Fig 1: Involvement of ubiquitin and IAP in SNIPER(ABL)‐39‐induced degradation of BCR‐ABL protein. (a) Turnover of BCR‐ABL proteins after SNIPER(ABL)‐39 treatment. K562 cells were treated with 10 μg/mL of cycloheximide (CHX) in the presence or absence of 30 nM of SNIPER(ABL)‐39 for the indicated periods. Numbers below the panels represent BCR‐ABL/GAPDH, Cyclin B1/GAPDH and MCL‐1/GAPDH ratios normalized by time 0 control as 100. Data in the graphs are means ± SD (n = 3). (b) Expression of Bcr‐Abl mRNA in K562 cells. Cells were incubated with the indicated concentration of SNIPER(ABL)‐39 for 6 h. Expression levels are relative to vehicle treatment, which was arbitrarily set to 1. Data in the bar graph are means ± SD (n = 3). (c) Effect of ubiquitin activating enzyme inhibitor MLN7243 on protein knockdown activity of SNIPER(ABL)‐39 in K562 cells. Cells were incubated with the indicated concentration of SNIPER(ABL)‐39 and/or MLN7243 for 6 h. (d) Silencing of both cIAP1 and XIAP expression attenuates SNIPER(ABL)‐39‐dependent BCR‐ABL protein degradation. In K562 cells, endogenous cIAP1 and/or XIAP were depleted by shRNA for 72 h. Then cells were treated with the indicated concentration of SNIPER(ABL)‐39 for 6 h. Numbers below the ABL panel represent BCR‐ABL/GAPDH, BCR‐ABL/β‐tubulin, or BCR‐ABL/β‐actin ratio normalized by vehicle control as 100.
Fig 2: 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 3: SNIPER(ABL) composed of various ABL inhibitors and IAP ligands. (a) Chemical structures of SNIPER(ABL). (b–e) The protein knockdown activities of imatinib‐conjugated (b), GNF5‐conjugated (c), HG‐7‐85‐01‐conjugated (d) and dasatinib‐conjugated SNIPER(ABL) (e) were evaluated. K562 cells were incubated with the indicated concentration of SNIPER or ligands mix (LM; indicated ABL inhibitor and IAP ligand) for 6 h. Numbers below the ABL panel represent BCR‐ABL/GAPDH or BCR‐ABL/β‐tubulin ratio normalized by vehicle control as 100. (f) List of SNIPER(ABL) compounds and their DC 50 values. The upper name represents the code number of the SNIPER(ABL) and the lower number shows the concentration of SNIPER(ABL) required to reduce BCR‐ABL protein by 50% (DC 50, nM).
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.
Fig 5: 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.
Supplier Page from Thermo Fisher Scientific for Recombinant Human ABL1, His Tag Protein