Fig 2: Chemical inhibition of PDGFR signalling alters the migratory behaviour of P-Rex1 expressing cells in both 2D and 3D environments.(A) Following a period of serum starvation, P-Rex1 expressing fibroblasts were stimulated with PDGF-BB, in the presence and absence of DMSO control or chemical inhibitors as indicated. Cells were the lysed and the levels of P-Rex1 and PDGFRβ were determined by Western blotting. The activity of the PI3K/Akt axis was determined by Western blotting for Akt which was phosphorylated at Thr473. (B) Quantification of the invasive migration of P-Rex1 expressing fibroblasts into Matrigel in the presence and absence of DMSO, PDGFRi (5 µM) or PI-103 (1 µM) as indicated (±SEM, ***P<0.0005 Mann-Whitney rank sum test, 3 independent experiments). (C) P-Rex1 expressing fibroblasts emerging from the migratory front of scratch-wound assays in the presence and absence of DMSO PDGFRi (5 µM) or PI-103 (1 µM) were analysed using time-lapse microscopy, with images captured every 10 minutes over a period of 18 hours. Individual cells were manually tracked and the migratory path of representative cells depicted as track plots. The phase contrast images correspond to the initial frame from the supplementary movies S5–S7 as indicated. (D-F) Influence of chemical inhibition on migratory behaviour was quantified as in Figure 2 by determination of mean cellular velocity (D), forward migratory index (E) and migratory persistence (F). (Bars 50 µm, *P<0.05, ***P<0.0005, Mann-Whitney rank sum test).

Fig 3: Chronic alcohol intake upregulated NLRP3 inflammasome expression in the pericytes after I/R injury. (A and B) Representative immunofluorescence images showing NLRP3 (red) or IL-1β (red) in pericytes (PDGFR-β: green) in each group (n = 3–5 rats for each group). Nuclei were stained with DAPI (blue). Scale bar = 10 µm. (C and D) Semiquantitative analysis of average NLRP3 or IL-1β fluorescence intensity in each group. *p < 0.05, ** p < 0.01, ***p<0.001, ****p<0.0001 versus control, ^p < 0.05, ^^ p < 0.01, ^^^p<0.001 versus tMCAO.

Fig 4: Chronic alcohol intake activates TLR4 in the pericytes. (A and B) Representative immunofluorescence images and semiquantitative analysis of TLR4 (red) in pericytes (PDGFR-β: green) in each group (n = 3–5 rats for each group). Nuclei were stained with DAPI (blue). Scale bar = 10 µm. *p < 0.05, ** p < 0.01, ***p<0.001 versus control, ^p < 0.05, ^^ p < 0.01 versus tMCAO.

Fig 5: Chronic alcohol intake upregulated NLRP3 inflammasome expression in the pericytes after I/R injury. (A and B) Representative immunofluorescence images showing NLRP3 (red) or IL-1β (red) in pericytes (PDGFR-β: green) in each group (n = 3–5 rats for each group). Nuclei were stained with DAPI (blue). Scale bar = 10 µm. (C and D) Semiquantitative analysis of average NLRP3 or IL-1β fluorescence intensity in each group. *p < 0.05, ** p < 0.01, ***p<0.001, ****p<0.0001 versus control, ^p < 0.05, ^^ p < 0.01, ^^^p<0.001 versus tMCAO.