Journal: Nucleic Acids Research

Article Title: hnRNPA2B1 induces HBV cccDNA degradation by recruiting APOBEC3B

doi: 10.1093/nar/gkag149

Figure Lengend Snippet: hnRNPA2B1 interacts with APOBEC3B to promote its recruitment to cccDNA. ( A ) HA-A2B1 was co-transfected with FLAG-APOBEC3s (A3A, A3B, A3C, A3D, A3F, or A3G) in Huh7 cells for 48 h. Co-IP assay was performed to analyze their interactions. The arrow indicates the target band. ( B ) Co-IP assay was performed with anti-A2B1 antibody or anti-A3B antibody in HLCZ01 cells to evaluate the endogenous interaction of hnRNPA2B1 and APOBEC3B. ( C ) hnRNPA2B1 overexpression was induced by Dox in HBV-infected HepG2 NTCP-A2B1 cells (left panel), while hnRNPA2B1 knockdown was accessed by shA2B1 lentivirus in HBV-infected HepG2 NTCP cells (right panel). The enrichment of A3B on cccDNA was analyzed by ChIP assay, and the immunoprecipitation efficiency was measured by western blot. HBV-infected HLCZ01 A2B1-Teton cells transfected with APOBEC3B-siRNA (siA3B) and treated with Dox for 72 h were subjected to: 3D-PCR with denaturation temperature gradient (85°C–83°C) ( D ); deep sequencing of 85°C-amplified cccDNA fragments showing C>T and G>A events and mutation frequency ( E ); and parallel assessments of cccDNA, HBsAg/HBeAg, and preC/pgRNA levels by Southern blot, qPCR, ELISA, and RT-qPCR, respectively ( F ). ( G ) Schematic diagram of FLAG-hnRNPA2B1 truncates (left panel). HA-A3B was co-transfected with different FLAG-hnRNPA2B1 truncates in Huh7 cells for 48 h. Co-IP assay was performed to analyze their interactions. ( H ) FLAG-A2B1 and A2B1ΔPrLD were transfected in HBV-infected HLCZ01 cells for 72 h, the enrichment of A3B on cccDNA was analyzed by ChIP assay, and the efficiency of immunoprecipitation was detected by western blot. Results are representative of two [(A, B) and (G)] or three [(C, D) to (F) and (H)] independent experiments. P -values were determined by one-way ANOVA (F) or two-way ANOVA (C, H); ** P < .01, NS: non-significant.

Article Snippet: Rabbit anti-hnRNPA2B1 polyclonal antibody (A1162, Abclonal, Wuhan, China), rabbit anti-HBc polyclonal antibody (B0586, Dako, Copenhagen, Denmark), rabbit anti-APOBEC3B polyclonal antibody (14559-1-AP, Proteintech, Chicago, IL, USA), mouse anti-β-actin monoclonal antibody (66009-1Ig, Proteintech), mouse anti-FLAG-tag monoclonal antibody (M185-3, MBL, Nagoya, Japan), mouse anti-HA monoclonal antibody (M180-3, MBL), mouse anti-ubiquitin monoclonal antibody (sc-8017, Santa), and rabbit anti-HBx polyclonal antibody (ab39716, Abcam).

Techniques: Transfection, Co-Immunoprecipitation Assay, Over Expression, Infection, Knockdown, Immunoprecipitation, Western Blot, Sequencing, Amplification, Mutagenesis, Southern Blot, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Computational and Structural Biotechnology Journal

Article Title: Integrated metabolome and transcriptome analysis reveals ferroptosis involvement in cisplatin resistance of esophageal squamous cancer cell

doi: 10.1016/j.csbj.2026.01.003

Figure Lengend Snippet: Analysis of TCGA datasets reveals that APOBEC3B inhibits ferroptosis by activating PD-L1 and suppressing CD8 + T cells. (A) Venn diagram analysis of significantly upregulated genes in KYSE30, KYSE30/DDP, and TCGA-ESCC (left), and scatter plot showing survival analysis of 12 genes (right). (B) Survival analysis indicates that high APOBEC3B expression is associated with poor patient prognosis in the TCGA dataset. (C) Boxplot showing higher APOBEC scores in the high APOBEC3B expression group compared to the low expression group in the TCGA dataset (left); APOBEC3B expression is elevated in the copy number amplification group compared to the non-amplification group (right). (D) KEGG pathway enrichment analysis of genes significantly positively correlated with APOBEC3B. (E) Heatmap showing expression levels of CD274, ferroptosis suppressor factors, and ferroptosis drivers (left). Pearson correlation analysis reveals that APOBEC3B expression is significantly positively correlated with CD274 and ferroptosis suppressor factors and negatively correlated with ferroptosis drivers. (F) CIBERSORT immune infiltration analysis based on the TCGA dataset, with a boxplot displaying proportions of 22 immune cell types. (G) Boxplot showing expression levels of L-Glutamate (left) and oxidized glutathione (right) in HEEC, KYSE30, and KYSE30/DDP. (H) Heatmap showing correlation analysis of glutathione metabolism-related metabolites with CD274 and ferroptosis suppressor factors. （* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns: no significance）.

Article Snippet: Knockdown constructs for APOBEC3B (si-APOBEC3B, pSLenti-U6-siRNA (APOBEC3B)-CMV-EGFP-F2A-Puro-WPRE) and the corresponding negative control (si-NC, pSLenti-U6-siRNA (NC)-CMV-EGFP-F2A-Puro-WPRE) were obtained from OBiO Technology.

Techniques: Expressing, Amplification

Journal: Computational and Structural Biotechnology Journal

Article Title: Integrated metabolome and transcriptome analysis reveals ferroptosis involvement in cisplatin resistance of esophageal squamous cancer cell

doi: 10.1016/j.csbj.2026.01.003

Figure Lengend Snippet: Knocking down APOBEC3B enhances ferroptosis and inhibits the glutathione metabolism signaling pathway in cells. (A) qPCR analysis showed demonstrated the effectiveness of using three siRNAs to knockdown APOBEC3B. in KYSE30/DDP cells after knockdown. (B) Western blot analysis revealed a reduction in APOBEC3B expression in KYSE30/DDP cells after knockdown. (C) An Ferro-Orange probes was used to measure cytoplasmic Fe 2+ levels in KYSE30, KYSE30/DDP, and APOBEC3B knockdown cells. (D) BODIPY-C11 staining for detection of lipid peroxidation levels.(E) ROS production in KYSE30, KYSE30/DDP, and APOBEC3B knockdown cells was analyzed using flow cytometry. (F) Levels of GSH, GSSG, and the GSH/GSSG ratio were measured in KYSE30, KYSE30/DDP, and APOBEC3B knockdown cells.

Article Snippet: Knockdown constructs for APOBEC3B (si-APOBEC3B, pSLenti-U6-siRNA (APOBEC3B)-CMV-EGFP-F2A-Puro-WPRE) and the corresponding negative control (si-NC, pSLenti-U6-siRNA (NC)-CMV-EGFP-F2A-Puro-WPRE) were obtained from OBiO Technology.

Techniques: Knockdown, Western Blot, Expressing, Staining, Flow Cytometry

Journal: Computational and Structural Biotechnology Journal

Article Title: Integrated metabolome and transcriptome analysis reveals ferroptosis involvement in cisplatin resistance of esophageal squamous cancer cell

doi: 10.1016/j.csbj.2026.01.003

Figure Lengend Snippet: APOBEC3B knockdown inhibits PD-L1, activates CD8 + T cells, and enhances ferroptosis. (A) Western blot analysis of PD-L1 expression in KYSE30 and KYSE30/DDP cells. (B) Western blot analysis of PD-L1 expression in KYSE30 cells treated with DDP for 0, 48, and 72 h, and untrested KYSE30/DDP cells. (C) Western blot analysis of PD-L1 expression in KYSE30 cells, and in KYSE30/DDP cells with or without APOBEC3B knockdown. (D) Immunofluorescence analysis of CD8 + T cell distribution in the tumor immune microenvironment of esophageal squamous cell carcinoma treated with si-NC, si-APOBEC3B, PD-L1 inhibitor, or a combination of si-APOBEC3B and PD-L1 inhibitor. (E) Expression of ROS, (F) LPO, and (G) MDA in esophageal squamous cell carcinoma tissues treated with si-NC, si-APOBEC3B, PD-L1 inhibitor, or a combination of si-APOBEC3B and PD-L1 inhibitor. (H) Western blot analysis of protein levels of the anti-ferroptosis gene FTL, pro-ferroptosis gene ACSL4, and glutathione metabolism-related genes GPX4 and GCLC in esophageal squamous cell carcinoma tissues after APOBEC3B knockdown.

Article Snippet: Knockdown constructs for APOBEC3B (si-APOBEC3B, pSLenti-U6-siRNA (APOBEC3B)-CMV-EGFP-F2A-Puro-WPRE) and the corresponding negative control (si-NC, pSLenti-U6-siRNA (NC)-CMV-EGFP-F2A-Puro-WPRE) were obtained from OBiO Technology.

Techniques: Knockdown, Western Blot, Expressing, Immunofluorescence

Journal: Computational and Structural Biotechnology Journal

Article Title: Integrated metabolome and transcriptome analysis reveals ferroptosis involvement in cisplatin resistance of esophageal squamous cancer cell

doi: 10.1016/j.csbj.2026.01.003

Figure Lengend Snippet: APOBEC3B acts as a key driver in KYSE30/DDP cells, simultaneously inducing PD-L1 expression and repressing glutathione metabolism, which collectively inhibits ferroptosis.

Article Snippet: Knockdown constructs for APOBEC3B (si-APOBEC3B, pSLenti-U6-siRNA (APOBEC3B)-CMV-EGFP-F2A-Puro-WPRE) and the corresponding negative control (si-NC, pSLenti-U6-siRNA (NC)-CMV-EGFP-F2A-Puro-WPRE) were obtained from OBiO Technology.

Techniques: Expressing