Fig. 5: Non-autophagic function of mitophagy induced by arachidonic acid determines mPT-triggered pyroptosis.

a Representative fluorescent images of BxPC3 cells with stable transfection of pLent-EF1a-cox8-eGFP-mCherry-CMV-Puro lentivirus. Cells were stimulated with 40 or 120 μM arachidonic acid for 1.5 h. n = 3 independent experiments, one representative image of 3 independent experiments is shown. Scale bars, 10 μm. b Immunoblots of LC3B, Tubulin, and HSP90 in cell lysates of arachidonic acid-treated BxPC3, SW1990, and Pan02 cells. Cells were stimulated with 40 or 120 μM arachidonic acid for 1.5 h. n = 3 independent experiments, one representative image of 3 independent experiments is shown. c, d Representative immunofluorescent images (c) and quantitative analysis (d) of SW1990 cells with transfection of LC3-eGFP and MitoTracker Red staining. Cells were stimulated with 120 μM arachidonic acid for 1.5 h. n = 3 independent experiments, one representative image of 3 independent experiments is shown, Pearson’s Correlation Coefficient (PCC) and Manders’ Colocalization Coefficient (MCC) were applied for Two-way ANOVA with Tukey’s multiple comparisons test. e mtDNA expression levels in BxPC3 cells treated with 40 or 120 μM arachidonic acid for 1.5 h. n = 3 independent experiments, One-way ANOVA with Dunnett’s multiple comparisons test. f Representative fluorescent images of pLent-EF1a-cox8-eGFP-mCherry-CMV-Puro lentivirus stably transfected BxPC3 cells pre-treated with BAPTA-AM/EGTA-AM. Cells were pre-treated with 20 μM BAPTA-AM/EGTA-AM for 2 h followed by 120 μM arachidonic acid treatment for 1.5 h. n = 3 independent experiments, one representative image of 3 independent experiments is shown. Scale bars, 10 μm. g Immunoblots of LC3B and Tubulin in cell lysates of BAPTA-AM/EGTA-AM pre-treated BxPC3 cells. BxPC3 cells were pre-treated with 2.5–20 μM BAPTA-AM/EGTA-AM for 2 h followed by 120 μM arachidonic acid treatment for 2 h. n = 3 independent experiments, one representative image of 3 independent experiments is shown. h Representative fluorescent images of pLent-EF1a-cox8-eGFP-mCherry-CMV-Puro lentivirus stably transfected BxPC3 cells pre-treated with chloroquine. Cells were pre-treated with 50 μM chloroquine for 24 h followed by 120 μM arachidonic acid for 1.5 h. n = 3 independent experiments, one representative image of 3 independent experiments is shown. Scale bars, 10 μm. i, j Representative images (i) and quantitative analysis (j) of pyroptotic morphology changes in chloroquine pre-treated BxPC3 cells. BxPC3 cells were pre-treated with 50 μM chloroquine for 24 h followed by 120 μM arachidonic acid for 75 min. The pyroptotic cells were marked with yellow arrowheads. n = 5 independent experiments, one representative image of 5 independent experiments is shown, One-way ANOVA with Dunnett’s multiple comparisons test. Scale bars, 50 μm. k Immunoblots of GSDME, Caspase 3, LC3B, and β-actin in cell lysates of chloroquine pre-treated BxPC3 cells. Cells were pre-treated with 50 μM chloroquine for 12 or 24 h followed by 40 or 120 μM arachidonic acid treatment for 2 h. n = 3 independent experiments, one representative image of 3 independent experiments is shown. l LDH release assay of chloroquine pre-treated BxPC3 cells. BxPC3 cells were pre-treated with 50 μM chloroquine for 24 h followed by 40 or 120 μM arachidonic acid treatment for 4 h. n = 3 independent experiments, Two-way ANOVA with Tukey’s multiple comparisons test. m Time courses of immunoblots of GSDME, Caspase 3, LC3B, and Tubulin in cell lysates. BxPC3 cells were pre-treated with 50 μM chloroquine for 24 h, followed by 120 μM arachidonic acid treatment. n = 3 independent experiments, one representative image of 3 independent experiments is shown. n Time courses of LDH release assay of chloroquine pre-treated BxPC3 cells. Experimental design as in (m). n = 3 independent experiments, Two-way ANOVA with Tukey’s multiple comparisons test. o, p Representative immunofluorescent images (o) and quantitative analysis (p) of PINK1-KD SW1990 cells with transfection of LC3-eGFP and MitoTracker Red staining. Cells were stimulated with 120 μM arachidonic acid for 1.5 h. n = 3 independent experiments, one representative image of 3 independent experiments is shown, Pearson’s Correlation Coefficient (PCC) and Manders’ Colocalization Coefficient (MCC) were applied for Two-way ANOVA with Tukey’s multiple comparisons test. q, r Representative images (q) and quantitative analysis (r) of pyroptotic morphology changes in PINK1-KD SW1990 cells. The pyroptotic cells were marked with yellow arrowheads. Cells were stimulated with 120 μM arachidonic acid for 2 h. n = 5 independent experiments, one representative image of 5 independent experiments is shown, One-way ANOVA with Dunnett’s multiple comparisons test. Scale bars, 25 μm.(s) Immunoblots of GSDME, Caspase 3, LC3B, and Tubulin in cell lysates of PINK1-KD SW1990 cells compared with N.C. group. Experimental design as in (q). n = 3 independent experiments, one representative image of 3 independent experiments is shown. t LDH release assay of PINK1-KD SW1990 cells compared with the N.C. group. Cells were stimulated with 120 μM arachidonic acid for 4 h. n = 3 independent experiments, One-way ANOVA with Dunnett’s multiple comparisons test. u Immunoblots of GSDME, Caspase 3, LC3B, and Tubulin in cell lysates of chloroquine and CCCP pre-treated BxPC3 cells. Cells were pre-treated with 50 μM chloroquine and 10 μM CCCP for 6 h followed by 120 μM arachidonic acid treatment for 2 h. n = 3 independent experiments, one representative image of 3 independent experiments is shown. v LDH release assay of chloroquine and CCCP pre-treated BxPC3 cells. Cells were pre-treated with 50 μM chloroquine and 10 μM CCCP for 6 h followed by 120 μM arachidonic acid treatment for 4 h. n = 3 independent experiments, One-way ANOVA with Dunnett’s multiple comparisons test. HSP90, Tubulin, β-actin, and GAPDH were used as loading controls. Densitometric quantification of cleaved GSDME (~ 30 kDa, lower band) and cleaved caspase 3 (17/19 kDa, lower band) normalized to loading control (Two distinct bands on the membrane). Bar graphs expressed as mean ± SEM. See also Supplementary Fig. 8. Source data are provided as a Source Data file.