Journal: Nature Cell Biology

Article Title: Gasdermin E mediates resistance of pancreatic adenocarcinoma to enzymatic digestion through a YBX1–mucin pathway

doi: 10.1038/s41556-022-00857-4

Figure Lengend Snippet: a , The expression of TET1-3 from AsPC-1 cells was determined by real-time PCR. b , TET1, TET2, TET3 (green color) and DAPI immunostaining in CCC-HPE-2, PANC-1, AsPC-1 and BxPC-3 cells. The mean fluorescence intensity (MFI) of TET1-3 was calculated by Image J. Scale bar, 5 μm. c , Western blot analysis of GSDME and TET2 expression from SGCTR and TET2 -SGs- PANC-1 or AsPC-1 cells. d , The expression of GSDME in SGCTR or TET2 -SGs was detected by real-time PCR. e , The knockout efficiency of GSDME in PANC-1, AsPC-1 or BxPC-3 cells was determined by western blot. f , The expression of GSDME in vector or Flag-GSDME-overexpressing PANC-1, AsPC-1 or BxPC-3 were determined by western blot. g , h , SGCTR or GSDME -SGs- PANC-1 cells (2 × 10 6 ) were subcutaneously injected into mice. Tumor growth was measured (g, n = 6/group). Tumors were presented photographically (h, left, n = 4/group) or weighed (h, right). i , j , SGCTR, GSDME -SGs or GSDME -SG/Flag- GSDME -PANC-1 cells (i, 5 × 10 5 cells) or AsPC-1 cells (j, 2.5 × 10 5 cells) were orthotopically injected into mice. Tumors were presented photographically (left) or weighed (right) (i, n = 5/group; j, n = 6/group). k , The knockout efficiency of Gsdme in Pan02 cells was determined by western blot. l , SGCTR or Gsdme -SGs-Pan02 cells (1 × 10 6 cells) were orthotopic injected into the pancreas of C57BL/6 mice. 40 days after injection, tumors were presented photographically (left) and weighted (right) (n = 4/group). The normal pancreas was served as control. Scale bar, 1 cm. m , The peripheral lymphocytes from humanized mice were analyzed by FACS using an anti-human CD45 antibody before the subsequent animal experiments (n = 6). n , SGCTR and GSDME -SG-AsPC-1 cells (2.5 × 10 5 cells) were orthotopic injected into the pancreas of humanized mice. Tumors were presented photographically (left) and weighted (right) (n = 6/group). Scale bar, 1 cm. In a-c and f , n = 3 biological independent experiments. ** P < 0.01, *** P < 0.001, by two-tailed one-way ANOVA Bonferroni’s test ( a , d , h-j , l and n ) or two-tailed student’s t-test ( b ). The data represent mean ± SD.

Article Snippet: The human pancreatic cancer cell lines PANC-1 (X100160), AsPC-1 (X100459) and BxPC-3 (X100441), the mouse pancreatic cancer cell line Pan02 (X100165), the embryonic pancreatic-tissue-derived cell line CCC-HPE-2 (X100418), HEK-293T cells (X100478) and Sf9 insect cells (X100118) were purchased from the China Center for Type Culture Collection.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunostaining, Fluorescence, Western Blot, Knock-Out, Plasmid Preparation, Injection, Two Tailed Test

Journal: Nature Cell Biology

Article Title: Gasdermin E mediates resistance of pancreatic adenocarcinoma to enzymatic digestion through a YBX1–mucin pathway

doi: 10.1038/s41556-022-00857-4

Figure Lengend Snippet: a , b , SGCTR or GSDME -SGs- AsPC-1 or PANC-1 cells were stimulated with lysate (20 μl/ml) for 72 hr. The viable cells were measured by TB staining (a) or ATP Cell Viability Assay (b). c , d , The same as (a), except that the AsPC-1 cells were treated with IGF-II (2 ng/ml), Insulin (1 μg/ml), glucagon (0.5 ng/ml) or lysate. Cell viability was detected by TB staining (c) or ATP cell viability assay (d). e , f , SGCTR or GSDME -SGs- AsPC-1 cells were treated with lysate, DMH (10 μM), Orlistat (12.5 μM), lysate + DMH or lysate + Orlistat for 72 hr. Cell viability was determined by TB staining (e) or ATP cell viability assay (f). g , SGCTR, YBX1 -SGs-AsPC-1 cells were treated with PBS, Try/Chy or Doxorubicin (50 μM) for 48 hr. The representative images were shown. Scale bar, 20μm. h , The expression of GSDME from CCC-HPE-2, PANC-1, AsPC-1, and BxPC-3 were analyzed by western blot. i , j , CCC-HPE-2, PANC-1, AsPC-1 or BxPC-3 cells were treated with trypsin/chymotrypsin (Try/Chy) for 72 hr. Cell viability was determined by TB staining (i) or ATP cell viability assay (j). k , SGCTR and GSDME -SG-Pan02 cells (1 × 10 6 cells) were orthotopic injected into the pancreas of Prss1 −/− C57BL/6JGpt mice. 40 days after injection, tumors were presented photographically (left) and weighted (right) (n = 4). Scale bar, 1 cm. In a-f, i and j, n = 3 biological independent experiments. NS, no significance. ** P < 0.01, *** P < 0.001, by one-way ANOVA Bonferroni’s test ( a-f ) or student’s t-test ( i , j and k ). The data represent mean ± SD.

Article Snippet: The human pancreatic cancer cell lines PANC-1 (X100160), AsPC-1 (X100459) and BxPC-3 (X100441), the mouse pancreatic cancer cell line Pan02 (X100165), the embryonic pancreatic-tissue-derived cell line CCC-HPE-2 (X100418), HEK-293T cells (X100478) and Sf9 insect cells (X100118) were purchased from the China Center for Type Culture Collection.

Techniques: Staining, Viability Assay, Expressing, Western Blot, Injection

Journal: Nature Cell Biology

Article Title: Gasdermin E mediates resistance of pancreatic adenocarcinoma to enzymatic digestion through a YBX1–mucin pathway

doi: 10.1038/s41556-022-00857-4

Figure Lengend Snippet: a , The expression of MUC1 and MUC13 from AsPC-1 cells transfected with SGCTR or GSDME -SGs was determined by western blotting. b , AsPC-1 cells transfected with SGCTR, MUC1 -SGs, MUC13 -SGs or MUC1/MUC13 -SGs were treated with PBS or Try/Chy for 72 h. Viable cells were measured by TB staining. c , The same as b , except that AsPC-1 cells transfected with GSDME -SG, GSDME -SG/Flag- MUC1 or GSDME -SG/Flag- MUC13 were used. d , AsPC-1 or BxPC-3 cells were treated with PBS or Try/Chy for 48 h. The expression levels of GSDME, MUC1 and MUC13 were analysed by western blotting. e , The same as d , except that CCC-HPE-2, PANC-1, AsPC-1 and BxPC-3 cells were used. f , The same as b , except that AsPC-1 cells were treated with benzyl-GalNAc (2 mM), Try/Chy or Try/Chy plus benzyl-GalNAc. g , AsPC-1 cells transfected with SGCTR, MUC1 -SGs, MUC13 -SGs or MUC1/MUC13 -SGs (2.5 × 10 5 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 – f , n = 3 biological independent experiments. P values were determined by one-way ANOVA Bonferroni’s test ( b , c , f , g ). The data represent the mean ± s.d.

Article Snippet: The human pancreatic cancer cell lines PANC-1 (X100160), AsPC-1 (X100459) and BxPC-3 (X100441), the mouse pancreatic cancer cell line Pan02 (X100165), the embryonic pancreatic-tissue-derived cell line CCC-HPE-2 (X100418), HEK-293T cells (X100478) and Sf9 insect cells (X100118) were purchased from the China Center for Type Culture Collection.

Techniques: Expressing, Transfection, Western Blot, Staining, Injection