Fig. 2: PTPN23 depletion activated NF- κB and triggered apoptosis, necroptosis, and pyroptosis.

a GSEA analysis showing that NF-κB signature was upregulated upon PTPN23 depletion at post-infection day 6. Statistical significance was assessed using Family-Wise Error Rate (FWER) correction. b Immunoblot of NF-κB and MAPK signaling in NOMO-1 cells harvested 6 days after sgNEG, N23-sg1, or N23-sg2 lentiviral infection. (n = 3 independent experiments). c Left, SYTOX AADvanced and fluorogenic substrate of Caspase-3/7 stains of PTPN23-dTAG NOMO-1 cells treated with 200 nM dTAG-13 for 4 days. Right, Quantification of necrotic and apoptotic cells from negative control, PTPN23 sgRNA infected cells. Necrotic cells are SYTOX AADvanced positive and cleaved Caspase-3/7 negative; apoptotic cells are cleaved Caspase-3/7 positive (n = 6, derived from 3 independent experiments, each performed with 2 replicates). d Immunoblot analysis of cleaved forms of PARP and caspases in PTPN23-dTAG cells after 4 days of dTAG-13 (200 nM) treatment (n = 3 independent experiments). e Immunoblotting analysis of RIPK3 and MLKL in PTPN23-dTAG cells after 5 days of dTAG-13 (200 nM) treatment. (n = 3 independent experiments). f Volcano plot of RNA-seq analysis to compare transcriptional changes of sgNEG and PTPN23 sgRNA infected cells at post-infection day 6. g ELISA assay measuring the amount of secreted IL-1β in PTPN23 deficient NOMO-1 cells. PTPN23-dTAG NOMO-1 cells were treated with dTAG-13 (200 nM) for 4 days, and seeded into 96-well plates. The supernatants were then collected after 24 h and 48 h in the presence of dTAG-13 (200 nM) for ELISA analyses (n = 3 biological replicates). h Immunoblotting analysis of CASP1 and GSDMD in PTPN23-dTAG NOMO-1 cells after 5 days of dTAG-13 (200 nM) treatment (n = 3 independent experiments). Each immunoblot was reproduced three times with similar results for (b, d, e, and h). i Representative images of immunofluorescence microscopy of ASC specks in PTPN23-dTAG NOMO-1 cells. Cells were treated with 200 nM dTAG-13 for 5 days. Scale bar: 5 μm. j GFP competition growth assays performed using CASP8/RIPK3/CASP1 or CASP8/RIPK3/GSDMD triple knockout cells. Two single-cell clones derived from each genetic background were pooled together (n = 3 independent experiments). Data are presented as mean ± SEM, statistical analysis for (c) by Two-way ANOVA, Dunnett’s multiple comparisons test; (g) by Two-way ANOVA, Tukey’s multiple comparisons test.