Figure 4. Upd-JAK-STAT signaling is affected with loss of Lin28. (A) In-situ hybridization of young (0-1 day old) testis. upd RNA level is decreased in lin28 mutant testis relative to wild-type and increase in upd levels in hub specific expression of Lin28. Scale 10µm. (B) quantitative RT-PCR of young (0-1day old) testis, upd mRNA level is decreased in lin28 mutants relative to wild-type. (C) RT-qPCR of young vs old testis showing reduced levels of upd mRNA in WT testis with age, while, maintained levels of upd in overexpression of Lin28 with age. (D) Young (0-1-day old) testis stained with Zfh1, TJ and FasIII antibodies. Zfh1 level, but not TJ and FasIII, is reduced in lin28 mutants. Scale 10µm. (E) Quantification of Zfh1 levels in CySCs shows reduced Zfh1 levels in mutants. [wildtype, n= 197; lin28EP, n=220; lin28df30, n= 185]. (F) Comparison of Zfh1 levels in young and old WT testes indicating that with age Zfh1 level goes down due to loss in STAT activity. [WT young, n= 197; WT old, n= 145]. (G) 50-day old testes stained with Zfh1, TJ and FasIII antibodies showing reduced levels of Zfh1 with age. Scale 10µm. (H) Comparison of Zfh1 levels in WT vs mutant [ wildtype, n= 145; lin28df30, n=125] (I) STAT92E protein level is reduced in the mutants. Scale 10µm. (J) Comparison of STAT92E levels in WT (n=115) vs mutants (lin28EP n=92; lin28df30, n=97). STAT92E protein level is reduced in lin28 mutants; * depicts the hub area. ** denotes P<0.01, *** denotes P<0.001, **** denotes P<0.0001, ns denotes not significant.