Figure 7.

FKN regulates OPC and microglia cell fates in vitro

(A) OPCs were cultured with or without microglia in OPC DM and VC/FKN for 1–3 days in vitro (DIV).

(B) Representative images of OPCs cultured in VC or FKN for 3DIV and immunostained for MBP (green) and OLIG2 (red). Arrows designate MBP+OLIG2+ oligodendrocytes.

(C–F) Quantification of % MBP+Olig2+ cells at 3DIV (C and D) and OPC proliferative index at 1DIV (E and F) in OPC cultures (C, E) or OPC-microglia co-cultures (D and F).

(G) Quantification of % MBP+OLIG2+ oligodendrocytes in co-cultures with VC- and/or FKN-pre-treated OPCs and microglia.

(H) Microglia were incubated with VC/FKN with or without pre-stimulation with IL-1β and IL-4/IL-13 cytokines or no cytokines (basal conditions).

(I) Representative images of microglia treated with VC/FKN for 3DIV and immunostained with IBA1 (purple) and Ki67 (green).

(J) Quantification of (I).

(K) Representative images of IL-1β-pre-stimulated microglia treated with VC/FKN and immunostained with CD16/32 (green), IBA1 (purple) and counterstained with Hoechst (white).

(L) Quantification of (K).

(M) qPCR analysis of Cx3cr1 mRNA expression normalized to Gapdh and Hnrnpab housekeeping genes and calibrated against basal VC-treated microglia. Scale bars, 20 μm. Error bars represent SEM. Each connected line corresponds to one biological replicate. Data were analyzed with Student’s paired t test, except graph in (G) was analyzed with one-way ANOVA followed by Dunnett’s post hoc test, graph in (L) was with multiple t test and graph in (M) with two-way ANOVA followed by Tukey post hoc test. ^∗∗p < 0.01, ^∗p < 0.05, n = 3–4 independent experiments.

See also Figure S6.