Figure 6. Hsf1 and 17-AAG induce transcriptional changes and promote proteostasis maintenance in ex vivo cultured HSCs.

(A) Strategy to compare gene expression profiles in Hsf1^fl/fl (WT) and Hsf1^fl/fl;Mx1-Cre⁺ (Hsf1^−/−) HSCs cultured for 4h in the presence or absence of 17-AAG. (B) Heat map showing differentially expressed transcripts (≥1.5-fold change; Padj<0.05) between WT and Hsf1^−/− HSCs cultured for 4h with 17-AAG or DMSO (n=2). (C) Volcano plot showing differentially expressed transcripts in cultured (4h) WT and Hsf1^−/− HSCs. Genes significantly upregulated in WT HSCs are shown in blue and in Hsf1^−/− HSCs are shown in red. (D) Heat map showing the subset of genes whose expression is significantly upregulated (≥1.5-fold change; Padj<0.05) in WT as compared to Hsf1^−/− cultured HSCs and that exhibit further increased expression (≥1.1-fold) in WT HSCs cultured with 17-AAG. (E) Protein synthesis in WT and Hsf1^−/− CD48⁻LSK cells cultured for 4h in basic HSC medium supplemented with DMSO or 17-AAG (n=5–6/genotype/condition). (F) Relative frequency of GFP⁺ HSCs after culture (18h) of Ub^G76V-GFP (DMSO and 17-AAG) and Hsf1^−/−;Ub^G76V-GFP HSCs (n=4–5/condition). Data represent mean ± SD (E,F). Significance was assessed using a t-test (E,F; *P<0.05; **P<0.01, ***P<0.001).