Fig 1: Effect of DSP and PPL variants on RhoA activity.a Active RhoA assays performed in EPC2 cells transduced with constructs encoding non-variant and mutated DSP or PPL. Statistics (versus non-variants): p.G46D, P = 0.132; p.R808C, P = 0.0691; p.Y895C, P = 0.0453; p.1067_1068del, P = 0.1013; p.N1215S, P = 0.2131; p.R1340C, P = 0.048; p.E1723Q, P = 0.0619; p.R108C, P = 0.4849; p.E632K, P = 0.0443; p.K1051V, P = 0.1589; p.L1154V, P = 0.0419; p.E1163K, P = 0.0035; p.V1377E, P = 0.8738. b Wound healing assays performed in EPC2 cells transduced with constructs encoding non-variant and mutated DSP (p.Y895C) treated with the Rho activator CN01 or the Rho kinase inhibitor Y27632. Quantification of the wound closure after 12 h was shown. Scale bar: 500 μM. Statistics: Non-variant vs. Variant, P = 0.0001; Non-variant vs. Variant + CN01, P = 0.7658; Non-variant vs. Variant + Y27632, P < 0.0001; Variant vs. Variant + CN01, P = 0.0002; Variant vs. Variant + Y27632, P = 0.0394; Variant + CN01 vs. Variant + Y27632, P < 0.0001. c Enzyme-linked immunosorbent assay of the level of active RhoA (RhoA-GTP) in protein lysates of biopsies from control individuals, non-familial patients with inactive EoE and non-familial patients with active EoE (Control, n = 3; Inactive EoE; n = 3; Active EoE, n = 5). Western blot analysis shows the expression of total RhoA in protein lysates of biopsies from each subject. Statistics: control vs. inactive EoE, P > 0.8803; control vs. active EoE, P = 0.0398; inactive EoE vs. active EoE, P = 0.0479. For panels a, b, data are representative of three experiments performed in duplicate and are presented as mean ± SEM. For panel c, data are presented as mean ± SEM, with markers representing biologically independent subjects. For panels a–c, two-tailed P-values were determined by the following tests: the one-way ANOVA test followed by a Dunnett’s multiple-comparison test (a) or Tukey’s multiple comparisons test (b, c). *P < 0.05, **P < 0.01, and ***P < 0.001. DSP desmoplakin, PPL periplakin, MW molecular weight, SEM standard error of the mean.
Fig 2: Influence of PPL on proliferation and apoptosis of OV cells. (A) PPL expression in OV cell lines detected by RT-qPCR; (B) influence of PPL siRNA low PPL in OV cell lines detected by RT-qPCR, *P<0.05; (C) influence of PPL on the proliferation ability of OV cells detected by MTT, *P<0.05; (D) influence of PPL knockdown on OV apoptosis detected by flow cytometry. NC, negative control; PPL, periplakin, OV, ovarian cancer. RT-qPCR, quantitative reverse transcription polymerase chain reaction; siRNA, small interfering RNA.
Fig 3: Influence of knockdown PPL on apoptosis-related protein expression detected by western blotting. (A) Western blotting detection of apoptosis-related protein expression in OV cells after silencing PPL; (B-E) expressions of cleaved-caspase-3, PPL, BAX, and BCL-2 after silencing PPL. Compared with that of the si-NC group, the expression of cleaved-caspase 3 and Bax proteins increased in the si-PPL-1 and si-PPL-3 groups, while BCL-2 protein expression decreased, with significant differences, **P<0.01, ***P<0.001. NC, negative control; PPL, periplakin; BAX, BCL-2-associated X protein; BCL-2, B-cell lymphoma 2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; OV, ovarian cancer.
Fig 4: PPL expression and prognostic analysis of gene in GEO database. (A) PPL expression in OV tissues analyzed by GEO database; (B,C) influence of PPL expression on OS and DFS of OV patients analyzed by Kaplan-Meier, indicating that PPL high expression was associated with poor survival in OV patients. PPL, periplakin; GEO, Gene Expression Omnibus; OV, ovarian cancer; OS, overall survival; PFS, progress-free survival; HR, hazard ratio.
Fig 5: Calpain 14–mediated desmosomal protein degradation.a Immunoblots of lysates from DSP (non-variant or mutant p.Y895C)–transfected HEK293T cells with CAPN14 or enzymatically inactive CAPN14-C101A co-transfection for changes in DSP levels following the addition of exogenous Ca2+ (left). Protein remaining after activation of CAPN14 was determined by the difference in band intensity between after and before activation [(after x 100)/before] (right). Statistics: DSP WT vs. DSP mutant, P = 0.0131. b Effect of DSP and PPL variants on protein degradation on activation of co-transfected CAPN14. Total protein remaining was determined for each mutation. Total protein (%) = (each protein remaining x 100)/average of non-variant cells. Statistics (versus non-variants): p.G46D, P = 0.8212; p.R808C, P = 0.0296; p.Y895C, P = 0.0352; p.1067_1068del, P = 0.0125; p.N1215S, P = 0.2675; p.R1340C, P = 0.2581; p.E1723Q, P = 0.6186; p.R108C, P = 0.0712; p.E632K, P = 0.0156; p.K1051V, P = 0.0946; p.L1154V, P = 0.0323; p.E1163K, P = 0.2167; p.V1377E, P = 0.9157. c Gene expression of CAPN14 in human normal tissues from the Human Protein Atlas (https://www.proteinatlas.org/). d Calpain inhibition results in rescue of DSP and PPL levels by CAPN14-mediated degradation. Total protein (%) = (each protein remaining x 100)/average of cells without exogenous Ca2+ and SNJ-1945. Statistics: DSP (without SNJ-1945 vs. with SNJ-1945, P = 0.0028); PPL (without SNJ-1945 vs. with SNJ-1945, P = 0.0188). For panels a, b, and d, data are representative of three experiments performed in duplicate and are presented as mean ± SEM, and two-tailed P-values were determined by the unpaired t-test (a and d) or one-way ANOVA test followed by a Dunnett’s multiple-comparison test (b). *P < 0.05 and **P < 0.01. CAPN14 calpain-14, DSP desmoplakin, PPL periplakin, SEM standard error of the mean, WT wild-type.
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