Fig. 4: HSF1 directly regulates Clu expression in BRCA-mut tumors.

(a–b) FFPE tumor sections from 14 BRCA-WT and 11 BRCA-mut PDAC patients were stained by MxIF for HSF1, CLU and αSMA. Images were analyzed by ImageJ. Pearson correlation between HSF1 mean intensity and CLU/αSMA ratio in the stroma of (a) BRCA-WT patients, and (b) BRCA-mut patients was calculated. (c–f) Expression levels of Clu (c), Muc5b (d) Acta2 (e) and Il6 (f) in primary PSCs freshly isolated from WT and Hsf1-null mice. n = 6 WT and 7 Hsf1-null mice, combined from 3 independent experiments. Statistical analysis was conducted via unpaired two-tailed t-test (g–j) Immortalized PSCs were seeded in Matrigel for 4 days. Conditioned media (CM) from KPC cells in which Brca2 was silenced by shRNA or non-targeting shControl (KPC) was then added for an additional 4 days, or cells were left in growth medium as control. 3 nM of the HSF1 inhibitor, CMLD011866 (aglaroxin C), or PBS control was added to the conditioned media (CM) every 2 days, for 4 days, after which the expression levels of Clu (g), Muc5b (h) Acta2 (i), and Il6 (j), were measured by qRT-PCR. For each condition n = 5-14 biologically independent culture domes combined from 3 independent experiments. Statistical analyses were conducted using one way ANOVA and Tukey’ test for multiple comparisons. (k–m) Immortalized PSCs were cultured with or without KPC-shControl-CM for 24 h. ChIP-PCR was performed for putative heat-shock elements of Hsp1a1 (k), Clu (l), and Hsp90aa (m), and for an intergenic region for normalization, following pulldown with anti-HSF1 antibody compared to IgG control. One-way ANOVA was performed on log2 values to compare between the group ratios of expression and Tukey’s test was performed to adjust for multiple comparisons. Data are presented as mean \(\pm\) SEM for each primer normalized to the intergenic control in (c–m) (n = 6 biologically independent culture wells combined from 3 independent experiments). (n) PSCs were cultured in the presence of boiled or unboiled CM from KPC-shControl (unboiled n = 4, boiled n = 6 biologically independent samples) and KPC-shBrca2 organoids (unboiled n = 6, boiled n = 5 biologically independent samples) as described in (g–j). Expression of Clu in PSCs were subsequently measured by qRT-PCR. Statistical analysis was conducted with two-way ANOVA. Data are presented as mean \(\pm\) SEM (o) Top 10 differentially expressed proteins (fold change of protein abundance based on mass-spectometry label-free quantification, shBrca2/shControl) from CM of KPC-shControl and KPC-shBrca2 organoids as measured by mass-spectrometry. Source data are provided as a Source Data file.