Fig. 6

The LLPS microenvironment formed by the KMT2D protein influences its functions and modulates the stability of WDR5. a Representative images of cell immunofluorescence detecting the KMT2D expression in wild-type PANC-1 cells following treatment with 1,6-hexanediol (HD). KMT2D protein expression was markedly reduced and scattered after HD treatment. KMT2D, 593 KDa; GAPDH, 37 KDa. b, c Western blotting analyses of the KMT2D-enzyme complex in PANC-1 cells treated with HD. The expression of WDR5 decreased following HD treatment in a concentration- and time-dependent manner, while the expressions of ASH2L and RBBP5 were not affected. ASH2L, 69 KDa; RBBP5, 59 KDa; WDR5, 36 KDa; H3K4me1, 17 KDa; GAPDH, 37 KDa. d RT-PCR assays of metastasis-associated markers in PANC-1 cells treated with HD. The transcription levels of CDH1 and EpCAM significantly increased, while those of the CDH2 and vimentin decreased (error bars denote standard deviation). e RT-PCR assays of WDR5 in PANC-1 cells treated with HD. After HD treatment, the gene expression of WDR5 did not change (error bars denote standard deviation). f Representative images of cell immunofluorescence determining the WDR5 expression in PANC-1 cells treated with HD. The protein expression of WDR5 significantly decreased. g Western blotting analyses of the WDR5 expression after treatment with HD and CHX. Following HD treatment for 6 h at a concentration of 10 mg/mL, the stability of the WDR5 protein was sensitive to CHX. h Representative images of cell immunofluorescence determining the H3K4me1 expression in PANC-1 cells treated with HD. The level of H3K4me1 significantly decreased in cells treated with HD. i Co-immunoprecipitation analyses of the interactions between KMT2D and other components in PANC-1 cells treated with HD for 6 h at a concentration of 10 mg/mL. The interactions between KMT2D and WDR5, ASH2L and RBBP5 were markedly weaker following HD treatment. All values are represented as means ± SD. Data are representative of three independent experiments