Journal: JCI Insight

Article Title: SPOP mediates apoptosis and protects against necroptosis by regulating ubiquitination of RIPK1 and RIPK3

doi: 10.1172/jci.insight.180655

Figure Lengend Snippet: ( A ) SPOP +/+ and SPOP –/– MEFs were pretreated with Nec-1s or DMSO for 1 hour and then treated with DMSO or T5z7 or T5z7+N for 6 hours; subsequently whole cell lysate (WCL) was harvested for immunoblot (IB) analysis. Values on left are in kilodaltons. C-, cleaved. ( B ) SPOP +/+ and SPOP –/– MEFs were pretreated with Nec-1s (N) or DMSO for 1 hour and then treated with DMSO or TSZ or TSZ+N or T5z7Z or T5z7Z+N for 4 hours; subsequently WCL was harvested for IB analysis. ( C ) SPOP +/+ and SPOP –/– MEFs were pretreated with GSK872 (G) or DMSO for 1 hour and then treated with DMSO or TSZ or TSZ+G for 4 hours; subsequently WCL was harvested for IB analysis. ( D ) IB analysis of WCL derived from RIPK3 +/+ and RIPK3 –/– MEFs with SPOP knockout through CRISPR technology. Parental MEFs were used as the control. Cells were treated with TSZ or DMSO for 4 hours; subsequently WCL was harvested for IB analysis. ( E ) A schematic illustration of RIPK1-mediated multimodal signaling events downstream of TNFR1. ( F ) SPOP +/+ and SPOP –/– MEFs were stimulated by TNF-α for the indicated periods of time. WCL was harvested for IB analysis. ( G ) SPOP +/+ and SPOP –/– MEFs were stimulated by FLAG–TNF-α for the indicated periods of time, and TNF-RSC was immunoprecipitated (IP) using anti-Flag resin and analyzed using the indicated antibodies. ( H ) SPOP +/+ and SPOP –/– MEFs were pretreated with Nec-1s or DMSO for 1 hour and then treated with DMSO or TSZ or TSZ+N for 4 hours. Necrosome was immunoprecipitated using RIPK1 antibody and analyzed using the indicated antibodies.

Article Snippet: The reagent and concentration used in the experiment are as follows: recombinant mouse TNF-α (Cell sciences, CRT192C, 100 ng/mL), SM-164 (MedChemExpress [MCE], HY-15989, 50 nM), Nec-1s (Selleck, S8641, 20 μM), 5z7 (Sigma, 09890, 500 nM), zVAD.fmk (Sigma, V116, 25 μM), cycloheximide (Sigma, C-6255, 1 μM), and GSK872 (MilliporeSigma, 530389, 10 μM).

Techniques: Western Blot, Derivative Assay, Knock-Out, CRISPR, Control, Immunoprecipitation

Journal: JCI Insight

Article Title: SPOP mediates apoptosis and protects against necroptosis by regulating ubiquitination of RIPK1 and RIPK3

doi: 10.1172/jci.insight.180655

Figure Lengend Snippet: ( A ) After knockdown of RIPK1 in 786-O, HepG2, and MDA-MB-231 cells, the protein levels of apoptotic markers mediated by TNF-α+SM164 (TS) stimulation were detected. ( B ) WT and RIPK3-overexpressing 786-O, HepG2, and MDA-MB-231 cells were treated with DMSO or TSZ for 6 hours; subsequently WCL was harvested for IB analysis. ( C ) Cell viability assay to assess the sensitivity of 786-O, HepG2, and MDA-MB-231 cells to SM164 treatment. ( D ) Cell viability assay for the effect of RIPK1 knockdown on the sensitivity of 786-O, HepG2, and MDA-MB-231 cells to sunitinib treatment. ( E ) Cell viability assay to assess the effect of combined use of SM164 on sunitinib sensitivity in 786-O, HepG2, and MDA-MB-231 cells. ( F ) Cell viability assay for the effect of RIPK1 knockdown on the sensitivity of 786-O, HepG2, and MDA-MB-231 cells to sunitinib alone, and sunitinib combined with SM164 treatment. ( G ) WT and RIPK1-knockdown 786-O, HepG2, and MDA-MB-231 cells were treated with DMSO, sunitinib alone, SM164 alone, or sunitinib combined with SM164 for 24 hours; subsequently WCL was harvested for IB analysis. Cell Counting Kit-8 (CCK-8) experiments were performed in triplicate and repeated 3 times independently. Data are presented as mean ± SD. The significance of differences was revealed based on paired Student’s t test.

Article Snippet: The reagent and concentration used in the experiment are as follows: recombinant mouse TNF-α (Cell sciences, CRT192C, 100 ng/mL), SM-164 (MedChemExpress [MCE], HY-15989, 50 nM), Nec-1s (Selleck, S8641, 20 μM), 5z7 (Sigma, 09890, 500 nM), zVAD.fmk (Sigma, V116, 25 μM), cycloheximide (Sigma, C-6255, 1 μM), and GSK872 (MilliporeSigma, 530389, 10 μM).

Techniques: Knockdown, Viability Assay, Cell Counting, CCK-8 Assay