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. Author manuscript; available in PMC: 2018 Jan 1.
Published in final edited form as: Cancer Res. 2017 May 18;77(13):3513–3526. doi: 10.1158/0008-5472.CAN-16-3424

Figure 3.

Figure 3

Knockdown of Bcl-xL, Bcl-2 and Mcl-1 sensitizes for G-TPP-mediated apoptosis. A, Representative flow plots of LN229 cells that were treated with n.t.-siRNA, Bcl-xL-, Bcl-2- or Mcl-1-siRNAs prior to additional treatment with either solvent or G-TPP. Staining for propidium iodide and flowcytometric analysis was performed to determine the fraction of subG1 cells. B, Knockdown of Bcl-xL, Bcl-2 and Mcl-1 was confirmed by capillary electrophoresis. Vinculin served as loading control. C, LN229 cells were treated with n.t.-siRNA or Bax/Bak-siRNA prior to treatment with solvent, ABT263, G-TPP or the combination as indicated. Staining for Annexin V and propidium iodide was performed prior to flowcytometric analysis. Representative flow plots are shown. Bax/Bak knockdown was confirmed by Western blot analysis (see figure 3D). D, Whole cell extracts were collected from LN229 cells treated with n.t.-siRNA or Bax/Bak-siRNA in the presence or absence of 0.25µM ABT263/1µM G-TPP followed by Western blot analysis for PARP, cPARP, cCP3, BAX and BAK. Equal loading was verified by Western blot analysis for Actin. E, LN229 cells were treated with solvent, 0.25µM ABT263, 1µM G-TPP or the combination. Immunoprecipitation (IP) for BAX (6A7) was performed prior to immunoblotting for Bax. IP with non-specific IgG was used as a negative control. F, LN229 cells were treated with n.t.-siRNA or BIM-siRNA in the presence or absence of 0.25µM ABT263/1µM G-TPP. Whole cell extracts were collected and Western blot analysis was performed for BIM and cCP3. Actin served as a loading control. G, LN229 cells were treated with solvent, 0.25µM ABT263, 1µM G-TPP or the combination. Whole cell extracts were collected prior to co-immunoprecipitation for Mcl-1. Immunoblots for Bim, Mcl1, Noxa and the IgG light chain (LC) were performed. The BIM L+S/Mcl-1 ratio was calculated based on densitometric analysis using ImageJ 1.47v (http://imagej.nih.gov/ij). H, Western blot analysis for Mcl-1, Bcl-xL, BIM, Bak, Bax and Noxa of LN229 cells treated as described for G. Actin served as loading control.