Figure 4: A role of DGKA in fibroblast activation and stress response.
From: Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis
(a,b) Collagen expression in untreated and TGF-beta1-stimulated NHDF (n=3) after siRNA silencing or inhibition of DGKA using the small molecule inhibitor R59949 (5.0 μM). Cells were pre-treated with drug or siRNA for 48 h, subsequently treated with TGFB1 (4 ng ml−1) and analysed 48 h later. Graphs show expression of collagen 1A1 COL1A1 mRNA (a) and total collagen release using hydroxyproline content as a surrogate marker (b). (c) Diacylglycerol kinase (DGK) activity and (d) DGKA mRNA expression in NHDF (n=3) on treatment for 48 h with model stress inducers, BLM, 40 μM bleomycin; BRE, 0.1 μM brefeldin A; ETO, 30 μM etoposide; TUN, 0.5 μM tunicamycin; P values refer to comparison with untreated controls. Dots show mean values from duplicate experiments for each fibroblast (triplicates for d), bars depict mean±s.e.m. in each group. (e) DAG levels in NHDF after inhibition of DGKA by R59949 (5.0 μM) or siRNA 48 h after co-exposure to radiation. (f) DGKA inhibition by R59949 and changes in DAG levels in NHDF co-exposed to TGFB1 (4 ng ml−1) for 48 h. (g,h) LPA (g) and PA (h) levels in NHDF after inhibition of DGKA by R59949 (5.0 μM) and co-exposure to radiation. Scale bars indicate normalized relative DAG or PA/LPA levels in fibroblasts compared with untreated controls (decrease, blue; increase, red). Data depict mean from duplicate experiments in NHDF (n=3). siCon, non-targeting siRNA control; siDGKA, siRNA directed against DGKA. *P<0.05, **P<0.01, ****P<0.0001, Student’s t-test.
