Journal: Frontiers in Oncology

Article Title: Hypoxia promotes pancreatic adenocarcinoma progression by stabilizing ID1 via TRIM21 suppression

doi: 10.3389/fonc.2025.1616968

Figure Lengend Snippet: ID1 overexpression promotes tumor aggressiveness and predicts poor outcomes in PAAD. (A) ID1 expression levels in normal and PAAD tissues based on UALCAN database analysis. (B) ID1 expression stratified by cancer stage in PAAD, showing significantly elevated levels in stage I and II tumors. (C) ID1 expression levels in PAAD stratified by tumor grade, with progressive increases observed from grade 1 to grade 3. (D) ID1 expression based on nodal metastasis status in PAAD patients. (E) Kaplan–Meier survival analysis of overall survival (OS) in PAAD patients with high versus low ID1 expression. (F) Kaplan–Meier survival analysis of relapse-free survival (RFS) in PAAD patients with high versus low ID1 expression. (G) Western blot analysis of ID1 protein expression in PAAD cell lines (AsPC-1, PANC-1, SW1990) and normal pancreatic duct epithelial cells (HPDEC). (H) Quantitative RT-PCR analysis of ID1 mRNA expression in PAAD cell lines (AsPC-1, PANC-1, SW1990) and normal pancreatic duct epithelial cells (HPDEC). *Statistical significance: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant .

Article Snippet: The primary antibodies used in western blot, immunohistochemistry and confocal assays included antibodies against ID1 (#67827, proteintech), DYKDDDDK Tag (#14793, Cell Signaling Technology), Ubiquitin (#20326, Cell Signaling Technology), β-Tubulin ((#10094, proteintech), TRIM21 (#12108, proteintech), GAPDH (#60004, proteintech) and β-ACTIN (# 66009-1, Proteintech).

Techniques: Over Expression, Expressing, Western Blot, Quantitative RT-PCR

Journal: Frontiers in Oncology

Article Title: Hypoxia promotes pancreatic adenocarcinoma progression by stabilizing ID1 via TRIM21 suppression

doi: 10.3389/fonc.2025.1616968

Figure Lengend Snippet: Hypoxia potentiates ID1-driven malignant progression in PAAD through enhanced proliferation and metastatic capacity. (A) ID1 was silenced in the indicated cell lines using ID1-targeting shRNA. (B) The indicated cells were cultured under normoxic (21% O 2 ) or hypoxic (3% O 2 ) conditions for the indicated durations. Cell proliferation was measured using the CCK-8 assay. “Relative cell viability (%)” represents viability normalized to the corresponding normoxic control at 24 h Data are presented as mean ± SD from five independent experiments (n = 5). (C) Colony formation assays were performed with PANC-1 and AsPC-1 cells cultured under normoxic or hypoxic conditions for two weeks. Colonies were stained and counted. Data are presented as mean ± SD from three independent experiments (n = 3). (D, E) Cell migration and invasion of PANC-1 and ASPC-1were assessed by Transwell assays under normoxic or hypoxic conditions. Representative images of migrated/invaded cells stained on the membrane are shown. Scale bar, 50 μm. *Statistical significance: *P < 0.05; **P < 0.01; ****P < 0.0001; ns, not significant .

Article Snippet: The primary antibodies used in western blot, immunohistochemistry and confocal assays included antibodies against ID1 (#67827, proteintech), DYKDDDDK Tag (#14793, Cell Signaling Technology), Ubiquitin (#20326, Cell Signaling Technology), β-Tubulin ((#10094, proteintech), TRIM21 (#12108, proteintech), GAPDH (#60004, proteintech) and β-ACTIN (# 66009-1, Proteintech).

Techniques: shRNA, Cell Culture, CCK-8 Assay, Control, Staining, Migration, Membrane

Journal: Frontiers in Oncology

Article Title: Hypoxia promotes pancreatic adenocarcinoma progression by stabilizing ID1 via TRIM21 suppression

doi: 10.3389/fonc.2025.1616968

Figure Lengend Snippet: Hypoxia stabilizes ID1 protein by inhibiting ubiquitin-mediated degradation in pancreatic cancer cells. (A) Immunoblot analysis of ID1 protein expression under normoxic (21% O 2 ) and hypoxic (3% O 2 ) conditions. (B) ID1 mRNA levels were measured by quantitative RT-PCR after 24-hour exposure to hypoxic (3% O 2 ) or normoxic (21% O 2 ) conditions. (n = 9). (C) Cycloheximide (CHX) chase assay (20 μg/mL) was conducted to evaluate ID1 protein stability. Cells were treated with CHX to block protein synthesis for 0, 6, 12, 18 hours after cells subjected to hypoxia for 12 hours, and ID1 levels were assessed at indicated time points to determine the degradation rate. (D) Immunoblot analysis of ID1 protein was performed following treatment with the proteasome inhibitor MG132 (20 μM) and CHX (20 μM) for 18 hours, after cells were subjected to hypoxia for 12 hours under normoxic and hypoxic conditions. (E) Ubiquitination assay was conducted by immunoprecipitating Flag-tagged ID1 using anti-Flag magnetic beads, followed by immunoblotting with anti-ubiquitin antibodies. The results show that hypoxia markedly reduces ID1 polyubiquitination, indicating that hypoxic conditions suppress its ubiquitin-mediated proteasomal degradation. ****P < 0.0001.

Article Snippet: The primary antibodies used in western blot, immunohistochemistry and confocal assays included antibodies against ID1 (#67827, proteintech), DYKDDDDK Tag (#14793, Cell Signaling Technology), Ubiquitin (#20326, Cell Signaling Technology), β-Tubulin ((#10094, proteintech), TRIM21 (#12108, proteintech), GAPDH (#60004, proteintech) and β-ACTIN (# 66009-1, Proteintech).

Techniques: Ubiquitin Proteomics, Western Blot, Expressing, Quantitative RT-PCR, Blocking Assay, Magnetic Beads

Journal: Frontiers in Oncology

Article Title: Hypoxia promotes pancreatic adenocarcinoma progression by stabilizing ID1 via TRIM21 suppression

doi: 10.3389/fonc.2025.1616968

Figure Lengend Snippet: Hypoxia-mediated TRIM21 silencing orchestrates ID1 stabilization to promote pancreatic tumorigenesis. (A) Immunoblot analysis of TRIM21 expression under hypoxic (3% O 2 ) versus normoxic (21% O 2 ) conditions. (B) Immunoblotting of ID1 protein levels in control and TRIM21-knockdown cells under normoxic and hypoxic conditions. (C) Co-immunoprecipitation (Co-IP) assay demonstrating the interaction between ID1 and TRIM21 under normoxia and hypoxia. Cell lysates from PANC-1 and AsPC-1 cells were immunoprecipitated using anti-Flag antibody to pull down Flag-tagged ID1. “Input” lanes show whole-cell lysates before immunoprecipitation; “IP: Flag” lanes indicate proteins co-precipitated with Flag-ID1. Lane 1 represents the negative control (IgG control IP), lane 2 corresponds to the normoxia-treated group, and lane 3 represents the hypoxia-treated group. (D) Immunofluorescence staining showing co-localization (yellow) of ID1 (red) and TRIM21 (green) in cells cultured under normoxia. Hypoxia reduced the degree of co-localization. Nuclei were counterstained with DAPI (blue). Scale bar: 20 μm. (E, F) Ubiquitination assays showing polyubiquitinated ID1 levels in control and TRIM21-knockdown cells under normoxic and hypoxic conditions. TRIM21 depletion decreased ID1 ubiquitination, particularly under normoxia.

Article Snippet: The primary antibodies used in western blot, immunohistochemistry and confocal assays included antibodies against ID1 (#67827, proteintech), DYKDDDDK Tag (#14793, Cell Signaling Technology), Ubiquitin (#20326, Cell Signaling Technology), β-Tubulin ((#10094, proteintech), TRIM21 (#12108, proteintech), GAPDH (#60004, proteintech) and β-ACTIN (# 66009-1, Proteintech).

Techniques: Western Blot, Expressing, Control, Knockdown, Co-Immunoprecipitation Assay, Immunoprecipitation, Negative Control, Immunofluorescence, Staining, Cell Culture, Ubiquitin Proteomics

Journal: Frontiers in Oncology

Article Title: Hypoxia promotes pancreatic adenocarcinoma progression by stabilizing ID1 via TRIM21 suppression

doi: 10.3389/fonc.2025.1616968

Figure Lengend Snippet: The TRIM21-ID1 regulatory axis controls pancreatic tumor growth through ubiquitin-mediated proteostasis. (A–D) PANC-1 cells with stable knockdown of ID1 and TRIM21 were implanted into athymic nude mice via subcutaneous injection. Tumor growth was examined 21 days after injection. Representative tumor xenografts were shown ( n = 5 mice per group) (A) . Tumor weights were calculated (B) . Tumor growth was measured every other day beginning on day 21, and tumor volumes were calculated (C) . Immunohistochemical analyses of tumor sections with the indicated antibodies were performed. Representative images are shown (D) . Scale bar, 100 μm. *Statistical significance: *P < 0.05; ***P < 0.001; ****P < 0.0001 .

Article Snippet: The primary antibodies used in western blot, immunohistochemistry and confocal assays included antibodies against ID1 (#67827, proteintech), DYKDDDDK Tag (#14793, Cell Signaling Technology), Ubiquitin (#20326, Cell Signaling Technology), β-Tubulin ((#10094, proteintech), TRIM21 (#12108, proteintech), GAPDH (#60004, proteintech) and β-ACTIN (# 66009-1, Proteintech).

Techniques: Ubiquitin Proteomics, Knockdown, Injection, Immunohistochemical staining