treatment human hcc cell lines hcclm3  (Procell Inc)

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: Additional human HCC cell lines HCCLM3 (CL-0042) and SMCC7721 (CL-0216) were obtained from Procell (Wuhan, China), and the MHCC97H (ZB001) cell line was acquired from Bei Zhi Creatures (Shanghai, China).

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: Additional human HCC cell lines HCCLM3 (CL-0042) and SMCC7721 (CL-0216) were obtained from Procell (Wuhan, China), and the MHCC97H (ZB001) cell line was acquired from Bei Zhi Creatures (Shanghai, China).

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: Additional human HCC cell lines HCCLM3 (CL-0042) and SMCC7721 (CL-0216) were obtained from Procell (Wuhan, China), and the MHCC97H (ZB001) cell line was acquired from Bei Zhi Creatures (Shanghai, China).

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

Journal: Frontiers in Pharmacology

Article Title: The SMAC Mimetic APG-1387 Sensitizes Immune-Mediated Cell Apoptosis in Hepatocellular Carcinoma

doi: 10.3389/fphar.2018.01298

Figure Lengend Snippet: APG-1387 treatment induced rapid cIAPs degradation but failed to induce cell death in HepG2 and HCCLM3 cells. (A,B) The mRNA expression of cancer stem cell (CSC) genes ( ABCG2, CD44 , and SOX2 ) and the percentages of side population (SP) cells in HepG2 and HCCLM3 cells, were measured by qRT-PCR and flow cytometry, respectively. (B) The proportion of SP cells in HepG2 and HCCLM3 cell lines were 0.16% and 32.2%, which reduced to 0% and 0.28% in the presence of verapamil. (C) The relative expression of mRNA of CSC and IAP genes between SP and the main population (MP) HCCLM3 cells were examined by qRT-PCR. (D) The basic protein levels of cIAP1, cIAP2, and XIAP in HepG2 and HCCLM3 cells were analyzed by Western blot. (E,F) The changes in protein levels of the IAPs and cleaved poly (ADP-ribose) polymerase (PARP) proteins, (G,H) and cell viability of HepG2 and HCCLM3 cells were detected after treatment with AGP-1387 as a single agent at indicated concentrations and time periods. Con, control; cl-PARP, cleaved-PARP; kDa, kilodalton. Error bars represented the mean ± SEM of triplicate representative experiments; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, by two-tailed unpaired t -test.

Article Snippet: A total of 20 male NOD-SCID mice (Charles River Laboratories, Wilmington, DE, United States) aged 4–6 weeks, were implanted with 5 × 10 6 human HCC cell line HCCLM3 cells, subcutaneously into the right axillary cavity.

Techniques: Expressing, Quantitative RT-PCR, Flow Cytometry, Western Blot, Control, Two Tailed Test

Journal: Frontiers in Pharmacology

Article Title: The SMAC Mimetic APG-1387 Sensitizes Immune-Mediated Cell Apoptosis in Hepatocellular Carcinoma

doi: 10.3389/fphar.2018.01298

Figure Lengend Snippet: APG-1387 treatment enhanced TNF-α and TRAIL mediated anti-cancer activities in the HCC cell lines. (A,B) HepG2 and (C,D) HCCLM3 cells were pre-inoculated in quadruplicate at 2,000 cells per well in 96-well plates for 12 h, cell viability was evaluated using a CCK-8 assay after 24 h of stimulation with TNF-α or TRAIL, or in combination with APG-1387. (E) HepG2 and (F) HCCLM3 cells were pre-seeded in 6-well plates at 1,000 cells per well for 12 h and then stimulated with 2 μM APG-1387, 100 ng/ml TNF-α, 20 ng/ml TRAIL or their combination. After incubating for 14 days, the colonies were stained with Giemsa dye and counted with ImageJ software. (G,H) After 24 h of stimulation with APG-1387 and TNF-α or TRAIL, the percentages of side population (SP) cells were analyzed by flow cytometry after staining with Hoechst 33342 fluorescence dye alone or in combination with verapamil, which was used as a control to block the efflux of Hoechst 33342. (I) Under indicated treatment of APG-1387 and TNF-α or TRAIL, lysates of HepG2 and HCCLM3 cells were harvested, then the level of Sox2 protein was evaluated using a Western blot assay. Con, control; A, 2 μM APG-1387; T, 100 ng/ml TNF-α; TR, 20 ng/ml TRAIL; kDa, kilodalton. Error bars represented the mean ± SEM of triplicate representative experiments; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, by two-tailed unpaired t -test.

Article Snippet: A total of 20 male NOD-SCID mice (Charles River Laboratories, Wilmington, DE, United States) aged 4–6 weeks, were implanted with 5 × 10 6 human HCC cell line HCCLM3 cells, subcutaneously into the right axillary cavity.

Techniques: CCK-8 Assay, Staining, Software, Flow Cytometry, Fluorescence, Control, Blocking Assay, Western Blot, Two Tailed Test

Journal: Frontiers in Pharmacology

Article Title: The SMAC Mimetic APG-1387 Sensitizes Immune-Mediated Cell Apoptosis in Hepatocellular Carcinoma

doi: 10.3389/fphar.2018.01298

Figure Lengend Snippet: APG-1387 treatment enhanced TNF-α- and TRAIL-induced cell death in HepG2 and HCCLM3 cells. (A–C) HepG2 and HCCLM3 cells were seeded at 1 × 10 6 cells per well in 6-well plates for 12 h. After 24 h of stimulation with APG-1387 and TNF-α or TRAIL, all of the cells were collected separately, and incubated with Annexin-V and 7-AAD for 15 min, then apoptosis (Annexin-V+ 7-AAD–) and necrosis (Annexin-V+ 7-AAD+) of HepG2 or HCCLM3 cells were detected by flow cytometry. (D) After 24 h of stimulation with APG-1387 and TNF-α or TRAIL, lysates of HepG2 and HCCLM3 cells were harvested. Western blot was performed to analyze the changes in levels of apoptosis-related proteins, including poly (ADP-ribose) polymerase (PARP), caspase-3, cleaved-caspase-8, cleaved-caspase-9, cIAP1, cIAP2, and XIAP and gasdermin E (GSDME). (E,F) HepG2 and HCCLM3 cells were pre-treated with a pan-caspase inhibitor (20 μM Z-VAD-fmk) or RIPK1 inhibitor (50 μM Nec-1) for 1 h. Then combination treatments involving APG-1387 with TNF-α or TRAIL were used, and the percentages of cell death were evaluated by flow cytometry. Con, control; A, 2 μM APG-1387; T, 100 ng/ml TNF-α; TR, 20 ng/ml TRAIL; cl-PARP, cleaved-PARP; Cas - 3, caspase-3; cl-Cas - 3, cleaved-caspase-3; cl-Cas - 8, cleaved-caspase-8; cl-Cas - 9, cleaved-caspase-9; GSDME-F, full-length GSDME; GSDME-N, N terminal fragment of GSDME; Nec-1, necrostatin-1; kDa, kilodalton. Error bars represented the mean ± SEM of triplicate representative experiments; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, by two-tailed unpaired t -test.

Article Snippet: A total of 20 male NOD-SCID mice (Charles River Laboratories, Wilmington, DE, United States) aged 4–6 weeks, were implanted with 5 × 10 6 human HCC cell line HCCLM3 cells, subcutaneously into the right axillary cavity.

Techniques: Incubation, Flow Cytometry, Western Blot, Control, Two Tailed Test

Journal: Frontiers in Pharmacology

Article Title: The SMAC Mimetic APG-1387 Sensitizes Immune-Mediated Cell Apoptosis in Hepatocellular Carcinoma

doi: 10.3389/fphar.2018.01298

Figure Lengend Snippet: APG-1387 sensitized HepG2 and HCCLM3 cells to NK cell-mediated killing in vitro. HepG2 or HCCLM3 cells were co-cultured with purified NK cells, which were stimulated with 10 ng/ml interleukin (IL)-12, 10 ng/ml IL-15, and 100 ng/ml IL-18, either alone or in the presence of 2 μM APG-1387. (A,B) After co-incubation for 24 h, HepG2 or HCCLM3 cells were collected for analysis of killing effect of NK cells by flow cytometry, (C) while supernatants were collected for the LDH assay. Con, control; A, 2 μM APG-1387; NK(+), NK cells co-cultured with 10 ng/ml IL-12, 10 ng/ml IL-15, and 100 ng/ml IL-18. Error bars represented the mean ± SEM of triplicate representative experiments; ∗ p < 0.05, ∗∗ p < 0.01; by paired t -test.

Article Snippet: A total of 20 male NOD-SCID mice (Charles River Laboratories, Wilmington, DE, United States) aged 4–6 weeks, were implanted with 5 × 10 6 human HCC cell line HCCLM3 cells, subcutaneously into the right axillary cavity.

Techniques: In Vitro, Cell Culture, Purification, Incubation, Flow Cytometry, Lactate Dehydrogenase Assay, Control

Journal: Frontiers in Pharmacology

Article Title: The SMAC Mimetic APG-1387 Sensitizes Immune-Mediated Cell Apoptosis in Hepatocellular Carcinoma

doi: 10.3389/fphar.2018.01298

Figure Lengend Snippet: APG-1387 sensitized HCCLM3 tumors toward NK cell-mediated killing in vivo. Four groups of NOD-SCID mice bearing human HCC cell line HCCLM3 tumors were injected intraperitoneally with APG-1387 at 20 mg/kg every 3 days for 4 weeks, and/or injected at the tumor site with 2 × 10 7 IL-12, IL-15, and IL-18-activated NK cells per mouse (or with the same volume of PBS) every 6 days for 4 weeks. (B,E) Tumor volume and body weight were measured every 3 days. (C,D) After 4 weeks of treatment, the mice were sacrificed, the tumors were harvested and weighed. (A) The expression of IAPs in tumors was measured by Western blot and (F) the relative proportion of cleaved caspase-3 was shown using confocal microscopy. Con, control; A, APG-1387; NK(+), NK cells co-cultured with 10 ng/ml IL-12, 10 ng/ml IL-15, and 100 ng/ml IL-18; cl-Cas-3, cleaved-caspase-3. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, N.S., not significant; by two-tailed unpaired t -test.

Article Snippet: A total of 20 male NOD-SCID mice (Charles River Laboratories, Wilmington, DE, United States) aged 4–6 weeks, were implanted with 5 × 10 6 human HCC cell line HCCLM3 cells, subcutaneously into the right axillary cavity.

Techniques: In Vivo, Injection, Expressing, Western Blot, Confocal Microscopy, Control, Cell Culture, Two Tailed Test