Fig. 2

Fig. 2 Inhibition of RNLTR12-int expression affects Mbp expression (A) RNLTR12-int expression was inhibited upon transfection of siRNA against RNLTR12-int (siRNLTR12-int) as determined by RT-PCR. PCR products were run on a 2% agarose gel (left). RT-qPCR analysis (right). Data were normalized to cytoplasmic beta-actin (Actb). Control siRNA: siGENOME non-targeting siRNA pool (Dharmacon). n = 3 independent experiments (each time 4 P7 brains were pooled for OPC isolation), mean ± SEM, ∗p < 0.05, Student’s t test (unpaired, two tailed) with Welch’s correction. (B) Left: immunofluorescence analysis using antibody to OLIG2 (oligodendrocyte lineage-specific marker) and to MBP (differentiation/maturation marker). Upon transfection, OPCs were maintained in differentiation media for 5 days. Scale bars, 60 μm. Right: quantification of matured OLs (MBP+ and OLIG2+), presented as a percentage of total OLIG2+ cells. n = 3 independent experiments (each time three replicates), mean ± SEM, ∗∗p < 0.0001, two-way ANOVA. (C) Effect of the inhibition of RNLTR12-int on RNA-level expression of Pdgfra, Cnp, and Mbp was determined by RT-qPCR. Data were normalized to Actb. n = 3 independent experiments, mean ± SEM, ∗p < 0.05, Student’s t test (unpaired, two tailed) with Welch’s correction. (D) Global changes of gene expression due to inhibition of RNLTR12-int in differentiating OPC as determined by RNA-seq (GEO: GSE237744). Left: volcano plot representing differential expression, up- (red dot) and downregulated (green dot) genes. Right: GO-term overrepresented in differentially expressed genes. p adj: adjusted p value (Benjamini-Hochberg FDR after Wald test). Genes involved in myelination (green bars) are downregulated. n = 4 (control siRNA), 3 (siRNLTR12-int) independent experiments. See also Figures S4 and S5.