Figure 5: HSF1 controls CHL synthesis and protein CHLation via AMPK.
(A and B) Quantitation of cellular CHL in A2058 cells and MEFs following HSF1 depletion or in A2058 cells transfected with 10μM HSF1 peptides twice in 7 days. (C) Establishment and validation of SHH CHLation sandwich ELISA. SFM: serum-free medium. (D and E) Quantitation of SHH-N CHLation in A2058 cells with inducible HSF1 knockdown for 6 days with either AMPKα knockdown or 50μM CHL supplementation in the medium for 24 hr. Secreted CHLated SHH-N were normalized against total SHH-N present in the medium. (F) Quantitation of SHH-N CHLation in Hsf1-deficient MEFs reconstituted with HSF1324–529. (G) Quantitation of SHH mRNAs in cells deficient for HSF1. (H) ChIP analysis of HSF1 binding to the SHH promoter in A2058 cells expressing inducible HSF1-targeting shRNAs. Alphoid DNA loci served as a negative control. (I) Left panel: visualization of GLI1 binding to genomic DNAs by PLA (red) in parental A2058 cells stimulated for 4 hr by the conditioned media collected from A2058 cells expressing inducible scramble or HSF1-targeting shRNAs. Scale bars: 10μm. Right panel: Quantitation of GLI1-gDNA binding PLA by FACS (data as geometric means of FL2-H).
Error bars are mean±SD, n=3 independent experiments, One-way ANOVA (A-I) or Student’s t test (A, C, and G, two-tailed). See also Figure S5.