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Fig. 4 | Journal of Neuroinflammation

Fig. 4

From: Interferon-γ blocks signalling through PDGFRβ in human brain pericytes

Fig. 4

Chronic IFNγ increases PDGFRβ membrane expression. a, b Detection of PDGF-BB in pericyte conditioned media. Human brain pericytes at 90 % confluence were left untreated (control (Con)) or serum starved for 2 h and then treated with vehicle, IFNγ (1 ng/mL), PDGF-BB (100 ng/mL), or both IFNγ and PDGF-BB for 24 h. Lysates were collected for western blot analysis with the indicated antibodies, and a representative blot is shown (n = 2) (a) Bands were quantified with Image Studio™ software and intensity was normalized to vehicle control (b); PDGF-BB was normalized to GAPDH. ce Pericytes were treated for 0, 24, 48, 72, or 96 h (more cytokines added once every 24 h to appropriate wells) with vehicle (Veh) or IFNγ (1 ng/mL). c Grey value intensities of PDGFRβ membrane staining are depicted in a pseudo-colour image according to the legend (right). d Representative images of membrane PDGFRβ (red) and Hoechst (blue) in pericytes after 96 h of vehicle or IFNγ treatment. Scale bar, 100 μm. e Quantification of total PDGFRβ (white bars) and membrane PDGFRβ (black bars) staining intensity per cell was normalized to 0 h time point, plotted as mean ± s.e.m. (n = 3), ****(p < 0.0001), ***(p < 0.001), **(p < 0.01) (ANOVA). f, g 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 (Veh) or PDGF-BB (100 ng/mL) for 30 min. Surface PDGFRβ expression was analysed using flow cytometry, and a representative plot is shown (f). Mean fluorescence intensity (MFI) of cell surface PDGFRβ is plotted as mean ± s.e.m. (n = 3) (g)

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