Fig 1: Reduction of miR-132-3p led to enhanced MUC13 expression in gastric cancer cells. (A) TargetScan prediction programs indicated that miR-132-3p and miR-212-3p may bind to the 3′UTR of MUC13. (B) Reverse transcription-quantitative polymerase chain reaction was performed to determine the level of miR-132-3p and miR-212-3p in gastric cancer tissues (GC) (n=5 independent samples) and normal control (NC). (C) Luciferase assay indicated that miR-132-3p decreased the relative luciferase activity of pmirGLO-MUC13–3′UTR. (D) RT-qPCR analysis of miR-132-3p levels following transfection of miR-132-3p mimics or inhibitors. (E) Transfection of miR-132-3p mimics significantly decreased the protein level of MUC13 in MKN28, and transfection of miR-132-3p inhibitors significantly decreased the protein level of MUC13. *P<0.05, **P<0.01, ***P<0.001 vs. control. MUC13, mucin 13; UTR, untranslated region; miR, microRNA; RLU, relative luciferase unit; NC, negative control.
Fig 2: 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 3: Expression of MUC13 was increased in gastric cancer tissues compared with normal tissues. (A) Western blot analysis. *P<0.05 vs. control. (B) Immunohistochemistry analysis. n=5 independent tissues. MUC13, mucin 13.
Fig 4: GSDME-YBX1-MUCs participated in regulating tumor growth in patients.a, The expression profile of MUC1 and MUC13 from the TCGA Research Network (http://cancergenome.nih.gov/). Data were presented by box plots, where the centre line shows the median, the bounds of the box show the first and third quantile, whiskers extend to the most extreme values within 1.5 interquartile range (1.5*IQR), and dots denote outliers reaching past 1.5 interquartile range. n = 179 for PDAC tissues and n = 171 for adjacent tissues. T, tumor tissues; N, adjacent normal tissues. b,c, Immunohistochemical staining of YBX1 (b) or MUC1 (c) from the pancreatic sections of PDAC patients (n = 10). Scale bar, 50 μm. **P < 0.01, ***P < 0.001, by two-tailed Mann-Whitney test (a-c). The data represent mean ± SD. Source data
Fig 5: 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
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