Fig 2: Knockdown of Etv1 in Schwann cells inhibits its migration ability. (A) The insert-based scratch migration assay. The histogram shows that Etv1 knockdown in SCs decreased its migration ability compared to SCs transfected with the scrambled vector. ***, P<0.001 vs. scramble; t-test; n=6. Representative phase contrast microscopy images (left) were selected from 6 independent experiments. Scale bar =100 μm. The schematic diagram showing the experimental procedure (top right). (B) The phase contrast microscopy images show that the SCs reaggregate on fasciculated DRG axons following treatment with Etv1-siRNA and scramble. The area inside the red dashed line represents the distance of SC migration. Scale bar =100 μm. The schematic diagram on the top right showing the experimental procedure. The histogram shows that Etv1 knockdown in SCs growing on fascicular DRG axons inhibits their migration out of the reaggregated SC spheres. **, P<0.01 vs. scramble; t-test; n=5. PLL, poly-L-lysine; siRNA small interfering RNA; DRG, dorsal root ganglion; SC, Schwann cell.

Fig 3: Etv1 knockdown reduces the proliferation of SCs. (A) The proliferation of SCs seeded on PLL transfected with Etv1-siRNA or scramble-transfected was detected using EdU labeling kits. Proliferating SCs are stained with EdU (green) and Hoechst (blue) is used to mark cell nuclei. Scale bar =50 µm. The histogram shows that knockdown Etv1 in SCs cultured on PLL significantly decreased their proliferation. ***, P<0.001 vs. scramble; t-test; n=12. (B) Proliferation ratio of SCs transfected with Etv1-siRNA or scramble seeded on fasciculated DRG axons. Proliferating SCs are stained with EdU (green), fasciculated DRG axons are stained with ß tubulin III (red), and Hoechst (blue) is used to mark cell nuclei. Scale bar =50 µm. Histograms showing that knockdown Etv1 in SCs cultured on fasciculated DRG axons significantly reduced their proliferation **, P<0.01 vs. scramble; t-test; n=6. PLL, poly-L-lysine; siRNA, small interfering RNA; DRG, dorsal root ganglion; SC, Schwann cell.

Fig 4: Screening for myelination-related TFs by analyzing the transcriptomic data of myelinating SCs. (A) The heatmap shows the top 20 signature TFs (rows) with FCs >2 compared to day 0 during SC myelination (columns: FC versus co-0 d). The blue dotted box indicates the genes, including Etv1, Egr2, Pou3f2, and Myrf, with the same expression trend. (B) qPCR showing the gene expression of Etv1, Egr2, Pou3f2, and Myrf in SCs during myelination. ###, P<0.001, one-way ANOVA; *, P<0.05, **, P<0.01, ***, P<0.001 vs. co-0 d, n=3 per group. (C) The regulatory network of the TFs. Nodes correspond to genes, triangles correspond to TFs, and dashed circles correspond to interactions. The over-represented TFs (triangles) are shown in purple, such as Etv1, Egr2, Sin3a, and Zeb1. The triangle size is based on its centrality. NS, no statistical difference; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TF, transcription factor; SC, Schwann cell; FC, fold change; qPCR, quantitative polymerase chain reaction; ANOVA, analysis of variance.

Fig 5: Etv1 knockdown in Schwann cells antagonizes its differentiation. (A) (a1) Representative images of phase contrast microscopy and ICC analysis, showing that dB-cAMP can effectively induce SC differentiation. Red staining represents MAG expression and cell nuclei (blue) are marked with Hoechst staining. Scale bar =100 µm. (a2) WB comparing the expression of MAG in SCs treated with dB-cAMP or vehicle for 24, 48, and 72 hours. ***, P<0.001; one-way ANOVA; n=3 per group. (B) (b1) Western blot comparing Etv1 levels in SCs differentiated for 24, 48, and 72 hours. ***, P<0.001; one-way ANOVA; n=3 per group. (b2) WB showing Etv1 and MAG expression in differentiated SCs transfected with Etv1-siRNA and scrambled siRNA. The histogram shows that the knockdown of Etv1 in SCs significantly reduced the expression of MAG after the induction of differentiation. Student’s t-test; **, P<0.01 vs. scramble; n=3 per group. MAG, myelin-associated glycoprotein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Ctrl, control; Dif, differentiation; siRNA, small interfering RNA; ICC, immunocytochemistry; SC, Schwann cell; WB, Western blotting; ANOVA, analysis of variance.