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

Fig. 4. Role of PRKCSH in TNFSF-mediated caspase-8 activation, ubiquitination, and phosphorylation.

Fig. 4

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-8 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-8 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-8 activation in lung cancer cells. Cells transfected with siPRK#2 or siCon were treated with TRAIL and/or caspase-8 inhibitor or zVAD-FMK for 24 h, and caspase-8 activation in cell lysates was analyzed by immunoblotting. d Immunoblot analysis of TRAIL-mediated ubiquitination of caspase-8 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. Cells were treated with 50 ng/mL TRAIL for the indicated times, and ubiquitinated caspase-8 in cell lysates was pulled down with anti-caspase-8 antibody, followed by immunoblot analysis with anti-ubiquitin antibody. e Immunoblot analysis of K63 or K48 ubiquitination of caspase-8 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. Cells were treated with 50 ng/mL TRAIL for the indicated times, and K63 or K48 ubiquitinated caspase-8 in cell lysates was pulled down with anti-caspase-8 antibody, followed by immunoblot analysis with anti-K63 or K48 ubiquitin antibody. f Immunoblot analysis of TNFSF-mediated caspase-8 phosphorylation 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. Cells were treated with 50 ng/mL TRAIL for the indicated times, and caspase-8 phosphorylation in cell lysates was analyzed by immunoblotting. Data are representative immunoblots and quantitative analysis of caspase-8 phosphorylation levels in each cell line. **p < 0.01, *p < 0.05.