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. 2022 Feb 8;17(9):2029–2035. doi: 10.4103/1673-5374.335165

Figure 2.

Figure 2

RUX reduces microglial activation and proliferation in vitro. Microglia were treated with 0.1, 0.5, or 1 mM RUX for 24 hours, followed by stimulation with IFN-γ (20 ng/mL) for 6 hours. (A) Representative immunofluorescence images of IBA1+ (green, Alexa Fluor 488)/iNOS+ (red, Alexa Fluor 594) microglia. IFN-γ stimulation increased iNOS expression in microglia, but RUX inhibited this increase. (B) Representative immunofluorescence images of IL-1β+ (green, Alexa Fluor 488) and IL-6+ (red, Alexa Fluor 594) microglia. IFN-γ stimulation increased IL-1β and IL-6 expression in microglia, but RUX reduced this IFN-γ-induced increase. (C) Western blot of iNOS and COX-2 expression in microglia. (D, E) Quantification of iNOS (D) and COX-2 (E) expression. Data are expressed as mean ± SD. The experiments were repeated five times. *P < 0.01, **P < 0.01, vs. Con group; #P < 0.05, vs. IFN-γ group (one-way analysis of variance followed by Tukey's post hoc test). (F) Representative EdU (red) images of microglia. IFN-γ stimulation increased microglia proliferation, but RUX restrained the IFN-γ-induced proliferation. (G) Representative immunofluorescence images of Ki67 (red, Alexa Fluor 594) expression in microglia. IFN-γ stimulation increased the expression of proliferation marker Ki67, but RUX inhibited its expression. Scale bars: 50 μm in A and B; 100 μm in F and G. COX-2: Cyclooxyganese-2; EdU: 5-ethynyl-2′-deoxyuridine; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IBA1: ionized calcium binding adaptor 1; IFN-γ: interferon-γ; IL: interleukin; iNOS: inducible nitric oxide synthase; RUX: ruxolitinib.