Journal: NPJ Precision Oncology

Article Title: METTL21A promotes hepatocellular carcinoma progression via methylating and stabilizing BAG3

doi: 10.1038/s41698-025-01021-5

Figure Lengend Snippet: A In silico analysis identified potential transcription factors within the METTL21A promoter region. B Spearman’s correlation analysis between METTL21A and CTCF mRNA expression in the TCGA-LIHC cohort. C The mRNA expression levels of METTL21A were assessed by RT-qPCR in Huh7 and SNU182 cells following CTCF silencing. D Protein expression levels of METTL21A were assessed by Western blot in Huh7 and SNU182 cells after CTCF silencing. E Spearman’s correlation analysis of METTL21A and CTCF mRNA expression in HCC clinical samples ( n = 45). F Dual-luciferase reporter gene assay in cells co-transfected with siRNA, pRL-TK, and pGL3-Basic /pGL3-METTL21A plasmids. G Primer design targeting regions in the METTL21A promoter: P1 (−1300 bp to −1100 bp), P2 (−100 bp to +100 bp relative to the transcription start site, TSS), and P3 (+400 bp to +600 bp). H ChIP-qPCR analysis of three METTL21A promoter regions (P1, P2, and P3) in CTCF-silenced HCC cells using anti-CTCF antibody (IgG as control). GAPDH exon 3 served as a negative control. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Human HCC cell lines (Huh7, SNU182, Hep3B, LM3, Li7, MHCC97H, SNU449, and SK-Hep-1) and HEK293T cells were obtained from Cellcook Biotechnology (Guangzhou, China), while PLC/PRF/5 cells were sourced from the American Type Culture Collection (ATCC, USA).

Techniques: In Silico, Expressing, Quantitative RT-PCR, Western Blot, Luciferase, Reporter Gene Assay, Transfection, ChIP-qPCR, Control, Negative Control

Journal: NPJ Precision Oncology

Article Title: METTL21A promotes hepatocellular carcinoma progression via methylating and stabilizing BAG3

doi: 10.1038/s41698-025-01021-5

Figure Lengend Snippet: A Validation of METTL21A knockdown efficiency in HCC cells by Western blot analysis. B Proliferation kinetics of METTL21A-deficient HCC cells measured by CCK-8 assay. C Clonogenic potential assessment of METTL21A-silenced HCC cells through colony formation assays. D Western blot confirmation of METTL21A overexpression in HCC cell lines. E Enhanced proliferative capacity of METTL21A-overexpressing HCC cells demonstrated by CCK-8 assay. F Quantitative analysis ( n = 3) of colony formation efficiency in METTL21A-overexpressing HCC cells. G Cell cycle distribution profiles analyzed by flow cytometry. H Apoptotic cell fractions quantified by flow cytometric analysis of Annexin V/PI staining. I - K In vivo tumorigenicity assessment showing representative tumor specimens ( I ), growth kinetics ( J ), and final tumor weights ( K ) in subcutaneous xenograft models ( n = 5). L Proliferation evaluation by Ki-67 IHC staining and quantification in xenograft tissues ( n = 5). Scale bars: 100 μm (overview), 50 μm (detail). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Human HCC cell lines (Huh7, SNU182, Hep3B, LM3, Li7, MHCC97H, SNU449, and SK-Hep-1) and HEK293T cells were obtained from Cellcook Biotechnology (Guangzhou, China), while PLC/PRF/5 cells were sourced from the American Type Culture Collection (ATCC, USA).

Techniques: Biomarker Discovery, Knockdown, Western Blot, CCK-8 Assay, Over Expression, Flow Cytometry, Staining, In Vivo, Immunohistochemistry

Journal: NPJ Precision Oncology

Article Title: METTL21A promotes hepatocellular carcinoma progression via methylating and stabilizing BAG3

doi: 10.1038/s41698-025-01021-5

Figure Lengend Snippet: A Migration and invasion capacity of METTL21A-knockdown HCC cells assessed by transwell assays. Quantification represents mean cell counts from five random fields per sample. Scale bar: 200 μm. B Enhanced metastatic potential of METTL21A-overexpressing HCC cells demonstrated by transwell migration and invasion assays ( n = 3). Cell counts were obtained from five representative microscopic fields. Scale bar: 200 μm. C Pulmonary metastasis evaluation in experimental metastasis models ( n = 5). Left panel: macroscopic lung sections with H&E staining showing metastatic nodules (scale bar: 200 μm [overview], 50 μm [detail]). Right panel: quantitative analysis of lung metastatic burden. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Human HCC cell lines (Huh7, SNU182, Hep3B, LM3, Li7, MHCC97H, SNU449, and SK-Hep-1) and HEK293T cells were obtained from Cellcook Biotechnology (Guangzhou, China), while PLC/PRF/5 cells were sourced from the American Type Culture Collection (ATCC, USA).

Techniques: Migration, Knockdown, Staining

Journal: NPJ Precision Oncology

Article Title: METTL21A promotes hepatocellular carcinoma progression via methylating and stabilizing BAG3

doi: 10.1038/s41698-025-01021-5

Figure Lengend Snippet: A Bioinformatics analysis identified the top 10 potential METTL21A-interacting proteins through integrated screening of HitPredict, BioGRID, and HINT databases. B Immunofluorescence confocal microscopy revealed significant co-localization (yellow signals) of endogenous METTL21A (red) and BAG3 (green) in Huh7 and SNU182 cells (scale bars: 20 µm). C Exogenous Co-IP in HEK293T cells transfected with Flag-METTL21A and Myc-BAG3 confirmed direct interaction, with anti-Flag/Myc antibodies showing reciprocal pulldown (IgG control negative). D Endogenous Co-IP in Huh7 and SNU182 cell lysates demonstrated a physiological interaction between METTL21A and BAG3 (IgG control negative). E The expression levels of BAG3 mRNA in control and METTL21A-silenced HCC cells were determined by RT-qPCR. F The expression levels of BAG3 protein in control and METTL21A-silenced HCC cells were assessed by Western blot analysis. G The BAG3 protein levels in the subcutaneous tumor model were measured using Western blotting. H The half-life of BAG3 was determined by CHX (20 μM) chase assay in METTL21A-silenced and control Huh7 cells. The BAG3 band intensities were measured over time and compared to the initial time point. * p < 0.05 and ** p < 0.01.

Article Snippet: Human HCC cell lines (Huh7, SNU182, Hep3B, LM3, Li7, MHCC97H, SNU449, and SK-Hep-1) and HEK293T cells were obtained from Cellcook Biotechnology (Guangzhou, China), while PLC/PRF/5 cells were sourced from the American Type Culture Collection (ATCC, USA).

Techniques: Immunofluorescence, Confocal Microscopy, Co-Immunoprecipitation Assay, Transfection, Control, Expressing, Quantitative RT-PCR, Western Blot

Journal: NPJ Precision Oncology

Article Title: METTL21A promotes hepatocellular carcinoma progression via methylating and stabilizing BAG3

doi: 10.1038/s41698-025-01021-5

Figure Lengend Snippet: A Western blot analysis confirmed BAG3 overexpression in METTL21A-silenced Huh7 and SNU182 HCC cell lines. B CCK-8 proliferation assays demonstrated that BAG3 overexpression partially rescued the growth inhibition caused by METTL21A knockdown. C Colony formation assays revealed that BAG3 overexpression restored the clonogenic potential of METTL21A-deficient HCC cells, with quantitative analysis of colony numbers. D Transwell migration and invasion assays showed that BAG3 overexpression mitigated the metastatic suppression induced by METTL21A silencing (scale bars: 200 μm), with cell counts from five representative fields. E Representative images of METTL21A and BAG3 IHC staining in the same HCC clinical sample were presented. Scale bars: 200 μm. F Correlation analysis revealed a significant positive relationship between METTL21A and BAG3 protein levels across HCC patient samples. G Kaplan–Meier survival curves stratified by combined METTL21A/BAG3 expression showed significantly worse OS and DFS for patients with high co-expression ( n = 30) vs low co-expression ( n = 28) (log-rank test). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Human HCC cell lines (Huh7, SNU182, Hep3B, LM3, Li7, MHCC97H, SNU449, and SK-Hep-1) and HEK293T cells were obtained from Cellcook Biotechnology (Guangzhou, China), while PLC/PRF/5 cells were sourced from the American Type Culture Collection (ATCC, USA).

Techniques: Western Blot, Over Expression, CCK-8 Assay, Inhibition, Knockdown, Migration, Immunohistochemistry, Expressing

Journal: NPJ Precision Oncology

Article Title: METTL21A promotes hepatocellular carcinoma progression via methylating and stabilizing BAG3

doi: 10.1038/s41698-025-01021-5

Figure Lengend Snippet: A Endogenous TRIM21 and BAG3 proteins were immunoprecipitated using specific antibodies and subsequently analyzed through Western blotting. B Immunofluorescence confocal microscopy revealed significant co-localization of endogenous TRIM21 and BAG3 in Huh7 and SNU182 cells (scale bars: 20 µm). C Cell lysates from control and METTL21A-silenced Huh7 cells were immunoprecipitated with anti-BAG3 antibody and analyzed by Western blotting. D Cell lysates from control and METTL21A-overexpressing Huh7 cells were immunoprecipitated with an anti-BAG3 antibody and analyzed via Western blotting. E BAG3 protein levels in TRIM21-silenced HCC cells were assessed by Western blotting. F Western blot analysis assessed BAG3 levels in TRIM21-silenced HCC cells following treatment with MG132 (10 μM) for 6 h. G The half-life of BAG3 was determined in TRIM21-silenced Huh7 cells using a CHX (20 μM) chase assay. The BAG3 band intensities were measured over time and compared to the initial time point. H Cells with TRIM21 silence were treated with MG132 (10 μM) for 6 h, then cell lysates were subjected to immunoprecipitation using an anti-BAG3 antibody and analyzed by Western blotting. I Control and TRIM21-silenced HEK293T cells were transfected with plasmids for 48 h, treated with MG132 (10 μM, 6 h), and cell lysates were immunoprecipitated with anti-Myc antibodies for Western blot analysis of Myc-BAG3 ubiquitination (WT, K11, K48, and K63-linked). J Western blot analysis of BAG3 protein in Huh7 cells co-transfected with siMETTL21A and/or siTRIM21 for 48 h.

Article Snippet: Human HCC cell lines (Huh7, SNU182, Hep3B, LM3, Li7, MHCC97H, SNU449, and SK-Hep-1) and HEK293T cells were obtained from Cellcook Biotechnology (Guangzhou, China), while PLC/PRF/5 cells were sourced from the American Type Culture Collection (ATCC, USA).

Techniques: Immunoprecipitation, Western Blot, Immunofluorescence, Confocal Microscopy, Control, Transfection, Ubiquitin Proteomics