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. 2021 Aug 25;40(19):e108041. doi: 10.15252/embj.2021108041

Figure 6. Sox4/11 overexpression and GSK3 inhibition both rescue differentiation defects of DKO NPCs.

Figure 6

  1. Schematic illustrating strategy to rescue DKO NPC differentiation defect by simultaneous lentiviral overexpression of Flag‐Sox4 and Flag‐Sox11 with two lentiviruses. Created with Biorender.com.
  2. IF for Tuj1 and Map2 (not shown) to identify newly formed neurons in NPCs after seven‐day culture in differentiation medium. Empty CAG‐IRES‐EGFP vector was used as control in cells not transduced with Sox4/11. Transduced cells were identified by IF for GFP (Co and DKO) or FLAG (Co + Sox4/11 and DKO + Sox4/11). Arrowheads indicate mature neurons, and asterisks denote immature neurons lacking neurite extension. Scale bar 20 μm.
  3. Quantification of transduced Tuj1+ cells from (B). Note the decrease in neuron number in DKO cultures compared to control, and the rescue in DKO cultures upon Sox4/11 expression (experiment performed twice in triplicates, representative experiment shown). Data are means ± SEM.
  4. Schematic illustrating the rescue DKO NPC differentiation defect with the GSK3 inhibitor CHIR99021 (chemical name: 6‐[2‐[[4‐(2,4‐dichlorophenyl)‐5‐(5‐methyl‐1H‐imidazol‐2‐yl)pyrimidin‐2‐yl]amino]ethylamino]pyridine‐3‐carbonitrile). Created with Biorender.com.
  5. IF for Tuj1 to identify newly formed neurons in NPCs after seven‐day culture in differentiation medium. Arrowheads indicate mature neurons, and asterisks denote immature neurons lacking neurite extension. Scale bar 20 μm.
  6. Quantification of Tuj1+ cells in control, control + CHIR (1 µM, 3 µM), DKO, and DKO + CHIR NPC cultures. Data are means ± SEM (experiment performed twice in triplicates, representative experiment is shown). Numbers below asterisks show fold change.

Data information: One‐way ANOVA test for all statistical analyses: ns, not significant; *P < 0.05, **P < 0.01 ***P < 0.001. P = 0.97 (C, yellow bar); P = 0.00010 (C, red bar); P = 0.0023 (C, purple bar); P = 0.031 (F, yellow bar); P = 5 × 10−6 (F, grey bar); P = 2 × 10−5 (F, purple bar); and P = 1 × 10−6 (F, green bar).

Source data are available online for this figure.