Fig. 4. p53 suppresses HSF1 activity via cyclin-dependent kinase inhibitor CDKN1A/p21 in human CRC cells.

a p21 silencing attenuates Nutlin-induced HSF1 suppression. Forty-eight hours after HCT116 cells were transfected with siRNAs against p21 and p53 or scrambled control siRNA, cells were treated with 10 µM Nutlin /DMSO for 24 h. b Rescue of p53-induced HSF1 target gene suppression by p21 depletion in RKO cells treated as in a. c WTp53-harboring CRC cell lines were treated with DMSO, 10 µM Palbociclib (Palbo), or 10 µM Nutlin for 24 h. Cell cycle inhibition was confirmed by Rb de(hypo)phosphorylation. pRb phospho-Rb. d HSF1 target gene repression after direct cell cycle inhibition. RKO cells were treated with 10 µM Palbociclib (CDK4/6 inhibitor), 10 µM Nutlin, or DMSO for 24 h. e, f Cell cycle inhibition by p53 inactivates HSF1 activity. HCT116 cells were treated with DMSO, 10 µM Nutlin, 10 µM Palbociclib, RG7112 (e) or RG7388/Idasanutlin (f) as indicated for 24 h. g Cell cycle inhibition prevents pSer326-HSF1 activation in stably HSF1-overexpressing HCT116 cells. HSF1c1 or empty vector clone (ORF) were treated with DMSO, H₂O, 10 µM Nutlin, or 10 µM Palbociclib for 24 h. h CDK4/6 inhibition causes HSF1 suppression. HCT116 cells were treated with DMSO, 10 µM Nutlin, 10 µM Palbociclib, 0.5 and 10 µM RO3306, and 20 µM Roscovitine (Rosco) for 24 h. i Representative immunohistochemistry of serial sections for pRB and pHSF1 from p53^Q/+ tumors treated with DMSO or Nutlin as in Fig. 2a (n = 6 tumors each). Scale bars, 100 μm. b, d qRT-PCR, normalized to RPLP0 mRNA. Relative values given in [ratio (2^−ddCT)]. Mean ± SD of ≥2 independent experiments, at least one with a technical replicate. Student’s t test, two-sided. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; ns not significant. a, c, e–h Immunoblot analyses. Actin, loading control. pHSF1/actin, pHSF1 densitometry normalized to loading control. Source data are provided as Source data file.