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. 2024 Jan 10;56(1):192–209. doi: 10.1038/s12276-023-01147-1

Fig. 5. Role of PRKCSH in TNFSF-mediated caspase-9 activation and Mcl-1 expression.

Fig. 5

a TNFSF-mediated tumor cell killing assay in tumor 3D spheroid culture (scale bar = 200 μm). The tumor organoids of A549-shCon and A549-shPRK cells were treated with caspase-9 inhibitor or pan-caspase inhibitor (zVAD-FMK) for 1 h, followed by treatment with 50 ng/mL TRAIL for 24 h. TNFSF-mediated cytotoxicity against tumor organoids was analyzed by measuring spheroid volume. Propidium iodide-stained cells indicate dead cells in tumor organoids. Data are shown as the means ± SDs of three independent assays. The statistical significance of differences between two groups was determined with the two-tailed Student’s t test. b TNFSF-mediated tumor cell killing assay in 2D cultured tumor cells. Lung cancer cells, including A549, H292R, and H1299 (p53 null) cells, were transfected with siPRK#2 or siCon, followed by treatment with 50 ng/mL TRAIL with or without caspase-9 inhibitor or zVAD-FMK for 24 h. TNFSF-mediated cytotoxicity against tumor cells was analyzed by propidium iodide and Annexin V staining assays. c Immunoblot analysis of TNFSF-mediated caspase-9 activation in lung cancer cells. Cells transfected with siPRK#2 or siCon were treated with TRAIL and/or a caspase-9 inhibitor or zVAD-FMK for 24 h, and caspase-9 activation in cell lysates was analyzed by immunoblotting. d Immunoblot analysis of Mcl-1 protein expression in lung cancer cells. Cells were transfected with siPRK#2 or siCon for 48 h, and the expression levels of Mcl-1 long (Mcl-1L) and small form (Mcl-1S) in cell lysates were analyzed by immunoblotting. Data are representative immunoblots and quantitative analysis of Mcl-1 protein expression levels in each cell line. e Analysis of Mcl-1 mRNA expression levels in lung cancer cells. The expression levels of Mcl-1L and Mcl-1S mRNA in A549-shCon and A549-shPRK cells or other lung cancer cells, including H292R and H1299 (p53 null) cells transfected with siPRK#2 or siCon, were analyzed by qPCR. Data are representative agarose gels and quantitative analysis of Mcl-1 mRNA expression in each cell line. f Analysis of TNFSF-induced Mcl-1 protein and mRNA expression in lung cancer cells. Cells were treated with 50 ng/mL TRAIL for the indicated times, and the expression levels of Mcl-1L and Mcl-1S protein and mRNA were analyzed by immunoblotting and qPCR. Data are representative immunoblots and quantitative analysis of Mcl-1 protein and mRNA expression in each cell line. g The effect of Mcl-1 overexpression on TNFSF-mediated tumor cell killing in PRKCSH-deficient cells. A549-shPRK cells were transfected with Mcl-1 cDNA plasmid, followed by treatment with TRAIL at the indicated concentrations for 24 h. TNFSF-mediated cytotoxicity was analyzed by MTT assay, and caspase-9 activation was analyzed by immunoblotting. h The effect of Mcl-1 knockdown on TNFSF-mediated tumor cell killing in lung cancer cells. H292R and H1299 (p53 null) cells were transfected with siRNA to Mcl-1, followed by treatment with TRAIL at the indicated concentrations for 24 h. TNFSF-mediated cytotoxicity was analyzed by MTT assay, and caspase-9 activation was analyzed by immunoblotting. **p < 0.01, *p < 0.05.