Research Paper Volume 14, Issue 7 pp 2989—3029

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.