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

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — 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 TRAIL or TNF-α at the indicated concentrations 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 A549 cells. A549 cells transfected with PRKCSH siRNA (siPRK#2) or control siRNA (siCon) were treated with TRAIL or TNF-α at the indicated concentrations for 24 h. TNFSF-mediated cytotoxicity against tumor cells was analyzed by MTT assay (for cell viability) and propidium iodide and Annexin V staining assay (for cell death). c Immunoblot analysis of TNFSF-mediated activation of caspases in A549-shCon and A549-shPRK cells. Cells were treated with TRAIL or TNF-α at the indicated concentrations for 24 h, and the activation of caspases in cell lysates was analyzed by immunoblotting. d TNFSF-mediated tumor cell killing assay in other tumor cells. Hepatoma (Huh-7), lung cancer (H1299, p53 null), and colon cancer (HCT116, p53 null) cells were transfected with siRNAs to PRKCSH (siPRK #1 or #2) or control siRNA (siCon), followed by treatment with TRAIL or TNF-α at the indicated concentrations for 24 h. TNFSF-mediated cytotoxicity against tumor cells was analyzed by MTT assay (for cell viability) and propidium iodide and Annexin V staining assay (for cell death). e Immunoblot analysis of TNFSF-mediated activation of caspases in other tumor cells. Hepatoma (Huh-7), lung cancer (H1299, p53 null), and colon cancer (HCT116, p53 null) cells were transfected with siRNAs to PRKCSH (siPRK #1 or #2) or control siRNA (siCon), followed by treatment with TRAIL or TNF-α at the indicated concentrations for 24 h, and activation of caspases in cell lysates was analyzed by immunoblotting. **p < 0.01, *p < 0.05.