Fig 1: ABL1 but not DDR1 knockdown reduces MLH1 protein level and function. (A) Quantification of western blots for ABL1 (left) and MLH1 (right) after transfection with siRNA duplexes against ABL1 or scrambled control in HEK293 cells. n = 3, statistical significance determined by 1-way ANOVA with Bonferroni post-test *p < 0.05 (B) Quantification of western blots for DDR1 (left) and MLH1 (right) after transfection with siRNA duplexes against DDR1 or scrambled control in HEK293 cells. n = 3, statistical significance determined by 1-way ANOVA with Bonferroni post-test *p < 0.05. (C) Cell survival measured via viable cell count after treatment with 6TG after siRNA knockdown of ABL1 using duplex C, n = 3 significance was determined by unpaired t-test *p < 0.05.
Fig 2: Inhibition of lysosomal degradation restores MLH1 levels reduced by nilotinib treatment. Western blot analysis and quantification of MLH1 protein expression after nilotinib or vehicle control co-treated with 100 nM Bafilomycin or vehicle control, n = 3 significance was determined by unpaired t-test *p < 0.05.
Fig 3: MLH1 binds to Hsp70 in the absence of ABL1 signaling and inhibition of Hsp70 restores MLH1 levels. (A) MLH1 immunoprecipitation from nuclear or cytoplasmic fractions in cells transfected with scrambled control or ABL1 siRNA followed by immunoblot for Hsp70 and MLH1. Cytoplasmic and nuclear fractionation purity was confirmed by GAPDH and Histone H3 immunoblots, respectively. Quantification of percent MLH1 bound to Hsp70 is shown. n = 3, significance was determined by unpaired t-test *p < 0.05. (B) MLH1 immunoprecipitation from whole cell lysate of HEK293 cells overexpressing MLH1 and treated with nilotinib or vehicle control, followed by immunoblot for Hsp70 and MLH1 (C) Immunoblot analysis and quantification of MLH1 protein expression in HEK293 cells after treatment with nilotinib or vehicle control after 24 h of co-treatment with 1 µM Hsp70 inhibitor, YM-01, or vehicle control. n = 3, significance was determined by unpaired t-test *p < 0.05.
Fig 4: ABL1 phosphorylates MLH1. (A) MLH1 mRNA fold change with ABL1 knockdown (left) or inhibition by imatinib (right) determined by RT-qPCR n = 3 (B) MLH1 immunoprecipitation in HEK293 cells with ABL1 and MLH1 overexpressed followed by immunoblot with anti-phosphotyrosine antibody. Treatment with 5 µM nilotinib reduces the phospho-tyrosine signal associated with MLH1. (C) Kinase assay using recombinant ABL1 (25 ng) and MLH1 (100 ng) protein incubated for 15 min, followed by SDS-PAGE and immunoblot analysis indicates tyrosine phosphorylation of MLH1 only in the presence of ABL1 kinase.
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