Figure 6. Defining the selectivity for HSF1 activating compounds identified through reporter based HTS.

A. Plot showing residuals calculated by comparing the expression of our stress-responsive gene panel between HEK293T cells treated with compound A3 (10 μM, 4 h) or vehicle. Calculation of residuals was performed as described in Fig. 2A. Genes are grouped by target stress-responsive signaling pathway. Statistics were calculated using one-way ANOVA. Significance shown reflects comparison to “Other” target transcript set. ****p<0.0001. See Table S3 for full ANOVA table.

B. Plot showing residuals calculated by comparing the expression of our stress-responsive gene panel between HEK293T cells treated with compound C1 (10 μM, 4 h) or vehicle. Calculation of residuals was performed as described in Fig. 2A. Genes are grouped by target stress-responsive signaling pathway. Statistics were calculated using one-way ANOVA. Significance shown reflects comparison to “Other” target transcript set. ****p<0.0001. See Table S3 for full ANOVA table.

C. Plot showing residuals calculated by comparing the expression of our stress-responsive gene panel between HEK293T cells treated with compound D1 (10 μM, 4 h) or vehicle. Calculation of residuals was performed as described in Fig. 2A. Genes are grouped by target stress-responsive signaling pathway. Statistics were calculated using one-way ANOVA. Significance shown reflects comparison to “Other” target transcript set. ****p<0.0001. See Table S3 for full ANOVA table.

D. Plot showing residuals calculated by comparing the expression of our stress-responsive gene panel between HEK293T cells treated with compound F1 (10 μM, 4 h) or vehicle. Calculation of residuals was performed as described in Fig. 2A. Genes are grouped by target stress-responsive signaling pathway. Statistics were calculated using one-way ANOVA. Significance shown reflects comparison to “Other” target transcript set. *p<0.05. See Table S3 for full ANOVA table.

E. Heat map of fold-change transcript levels from whole-transcriptome RNAseq (relative to vehicle treated cells) for the 100 genes most significantly altered by A3 treatment in HEK293T cells (82 positive regulation, top; 10 negative regulation, bottom). The changes in these genes are also shown for HEK293T cells treated with C1, D1, and F4 (10uM for 4 hours).

F. Venn diagrams showing overlap of upregulated genes from whole-transcriptome RNAseq of HEK293T cells treated with A3 (10μM 4 hours) and HeLa cells under heat shock (left)⁶, or dox-inducible cHSF1 (right) in HEK293^TREX cells¹⁷. Select genes identified in the overlap are highlighted.

G. Numbers of genes from GO analysis of significantly altered transcripts in HEK293T cells treated with compound A3 (10 μM, 4 h) relative to vehicle-treated cells. GO analysis was performed using David⁶¹. Entire GO analysis is reported in Supplementary Table S5.