Extended Data Fig. 7: TRIM21 regulates MCM8 ubiquitination and cytosolic export.
a, Immunoblot showing nuclear and cytosolic MCM8 when treated with Exportin1 inhibitor Leptomycin B (10 nM) under indicated conditions. b, Immunoblot showing mitoLC3B, as well as MCM8 and other mitochondrial proteins (TOM20, MFN2, PINK1) in THP-1 cells treated with leptomycin B under indicated conditions. c, Immunoblot showing nuclear and cytosolic MCM8 when treated with the ubiquitin activating enzyme inhibitor TAK-243 (1 μM) under indicated conditions. d, Immunoprecipitation and immunoblot showing interaction of MCM8 with LC3 in Flag-MCM8 transfected HeLa cells when treated with TAK-243 under indicated conditions. e, f, Immunoprecipitation and immunoblot showing interaction of HA-MCM8 with Flag-TRIM21 (e) or endogenous interaction of MCM8 with TRIM21 (f) in HeLa cells under indicated conditions. g, Immunoblot showing ubiquitination of MCM8 in ctrl- or TRIM21-siRNA transfected HeLa cells over-expressing Flag-MCM8 and HA-Ubiquitin under treatment with poly(dA:dT). h, i, Immunoblot showing endogenous ubiquitination of MCM8 in ctrl- or TRIM21-siRNA transfected HeLa (h) or THP-1 (i) cells. j, Bar plots showing the relative abundance of Dloop in the plasma from WT or Mcm8−/− mice under PBS (WT, n = 12 animals; Mcm8−/−, n = 9 animals) or LCWE (WT, n = 18 animals; treatment (WT, n = 18 animals; Mcm8−/−, n = 15 animals). Fold change is relative to PBS treated WT mice. k, Representative H&E images and coronary artery thickness of WT (PBS, n = 4 animals; LCWE, n = 6 animals) or Trim21−/− (PBS, n = 4 animals; LCWE, n = 6 animals) mouse heart sections under PBS or LCWE treatment. For box plots, the box extends from the Q1 to Q3 quartile values of the data, with a line at the median. The whiskers extend from minimum to maximum value of data. l, Representative staining for Ifna4 and CD31 on heart tissues of WT (PBS, n = 4 animals; LCWE, n = 6 animals) and Trim21−/− (PBS, n = 4 animals; LCWE, n = 6 animals) mice under PBS or LCWE treated conditions. Data are mean ± s.d. P values were calculated by two-tailed Student’s t test.
