Fig. 4: p53-mediated Type D cell death is dependent on specific cyclophilin proteins. | Nature Communications

Fig. 4: p53-mediated Type D cell death is dependent on specific cyclophilin proteins.

From: p53 restoration in small cell lung cancer identifies a latent cyclophilin-dependent necrosis mechanism

Fig. 4: p53-mediated Type D cell death is dependent on specific cyclophilin proteins.The alternative text for this image may have been generated using AI.

a Schematic of LentiCRISPRv2-mCherry transduction of Type D cells for positive selection assay. Type V cells are infected with mCherry-expressing lentiviral vectors expressing Cas9 and sgRNAs targeting specific cell death mediators or cyclophilins. Baseline mCherry expression is measured in viable (DAPI-) control cells using flow cytometry. Upon p53 restoration, changes in the proportion of mCherry-positive cells quantified in live (DAPI-) cells. Data plotted as fold change mCherry+ population relative to Control (vehicle treated) cells. b Fold change in the proportion of mCherry-positive cells 4 days after 4-OHT treatment in representative Type D (4716-11) cell line expressing sgRNAs targeting distinct cyclophilins. Empty Vector, β-Gal, and a p53 targeting sgRNA used as controls. Each symbol represents a technical replicate (n = 3). Error bars represent mean ± s.d. Experiment was conducted in n = 2 independent Type D cell lines. c, d Validation of results in (b) using an independent Type D (4711-18) cell line and additional sgRNAs targeting exons 3 and 4 of the Ppia gene (c) or exons 7 and 10 of the Ppie gene (d). Each symbol represents a technical replicate (n = 3).Error bars represent mean ± s.d. e Fold change in the proportion of mCherry-positive cells 4 days after 4-OHT treatment in Type V (4716-10) cell line expressing sgRNAs targeting cyclophilin A and cyclophilin E. Empty Vector, β-Gal, and a p53 targeting sgRNAs used as controls. Each symbol represents a technical replicate (n = 3). Error bars represent mean ± s.d. Experiment was conducted twice, with similar results. f Flow cytometry-assisted cell viability assay in Type D (4711-18) cell line expressing sgRNAs targeting cyclophilin A, cyclophilin E, or both, 5 days after 4-OHT treatment. Live cell percentage determined by quantification of DAPI negative population. β-Gal, and a p53 targeting sgRNA used as controls. Each symbol represents a technical replicate (n = 3). Error bars represent mean ± s.d. Experiment was conducted in n = 2 independent Type D cell lines. DKO double-knockout. g Immunoblot analysis for PPIA, PPIE and p53 expression from whole cell extract (WCE) or immunoprecipitated FLAG-IP samples from HEK293T cells overexpressing FLAG-Ppia or FLAG-Ppie, and HA-Trp53. h Immunoblot analysis for PPIA, PPIE and p53 expression 24 h after CsA treatment from whole cell extracts (WCE) or immunoprecipitated (FLAG-IP) samples from HEK293T cells overexpressing FLAG-Ppia or FLAG-Ppie, and HA-Trp53. i Type D (4711-18) cells and Type V (4716-10) were treated with 4-OHT and CsA for 48 h and p53-Cyclophilin A (CypA) or p53-Cyclophilin E (CypE) interactions were assessed using proximity ligation assay. Representative insets are shown for each condition. Scale bars: 6 μm. j Quantification of the average number of PLA signals per cell shown in (i). Values are expressed as mean ± s.d. n = 3–6 random fields of view (>100 cells). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Source data are provided as a Source Data file.

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