Research Paper Volume 14, Issue 7 pp 2989—3029

The tobacco phosphatidylethanolamine-binding protein NtFT4 increases the lifespan of Drosophila melanogaster by interacting with the proteostasis network


Figure 5. Transfection and heat stress response of heat shock proteins in S2 cells. Expression of stress-responsive (Hsp22, Hsp23, Hsp26, Hsp27, Hsp70Aa and Hsc70–4) (A) and non-responsive (HspB8 and l(2)efl) (B) heat shock protein genes in S2 cells after transient transfection with HA-EGFP, HA-NtFT4 or HA-EGFP-NtFT4 compared to non-transfected cells. After transfection and induction of gene expression, cells were cultivated at 27°C (–, white bars) or stressed by heat shock at 37°C for 1 h (+, gray bars show controls and red bars show NtFT4). Relative gene expression was analyzed by qRT-PCR using Gapdh2 as a reference. Data are means ± SEM (n = 3). Significance was tested by one-way ANOVA and Tukey’s post hoc test for responses to transfection (untransfected vs. HA-EGFP vs. HA-NtFT4 vs. HA-EGFP-NtFT4; a = significant compared to nontransfected cells, p < 0.1; b = significant compared to nontransfected cells, p < 0.05; Abbreviation: NS: not significant including all remaining comparisons) and using a t-test for pairwise comparisons of individual responses to heat shock (****p < 0.001, ***p < 0.01, **p < 0.05, *p < 0.1, Abbreviation: NS: not significant). (C) Immunodetection of HSP26 following the transient transfection of S2 cells with HA-EGFP, HA-NtFT4 or HA-EGFP-NtFT4 compared with nontransfected cells. The response of HSP26 to transfection and to heat shock at 37°C was analyzed 1 h after treatment by extracting proteins for immunodetection using anti-HSP26 antibodies (top right). The transient expression of HA-EGFP, HA-NtFT4 or HA-EGFP-NtFT4 was confirmed using anti-HA antibodies (bottom, arrowheads). All p-values are provided in Supplementary Table 9.