Fig. 2

Klf4 regulates NPC neuronal differentiation and proliferation. a (Upper) Immunostaining with Tuj1 (red) or Map2 (red) antibodies in NPCs infected with pLKO.1-shScramble or pLKO.1-shKlf4 (#1, #2, and #3) lentivirus. DAPI (blue). Scale bars, 50 μm. (Lower left) NPCs expressing Flag-Klf4 were transfected with pLKO.1-shScramble or pLKO.1-shKlf4 #1, #2, and #3, and one day later, Flag and α-tubulin in lysates were detected by immunoblotting (n = 2). (Lower right) Quantification of the proportion of Tuj1⁺ or MAP2⁺ cells in the analysis shown above. b (Left) Immunostaining of samples equivalent to those shown in a with Nestin or Ki67 (both red) antibodies. DAPI (blue). Scale bars, 50 μm. (Right) Quantification of Ki67⁺ (n = 2 or 3) or Nestin⁺ (n = 3) cells in samples analyzed at left. c qPCR analysis of indicated transcripts in NPCs transfected with pLKO.1-shScramble or pLKO.1-shKlf4 lentiviral vector and grown 2 days in N2 medium without bFGF (n = 3). d (Left) Klf4 cKO NPCs transfected with Control-EGFP vector or Klf4-EGFP vector. GFP⁺ cells were assessed after 1 or 2 days of culture in N2 medium. (Right) Analysis of neurite length in GFP-positive NPCs on 1 or 2 days of differentiation. e qPCR analysis of indicated transcripts in samples equivalent to those shown in d (n = 3). f (Upper) Immunostaining with Tuj1 (green) or Map2 (red) antibodies in NPCs infected pCDH-Klf4 or pCDH-control lentivirus. DAPI (blue). Scale bars, 50 μm. (Lower left) NPCs were infected with pCDH-Flag-Klf4 or pCDH-control lentivirus, and one day later, Flag and α-tubulin in lysates were detected by immunoblotting (n = 2). (Lower) Quantification of results shown above. g (Left) Immunostaining with Nestin or Ki67 (both red) antibodies in samples shown in f. DAPI (blue). Scale bars, 50 μm. (Right) Quantification of results shown at left. Data are presented as mean ± SD. t test analysis was performed to calculate statistical significance (*P < 0.05, **P < 0.005, ***P < 0.0005). See also Supplementary Fig. 7a–d and 16 (upper panel)