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. 2020 Dec 24;69(5):1184–1203. doi: 10.1002/glia.23957

FIGURE 2.

FIGURE 2

Deleting Kif3a from cultured OPCs reduces their proliferation. (a) Image of a Western blot detecting Kif3A and β‐actin protein expression in lysates generated from Pdgfrα‐histGFP or Pdgfrα‐histGFP:: Kif3a fl/fl OPCs 3 days after exposure to 0 or 2 μM tat‐Cre for 90 min. (b) Quantification of Kif3a expression (relative to β‐actin protein expression) in lysates generated from Pdgfrα‐histGFP or Pdgfrα‐histGFP:: Kif3a fl/fl OPCs 3 days after exposure to 0 or 2 μM tat‐Cre for 90 min. Data are expressed as mean ± SD for n = 3 independent cultures per treatment. One way ANOVA (F = 26.34, p = .001) with a Bonferroni post‐test **p < .01, ***p < .001. (c, d) Confocal image of 0 μM (c) and 2 μM (d) tat‐Cre‐treated Pdgfrα‐histGFP:: Kif3a fl/fl OPC cultures stained to detect γ‐tubulin (green), Arl13b (red) and GFP (blue). White arrow with solid arrow head identifies a γ‐tubulin+ Arl13b+ primary cilium. White arrows indicate γ‐tubulin+ puncta without assembled primary cilia. Insets show an example primary cilia (c) and γ‐tubulin+ puncta (d) at higher magnification. (e) Quantification of the proportion of GFP+ OPCs that have a γ‐tubulin+ basal body associated with an assembled Arl13b+ primary cilium in 0 and 2 μM tat‐Cre‐treated Pdgfrα‐histGFP:: Kif3a fl/fl OPC cultures. Data is expressed as mean ± SD for n = 4 independent cultures per treatment. Unpaired two‐tailed t test (t = 25.7, df = 6), ***p < .0001. (f–i) Confocal images (single z‐planes) of 0 μM (f, g) or 2 μM (h, i) tat‐Cre‐treated Pdgfrα‐histGFP:: Kif3a fl/fl OPCs that were exposed to EdU for 12 hr and immunostained to detect GFP (green), PDGFRα (red) and EdU (blue). White arrowheads indicate example GFP+ PDGFRα+ EdU+ cells. (j) Quantification of the proportion of OPCs that were EdU‐labeled in 0 and 2 μM tat‐Cre‐treated Pdgfrα‐histGFP:: Kif3a fl/fl OPC cultures. Data is expressed as mean ± SD for n = 3 independent cultures. Unpaired two‐tailed t test (t = 4.3, df = 4), *p = .01. Scale bars represent 25 μm (c, d) and 40 μm (f–i) [Color figure can be viewed at wileyonlinelibrary.com]