human hepatoma cell lines hepg2  (ATCC)

Journal: Tumour Virus Research

Article Title: PRMT5–mediated symmetric dimethylation of SHBs at Arg169 stabilizes SHBs and promotes angiogenesis and tumor growth

doi: 10.1016/j.tvr.2026.200340

Figure Lengend Snippet: SHBs is symmetrically dimethylated at arginine 169. (A–C) Huh7 and HepG2 cells were transfected with plasmids encoding SHBs–Strep–Flag or Strep–Flag control. Strep pull–down (IP:Strep) was performed, followed by Western blot (WB) with antibodies against (A) monomethylarginine (MMA), (B) asymmetric dimethylarginine (ADMA), or (C) symmetric dimethylarginine (SDMA). SHBs in the IP fraction and SHBs/β–actin in input lysates are shown as controls. (D) Cells expressing SHBs–Strep–Flag were treated with adenosine dialdehyde (ADOX, 40 μM) for 36 h, followed by Strep pull–down and WB for SDMA and SHBs. Densitometric ratios (SDMA/IP–SHBs and SHBs/β–actin) are shown above/below the blots. (E) Huh7 cells were transfected with plasmids encoding SHBs–Strep–Flag or the indicated R→K mutants (R73K, R78K, R79K, R169K). SDMA on immunoprecipitated SHBs was assessed by Strep pull–down and WB; densitometric SDMA/IP–SHBs ratios are shown above the blots. (F–G) HepG2 cells were transfected with plasmids encoding SHBs–Strep–Flag or SHBs/R169K–Strep–Flag (F) and SHBs/R169A–Strep–Flag (G) and analyzed by Strep pull–down and WB as in (E).

Article Snippet: Human hepatoma cell lines HepG2 (ATCC, HB–8065) and Huh7 (JCRB, JCRB0403), endothelial cell line EA.hy926 (ATCC, CRL–2922TM), and HEK293T cells (ATCC, CRL–3216) were obtained from the American Type Culture Collection (ATCC) and the Japanese Collection of Research Bioresources Cell Bank (JCRB, Japan).

Techniques: Transfection, Control, Western Blot, Expressing, Immunoprecipitation

Journal: Tumour Virus Research

Article Title: PRMT5–mediated symmetric dimethylation of SHBs at Arg169 stabilizes SHBs and promotes angiogenesis and tumor growth

doi: 10.1016/j.tvr.2026.200340

Figure Lengend Snippet: PRMT interacts with SHBs. (A) Huh7 cells were co–transfected with plasmids encoding SHBs–Strep–Flag (or Strep–Flag control) together with Flag–PRMT9. Strep pull–down was followed by WB with anti–Flag and anti–SHBs to assess co–precipitation. (B) Huh7 and HepG2 cells were co–transfected with plasmids encoding SHBs–Strep–Flag (or Strep–Flag control) together with Flag–PRMT5 and analyzed by Strep pull–down and WB as in (A). (C) Huh7 and HepG2 cells were co–transfected with plasmids encoding Strep–Flag–PRMT5 and SHBs–myc. Strep pull–down was performed and precipitates were immunoblotted for SHBs and Flag to validate the interaction. (D) Direct interaction between SHBs and PRMT5 was tested by GST pull–down. Purified GST or GST–PRMT5 (Coomassie–stained gel, left) was incubated with in vitro–translated SHBs–Flag, and bound SHBs was detected by WB using anti–Flag (right). (E) Confocal microscopy showing subcellular localization of SHBs (red) and PRMT5 (green) with nuclear DAPI staining (blue). Merged images and a representative line–scan fluorescence intensity profile (right) are shown.

Article Snippet: Human hepatoma cell lines HepG2 (ATCC, HB–8065) and Huh7 (JCRB, JCRB0403), endothelial cell line EA.hy926 (ATCC, CRL–2922TM), and HEK293T cells (ATCC, CRL–3216) were obtained from the American Type Culture Collection (ATCC) and the Japanese Collection of Research Bioresources Cell Bank (JCRB, Japan).

Techniques: Transfection, Control, Purification, Staining, Incubation, In Vitro, Confocal Microscopy, Fluorescence

Journal: Tumour Virus Research

Article Title: PRMT5–mediated symmetric dimethylation of SHBs at Arg169 stabilizes SHBs and promotes angiogenesis and tumor growth

doi: 10.1016/j.tvr.2026.200340

Figure Lengend Snippet: PRMT5 stabilizes SHBs protein expression in an Arg169–dependent manner. (A) Huh7 and HepG2 cells were co–transfected with plasmids encoding SHBs–Strep–Flag or SHBs/R169K–Strep–Flag together with increasing amounts of Flag–PRMT5 (0, 1, 3 μg). Whole–cell lysates were immunoblotted for SHBs, Flag, and β–actin; SHBs/β–actin ratios are shown above the blots. (B) Cells expressing SHBs–Strep–Flag or SHBs/R169K–Strep–Flag were transfected with NC or PRMT5 siRNAs (#1, #2). Lysates were immunoblotted for SHBs, PRMT5, and β–actin; SHBs/β–actin ratios are shown. (C–D) Cycloheximide (CHX) chase assays in (C) Huh7 and (D) HepG2 cells. Cells expressing SHBs or SHBs/R169K with vector or Flag–PRMT5 were treated with CHX for the indicated times (0–120 min), followed by WB for SHBs, Flag, and β–actin. Plots show relative SHBs levels normalized to time 0 with fitted linear regression (equations displayed). (E) HepG2 cells were co–transfected with plasmids encoding SHBs–Strep, HA–K48Ub, together with or without Flag–PRMT5, and treated with MG132 (20 μM) for 8 h, the ubiquitination levels of SHBs was evaluated via ubiquitination assay analysis. (F) HepG2 cells were co–transfected with plasmid encoding SHBs–Strep and TRIM21–myc (or control vector) and Flag–PRMT5 (or control vector), the cell lysates were subjected to immunoprecipitation using Strep–Tactin and analyzed by immunoblotting.

Article Snippet: Human hepatoma cell lines HepG2 (ATCC, HB–8065) and Huh7 (JCRB, JCRB0403), endothelial cell line EA.hy926 (ATCC, CRL–2922TM), and HEK293T cells (ATCC, CRL–3216) were obtained from the American Type Culture Collection (ATCC) and the Japanese Collection of Research Bioresources Cell Bank (JCRB, Japan).

Techniques: Expressing, Transfection, Plasmid Preparation, Ubiquitin Proteomics, Control, Immunoprecipitation, Western Blot

Journal: Tumour Virus Research

Article Title: PRMT5–mediated symmetric dimethylation of SHBs at Arg169 stabilizes SHBs and promotes angiogenesis and tumor growth

doi: 10.1016/j.tvr.2026.200340

Figure Lengend Snippet: Arg169 symmetric dimethylation is required for SHBs–driven angiogenesis and tumor growth. (A) WB analysis of SHBs and BIP expression in stably transduced Huh7 and HepG2 cells (Vector, SHBs, and SHBs/R169K). (B) ELISA measurement of VEGFA levels in the supernatants of Huh7/HepG2–Vector, Huh7/HepG2–SHBs, or Huh7/HepG2–SHBs/R169K cells. (C) Endothelial tube formation assay. EA.hy926 cells were cultured with conditioned media (CM) from Huh7 or HepG2 stable lines (Vector, SHBs, SHBs/R169K). Representative images and quantification of mesh numbers are shown. (D) Transwell migration assay. EA.hy926 cells were assessed for migration in response to CM from the indicated stable lines. Representative images and quantification of migrated cell numbers per field are shown. (E) Representative images of excised subcutaneous xenograft tumors derived from Huh7–Vector, Huh7–SHBs, or Huh7–SHBs/R169K cells. (F) Tumor growth curves (tumor volume over time) for the indicated xenograft groups. (G) Tumor weights at endpoint. (H) Representative immunohistochemical staining of xenograft tumors for CD31 and SHBs, with quantification of microvessel density (MVD) based on CD31 staining. Data are presented as mean ± SD; ∗ P < 0.05 as indicated.

Article Snippet: Human hepatoma cell lines HepG2 (ATCC, HB–8065) and Huh7 (JCRB, JCRB0403), endothelial cell line EA.hy926 (ATCC, CRL–2922TM), and HEK293T cells (ATCC, CRL–3216) were obtained from the American Type Culture Collection (ATCC) and the Japanese Collection of Research Bioresources Cell Bank (JCRB, Japan).

Techniques: Expressing, Stable Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Endothelial Tube Formation Assay, Cell Culture, Transwell Migration Assay, Migration, Derivative Assay, Immunohistochemical staining, Staining