SOX11 was regulated by RBM3 and promoted neuronal differentiation of cultured hNSCs
(A) Heatmap of CLIP-seq showing differentially RBM3-bound mRNA with 35°C- and 37°C-treatment.
(B) GO enrichment analysis showing 12 enriched BP terms related to neuron differentiation and neurogenesis on differentially RBM3-bound mRNA.
(C) Venn diagram showing 13 differentially expressed genes in both CLIP-seq and scRNA-seq.
(D) Violin maps of the 13 differentially expressed genes in scRNA-seq.
(E) GO enrichment analysis on the 13 differentially expressed genes.
(F) t-SNE plot highlighting the SOX11 expression in cultured hNSCs at Day 14.
(G) (Left) Western blots analysis showing the protein expression of SOX11 and RBM3 at Day 14 after scramble or RBM3 siRNA transfection. β-actin was used as the loading control. (Right) Normalized SOX11 to corresponding loading control were summarized for three independent trials.
(H) (Left) Western blots analysis showing the protein expression of SOX11 and RBM3 at Day 14 after vector or RBM3 overexpression by lentivirus. β-actin was used as the loading control. (Right) Normalized SOX11 to corresponding loading control were summarized for three independent trials.
(I) qRT-PCR analysis showing the mRNA level of SOX11 in cultured hNSCs at Day 14. (Left 4 columns) hNSCs were transfected with scramble or RBM3 siRNA with 35°C-treatment. (Right 3 columns) hNSCs were transducted with vector or RBM3 by lentivirus with 37°C-treatment.
(J) Representative immunofluorescence images of β-tubulin III positive cells and SOX11 expression at Day 14. (Left 4 panels) hNSCs were transfected with scramble or SOX11 siRNA with 35°C-treatment. (Right 3 panels) hNSCs were transducted with vector or SOX11 by lentivirus with 37°C-treatment.
(K) The percentage of β-tubulin III positive cells in (J).
(L) qRT-PCR analysis showing the mRNA level of TUBB3, DCX, GFAP, SOX11, and RBM3 at Day 14. (Left 4 columns) hNSCs were transfected with scramble or SOX11 siRNA with 35°C-treatment. (Right 3 columns) hNSCs were transducted with vector or SOX11 by lentivirus with 37°C-treatment. All data presented as mean ± SD. One-way ANOVA tests were used in (G), (I), (K) and (L). Student’s t test was used in (H). NS, not significant; ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Scale bars represent 50 μm in (J).
See also Tables S4, S5, and S6 and Figure S4.