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. Author manuscript; available in PMC: 2020 Jul 1.
Published in final edited form as: Mol Cancer Ther. 2019 May 15;18(7):1265–1277. doi: 10.1158/1535-7163.MCT-18-1241

Figure 5.

Figure 5.

TRAIL response is restored by the shBECN1 knockdown in the DLDR-1 cells.

(A) Western blot of BECN1, MAP1LC3B, SQSTM1, CASP8 full-length (FL), cleaved CASP8, CASP3, cleaved CASP3, CASP7, cleaved CASP7, and PARP after DLD1-R cells were treated with 20 ng/mL TRAIL for 8 hours. Cells were infected with shCTL (negative control) or shBECN1 lentiviral particles to generate stable knockdown cell lines before treatment with TRAIL. (B-D) Dose-response curves of TRAIL on the activities of caspase-8 (B), caspase-3/7 (C) and ATP content of cell viability (D) in the DLD1 cells, DLD1-R cells with BECN1 knockdown, and/or control DLD1-R cells treated with TRAIL. (E) Immunofluorescent images of BECN1 knockdown or control DLD1-R cells treatment with 100 nM Baf.A1 for 24 hours followed by staining for CASP8 (red), MAP1LC3B (green), and nuclei (blue). Scale bar, 10 μm. (F) Quantification of the average intensity of CASP8 (top panel), the average of MAP1LC3B intensity (middle panel), and the number of the CASP8 puncta merge with MAP1LC3B (bottom panel) in E, separately. (G-I, left panels) Dose-response curves of TRAIL in the absence or presence of 0.5/1 μM 17-HW on cell viability of Caco-2 cells (G), LS174T cells (H), and LS180 cells (I). Western blot of CASP8 and cleaved CASP8 after treatment of Caco-2 (G), LS174T (H) and LS180 (I) cells with 20 ng/mL TRAIL in the absence or presence of 0.5 μM 17-HW for 8 hours (right panels). (J-L, left panels) Dose-response curves of TRAIL on cell viability in the cells after the BECN1 knockdown compared to control Caco-2 cells (J), LS174T cells (K) and LS180 cells (L). Western blot of CASP8, cleaved CASP8 and BECN1 in the cells after the BECN1 knockdown or in control Caco-2 cells (G), LS174T cells (H) and LS180 cells (I) treated with 20 ng/mL TRAIL for 8 hours (right panels). ACTB was used as a loading control in the Western blot experiments. All values represent the mean ± SEM (n = 3 replicates) with a representative western blot shown. Statistical analysis was performed using two-tailed t-test (no significance, ns, p > 0.05; p < 0.01, **; p < 0.001, ***).