Fig. 2: Butyrylation of HSP90 at K754 is essential for cancer chemoresistance.

a The MS/MS spectrum of the modified HSP90 peptide. b KYSE150 and KYSE410 were transiently transfected with the pcDNA3.1-Flag plasmid expressing wild-type HSP-90 (HSP90-WT) or the HSP90-K754R mutant and were then subjected to immunoprecipitation/immunoblotting (IB) with the indicated antibodies. c The sequences surrounding K754 in HSP90 among seven species were aligned. Lysine 754 of HSP90 was colored in red. d KYSE150 and KYSE410 were transiently transfected with the pcDNA3.1-Flag plasmid expressing HSP90-WT or the HSP90-K754R mutant. Colony formation assay showing that the HSP90 K754R mutation abolished the promoting effect of HSP90-WT on 5-FU resistance in ESCC cells. e Representative images and quantitative analyses of tumor xenografts established with ESCC cells as indicated in the presence or absence of 5-FU. HSP90-WT or the HSP90-K754R mutant was re-overexpressed in HSP90-deficient ESCC cells. f, g The effect of HSP90 K754R mutation on its interaction with client proteins and cochaperones. h A CHX (100 μg/mL) chase assay was used to compare the stability of the wild-type HSP90 (HSP90-WT) and HSP90-K754R mutant proteins in ESCC cells. i Representative images and expression pattern of HSP90 in 170 ESCC and 145 paired adjacent normal tissues. j Kaplan–Meier analysis of overall survival for 170 ESCC patients stratified by the tumor HSP90 level. Bars, SDs; *P < 0.05; **P < 0.01; ***P < 0.001.