Fig. 5

Chronic IFNγ treatment and PDGFRβ knockdown blocks PDGF-BB-induced proliferation in pericytes. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle (Veh) or IFNγ (1 ng/mL). b, c After 48 h of cytokine treatment, cells were treated with either vehicle or PDGF-BB (10 ng/mL) to measure the PDGF-induced proliferative response. This was done in two ways: after 96 h total treatment, cells were fixed, labelled with a Ki67 antibody and Hoechst (b); alternatively, EdU was added to measure cell proliferation over the final 24 h of the experiment (c). Positive cells of the total cells measured by Hoechst (d) were quantified and plotted as mean ± s.e.m. (n = 3), ****, ^####(p < 0.0001), ***(p < 0.001), *(p < 0.05) from a two-way ANOVA. e, f LDH release (e) and AlamarBlue reduction (f) were also measured as cell death and cell health outputs, respectively, from the above proliferation experiments. g Knockdown of PDGFRβ in pericytes with siRNA after 96 h immunolabelled for PDGFRβ (green) and Hoechst (blue), scale bar, 100 μm. h, i Quantification of pericytes positive for PDGFRβ after siRNA knockdown. Percent positive for PDGFRβ (h) of the total cells measured by Hoechst (i), mean (per well) ± s.e.m. (n = 1). j Representative western blot of PDGF-BB response in PDGFRβ deficient pericytes (n = 2). k–m Proliferation response to PDGF-BB in PDGFRβ deficient pericytes after 48 h. Ki67 (k), EdU (l) positive, and total cells (m) mean ± s.e.m. (n = 5) were plotted and analysed with two-way ANOVA, ****(p < 0.0001), ^###(p < 0.001), ***(p < 0.001), and *(p < 0.05)