Figure 2
From: The RNA-binding protein MARF1 promotes cortical neurogenesis through its RNase activity domain
sMARF1 promotes the differentiation of cortical progenitor cells in vitro. (a) Schematic of the in vitro culture system of E12.5 mouse cortical progenitor cells. (b) Immunostaining for GFP (green) and Tuj1 (red) in E12.5 cortical progenitor cells 3 days after transfection with pCAGIG (Control) or an sMarf1 expression vector (sMARF1 o/e). White arrowheads indicate GFP-labeled Tuj1+ post-mitotic neurons. Scale bar, 50 μm. (c) Quantification of the GFP-labeled Tuj1+ cells. *p < 0.05 (n = 3, Student’s t-test). (d) Expression of Marf1 mRNA in E15.5 cortical neurons 2 days after nucleofection with scrambled (Control) or Marf1-specific shRNA vector (sMARF1 shRNA). **p < 0.01 (n = 3, Student’s t-test). (e) Immunostaining for GFP (green) and Ki67 (red) in E12.5 cortical progenitor cells 2 days after transfection with scrambled (Control) or Marf1-specific shRNA vector (sMARF1 shRNA). White arrowheads indicate GFP-labeled Ki67+ progenitor cells. Scale bar, 50 μm. (f) Quantification of the GFP-labeled Ki67+ cells. *p < 0.05 (n = 3, Student’s t-test). (g) Immunostaining for GFP (green) and Pax6 (red) in E12.5 cortical progenitor cells 2 days after transfection with scrambled (Control) or Marf1-specific shRNA vector (sMARF1 shRNA). White arrowheads indicate GFP-labeled Pax6+ radial glial cells. Scale bar, 50 μm. (h) Quantification of the GFP-labeled Pax6+ cells. **p < 0.01 (n = 3, Student’s t-test). (i) Immunostaining for GFP (green) and Tuj1 (red) in E12.5 cortical progenitor cells 3 days after transfection with scrambled (Control) or Marf1-specific shRNA vector (sMARF1 shRNA). White arrowheads indicate GFP-labeled Tuj1+ post-mitotic neurons. Scale bar, 50 μm. (j) Quantification of the GFP-labeled Tuj1+ cells. *p < 0.05 (n = 3, Student’s t-test). (k) Immunostaining for DAPI (blue), GFP (green) and Tuj1 (red). White arrowheads indicate Tuj1+ living neurons. Black arrowheads indicate Tuj1+ apoptotic neurons. Black arrows indicate Tuj1− apoptotic progenitors. (Left panels) White arrows indicate Tuj1− living progenitors. (Right panels) Scale bar, 50 μm. (l,m) Quantification of the apoptotic GFP-labeled Tuj1− progenitors (l) or Tuj1+ neurons (m) transfected with pCAGIG (Control), sMarf1 expression vector (sMARF1 o/e), scrambled (scr) or Marf1-specific shRNA vector (sMARF1 shRNA) (n = 3, one-way ANOVA, Tukey-Kramer test).
