account when interpreting the effects of transgene over-expression. Other studies [22], including ones from our own laboratory using the Act-GS-255B driver [36], found no negative effects of RU486 on adult fly life span. We conclude that the small negative effects observed here result from differences in the lot of RU486 drug, and/or small differences in effective concentrations due to specifics of media preparation. To confirm that the Act-GS-255B driver can produce increased life span, it was used to drive over-expression of the dominant p53 allele (p53-259H). Over-expression of p53-259H in adult flies using the ubiquitous Act-GS-255B driver produced increased median life span in females (+8%) but not males (-2.8%), and no life span increase when expressed in larvae (Table 3). These results are consistent with previous studies showing that expression of p53-259H in the adult nervous system with the Elav-GS driver can cause increased life span in females [14], and confirms that the Act-GS-255B driver can indeed produce increased life span when combined with an appropriate target gene.

Figure 3. Effect of transgene over-expression on survival of adult flies. Apoptosis and senescence-related genes wingless, Ras85D, and Ras85D activated form were over-expressed during larval development or in adults, and assayed for effects on adult life span in male and female flies, as indicated. The life span assays were performed at 29°C. Open circles represent the no-drug control ("-"). Solid squares represent adults treated with drug ("A"). Grey triangles represent larvae on drug ("L"). Survival curves are plotted as a function of adult age in days. Median life span of each cohort is presented along with p value for log rank test (in parentheses). (A, C, E, G) male flies. (B, D, F, H) female flies. (A, B) Control flies containing the driver and no target transgene. (C, D) Ras85D activated form. (E, F) Ras85D wild-type. (G, H) wingless.

Figure 4. Effect of Baculovirus p35 over-expression on survival of adult flies. Baculovirus p35 transgenes inserted on the X chromosome, chromosome 2, and chromosome 3 were over-expressed during larval development or adult stage, as indicated. The life span assays were performed at 25°C. Open circles represent the no-drug control ("-"). Solid squares represent adults treated with drug ("A"). Grey triangles represent larvae on drug ("L"). Survival curves are plotted as a function of adult age in days. Median life span of each cohort is presented along with p value for log rank tests (in parentheses). (A, C, E, G) male flies. (B, D, F, H) female flies. (A,B) Control flies containing the driver and no target transgene. (C, D) Baculovirus p35 transgene on X chromosome. (E, F) Baculovirus p35 transgene on second chromosome. (G, H) Baculovirus p35 transgene on third chromosome.

Most of the genes tested by over-expression with the ubiquitous Act-GS-255B driver did not affect life span to an extent greater than the small changes observed with the control flies. However, Ras activated form transgene was lethal when expressed in larvae, and reduced both male and female life span by -80% when expressed in adults (Figure 3C, D; Table 2). Over-expression of wild-type Ras or a Ras dominant-negative allele was not lethal to larvae, and produced only small decreases (-4% to -12%) in both male and female adult life span (Figure 2 E, F; Table 2), thereby in the range of negative effects observed with control flies. Over-expression of the wingless gene was found to be lethal to male and female larvae, using two independent wingless transgenes (Table 2). Over-expression of wingless in adult flies produced significant reductions in both male and female life span: ~-42% with one wingless transgene (Figure 3 G, H) and ~-10% with the other transgene (Table 2).

Finally, the tissue-general Act-GS-255B driver was used to over-express three different transgenes encoding the caspase inhibitor Baculovirus p35, during larval development and in adult flies (Figure 4; Table 3). Over-expression of Baculovirusp35 in adult flies using the tissue-general Act-GS-255B driver produced only small decreases in life span that were within the range observed with control flies, suggesting there were no significant effects in adults. In contrast, when Baculovirus p35 was over-expressed during larval development using the tissue-general driver, it reduced the mean life span of male and female adults by -20% to -50%. Interestingly, over-expression of each of the three independent Baculovirus p35 transgenes during larval development produced an unusual biphasic-shaped survival curve in adult females (Figure 4 D, F, H), suggesting the presence of a subset of adult female flies with unchanged or even increased life span. A Gompertz-Makeham model was found to give the best fit to the life span data for females in which Baculovirus p35 was over-expressed during larval development (Figure 5; Table 4). This analysis revealed that the decrease in mean life span was due to increased age-independent mortality. When the age-independent mortality was removed and the data re-plotted, it revealed a subset of female flies with unchanged (Figure 5 B, F) or increased life span (Figure 5D).

Figure 5. Mortality rate analysis of female larvae with and without Baculovirus p35 transgene expression. Open circles represent the no-drug control ("-"). Solid squares represent larvae cultured with drug ("L"). (A, B) Baculovirus p35 transgene on X chromosome. (C, D) Baculovirusp35 transgeneon second chromosome. (E, F) Baculovirus p35 transgene on third chromosome. (A, C, E) Plots of natural-log mortality rate vs. age in days. (B, D, F) The data were fitted to the Gompertz-Makeham model, which best described the mortality rate. The age-independent mortality was removed and the survival curves were re-drawn using only the Gompertz components. Mortality rate analysis showed that age-independent mortality was significantly higher for female larvae on drug versus control for all three Baculovirus p35 lines (Table 4).

Two independent Baculovirus p35 transgenes were also over-expressed in adult flies using the head-fat-body driver S₁-32, and the whole-body fat-body driver (S₁-32 plus S₁-106), and during larval development using the whole-body fat-body driver, however no consistent effects on life span were observed (Table 3).

The nervous system-specific Elav-GS driver was also used to over-express two baculovirus p35 transgenes. In adults the Elav-GS driver itself had little to no effect on life span, and over-expression of baculovirus p35 in adults using Elav-GS had no consistent effects on life span (Table 3). In contrast, when drug was administered to larvae, the Elav-GS driver itself was associated with significant decreases in life span in both males (~-30% to -40%) and females (~-25%), and significantly reduced the number of male adults, and no effects of the baculovirus p35 transgenes on life span could be identified in this background (Table 3). In an attempt to reduce this background toxicity and allow assay of baculovirus p35 transgenes with the Elav-GS driver in larvae, a 1:10 dilution of drug was used. Under these conditions the life span reductions caused by drug in males and females were smaller (~-2% to -12%), and the number of males obtained was approximately normal, however no increases in life span were observed upon over-expression of baculovirus p35 (Table 3).

The muscle-specific MHC-GS driver was used to drive over-expression of several transgenes in adult flies, however the MHC-GS driver itself was found to cause a significant RU486-dependent decrease in life span in both males and females (~-20% to -30%), and none of the target transgenes tested produced a significant life span increase in this background (Table 3).

Table 1. Starting Stocks

St#	Genotype	Notes	Abbreviation
1	w; GS-Actin255-B:+	Ubiquitous GeneSwitch 255B Driver	255B
2	w; GS-Actin255-A;+	Ubiquitous GeneSwitch 255A Driver	255A
3	w; P{Switch}bun[Switch 1-32];+	GeneSwitch Head Fat Body Driver	S32
4	w; P{Switch}S1-106 P{Switch}bun[Switch 1-32];+	GeneSwitch Head & Thorax-Abdomen Fat Body Driver	S106 S32
5	yw; +; GS-Elav	GeneSwitch Elav Driver	Elav
6	yw; Sp/CyO,FLP.lacZ; MHC:GS	GeneSwitch Muscle Driver	Sp/CyO, MHC
7	Oregon R ( +; +; +)	wild type
8	w1118; +; +	wild type
9	P{UAS.p35.H}BH3,w*;+;+	UAS-p35 on chromosome 1	p35
10	w*; P{UAS.p35.H}BH1;+	UAS-p35 on chromosome 2	p35
11	w*; +; P{UAS.p35.H}BH2	UAS-p35 on chromosome 3	p35
12	w1118; +; P{UAS-Ras85D.V12}TL1	UAS-Ras85D activated form	Ras act
13	w*; P{UAS-Ras85D.K}5-1;+	UAS-Ras85D WT form	Ras WT
14	P{UAS-Ras85D.N17}TL1, w1118; +; +	UAS-Ras85D DN form	Ras DN
15	w*; P{UAS-wg.H.T:HA1}3C;+	UAS-wg on chromosome 2	wga
16	w*; +; P{UAS-wg.H.T:HA1}6C	UAS-wg on chromosome 3	wgb
17	y1 w67c23; +;P{EPgy2}EY04093	EP-Pk61C	Pk61Ca
18	w; +; P{EP}Pk61CEP3644/TM6,Tb	EP-Pk61C	Pk61Cb
19	w*; +; P{UAS-DIAP1.H}3	UAS-DIAP1	DIAP1
20	y1 w67c23; P{EPgy2}EY00935	EP-nmo	nmo
21	y1 w*; +; P{UAS-Egfr.B}32-26-1	UAS-Egfr	Egfr
22	y1 w67c23; +; P{EPgy2}pntEY03254	EP-pnt	pnt
23	y1 w67c23; P{EPgy2}Mmp2EY08942/CyO; +	EP-Mmp2	Mmp2
24	y1 w67c23; +; P{EPgy2}Stat92EEY14209/TM3, Sb1 Ser1	EP-Stat92E	Stat
25	w*; +; P{EPgy2}pucEY09772/TM6C	EP-puc	puc
26	y1 w67c23; +; P{EPgy2}scramb2EY01180	EP-Sk2	Sk2
27	y1 w67c23; +; P{EPgy2}EY06207	EP-ban	ban
28	w1118; +; PBac{WH}CG14544f01091/TM6B, Tb1	XP-CG14544	CG14544
29	w1118; +; P{GUS-p53.259H}3.1	UAS-p53 point mutation	p53.259H

Table 2. Life span data of apoptosis-related gene experiments, with means, standard deviations, medians, percent change in mean and median, and log rank p value.

CrossMxF	RU486	Genotype	Sex	N	Meana	Median	%Change in Mean	%Change in Median	Log Rank p Value
Exp1 Life span assay using GS255B driver at 29C
8-1	-	w/Y; 255B/+; +	M	115	51.53±8.66	53	---------	---------	---------
	A	w/Y; 255B/+; +	M	120	47.29±11.06	50	-8.23	-5.66	0.001
	L	w/Y; 255B/+; +	M	40	47.28±12.15	51	-8.26	-3.77	0.002
	-	w/w; 255B/+; +	F	128	54.18±8.26	56	---------	---------	---------
	A	w/w; 255B/+; +	F	120	51.74±3.89	52	-4.5	-7.14	4.38E-09
	L	w/w; 255B/+; +	F	120	48.18±8.38	50	-11.07	-10.71	6.30E-11
12-1	-	w/Y; 255B/+; Ras act/+	M	122	51.11±11.58	55.5	---------	---------	---------
	A	w/Y; 255B/+; Ras act/+	M	128	9.45±3.42	10	-81.5	-81.98	0
	L	w/Y; 255B/+; Ras act/+	M	0	NA	NA	---------	---------	---------
	-	w/w*; 255B/+; Ras act/+	F	123	54.19±13.26	59	---------	---------	---------
	A	w/w*; 255B/+; Ras act/+	F	123	12.11±2.8	12	-77.64	-79.66	0
	L	w/w*; 255B/+; Ras act/+	F	0	NA	NA	---------	---------	---------
13-1	-	w/Y; 255B/Ras WT;+	M	124	46.65±9.01	47	---------	---------	---------
	A	w/Y; 255B/Ras WT;+	M	122	42.3±9.13	42	-9.32	-10.64	1.43E-04
	L	w/Y; 255B/Ras WT;+	M	47	42.06±10.37	43	-9.84	-8.51	0.004
	-	w/w*; 255B/Ras WT;+	F	126	51.31±8.64	52	---------	---------	---------
	A	w/w*; 255B/Ras WT;+	F	126	46.84±5.17	46	-8.71	-11.54	8.15E-12
	L	w/w*; 255B/Ras WT;+	F	118	43.66±8.85	46	-14.91	-11.54	0
1-14	-	Ras DN, w/Y; 255B/+; +	M	127	47.89±9.88	50	---------	---------	---------
	A	Ras DN, w/Y; 255B/+; +	M	123	43.64±6.87	44	-8.87	-12	5.78E-08
	L	Ras DN, w/Y; 255B/+; +	M	79	44.76±12.49	48	-6.54	-4	0.14
	-	Ras DN, w/w; 255B/+; +	F	121	51.65±14.25	57	---------	---------	---------
	A	Ras DN, w/w; 255B/+; +	F	125	51.82±8.3	53	0.32	-7.02	5.09E-04
	L	Ras DN, w/w; 255B/+; +	F	125	45.39±13.16	49	-12.12	-14.04	1.98E-09
15-1	-	w/Y; 255B/wga; +	M	130	52.56±8.37	55	---------	---------	---------
	A	w/Y; 255B/wga; +	M	122	29.83±9.32	32	-43.25	-41.82	0
	L	w/Y; 255B/wga; +	M	0	NA	NA	---------	---------	---------
	-	w/w*; 255B/wga; +	F	122	55.78±12.01	60	---------	---------	---------
	A	w/w*; 255B/wga; +	F	125	33.61±10.55	34	-39.75	-43.33	0
	L	w/w*; 255B/wga; +	F	0	NA	NA	---------	---------	---------
16-1	-	w/Y; 255B/+;wgb/+	M	124	52.31±8.81	56	---------	---------	---------
	A	w/Y; 255B/+;wgb/+	M	131	45.02±8.04	47	-13.94	-16.07	0
	L	w/Y; 255B/+;wgb/+	M	0	NA	NA	---------	---------	---------
	-	w/w*; 255B/+;wgb/+	F	120	51.29±10.38	53	---------	---------	---------
	A	w/w*; 255B/+;wgb/+	F	123	47.55±7.23	49	-7.29	-7.55	3.24E-10
	L	w/w*; 255B/+;wgb/+	F	0	NA	NA	---------	---------	---------
17-1	-	w/Y; 255B/+; Pk61Ca/+	M	122	47.9±9.67	47	---------	---------	---------
	L	w/Y; 255B/+; Pk61Ca/+	M	21	42.81±13.89	47	-10.63	0	0.224
	-	w/yw; 255B/+; Pk61Ca/+	F	127	49.82±16.97	56	---------	---------	---------
	L	w/yw; 255B/+; Pk61Ca/+	F	121	50.24±11.55	52	0.84	-7.14	8.57E-04
18-1	-	w/Y; 255B/+; Pk61Cb/+	M	126	57.29±8.23	59	---------	---------	---------
	L	w/Y; 255B/+; Pk61Cb/+	M	24	48.08±10.99	51	-16.06	-13.56	4.11E-12
	-	w; 255B/+; Pk61Cb/+	F	124	54.51±9.89	56.5	---------	---------	---------
	L	w; 255B/+; Pk61Cb/+	F	121	45.28±13.38	51	-16.93	-9.73	1.79E-14
19-1	-	w/Y; 255B/+; DIAP1/+	M	120	56.23±8.68	59	---------	---------	---------
	L	w/Y; 255B/+; DIAP1/+	M	93	47.68±17.07	53	-15.22	-10.17	0.002
	-	w/w*; 255B/+; DIAP1/+	F	120	54.64±11.2	57.5	---------	---------	---------
	L	w/w*; 255B/+; DIAP1/+	F	117	46.74±14.76	51	-14.45	-11.3	1.97E-07
7-1	-	w/Y; 255B/+; +	M	124	52.97±7.68	56	---------	---------	---------
	A	w/Y; 255B/+; +	M	124	49.73±4.6	50	-6.11	-10.71	6.19E-13
	L	w/Y; 255B/+; +	M	22	44.5±16.86	52	-15.99	-7.14	2.94E-05
	-	w/+; 255B/+; +	F	118	58.39±5.25	59	---------	---------	---------
	A	w/+; 255B/+; +	F	122	52.66±4.09	52	-9.81	-11.86	0
	L	w/+; 255B/+; +	F	122	50.45±9.78	53.5	-13.6	-9.32	1.18E-13
Ex 2 Life span assay using GS255B driver at 25C
7-1	-	w/Y; 255B/+; +	M	94	73.17±15.64	78	---------	---------	---------
	A	w/Y; 255B/+; +	M	93	69.97±12.33	72	-4.38	-7.69	5.22E-04
	-	w/+; 255B/+; +	F	92	87.2±18.44	92	---------	---------	---------
	A	w/+; 255B/+; +	F	91	91.93±7.76	94	5.43	2.17	0.940
20-1	-	w/Y; 255B/+; nmo/+	M	95	66.74±16.11	68	---------	---------	---------
	A	w/Y; 255B/+; nmo/+	M	90	64.66±14.3	66	-3.12	-2.94	0.102
	-	w/yw; 255B/+; nmo/+	F	97	67.59±28.66	74	---------	---------	---------
	A	w/yw; 255B/+; nmo/+	F	95	68.79±30.17	80	1.78	8.11	0.878
15-1	-	w/Y; 255B/wga; +	M	96	72.88±10.31	74	---------	---------	---------
	A	w/Y; 255B/wga; +	M	92	53.14±18.01	56	-27.08	-24.32	0
	-	w/w*; 255B/wga; +	F	97	78.89±19.38	84	---------	---------	---------
	A	w/w*; 255B/wga; +	F	97	53.72±22.13	52	-31.9	-38.1	0
17-1	-	w/Y; 255B/+; Pk61Ca/+	M	91	64.11±13.4	64	---------	---------	---------
	A	w/Y; 255B/+; Pk61Ca/+	M	94	62.85±13.08	66	-1.96	3.13	0.555
	-	w/yw; 255B/+; Pk61Ca/+	F	98	70.73±26.23	78	---------	---------	---------
	A	w/yw; 255B/+; Pk61Ca/+	F	94	79.81±23.77	90	12.83	15.38	0.149
21-1	-	w/Y; 255B/+; Egfr/+	M	89	62.38±11.19	66	---------	---------	---------
	A	w/Y; 255B/+; Egfr/+	M	97	62.06±9.18	64	-0.51	-3.03	0.166
	-	w/y w*; 255B/+; Egfr/+	F	95	65.71±21.16	68	---------	---------	---------
	A	w/y w*; 255B/+; Egfr/+	F	100	63.52±17.9	65	-3.33	-4.41	0.076
19-1	-	w/Y; 255B/+; DIAP1/+	M	102	76.57±13.04	78	---------	---------	---------
	A	w/Y; 255B/+; DIAP1/+	M	94	73.4±9.53	74	-4.13	-5.13	0.002
	-	w/w*; 255B/+; DIAP1/+	F	98	78.9±18.26	84	---------	---------	---------
	A	w/w*; 255B/+; DIAP1/+	F	95	81.39±19.17	88	3.16	4.76	0.011
22-1	-	w/Y; 255B/+; pnt/+	M	96	62.6±9.74	64	---------	---------	---------
	A	w/Y; 255B/+; pnt/+	M	94	59.15±10.7	60	-5.52	-6.25	0.077
	-	w/yw; 255B/+; pnt/+	F	92	74.32±27.19	85	---------	---------	---------
	A	w/yw; 255B/+; pnt/+	F	95	79.77±20.03	88	7.34	3.53	0.402
Exp3 Life span assay using GS255B driver at 25C
7-1	-	w/Y; 255B/+; +	M	100	81.01±15.38	86	---------	---------	---------
	A	w/Y; 255B/+; +	M	92	80.46±10.42	82	-0.68	-4.65	0.039
	-	w/+; 255B/+; +	F	85	92.49±11.86	94	---------	---------	---------
	A	w/+; 255B/+; +	F	99	92.05±13.07	94	-0.48	0	0.571
13-1	-	w/Y; 255B/Ras WT;+	M	95	75.87±12.26	78	---------	---------	---------
	A	w/Y; 255B/Ras WT;+	M	98	67.92±14.13	70	-10.48	-10.26	1.62E-06
	-	w/w*; 255B/Ras WT;+	F	96	83.43±12.26	86	---------	---------	---------
	A	w/w*; 255B/Ras WT;+	F	98	79.59±10.58	82	-4.6	-4.65	0.001
23-1	-	w/Y; 255B/Mmp2; +	M	96	69.77±13.03	70	---------	---------	---------
	A	w/Y; 255B/Mmp2; +	M	96	68.81±10.06	70	-1.37	0	0.117
	-	w/yw; 255B/Mmp2; +	F	98	85.94±18.45	91	---------	---------	---------
	A	w/yw; 255B/Mmp2; +	F	101	84.85±18.67	90	-1.27	-1.1	0.109
24-1	-	w/Y; 255B/+; Stat/+	M	96	64.31±10.06	65	---------	---------	---------
	A	w/Y; 255B/+; Stat/+	M	99	65.08±13.33	68	1.19	4.62	0.325
	-	w/yw; 255B/+; Stat/+	F	99	70.48±21.48	78	---------	---------	---------
	A	w/yw; 255B/+; Stat/+	F	96	62.29±24.99	74	-11.62	-5.13	0.076
25-1	-	w/Y; 255B/+; puc/+	M	97	70.78±14.98	70	---------	---------	---------
	A	w/Y; 255B/+; puc/+	M	96	68.96±13.62	68	-2.58	-2.86	0.269
	-	w/w*; 255B/+; puc/+	F	84	94.07±15.00	98	---------	---------	---------
	A	w/w*; 255B/+; puc/+	F	97	98.1±7.86	100	4.29	2.04	0.135
Exp4 Life span assay using GS255B driver at 25C
7-1	-	w/Y; 255B/+; +	M	92	73.76±18.31	78	---------	---------	---------
	A	w/Y; 255B/+; +	M	86	71.84±10.87	74	-2.61	-5.13	1.43E-04
	-	w/+; 255B/+; +	F	86	86.28±15.46	90	---------	---------	---------
	A	w/+; 255B/+; +	F	101	86.18±10.02	88	-0.12	-2.22	0.035
26-1	-	w/Y; 255B/+; Sk2/+	M	90	67.22±16.97	72	---------	---------	---------
	A	w/Y; 255B/+; Sk2/+	M	95	69.85±12.26	72	3.91	0	0.953
	-	w/yw; 255B/+; Sk2/+	F	101	73.29±24.6	84	---------	---------	---------
	A	w/yw; 255B/+; Sk2/+	F	106	78.08±19.61	86	6.53	2.38	0.84
27-1	-	w/Y; 255B/+; ban/+	M	98	66.59±21.91	70	---------	---------	---------
	A	w/Y; 255B/+; ban/+	M	95	61.56±18.15	62	-7.56	-11.43	0.003
	-	w/yw; 255B/+; ban/+	F	94	76.36±28.78	88	---------	---------	---------
	A	w/yw; 255B/+; ban/+	F	96	81.56±18.03	88	6.81	0	0.023
28-1	-	w/Y; 255B/+;CG14544/+	M	91	75.03±12.8	76	---------	---------	---------
	A	w/Y; 255B/+;CG14544/+	M	97	77.01±9.82	78	2.64	2.63	0.844
	-	w/w; 255B/+;CG14544/+	F	101	70.99±26.75	82	---------	---------	---------
	A	w/w; 255B/+;CG14544/+	F	96	69.33±20.57	79	-2.33	-3.66	2.28E-05
Exp5 Life span assay using GS255B driver, and MHC GS driver at 25C
7-1	-	w/Y; 255B/+; +	M	98	75.06±11.65	79	---------	---------	---------
	A	w/Y; 255B/+; +	M	97	73.96±12.27	78	-1.47	-1.27	0.161
	-	w/+; 255B/+; +	F	100	87.88±7.74	88	---------	---------	---------
	A	w/+; 255B/+; +	F	101	85.33±12.78	88	-2.91	0	0.014
8-1	-	w/Y; 255B/+; +	M	99	66.55±11.82	68	---------	---------	---------
	A	w/Y; 255B/+; +	M	97	69.84±10.57	72	4.94	5.88	0.14
	-	w/w; 255B/+; +	F	100	79.6±14.08	84	---------	---------	---------
	A	w/w; 255B/+; +	F	97	81.69±3.82	82	2.63	-2.38	0.005
17-1	-	w/Y; 255B/+; Pk61Ca/+	M	99	64.87±12.41	64	---------	---------	---------
	A	w/Y; 255B/+; Pk61Ca/+	M	98	62.16±12.33	64	-4.17	0	0.113
	-	w/yw; 255B/+; Pk61Ca/+	F	98	81.96±13.91	86	---------	---------	---------
	A	w/yw; 255B/+; Pk61Ca/+	F	99	80.46±11.15	82	-1.82	-4.65	0.001
18-1	-	w/Y; 255B/+; Pk61Cb/+	M	100	71.2±9.32	74	---------	---------	---------
	A	w/Y; 255B/+; Pk61Cb/+	M	101	73.33±8.19	74	2.99	0	0.135
	-	w; 255B/+; Pk61Cb/+	F	98	80.27±12.63	82	---------	---------	---------
	A	w; 255B/+; Pk61Cb/+	F	100	82.02±4.89	82	2.19	0	0.399
19-1	-	w/Y; 255B/+; DIAP1/+	M	101	72.97±11.13	76	---------	---------	---------
	A	w/Y; 255B/+; DIAP1/+	M	101	71.15±11.37	74	-2.5	-2.63	0.425
	-	w/w*; 255B/+; DIAP1/+	F	98	83.02±7.52	84	---------	---------	---------
	A	w/w*; 255B/+; DIAP1/+	F	106	79.47±12.78	82	-4.27	-2.38	0.011
7-6	-	yw/Y;+/CyO; MHC/+	M	96	71.31±13.18	76	---------	---------	---------
	A	yw/Y;+/CyO; MHC/+	M	98	58.92±12.84	60	-17.38	-21.05	0
	-	yw/+;+/CyO; MHC/+	F	96	78.04±15.76	84	---------	---------	---------
	A	yw/+;+/CyO; MHC/+	F	114	52.84±13.75	58	-32.29	-30.95	0
8-6	-	yw/Y;+/CyO; MHC/+	M	99	56.42±16.69	60	---------	---------	---------
	A	yw/Y;+/CyO; MHC/+	M	92	43.26±15	48	-23.33	-20	1.61E-12
	-	yw/w;+/CyO; MHC/+	F	93	60.73±18.82	68	---------	---------	---------
	A	yw/w;+/CyO; MHC/+	F	99	45.21±13.63	50	-25.55	-26.47	2.22E-16
17-6	-	yw/Y;+/CyO; MHC/Pk61Ca	M	100	54.32±14.66	58	---------	---------	---------
	A	yw/Y;+/CyO; MHC/Pk61Ca	M	101	52.46±12.42	56	-3.43	-3.45	0.004
	-	yw;+/CyO; MHC/Pk61Ca	F	100	61.3±19.84	65	---------	---------	---------
	A	yw;+/CyO; MHC/Pk61Ca	F	99	39.37±14.22	36	-35.77	-44.62	0
18-6	-	yw/Y;+/CyO; MHC/Pk61Cb	M	96	56.44±18.89	62	---------	---------	---------
	A	yw/Y;+/CyO; MHC/Pk61Cb	M	108	53.56±10.21	56	-5.11	-9.68	1.75E-07
	-	yw/w;+/CyO; MHC/Pk61Cb	F	96	55.25±21.21	56	---------	---------	---------
	A	yw/w;+/CyO; MHC/Pk61Cb	F	90	39.42±12.45	38	-28.65	-32.14	1.40E-11
19-6	-	yw/Y;+/CyO; MHC/DIAP1	M	98	66.71±12.27	68	---------	---------	---------
	A	yw/Y;+/CyO; MHC/DIAP1	M	98	54.92±10.4	56	-17.68	-17.65	6.66E-15
	-	yw/w*;+/CyO;MHC/DIAP1	F	108	64.89±18.19	71	---------	---------	---------
	A	yw/w*;+/CyO;MHC/DIAP1	F	104	57.04±14.85	58	-12.1	-18.31	6.72E-08
7-6	-	yw/Y;+/Sp; MHC/+	M	99	68.67±13.38	74	---------	---------	---------
	A	yw/Y;+/Sp; MHC/+	M	98	61.76±12.62	64	-10.07	-13.51	5.34E-08
	-	yw/+;+/Sp; MHC/+	F	94	73.66±13.99	78	---------	---------	---------
	A	yw/+;+/Sp; MHC/+	F	102	59.98±8.45	62	-18.57	-20.51	0
8-6	-	yw/Y;+/Sp; MHC/+	M	96	61.6±14.81	66	---------	---------	---------
	A	yw/Y;+/Sp; MHC/+	M	94	54.55±15.5	56	-11.45	-15.15	2.47E-06
	-	yw/w;+/Sp; MHC/+	F	93	58.41±17.34	62	---------	---------	---------
	A	yw/w;+/Sp; MHC/+	F	102	47.59±12.97	52	-18.53	-16.13	2.70E-14
17-6	-	yw/Y;+/Sp; MHC/Pk61Ca	M	95	49.31±12.75	54	---------	---------	---------
	A	yw/Y;+/Sp; MHC/Pk61Ca	M	100	45.14±11.03	48	-8.45	-11.11	1.95E-04
	-	yw;+/Sp; MHC/Pk61Ca	F	99	42.99±20.17	40	---------	---------	---------
	A	yw;+/Sp; MHC/Pk61Ca	F	100	35.78±16.13	30	-16.77	-25	0.003
18-6	-	yw/Y;+/Sp; MHC/Pk61Cb	M	100	56.36±12.56	60	---------	---------	---------
	A	yw/Y;+/Sp; MHC/Pk61Cb	M	98	51.06±9.77	52	-9.4	-13.33	2.13E-06
	-	yw/w;+/Sp; MHC/Pk61Cb	F	97	56.6±16.25	60	---------	---------	---------
	A	yw/w;+/Sp; MHC/Pk61Cb	F	94	41.79±11.81	42	-26.17	-30	1.67E-15
19-6	-	yw/Y;+/Sp; MHC/DIAP1	M	99	61.21±10.04	62	---------	---------	---------
	A	yw/Y;+/Sp; MHC/DIAP1	M	97	55.51±9.17	58	-9.32	-6.45	5.02E-08
	-	yw/w*;+/Sp; MHC/DIAP1	F	103	71.13±17.02	78	---------	---------	---------
	A	yw/w*;+/Sp; MHC/DIAP1	F	101	62.3±13.23	66	-12.41	-15.38	6.66E-16

Table 3. Life span data for baculovirus p35 experiments, with means, standard deviations, medians, percent change in mean and median, and log rank p value.

CrossMxF	RU486	Genotype	Sex	N	Meana	Median	%Change in Mean	%Change in Median	Log Rank p Value
Exp1 Life span assay of three UAS-p35 lines and UAS-p53.259H with GS255B driver at 25C
7-1	-	w/Y; 255B/+; +	M	120	84.6±14.25	90	---------	---------	---------
	A	w/Y; 255B/+; +	M	119	83.08±10.94	86	-1.8	-4.44	0.014
	L	w/Y; 255B/+; +	M	123	78.44±22.48	86	-7.28	-4.44	0.244
	-	w/+; 255B/+; +	F	116	92.02±9.64	94	---------	---------	---------
	A	w/+; 255B/+; +	F	121	94.69±8.61	94	2.9	0	0.009
	L	w/+; 255B/+; +	F	124	91.97±15.74	94	-0.05	0	0.047
1-9	-	p35,w*/Y; 255B/+; +	M	120	68.93±12.49	70	---------	---------	---------
	A	p35,w*/Y; 255B/+; +	M	123	68.62±11.76	70	-0.45	0	0.681
	L	p35,w*/Y; 255B/+; +	M	98	33.1±22.91	26	-51.98	-62.86	0
	-	p35,w*/+; 255B/+; +	F	122	83.28±15.13	86	---------	---------	---------
	A	p35,w*/+; 255B/+; +	F	130	77.15±20.92	82	-7.36	-4.65	0.11
	L	p35,w*/+; 255B/+; +	F	125	57.52±35.8	70	-30.93	-18.6	0.001
10-1	-	w/Y; 255B/p35; +	M	117	54.48±13	54	---------	---------	---------
	A	w/Y; 255B/p35; +	M	121	57.26±8.45	58	5.1	7.41	0.583
	L	w/Y; 255B/p35; +	M	110	34.25±19.5	34	-37.13	-37.04	5.60E-10
	-	w/w*; 255B/p35; +	F	120	64.05±14.63	66	---------	---------	---------
	A	w/w*; 255B/p35; +	F	126	60.79±16.68	66	-5.09	0	0.188
	L	w/w*; 255B/p35; +	F	123	49.37±32.2	54	-22.92	-18.18	0.436
11-1	-	w/Y; 255B/+; p35/+	M	133	86.03±12.51	90	---------	---------	---------
	A	w/Y; 255B/+; p35/+	M	122	81.31±13.87	84	-5.49	-6.67	8.92E-06
	L	w/Y; 255B/+; p35/+	M	56	46.18±24.21	44	-46.32	-51.11	0
	-	w/w*; 255B/+; p35/+	F	126	87.54±10.04	90	---------	---------	---------
	A	w/w*; 255B/+; p35/+	F	127	82.19±9.91	82	-6.11	-8.89	8.60E-05
	L	w/w*; 255B/+; p35/+	F	126	64.63±29.62	75	-26.17	-16.67	4.67E-07
29-1	-	w/Y; 255B/+;p53.259H/+	M	118	71.54±13.86	72	---------	---------	---------
	A	w/Y; 255B/+;p53.259H/+	M	125	68.90±10.41	70	-3.70	-2.78	0.002
	L	w/Y; 255B/+;p53.259H/+	M	119	67.73±16.92	70	-5.33	-2.78	0.069
	-	w; 255B/+; p53.259H/+	F	119	75.40±8.50	76	---------	---------	---------
	A	w; 255B/+; p53.259H/+	F	119	80.66±10.98	82	6.98	7.89	4.05E-08
	L	w; 255B/+; p53.259H/+	F	125	70.24±22.02	76	-6.84	0	0.202
Exp2 Life span assay of three UAS-p35 lines with head FB driver, whole body FB driver and GS255A driver at 25C
3-7	-	+/Y; S32/+; +	M	75	59.23±14.11	64	---------	---------	---------
	A	+/Y; S32/+; +	M	63	55.21±15.74	60	-6.79	-6.25	0.013
	-	w/+; S32/+; +	F	111	59.91±18.96	60	---------	---------	---------
	A	w/+; S32/+; +	F	115	63.77±17.71	66	6.45	10	0.263
3-9	-	p35,w*/Y; S32/+; +	M	122	62.69±10.62	64	---------	---------	---------
	A	p35,w*/Y; S32/+; +	M	105	58.3±13.45	60	-6.99	-6.25	0.022
	-	p35,w*/w; S32/+; +	F	112	59.95±25	72	---------	---------	---------
	A	p35,w*/w; S32/+; +	F	108	59±24.86	68	-1.58	-5.56	0.974
3-10	-	w*/Y; S32/p35; +	M	123	45.19±7.61	46	---------	---------	---------
	A	w*/Y; S32/p35; +	M	120	41.52±6.84	42	-8.12	-8.7	1.96E-04
	-	w*/w; S32/p35; +	F	121	61.62±8.71	62	---------	---------	---------
	A	w*/w; S32/p35; +	F	105	63.28±10.6	66	2.69	6.45	0.036
3-11	-	w*/Y; S32/+; p35/+	M	125	62.67±12.41	64	---------	---------	---------
	A	w*/Y; S32/+; p35/+	M	125	60.78±14.07	62	-3.03	-3.13	0.174
	-	w*/w; S32/+; p35/+	F	109	68.44±17.09	74	---------	---------	---------
	A	w*/w; S32/+; p35/+	F	113	70.52±18.12	76	3.04	2.7	0.043
4-7	-	+/Y; S106 S32/+; +	M	116	54.12±9.89	56	---------	---------	---------
	A	+/Y; S106 S32/+; +	M	118	52.68±9.77	52	-2.67	-7.14	0.208
	-	w/+; S106 S32/+; +	F	110	58.82±14.95	62	---------	---------	---------
	A	w/+; S106 S32/+; +	F	120	58.07±16.45	63	-1.28	1.61	0.569
4-9	-	p35,w*/Y; S106 S32/+; +	M	121	47.21±8.48	46	---------	---------	---------
	A	p35,w*/Y; S106 S32/+; +	M	110	47.67±10.28	48	0.99	4.35	0.263
	-	p35,w*/w; S106 S32/+; +	F	119	55.18±22.95	66	---------	---------	---------
	A	p35,w*/w; S106 S32/+; +	F	126	47.79±24.9	62	-13.38	-6.06	0.01
4-10	-	w*/Y; S106 S32/p35; +	M	125	33.39±4.44	34	---------	---------	---------
	A	w*/Y; S106 S32/p35; +	M	125	32.3±6.16	32	-3.26	-5.88	0.475
	-	w*/w; S106 S32/p35; +	F	121	49.55±8.14	50	---------	---------	---------
	A	w*/w; S106 S32/p35; +	F	121	50.84±8.85	50	2.6	0	0.107
4-11	-	w*/Y; S106 S32/+; p35/+	M	125	47.15±6.81	48	---------	---------	---------
	A	w*/Y; S106 S32/+; p35/+	M	117	48.6±8.42	48	3.07	0	0.072
	-	w*/w; S106 S32/+; p35/+	F	125	56.81±13.02	60	---------	---------	---------
	A	w*/w; S106 S32/+; p35/+	F	116	60.69±11.7	64	6.83	6.67	0.004
2-7	-	+/Y; 255A/+; +	M	114	66.04±8.95	67	---------	---------	---------
	A	+/Y; 255A/+; +	M	117	58.97±15.36	62	-10.69	-7.46	1.48E-05
	-	w/+; 255A/+; +	F	114	72.65±13.95	78	---------	---------	---------
	A	w/+; 255A/+; +	F	116	75.02±13.19	78	3.26	0	0.064
2-9	-	p35,w*/Y; 255A/+; +	M	111	65.98±14.65	66	---------	---------	---------
	A	p35,w*/Y; 255A/+; +	M	115	59.82±13.31	60	-9.34	-9.09	3.78E-05
	-	p35,w*/w; 255A/+; +	F	113	58.95±20.26	64	---------	---------	---------
	A	p35,w*/w; 255A/+; +	F	117	69.21±17.4	72	17.4	12.5	1.32E-06
2-10	-	w*/Y;255A/p35; +	M	113	48.98±9.74	48	---------	---------	---------
	A	w*/Y;255A/p35; +	M	125	47.66±7.19	48	-2.69	0	0.03
	-	w*/w; 255A/p35; +	F	115	60.57±16.71	66	---------	---------	---------
	A	w*/w; 255A/p35; +	F	118	62±17.79	70	2.35	6.06	0.052
2-11	-	w*/Y; 255A/+; p35/+	M	115	63.66±11.4	64	---------	---------	---------
	A	w*/Y; 255A/+; p35/+	M	114	64.92±9.41	64	1.98	0	0.776
	-	w*/w; 255A/+; p35/+	F	120	67.05±11.58	70	---------	---------	---------
	A	w*/w; 255A/+; p35/+	F	120	68.75±9.08	70	2.54	0	0.41
Exp3 Life span assay of two UAS-p35 lines with whole body FB driver at 29C
7-4	-	w/Y; S106 S32/+; +	M	124	49.15±12.5	54	---------	---------	---------
	L	w/Y; S106 S32/+; +	M	121	49.11±10.75	52	-0.08	-3.7	0.655
	-	w/+; S106 S32/+; +	F	121	51.95±10.82	54	---------	---------	---------
	L	w/+; S106 S32/+; +	F	118	55.29±10.06	60	6.42	11.11	0.029
8-4	-	w/Y; S106 S32/+; +	M	121	47.16±10.27	48	---------	---------	---------
	L	w/Y; S106 S32/+; +	M	118	42.85±12.89	44	-9.14	-8.33	0.002
	-	w/w; S106 S32/+; +	F	124	50.48±11.91	56	---------	---------	---------
	L	w/w; S106 S32/+; +	F	125	51.63±8.47	54	2.27	-3.57	0.196
10-4	-	w/Y; S106 S32/p35; +	M	121	50.43±6.73	52	---------	---------	---------
	L	w/Y; S106 S32/p35; +	M	121	46.5±8.28	48	-7.8	-7.69	4.23E-05
	-	w*/w; S106 S32/p35; +	F	120	50.4±12.84	56	---------	---------	---------
	L	w*/w; S106 S32/p35; +	F	129	48.57±8.9	50	-3.62	-10.71	1.45E-05
11-4	-	w/Y; S106 S32/+; p35/+	M	126	44.03±6.5	46	---------	---------	---------
	L	w/Y; S106 S32/+; p35/+	M	122	41.92±10.42	46	-4.8	0	0.208
	-	w*/w; S106 S32/+; p35/+	F	122	58.03±6.59	60	---------	---------	---------
	L	w*/w; S106 S32/+; p35/+	F	124	54.81±8.05	56	-5.56	-6.67	8.84E-07
Exp4 Life span assay of two UAS-p35 lines with Elav driver at 29C
7-5	-	yw/Y; +/+; Elav/+	M	131	53.92±7.15	54	---------	---------	---------
	A	yw/Y; +/+; Elav/+	M	129	52.33±8.14	53	-2.95	-1.85	0.083
	L	yw/Y; +/+; Elav/+	M	59	35.85±10.58	38	-33.51	-29.63	0
	-	yw/+; +/+; Elav/+	F	127	58.15±7.22	60	---------	---------	---------
	A	yw/+; +/+; Elav/+	F	129	57.11±5.19	58	-1.79	-3.33	0.013
	L	yw/+; +/+; Elav/+	F	120	43.46±7.94	44	-25.26	-26.67	0
8-5	-	yw/Y; +/+; Elav/+	M	126	44.08±8.36	44.5	---------	---------	---------
	A	yw/Y; +/+; Elav/+	M	120	43.32±8.76	45	-1.73	1.12	0.186
	L	yw/Y; +/+; Elav/+	M	102	26.24±8.51	26	-40.48	-41.57	0
	-	yw/w; +/+; Elav/+	F	124	46.88±9.92	50	---------	---------	---------
	A	yw/w; +/+; Elav/+	F	124	48.13±7.5	49.5	2.67	-1	0.406
	L	yw/w; +/+; Elav/+	F	114	34.82±10.34	36	-25.73	-28	0
10-5	-	yw/Y; p35/+; Elav/+	M	125	42.34±6.38	44	---------	---------	---------
	A	yw/Y; p35/+; Elav/+	M	122	43.34±10.34	46	2.38	4.55	0.007
	L	yw/Y; p35/+; Elav/+	M	9	20.89±10.3	26	-50.66	-40.91	0
	-	yw/w*; p35/+; Elav/+	F	121	49±10.63	52	---------	---------	---------
	A	yw/w*; p35/+; Elav/+	F	126	50.16±6.4	51	2.36	-1.92	0.014
	L	yw/w*; p35/+; Elav/+	F	9	28.22±10.27	32	-42.4	-38.46	1.60E-14
11-5	-	yw/Y; +; Elav/p35	M	120	51.24±10.46	54	---------	---------	---------
	A	yw/Y; +; Elav/p35	M	121	48.62±10.1	52	-5.12	-3.7	1.22E-06
	L	yw/Y; +; Elav/p35	M	1	10±NA	10	-80.48	-81.48	5.60E-10
	-	yw/w*; +; Elav/p35	F	118	56.77±3.89	58	---------	---------	---------
	A	yw/w*; +; Elav/p35	F	131	52.67±5.08	54	-7.22	-6.9	3.51E-13
	L	yw/w*; +; Elav/p35	F	0	NA	NA	---------	---------	---------
Exp5 Life span assay of two UAS-p35 lines with GS255B driver at 25C
8-1	-	w/Y; 255B/+; +	M	121	62.33±18.12	68	---------	---------	---------
	L	w/Y; 255B/+; +	M	119	62.57±16.22	68	0.39	0	0.478
	L1-10	w/Y; 255B/+; +	M	120	66.02±19.38	72	5.91	5.88	7.82E-04
	-	w/w; 255B/+; +	F	123	75.95±9.37	78	---------	---------	---------
	L	w/w; 255B/+; +	F	124	69.02±12.88	74	-9.13	-5.13	7.69E-07
	L1-10	w/w; 255B/+; +	F	124	78.18±9.17	80	2.93	2.56	7.84E-04
10-1	-	w/Y; 255B/p35; +	M	111	56.32±25.51	66	---------	---------	---------
	L	w/Y; 255B/p35; +	M	4	16±21.6	7	-71.59	-89.39	6.47E-05
	L1-10	w/Y; 255B/p35; +	M	117	58.56±17.95	62	3.98	-6.06	0.528
	-	w/w*; 255B/p35; +	F	119	68.47±13.26	72	---------	---------	---------
	L	w/w*; 255B/p35; +	F	30	27.47±16.58	24	-59.89	-66.67	0
	L1-10	w/w*; 255B/p35; +	F	124	64.5±16.45	70	-5.8	-2.78	0.757
11-1	-	w/Y; 255B/+; p35/+	M	117	66.15±9.97	68	---------	---------	---------
	L	w/Y; 255B/+; p35/+	M	1	14±NA	14	-78.84	-79.41	3.38E-14
	L1-10	w/Y; 255B/+; p35/+	M	123	64.98±15.73	70	-1.78	2.94	0.099
	-	w/w*; 255B/+; p35/+	F	123	74.41±5.98	76	---------	---------	---------
	L	w/w*; 255B/+; p35/+	F	0	NA	NA	---------	---------	---------
	L1-10	w/w*; 255B/+; p35/+	F	123	74.37±11.95	78	-0.04	2.63	0.003
Exp6 Life span assay of two UAS-p35 lines with Elav driver at 25C
8-5	-	yw/Y; +/+; Elav/+	M	108	61.69±17.95	67	---------	---------	---------
	L1-10	yw/Y; +/+; Elav/+	M	115	54.17±15.55	58	-12.18	-13.43	1.80E-08
	-	yw/w; +/+; Elav/+	F	120	57.42±14.79	64	---------	---------	---------
	L1-10	yw/w; +/+; Elav/+	F	117	56.41±10.82	58	-1.75	-9.38	0.004
10-5	-	yw/Y; p35/+; Elav/+	M	121	46.6±7.2	46	---------	---------	---------
	L1-10	yw/Y; p35/+; Elav/+	M	115	37.81±9.05	38	-18.86	-17.39	7.06E-14
	-	yw/w*; p35/+; Elav/+	F	123	50.63±15.32	54	---------	---------	---------
	L1-10	yw/w*; p35/+; Elav/+	F	121	49.19±12.16	50	-2.85	-7.41	0.035
11-5	-	yw/Y; +; Elav/p35	M	120	54.32±13.04	56	---------	---------	---------
	L1-10	yw/Y; +; Elav/p35	M	111	52.31±10.98	52	-3.7	-7.14	0.037
	-	yw/w*; +; Elav/p35	F	123	52.7±13.97	54	---------	---------	---------
	L1-10	yw/w*; +; Elav/p35	F	118	56.63±10.73	58	7.45	7.41	0.091

Table 4. Parameters for Gompertz-Makeham model and likelihood ratio test results.

	Parameters	L	-		chi2	df	p Value	chi2	df	p Value
Females
		one parameter compared at each time
p35 (X)								Both a and b are constrained
	a	5.39 x 10-9	3.96 x 10-7	1.789		1	0.181
	b	3.89 x 10-1	3.00 x 10-1	1.516		1	0.218
	c	2.08 x 10-2	1.63 x 10-3	59.967		1	<0.001	57.983	1	<0.001
p35 (2)								b is constrained
	a	7.71 x 10-6	2.10 x 10-4	5.234		1	0.022	50.203	1	<0.001
	b	2.41 x 10-1	1.92 x 10-1	1.700		1	0.192
	c	2.52 x 10-2	1.37 x 10-3	50.610		1	<0.001	50.154	1	<0.001
p35 (3)								Both a and b are constrained
	a	3.31 x 10-6	3.00 x 10-6	0.003		1	0.958
	b	2.50 x 10-1	2.46 x 10-1	0.009		1	0.923
	c	1.36 x 10-2	1.80 x 10-4	46.090		1	<0.001	66.787		<0.001

Acknowledgments

We thank Morris Waskar for constructing the "WB-FB-GS" driver strain, and thank Scott Pletcher for advice on use of WinModest, and Laurent Seroude for helpful comments. This work was supported by grants from the Department of Health and Human Services to JT (AG11833) and to ST (HG02790).

Conflicts of Interest

The authors in this manuscript have no conflict of interest to declare.

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