Journal: bioRxiv

Article Title: ZBP1 sequesters cGAS in the cytoplasm and sustains type I interferon responses to mitochondrial DNA

doi: 10.1101/2022.05.30.493783

Figure Lengend Snippet: A, B, Western blots of cytosolic (Cyto.) and nuclear (Nuc.) proteins in WT and Tfam +/- MEFs with or without IFNβ treatment (10 ng/mL, 6 h) ( A ). Percentages of cytosolic and nuclear cGAS in WT and Tfam +/- MEFs are quantified in ( B ). 4 different MEF lines of each genotype were used for quantification. C, qRT-PCR analysis (n = 3) of ISGs in HFF transfected with Ctrl or TFAM siRNA for 72 h. D, E, Western blots of cytosolic and nuclear proteins in vehicle (veh) or 50 nM Doxorubicin (Doxo) treated WT MEFs ( D ). Percentages of cytosolic and nuclear cGAS in vehicle or Doxo treated WT MEFs are quantified in ( E ). 2 different MEF lines are shown in the western blots, n = 3 different MEF lines were used for quantification. F, Schematic representation of the mouse cGAS full-length (FL) and truncations used in G-H . G, H, co-IP showing interactions between mouse ZBP1 and mouse cGAS FL and truncations in 293FT. Statistical significance was determined using analysis of variance (ANOVA) and Tukey ( B ) post hoc test or unpaired t-test ( C, E ). *P < 0.05, **P < 0.01, and ***P < 0.001. Error bars represent SEM.

Article Snippet: Doxorubicin (Adriamycin) HCl (S1208, Selleck Chemicals); Hydralazine Hydrochloride (H1289, Spectrum); ISD (tlrl-isdn), poly(dA:dT) (tlrl-patn), poly(dG:dC) (tlrl-pgcn) (InvivoGen); polyethylenimine, PEI (43896, Alfa Aesar); Human IFN Alpha Hybrid Protein (11200-1, PBL Assay Science); Recombinant Human IFN-beta Protein (8499-IF-010), Recombinant Mouse IFN-beta Protein (8234-MB-010) (R&D Systems); Recombinant Mouse IFN-α (752806, Biolegend); InVivo Plus mouse IgG1 isotype control (BP0083), InVivo MAb anti-mouse IFNAR-1 (BE0241) (Bio X Cell); 4-Hydroxytamoxifen (HB6040, hellobio); 2’3’-cGAMP ELISA Kit (501700, Cayman); LDH Cytotoxicity Detection Kit (MK401, TaKaRa).

Techniques: Western Blot, Quantitative RT-PCR, Transfection, Co-Immunoprecipitation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: BAP31 Promotes Tumor Cell Proliferation by Stabilizing SERPINE2 in Hepatocellular Carcinoma

doi: 10.3389/fcell.2020.607906

Figure Lengend Snippet: Inhibition of SERPINE2 attenuates BAP31-promoted cell proliferation. (A) Colony formation assay using Hep3b and MHCC97h cells that were transiently transfected with SERPINE2 siRNA or control siRNA. Representative images were acquired, and the colony numbers were counted. (B) Cell viability of transiently transfected Hep3b and MHCC97h cells detected by the cell counting kit-8 assay. (C) Protein levels of SERPINE2, BAP31, β-actin, Erk1/2, phospho-Erk1/2, p38, and phospho-p38 were monitored by western blot. (D) Colony formation assay using BAP31-overexpressing HCC cells that were transiently transfected using SERPINE2 siRNA and control siRNA. (E) Cell viability of transiently transfected Hep3b and MHCC97h cells detected by the cell counting kit-8 assay. ** p < 0.01. BAP31, B-cell receptor-associated protein 31; SERPINE2, serpin family E member 2.

Article Snippet: After blocking with 5% non-fat milk (BD Biosciences, USA), the membranes were incubated with antibodies against BAP31 (Proteintech, China; 11200-1-AP; 1:2,000), SERPINE2 (Proteintech; 66203-1-Ig; 1:1,000), β-actin (Proteintech; 60008-1-Ig; 1:5,000), Erk1/2, p38 (Cell Signaling Technology, USA; 9926; 1:1,000), and phospho-Erk1/2, phospho-p38 (Cell Signaling Technology; 9910; 1:1,000).

Techniques: Inhibition, Colony Assay, Transfection, Control, Cell Counting, Western Blot