Fig. 1: Increased ACLY activity is necessary for myofibroblast differentiation.
a, Western blot of mouse CFs with and without TGFβ treatment for 48 h. Immunoblots of pSer455–ACLY (pACLY), ACLY and αTubulin (αTub), loading control (load con). b, pACLY to total ACLY (tACLY) protein expression, band intensity (arbitrary units) ratio normalized to αTub from a. Unpaired two-sided t-test. c, Western blot of CFs treated with and without TGFβ for 0–24 h. Immunoblots for pACLY, ACLY, POSTN, COL1A1 and αTub. d, Correlation of POSTN and the pACLY:tACLY ratio (n = 3 biological replicates per timepoint). e, Western blot of COL1A1, POSTN and αTub (load con). CFs were transduced with adenovirus expressing Cre or βgal and treated with TGFβ or vehicle (Con). f, COL1A1 and POSTN protein expression normalized to αTub. g, Myofibroblasts identified by αSMA immunostaining (red), co-stained with DAPI (blue). Scale bars, 50 μm. h, Percentage of αSMA+ cells. i, qPCR mRNA expression of Acta2 and Col1a1. j, CFs treated with ACLYi (BMS-303141, 4 μM) or veh, with or without 10 ng ml−1 TGFβ, for 48 h; immunoblots of COL1A1 and αTub (load con). k, COL1A1 protein expression normalized to αTub. l, Myofibroblasts identified by αSMA immunostaining (red), co-stained with DAPI (blue). Scale bars, 50 μm. m, Percentage of αSMA+ cells. n, qPCR mRNA expression of Acta2 and Postn. n = 3 biological replicates. All data are depicted as mean ± s.e.m. Two-way ANOVA with Tukey honestly significant difference (HSD) for multiple comparisons. Veh, vehicle. Full-length western blots are available as source data.
