Fig. 5

KMT2D LCDs modulate the stability of WDR5 protein and influence the formation of the KMT2D-enzyme complex. a Representative images of cell immunofluorescence detecting the WDR5 expression in modified-PANC-1 cells. The WDR5 level had markedly reduced in the LCD-depleted cells, but normal expression was recovered in OE1 and OE2 cells. b Western blotting analyses of the KMT2D-enzyme complex, including WDR5, ASH2L, and RBBP5, in HEK293T and PANC-1 cells. Knockout of KMT2D LCDs significantly attenuated WDR5 expression, but the expressions of ASH2L and RBBP5 were not affected. In OE1 and OE2 cells, WDR5 expression is restored. ASH2L, 69 KDa; RBBP5, 59 KDa; WDR5, 36 KDa; GAPDH, 37 KDa. c, d RT-PCR analyses of WDR5 gene expression. After LCD deletion in HEK293T and PANC-1 cells, the gene expression of WDR5 was similar to that in OE1 cells, OE2 cells, and control group (vehicle; error bars denote standard deviation). e, f Western blotting analyses of WDR5 protein expression with CHX or MG132 treatment. The WDR5 protein level in KMT2D LCD knockout cells gradually decreased with the time after CHX treatment compared with the wild-type cells, indicating that the stability of WDR5 protein is closely related KMT2D LCDs. g Co-immunoprecipitation analyses of the interactions between KMT2D and other components. The interactions between KMT2D and WDR5 were significantly reduced in KMT2D LCD-depleted PANC-1 cells. Similar results were found for the interactions of KMT2D with ASH2L and RBBP5. In OE1 and OE2 cells, these interactions were restored. All values are represented as means ± SD. Data are representative of three independent experiments