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. 2020 May 14;11:2396. doi: 10.1038/s41467-020-16271-z

Fig. 3. HSP70 R469 is methylated by PRMT7 in cells.

Fig. 3

a PRMT7 (P7) knockout (KO) or knockdown (KD) reduces HSP70 methylation in various cell lines. 94A, 21B—HCT116 CRISPR PRMT7 KO clones; P parental C2C12; C-C2C12 expressing control guide RNA; 32 C2C12 CRISPR clone expressing PRMT7 catalytic mutant (delY35,A35S); 4,57,74—C2C12 CRISPR Prmt7 KO clones. PRMT7 was knocked down in HEK293T and MCF7 cells using siRNA, Con control, P7KD PRMT7 knockdown. b Monomethylation of inducible and constitutive HSP70 is PRMT7-dependent. MDA-MB-231 cells were transfected with PRMT7 siRNA for 3 days, heat-shocked for 1 h at 42 °C and analyzed 24 h after heat shock. c Only wild-type PRMT7 is able to rescue the HSP70 arginine monomethylation in HCT116 PRMT7 KO cells. Cells were transfected with GFP-tagged PRMT7 WT or catalytic mutant (R44A). d HSP70 R469A mutation blocks PRMT7-mediated methylation of HSPA8 and HSPA1. HCT116 PRMT7 KO cells were co-transfected with FLAG-tagged PRMT7 WT or R44A mutant and GFP-tagged HSPA8 or HSPA1 WT or R469A mutant. HSPA1/8-GFP was immunoprecipitated and analyzed for arginine monomethylation levels. The HSP70 methylation in MCF7, HCT116, and HEK293T cells was analyzed in cytoplasmic fraction to avoid unspecific band overlap. The experiments in a–d were repeated independently at least three times with similar results. Source data are provided as a Source Data file.