david chan  (Addgene inc)

Journal: eBioMedicine

Article Title: Environmental ciprofloxacin triggers pregnancy loss: senescence-driven miscarriage via TRIM21-mediated MFF degradation

doi: 10.1016/j.ebiom.2026.106146

Figure Lengend Snippet: TRIM21 was an E3 ubiquitin ligase of MFF and promoted its ubiquitination degradation. (A and B) The levels of the remained MFF protein in 40 μg/mL CIP-exposed HTR-8/SVneo cells and with 10 μM CHX treatment within 10 h and its relative quantification (n = 3 independent experiments, Student's t-test). (C) The protein levels of MFF in 40 μg/mL CIP-exposed HTR-8/SVneo cells treated with 10 μM MG132 or 50 μM CQ and co-treated with 10 μM CHX and its relative quantification (n = 3 independent experiments, one-way ANOVA). (D and E) IP assay analysis of the protein levels of MFF-Ub pulled down by MFF in 40 μg/mL CIP-exposed HTR-8/SVneo cells and its relative quantification (n = 3 independent experiments, Student's t-test). (F) MFF IP-MS assay analysis of the proteins that were pulled down by MFF in HTR-8/SVneo cells. (G–K) IP assay analysis of the protein levels of TRIM21 and MFF-Ub pulled down by MFF in HTR-8/SVneo cells with overexpression (Student's t-test) or knockdown (one-way ANOVA) of TRIM21, with MFF and TRIM21 protein levels in cell lysates and their relative quantification (n = 3 independent experiments).

Article Snippet: The lysates were incubated with MFF antibody (17090-1-AP, proteintech, dilution 1:200, RRID: AB_2142463 ) or TRIM21 antibody (67136-1-Ig, proteintech, dilution 1:200, RRID: AB_2882435 ) at 4 °C overnight, with equal weight of IgG antibody (ab172730, Abcam, dilution 1:200, RRID: AB_2687931 ) as negative control, and then incubated with Protein A/G magnetic beads (HY–K0202, MedChemExpress) for another 6 h to form bead-immunoprecipitate complex.

Techniques: Ubiquitin Proteomics, Quantitative Proteomics, Protein-Protein interactions, Over Expression, Knockdown

Journal: eBioMedicine

Article Title: Environmental ciprofloxacin triggers pregnancy loss: senescence-driven miscarriage via TRIM21-mediated MFF degradation

doi: 10.1016/j.ebiom.2026.106146

Figure Lengend Snippet: The joint roles of CIP and TRIM21 in MFF ubiquitination degradation. (A) TRIM21 mRNA levels in 0, 1, 10, 20, or 40 μg/mL CIP-exposed HTR-8/SVneo cells (n = 3 independent experiments, one-way ANOVA). (B) TRIM21 protein levels in 0, 1, 10, 20, or 40 μg/mL CIP-exposed HTR-8/SVneo cells and its relative quantification (n = 3 independent experiments, one-way ANOVA). (C and D) IP assay analysis of the protein levels of MFF pulled down by TRIM21 in 40 μg/mL CIP-exposed HTR-8/SVneo cells with MFF and TRIM21 protein levels in cell lysates and its relative quantification (n = 3 independent experiments, Student's t-test). (E–I) IP assay analysis of the protein levels of TRIM21 and MFF-Ub pulled down by MFF in 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown, with MFF and TRIM21 protein levels in cell lysates and their relative quantification (n = 3 independent experiments, one-way ANOVA).

Article Snippet: The lysates were incubated with MFF antibody (17090-1-AP, proteintech, dilution 1:200, RRID: AB_2142463 ) or TRIM21 antibody (67136-1-Ig, proteintech, dilution 1:200, RRID: AB_2882435 ) at 4 °C overnight, with equal weight of IgG antibody (ab172730, Abcam, dilution 1:200, RRID: AB_2687931 ) as negative control, and then incubated with Protein A/G magnetic beads (HY–K0202, MedChemExpress) for another 6 h to form bead-immunoprecipitate complex.

Techniques: Ubiquitin Proteomics, Quantitative Proteomics, Knockdown

Journal: eBioMedicine

Article Title: Environmental ciprofloxacin triggers pregnancy loss: senescence-driven miscarriage via TRIM21-mediated MFF degradation

doi: 10.1016/j.ebiom.2026.106146

Figure Lengend Snippet: Verification of TRIM21/MFF signalling in human villous tissues. (A and B) TRIM21 protein levels in HC and UM villous tissues and its relative quantification (n = 12, Student's t-test). (C–G) IP assay analysis of the protein levels of TRIM21 and MFF-Ub that were pulled down by MFF in HC and UM villous tissues, with MFF and TRIM21 protein levels in tissue lysates and their relative quantification (each n = 12, Student's t-test). (H) Multivariate logistic regression analysis of forest plot of odds ratio (OR) of TRIM21 protein levels in villous tissues with its 95% confidence interval (95% CI) (n = 12).

Article Snippet: The lysates were incubated with MFF antibody (17090-1-AP, proteintech, dilution 1:200, RRID: AB_2142463 ) or TRIM21 antibody (67136-1-Ig, proteintech, dilution 1:200, RRID: AB_2882435 ) at 4 °C overnight, with equal weight of IgG antibody (ab172730, Abcam, dilution 1:200, RRID: AB_2687931 ) as negative control, and then incubated with Protein A/G magnetic beads (HY–K0202, MedChemExpress) for another 6 h to form bead-immunoprecipitate complex.

Techniques: Quantitative Proteomics

Journal: eBioMedicine

Article Title: Environmental ciprofloxacin triggers pregnancy loss: senescence-driven miscarriage via TRIM21-mediated MFF degradation

doi: 10.1016/j.ebiom.2026.106146

Figure Lengend Snippet: Verification of murine Trim21/Mff signalling in CIP-exposed mouse placental tissues. (A) Murine Trim21 mRNA levels in placental tissues of CIP-exposed mice (n = 6, one-way ANOVA). (B and C) Murine Trim21 protein levels in placental tissues of CIP-exposed mice and its relative quantification (n = 6, one-way ANOVA). (D) IP assay analysis of the protein levels of Trim21 and Mff-Ub that were pulled down by Mff in placental tissues of 160 mg/kg/d CIP-exposed mice, with Mff and Trim21 protein levels in tissue lysates (each n = 6). (E) Schematic diagram of CIP-exposed mouse model with Trim21 knockdown. Pregnant mice were treated with saline or 160 mg/kg/d CIP and also treated with 10 mg/kg/3 d AS-Trim 21 (with AS–NC as control). (F and G) Embryo resorption (indicated by red arrows) and the average miscarriage rates in 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (each n = 6, one-way ANOVA). (H) IP assay analysis of the protein levels of Trim21 and Mff-Ub that were pulled down by Mff in 160 mg/kg/d CIP-exposed mice with Trim21 knockdown, with Mff and Trim21 protein levels in tissue lysates (each n = 6). (I) The quantification of positive intensity of SA-β-Galactose staining of placental tissues in 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (each n = 6, one-way ANOVA). (J) The protein levels of p16, p21, p38, and β-gal in placental tissues of 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (each n = 6). (K) The relative mtDNA copy number in placental tissues of 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (n = 6, one-way ANOVA). (L) The NAD + /NADH ratios in placental tissues of 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (n = 6, one-way ANOVA). (M) The MMP levels in placental tissues of 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (n = 6, one-way ANOVA).

Article Snippet: The lysates were incubated with MFF antibody (17090-1-AP, proteintech, dilution 1:200, RRID: AB_2142463 ) or TRIM21 antibody (67136-1-Ig, proteintech, dilution 1:200, RRID: AB_2882435 ) at 4 °C overnight, with equal weight of IgG antibody (ab172730, Abcam, dilution 1:200, RRID: AB_2687931 ) as negative control, and then incubated with Protein A/G magnetic beads (HY–K0202, MedChemExpress) for another 6 h to form bead-immunoprecipitate complex.

Techniques: Quantitative Proteomics, Knockdown, Saline, Control, Staining

Journal: eBioMedicine

Article Title: Environmental ciprofloxacin triggers pregnancy loss: senescence-driven miscarriage via TRIM21-mediated MFF degradation

doi: 10.1016/j.ebiom.2026.106146

Figure Lengend Snippet: Miscarriage treatment. (A) Schematic diagram of CIP-exposed mouse model with F + Q treatment. Pregnant mice were treated with saline or 160 mg/kg/d CIP and also treated with 50 mg/kg/2 d F and 10 mg/kg/2 d Q. (B–D) The protein levels of Trim21 and Mff in placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment and their relative quantification (each n = 6, Student's t-test). (E and F) Embryo resorption (indicated by red arrows) and the average miscarriage rates in 160 mg/kg/d CIP-exposed mice with F + Q treatment (each n = 6, Student's t-test). (G) IP assay analysis of the protein levels of Trim21 and Mff-Ub that were pulled down by Mff in 160 mg/kg/d CIP-exposed mice with F + Q treatment, with Mff and Trim21 protein levels in tissue lysates (each n = 6). (H) The quantification of positive intensity of SA-β-Galactose staining of placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment (each n = 6, Student's t-test). (I) The protein levels of p16, p21, p38, and β-gal in placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment (each n = 6). (J) The relative mtDNA copy number in placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment (n = 6, Student's t-test). (K) The NAD + /NADH ratios in placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment (n = 6, Student's t-test). (L) The MMP levels in placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment (n = 6, Student's t-test). (M) The proposed mechanisms.

Article Snippet: The lysates were incubated with MFF antibody (17090-1-AP, proteintech, dilution 1:200, RRID: AB_2142463 ) or TRIM21 antibody (67136-1-Ig, proteintech, dilution 1:200, RRID: AB_2882435 ) at 4 °C overnight, with equal weight of IgG antibody (ab172730, Abcam, dilution 1:200, RRID: AB_2687931 ) as negative control, and then incubated with Protein A/G magnetic beads (HY–K0202, MedChemExpress) for another 6 h to form bead-immunoprecipitate complex.

Techniques: Saline, Quantitative Proteomics, Staining