mhcc97h cells  (Servicebio Inc)

Journal: Frontiers in Immunology

Article Title: PSMD12 promotes hepatocellular carcinoma progression by stabilizing CDK1

doi: 10.3389/fimmu.2025.1581398

Figure Lengend Snippet: Effects of PSMD12 on HCC cell growth and migration. (A, B) Representative images and quantitative analysis of colony formation assays in hepatocellular carcinoma cells after PSMD12 knockdown or overexpression. (C-D) CCK-8 experiment was performed to measure the cell viability. (E, F) EdU assay demonstrating the proliferative capacity of cells with varying PSMD12 expression levels (scale bar: 200 μm). (G, H) Representative images of wound-healing assays in hepatocellular carcinoma cells following knockdown or overexpression of PSMD12 (scale bar: 200 μm). (I, J) Representative images of transwell assays in HCC cells following PSMD12 knockdown or overexpression (scale bar: 200 μm). Data are presented as mean ± standard deviation from three independent experiments. For two groups, Student’s t-tests were applied; for more than two groups, one-way ANOVA with Tukey’s multiple comparisons test was used (*p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant). (K-M) MHCC97H (shNC or shPSMD12#1) cells were subcutaneously injected into BALB/c nude mice. Tumor volumes were measured at specified time points, and at the experiment’s conclusion, tumors were excised, photographed, and weighed. Tumor volume (V) was calculated as V = 0.52 × length × width2. Tumor volume and weight were analyzed using one-way ANOVA followed by Tukey’s test. Tumor volumes are presented as mean ± SD, n = 4, **p < 0.01, ***p < 0.001. (N, O) Representative H&E staining showcases tumor tissues extracted from shPSMD12 and control nude mice. Tumor tissues were subjected to Ki67 staining, and the cell proliferation index was determined by quantifying Ki67-positive nuclei (n = 4, magnification: 200 ×, inset magnification: 400 ×; ***p < 0.001. Scale bar: 50μm).

Article Snippet: Four-week-old female BALB/c nude mice were purchased from Hangzhou Zhiyuan Laboratory Animal Technology Co., Ltd. MHCC97H cells (5×10 6 ) stably transfected with shNC, shPSMD12#1, shNC+CDK1, or shPSMD12#1+CDK1 were resuspended in 200 μL of PBS and subcutaneously injected into the dorsal region of the nude mice.

Techniques: Migration, Knockdown, Over Expression, CCK-8 Assay, EdU Assay, Expressing, Standard Deviation, Injection, Staining, Control

Journal: Frontiers in Immunology

Article Title: PSMD12 promotes hepatocellular carcinoma progression by stabilizing CDK1

doi: 10.3389/fimmu.2025.1581398

Figure Lengend Snippet: PSMD12 promotes HCC cell cycle progression and interacts with CDK1. (A) GSEA analysis of the TCGA-HCC cohort highlighting cell cycle signaling pathways. (B-E) Cell cycle distribution in HCC cells following PSMD12 knockdown or overexpression, presented as peak plots and quantitative analysis of cell distribution across G0/G1, S, and G2/M phases. (F, G) Western blot analysis of PSMD12, PCNA, Cyclin D1, CDK1, and GAPDH protein expression in PSMD12-silenced MHCC97H cells (F) and PSMD12-overexpressing HCCLM3 cells (G) , with GAPDH as the control. (H) GSEA analysis of the TCGA-HCC cohort revealing the Ubiquitin-mediated proteolysis and G2/M checkpoint signaling pathways. (I, J) Mass spectrometry detected co-precipitated PSMD12 and CDK1 proteins. (K-N) Co-immunoprecipitation (Co-IP) assays in MHCC97H and HCCLM3 cells confirmed the interaction between PSMD12 and CDK1. (O, P) Co-localization of PSMD12 (red) and CDK1 (green) in HCC cells, followed by DAPI nuclear counterstaining (blue). Scale bar: 20 μm. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

Article Snippet: Four-week-old female BALB/c nude mice were purchased from Hangzhou Zhiyuan Laboratory Animal Technology Co., Ltd. MHCC97H cells (5×10 6 ) stably transfected with shNC, shPSMD12#1, shNC+CDK1, or shPSMD12#1+CDK1 were resuspended in 200 μL of PBS and subcutaneously injected into the dorsal region of the nude mice.

Techniques: Protein-Protein interactions, Knockdown, Over Expression, Western Blot, Expressing, Control, Ubiquitin Proteomics, Mass Spectrometry, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Frontiers in Immunology

Article Title: PSMD12 promotes hepatocellular carcinoma progression by stabilizing CDK1

doi: 10.3389/fimmu.2025.1581398

Figure Lengend Snippet: PSMD12 regulates HCC proliferation through CDK1. (A) Western blot analysis of PSMD12, CDK1, PLK1, p-PLK1, AKT, p-AKT, and GAPDH expression in MHCC97H cells transfected with shNC, Vector, shPSMD12#1, or CDK1. (B) Western blot analysis showing protein expression of PSMD12, CDK1, PLK1, p-PLK1, AKT, p-AKT, and GAPDH in HCCLM3 cells transfected with Vector, PSMD12, or shCDK1. (C) CCK-8 assays demonstrated that restoration of CDK1 expression counteracted the growth-inhibitory effect of PSMD12 knockdown in MHCC97H cells. (D) Knockdown of CDK1 expression inhibited the pro-proliferative effect induced by PSMD12 overexpression in HCCLM3 cells, as assessed by CCK-8. (E, F) Representative images and quantification of colony formation assays in hepatocellular carcinoma cells following PSMD12 or CDK1 knockdown or overexpression. (G, H) EdU assays were performed to assess the proliferative capacity of cells (scale bar: 200 μm). (I, J) Quantification of EdU assays in hepatocellular carcinoma cells following PSMD12 or CDK1 knockdown or overexpression. Data are presented as mean ± standard deviation from three independent experiments and analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test. **p < 0.01, ***p <0.001. (K) Representative tumor morphology in BALB/c nude mice. (L, M) Statistical analysis of tumor volume (L) and tumor weight (M) across different groups. Tumor volume (V) was calculated as V = 0.52 × length × width2, and data were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. Tumor volumes are presented as mean ± SD, n = 4, **p < 0.01, ***p < 0.001. (N) Representative H&E and IHC staining of PSMD12, CDK1, and Ki67 in tumor tissues from different nude mouse groups. Scale bar: 50 μm.

Article Snippet: Four-week-old female BALB/c nude mice were purchased from Hangzhou Zhiyuan Laboratory Animal Technology Co., Ltd. MHCC97H cells (5×10 6 ) stably transfected with shNC, shPSMD12#1, shNC+CDK1, or shPSMD12#1+CDK1 were resuspended in 200 μL of PBS and subcutaneously injected into the dorsal region of the nude mice.

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, CCK-8 Assay, Knockdown, Over Expression, Standard Deviation, Immunohistochemistry

Journal: Frontiers in Immunology

Article Title: PSMD12 promotes hepatocellular carcinoma progression by stabilizing CDK1

doi: 10.3389/fimmu.2025.1581398

Figure Lengend Snippet: PSMD12 stabilizes CDK1 protein levels by reducing ubiquitin-mediated degradation of CDK1 in HCC cells. (A, B) Western blot analysis of PSMD12 and CDK1 protein levels in HCC cells following PSMD12 knockdown or overexpression, with GAPDH as a loading control. (C, D) qRT-PCR assessment of PSMD12 and CDK1 mRNA levels in HCC cells after PSMD12 knockdown or overexpression, with GAPDH as a control. (E, F) MHCC97H and HCCLM3 cells were treated with cycloheximide (CHX, 50 μg/mL), and Western blot analysis was performed to detect CDK1 protein levels at different time points. (G, H) MHCC97H and HCCLM3 cells were treated with CHX (50 μg/mL) for specified durations, with or without the addition of the PSMD12 overexpression plasmid, followed by Western blot analysis to assess CDK1 protein levels. (I, J) HCC cells were treated with 15 μmol/L MG132 and collected at 0/3/6/9 hours, followed by Western blot to analyze protein expression levels. (K, L) MG132 (15 μM) treatment of MHCC97H and HCCLM3 cells altered PSMD12 expression, with PSMD12 and CDK1 protein levels assessed by Western blot analysis. (M, N) Co-immunoprecipitation (Co-IP) assays in MHCC97H and HCCLM3 cells confirmed the interaction between ubiquitin (Ub) and CDK1. (O) Colocalization studies in HCC cells using an anti-CDK1 antibody (1:100, red) and anti-ubiquitin antibody (1:100, green), followed by DAPI nuclear counterstaining (blue). Scale bar: 20 mm. (P, Q) MG132 (15 μM) was added to MHCC97H and HCCLM3 cells, which were simultaneously transfected with shPSMD12#1 or HA-PSMD12 plasmids. Co-IP assays were performed to detect ubiquitin binding to CDK1. (R) After 48 hours of transfection of Myc-PSMD12, Flag-CDK1, and HA-Ub WT/K48/K63, cells were lysed, and then immunoprecipitation was performed with Flag antibody, and immunoblot analysis was performed with HA antibody. (S) The docking conformation and three-dimensional structure of PSMD12 and CDK1.PSMD12 and CDK1 are shown in orange and cyan respectively. Statistical significance was determined using Student's t-tests and one-way ANOVA followed by Tukey's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

Article Snippet: Four-week-old female BALB/c nude mice were purchased from Hangzhou Zhiyuan Laboratory Animal Technology Co., Ltd. MHCC97H cells (5×10 6 ) stably transfected with shNC, shPSMD12#1, shNC+CDK1, or shPSMD12#1+CDK1 were resuspended in 200 μL of PBS and subcutaneously injected into the dorsal region of the nude mice.

Techniques: Ubiquitin Proteomics, Western Blot, Knockdown, Over Expression, Control, Quantitative RT-PCR, Plasmid Preparation, Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Binding Assay