Fig. S4

Fig. S4 Functional impact of RNLTR12-int inhibition on oligodendrocyte morphology, cell fate, gene expression in Schwann cells, and elimination of any off-target effect, related to Figure 2 and STAR Methods (A) Absence of complex oligodendrocyte morphology due to inhibition of RNLTR12-int. Left: immunofluorescence analysis of O4 immunostaining 5 days after transfection of siRNA. Scale bars, 26 μm. siRNLTR12-int: siRNA against RNLTR12-int, control siRNA: siGENOME non-targeting siRNA pool (Dharmacon). Right: quantification of oligodendrocyte average outgrowth area (O4 immuno stained) and the total number of cells (O4 immuno positive) counted were plotted. Mean ± SEM, n = 3 independent experiments. ∗∗p < 0.01, Student’s t test with Welch’s correction (unpaired and two tailed). (B) Inhibition of RNLTR12-int did not initiate astrocytic fate. Immunofluorescence analysis of GFAP immunostaining 5 days after transfection of siRNA. Scale bars, 60 μm. Percentage of GFAP+ cell in total DAPI+ cells were plotted. n = 3 independent experiments (each time three replicates), mean ± SEM, p > 0.05, two-way ANOVA. siRNLTR12-int: siRNA against RNLTR12-int, control siRNA: siGENOME non-targeting siRNA pool (Dharmacon). (C) Effect of inhibition of RNLTR12-int on RNA-level expression of P0 and Mbp in Schwann sells (SCs). RNA-level expressions were determined by RT-qPCR. Upon transfection (siRNLTR12-int: siRNA against RNLTR12-int or control siRNA: siGENOME non-targeting siRNA pool [Dharmacon]), SC were maintained in differentiation media for 4 days, and RNA was harvested for RT-qPCR. n = 3 independent experiments (each time 2–3 replicates), mean ± SEM, ∗∗p < 0.01, ∗p < 0.05, two-way ANOVA. (D) Exclusion of any off-target effect. siRNA-mediated inhibition of RNLTR12-int in differentiating OPC does not alter expression of genes whose intronic region (either in the same or the opposite strand) contains fragment of RNLTR12-int. Normalized values from RNA-seq data plotted, mean ± SEM, ns: p > 0.05, Benjamini-Hochberg FDR after Wald test.