Additional file 7: Figure S7. of Interferon-γ blocks signalling through PDGFRβ in human brain pericytes

(A, B) Pericytes were treated for four consecutive days (once every 24 h) with either vehicle (Veh), TNFα (5 ng/mL), or IL-1β (1 ng/mL). After 48 h of cytokine treatment, cells were treated with either vehicle or PDGF-BB (10 ng/mL) to measure PDGFRβ and αSMA expression by immunocytochemistry. Quantification of PDGFRβ (A) and αSMA (B) staining, mean ± s.e.m. (n = 3), ****(p < 0.0001), ***(p < 0.001), *(p < 0.05) (two-way ANOVA). (C) Pericytes were treated for three or four consecutive days (once every 24 h) with either vehicle (Veh), TNFα (5 ng/mL), or IL-1β (1 ng/mL). After 48 h, cells were treated with PDGF-BB (10 ng/mL) for either 24 or 48 h. Western blot band intensity of PDGFRβ, αSMA, and GAPDH were quantified, normalized to GAPDH, and plotted as mean ± s.e.m. (n = 3), and differences were not significant (two-way ANOVA). (TIF 1163 kb

Additional file 6: Figure S6. of Interferon-γ blocks signalling through PDGFRβ in human brain pericytes

Pericytes were treated for four consecutive days (once every 24 h) with either vehicle (Veh), TNFα (5 ng/mL), or IL-1β (1 ng/mL). After 48 h of cytokine treatment, cells were treated with either vehicle or PDGF-BB (10 ng/mL) to measure the PDGF-BB-induced proliferative response (A, B). This was done in two ways: after 96 h total treatment, cells were fixed, labelled with a Ki67 antibody and Hoechst (A, C); alternatively, EdU was added to measure cell proliferation over the final 24 h of the experiment (B, C). Positive cells of the total cells measured by Hoechst 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. (TIF 1034 kb

Additional file 3: Figure S3. of Interferon-γ blocks signalling through PDGFRβ in human brain pericytes

Repeats of additional cases from Fig. 3(b–e): Pericytes were treated for four consecutive days (once every 24 h) with either vehicle (Veh) or IFNγ (1 ng/mL). After 96 h total treatment, cells were serum starved for 2 h and then treated with vehicle (−) or PDGF-BB (100 ng/mL) for 30 min as in Fig. 3. (a, c) Representative western blots of treated pericyte from two additional cases. (B, D) Bands were quantified with Image Studio™ and normalized to vehicle control. p-PDGFRβ was normalized to total PDGFRβ, and p-Akt and p-ERK were normalized to total Akt and ERK, respectively. (TIF 2398 kb

Additional file 4: Figure S4. of Interferon-γ blocks signalling through PDGFRβ in human brain pericytes

Pericytes were treated for four consecutive days (once every 24 h) with either vehicle (Veh), TNFα (5 ng/mL), or IL-1β (1 ng/mL). After 96 h total treatment, cells were serum starved for 2 h and then treated with vehicle (−) or PDGF-BB (100 ng/mL) for 30 min. PDGFRβ puncta were quantified using MetaXpress™ software and normalized to cell number and vehicle control and plotted as mean ± s.e.m. (n = 3), ***(p < 0.001), *(p < 0.05) (two-way ANOVA). Note: Control data are from the same experiments as Fig. 3g. (TIF 723 kb