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. Author manuscript; available in PMC: 2019 Nov 17.
Published in final edited form as: Biochem Pharmacol. 2017 Dec 5;148:13–26. doi: 10.1016/j.bcp.2017.11.022

Fig. 6.

Fig. 6.

Upregulation of Bim and downregulation of Mcl-1 by combined VS-5584 and SCH772984 treatment is accompanied by increased association of Bim with Bcl-2. (A) Experimental outline for the combination of VS-5584 (VS), SCH772984 (SCH), and ABT-199 (ABT). (B) THP-1 cells were treated with VS + SCH for 12h, and then ABT was added for an additional 12 h. Representative Western blots, using whole cell lysates, are shown with the corresponding normalized densitometry measurements below. # indicates p < .05 compared to combined VS + SCH (n = 3, data not shown). (C, D) THP-1 cells were treated as shown in panel A. Bcl-2 (panel C) or Bim (panel D) was immunoprecipitated from whole cell lysates. Western blots were probed with anti-Mcl-1, Bim, or Bcl-2 antibody. The fold changes for Bcl-2, Mcl-1 and Bim densitometry measurements, compared to no drug treatment control, are indicated. For panel C, # indicates p < .05 compared to combined VS + SCH (n = 3, data not shown). For panel D, # indicates p < .05 compared to VS + SCH (Bcl-2 blot) or ABT alone (Mcl-1 blot). (E) THP-1, MOLM-13, OCI-AML3, and U937 cells were treated as shown in panel A. Annexin V-FITC/PI staining and flow cytometry analysis results are shown. *** indicates p < .001. (F, G) THP-1 cells infected with NTC or Bim shRNA lentivirus were subjected to Western blotting and probed with the indicated antibodies. The fold changes for the Bim densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated (panel F). * indicates p < .05 compared to NTC (n = 3, data not shown). THP-1 NTC and Bim shRNA cells were treated as shown in panel A and subjected to Annexin V/PI staining and flow cytometry analysis (panel G). *** indicates p < .001. (H) In the presence or absence of 50 μM Z-VAD-FMK, THP-1 cells were pretreated with VS + SCH for 12 h, then ABT-199 was added for an additional 12 h, and then subjected to Annexin V/PI staining and flow cytometry analysis. *** indicates p < .001.