Fig 2: The GSDME–YBX1–mucin pathway mediates tumour progression in patients with PDAC.a, The expression profile of GSDME and YBX1 from the TCGA Research Network (https://cancergenome.nih.gov/). Data are presented as box plots, where the centre line shows the median, the bounds of the box show the first and third quartiles, whiskers extend to the most extreme values within 1.5-times the interquartile range, and dots denote outliers reaching past the 1.5 interquartile range. n = 179 for PDAC tissues and n = 171 for adjacent tissues. N, normal adjacent tissue; T, tumour tissues; TPM, transcripts per million. b,c, Immunohistochemical images (left) and quantification (right) of GSDME (b) and MUC13 (c) from sections of tumour tissues and adjacent tissues of patients (P1–P3) with PDAC (n = 10). Scale bars, 50 µm. d–f, Correlation analysis between the level of GSDME methylation (d), YBX1 (e) or MUC1 (f) and overall survival of patients with PDAC (n = 177). g, Schematic of the GSDME–YBX1–mucin pathway to regulate the resistance of PDAC to digestive enzymes. P values were determined by two-tailed Mann–Whitney test (a–c) or two-sided Pearson’s correlation test (d–f). The data represent the mean ± s.d.Source data

Fig 3: GSDME binds YBX1 and translocates into the nucleus.a, AsPC-1 cells transfected with SGCTR, GSDME-SG, GSDME-SG/WT-GSDME, GSDME-SG/NES-GSDME or GSDME-SG/NLS-GSDME were treated with Try/Chy for 72 h. Viable cells were measured by TB staining. b, AsPC-1 cells were treated with Try/Chy for 36 h. The cytosolic or nuclear fraction was extracted to perform an immunoprecipitation (IP) assay with anti-Flag-GSDME antibody (left). The expression of YBX1 was analysed by western blotting (right). c, AsPC-1 cells transfected with GSDME-SG/Vector or GSDME-SG/Flag-GSDME were treated with Try/Chy for 24 h. The cells were stained with anti-Flag and YBX1 antibodies and observed under a Stedycon super-resolution microscope. The plane projection of the Flag-GSDME AsPC-1 cell is indicated on the right, and the yellow spots represent colocalization of Flag-GSDME and YBX1. Scale bar, 1 µm. d, Binding (KD) between GSDME and YBX1 was measured by bio-layer interferometry. GSDMB was used as a negative control. e, AsPC-1 cells transfected with SGCTR, GSDME-SG, GSDME-SG/WT-GSDME, GSDME-SG/D270A or GSDME-SG/GSDMB (2.5 × 105 cells) were orthotopically injected into the pancreas of NSG mice. At 30 days after injection, tumours were photographed (left) and weighed (right) (n = 6 per group). Scale bar, 1 cm. f, AsPC-1 cells transfected with GSDME-SG/Vector, GSDME-SG/WT-GSDME, GSDME-SG/D270A, GSDME-SG/N320, GSDME-SG/N394, GSDME-SG/N419 or GSDME-SG/GSDMB were treated with PBS or Try/Chy for 48 h. Cell viability was measured. g, The same as a, except that AsPC-1 cells transfected with SGCTR, YBX1-SGs or YBX1-SG/Flag-YBX1 were used. h, AsPC-1 cells transfected with SGCTR, YBX1-SG or YBX1-SG/Flag-MUC1/MUC13 (2.5 × 105 cells) were orthotopically injected into the pancreas of mice. Tumours were photographed (left) and weighed (right) (n = 6 per group). Scale bar, 1 cm. For a–d, f and h, n = 3 biological independent experiments. P values were determined by one-way ANOVA Bonferroni’s test (a,e–h). The data represent the mean ± s.d.Source data

Fig 4: YBX1 mediated MUCs expression.a, The expression of YBX1, MUC1 or MUC13 from vector or Flag-YBX1-AsPC-1 cells treated with or without Trp/Chy for 48 hr was analyzed by western blot. b, The knockout efficiency of YBX1 from BxPC-3 cells was determined by western blot. c-e, The expression of MUC1 and MUC13 from SGCTR, Flag-YBX1, YBX1-SG, YBX1-SG/NLS-YBX1 or YBX1-SG/NES-YBX1-AsPC-1 cells stimulated by Try/Chy for 24 hr (c, d) or 48 hr (e) was determined by real-time PCR (c, d) or western blot (e). f, The log2TPM + 1 expression of GSDME in HEK239T cell line and human PDAC cell lines was quantified by RNA sequencing. g, The level of GSDME in 293 T and AsPC-1 cells were analyzed by Western blot. h, Immunostaining of GSDME from SGCTR or YBX1-SG-AsPC-1 cells treated with Trp/Chy for 36 hr. Scale bar, 5 µm. i, Immunostaining of Flag from Flag-GSDME-AsPC-1 cells treated with Try/Chy or/and nuclear pore inhibitor WGA (100 ng/mL) for 48 hr, following SLO pretreatment, was observed under the confocal microscopy. The mean MFI of nuclear Flag was calculated by Image J. Scale bar, 10 µm. j, The cell viability from SGCTR, GSDME-SG and GSDME-SG/NLS-YBX1-PANC-1 cells treated with or without Try/Chy was determined by TB staining. k, SGCTR, YBX1-SG or YBX1-SG/NLS-GSDME-AsPC-1 or BxPC-3 cells were treated with or without Try/Chy for 72 hr. The viable cells were calculated by TB staining. l, Immunostaining of Flag and Nup153 from GSDME-SG/Vector, GSDME-SG/Wt-GSDME, GSDME-SG/GSDME-D270A, GSDME -SG/GSDME-N320, GSDME-SG/GSDME-N394 or GSDME-SG/GSDME-N419- AsPC-1 cells treated with Try/Chy for 48 hr were observed under the STEDYCON super-resolution microscope. Scale bar, 1 µm. In a-k, n = 3 biological independent experiments. ***P < 0.001, by one-way ANOVA Bonferroni’s test (c,d,h-k). The data represent mean ± SD. Source data

Fig 5: GSDME-YBX1-MUCs pathway regulated tumor growth.a, Immunostaining of GSDME in Trp/Chy–treated AsPC-1 and BxPC-3 cells. The mean fluorescence intensity (MFI) or fluorescence intensity of nucleus/cytosol was calculated by Image J. Scale bars, 10 µm. b, SGCTR, GSDME-SG, GSDME-SG/NES-GSDME and GSDME-SG/NLS-GSDME-PANC-1 cells were treated with Try/Chy for 72 hr. The viable cells were measured by TB staining. c, SGCTR, GSDME-SG, GSDME-SG/NES-GSDME or GSDME-SG/NLS- GSDME-AsPC-1 or PANC-1 cells were treated with Try/Chy for 48 hr. The expression of MUC1 and MUC13 were analyzed by western blot. d, Vector and Flag-GSDME-AsPC-1 cells were treated with PBS or Try/Chy for 48 hr. RNA immunoprecipitation assays were performed using an antibody against Flag and relative precipitated RNA of MUC1 (left) and MUC13 (right) levels were normalized. Antibody against IgG served as the negative control. e, Vector or Flag-GSDME-AsPC-1 cells were treated with Try/Chy for 24 hr, followed by the addition of cycloheximide (20 µM) for indicated time (0, 4, 8 or 12 hr). Cells were lysed to perform western blot analysis with an anti-Muc1 or anti-Muc13 antibody (left). The level of Muc1 (middle) or Muc13 (right) was quantified. f, GSDME-SG/Flag-GSDME-AsPC-1 cells were treated with Try/Chy for 48 hr. Cell lysates were immunoprecipitated with anti-Flag for mass spectrometry. Identified transcriptional regulatory proteins were listed. g, GSDME-SG/Vector or GSDME-SG/Flag-GSDMB-AsPC-1 cells were treated with Try/Chy for 24 hr. The cells were stained with anti-Flag and YBX1 antibodies and observed under the STEDYCON super-resolution microscope. Scale bar, 1 µm. h, Representative images of PLA in GSDME-SG/Vector, GSDME-SG/Wt-GSDME, GSDME-SG/D270A, GSDME-SG/N320, GSDME-SG/N394, GSDME-SG/N419 or GSDME-SG/GSDMB-AsPC-1 cells were treated with PBS or Try/Chy for 48 hr. The red spots are regions of signal amplification denoting GSDME and YBX1 interaction. The PLA signals were quantified and analyzed. Scale bars, 10 µm. i, The same as (a), except that cells were stained with anti-YBX1 antibody. Scale bars, 10 µm. j, The knockout efficiency of YBX1 in AsPC-1 or PANC-1 cells was determined by western blot. k, The same as (b), except that SGCTR, YBX1-SGs and YBX1-SG/Flag-YBX1- PANC-1 cells were used. l, SGCTR, YBX1-SGs-AsPC-1 cells were treated with PBS, Try/Chy or Doxorubicin (50 µM) for 48 hr. The expression of GSDME was analyzed by Western blot. m, SGCTR or YBX1-SGs-AsPC-1 cells (2.5 × 105 cells) were orthotopically injected into mice. Tumors were presented photographically (left) or weighed (right) (n = 5/group). Scale bars, 1 cm. In a-e and h, n = 3 biological independent experiments. **P < 0.01, ***P < 0.001, by two-tailed Student’s t-test (a,i) or one-way ANOVA Bonferroni’s test (b,k and m). The data represent mean ± SD. Source data