Fig 2: p73 protein induction in response to teriflunomide stimulation. a–c”’ Double immunostaining and its quantification confirmed that early teriflunomide pulses (scheme II, see Fig. 3) result in cells displaying strong TAp73 signals (arrowheads in b) correlating also with MBP positivity (dashed bars in a). Late teriflunomide pulses (scheme III) could not boost both protein markers. Scale bars, 20 µm. d–g Western blot analysis confirmed that early short-term teriflunomide pulses (scheme II) result in a strong induction of CNPase (e), MBP (f), and PLP levels (g), as well as of TAp73 (d) protein levels. Late pulses (scheme III) did not upregulate TAp73 or myelin marker expression (as quantified for TAp73 in d). Actin was used for normalization and protein molecular weights are indicated in kilodalton. Data are shown as mean, and error bars represent SEM. t test (ns, not significant, *p < 0.05). Number of experiments: n = 3 for (a, d, f, g), n = 2 for (e)

Fig 3: Expression of BTEB-like Klf factors in oligodendroglial cells. (A–D) Transcript levels for Klf9, Klf13, Klf14 and Klf16 in neuroectodermal cell types (A, astrocyte; N, neuron; OL, newly formed oligodendrocyte) after immunopanning at the time of birth (expressed as FPKM) according to GSE 52564 (30). (E) Transcript levels for Klf9, Klf13, Klf14 and Klf16 in oligodendroglial cells from spinal cord at P6 (expressed as read counts) according to GSE119127 (27). (F) Relative transcript levels for Klf9, Klf13, Klf14, Klf16, Mbp and Pdgfra in primary rat oligodendroglial cultures kept under proliferative conditions (prol) or undergoing differentiation for 3 or 6 days (3d, 6d) as determined by qRT-PCR. Normalized transcript levels for Klf9, Mbp and Pdgfra under proliferating conditions were set to 1, and levels in differentiating oligodendrocytes expressed relative to it (n = 3). (G, H) Protein amounts of KLF9, KLF13, MBP and PDGFRA in whole cell extracts from rat oligodendroglial cells kept under proliferating or differentiating conditions as determined by Western blot (G). Numbers on the right of the panels represent approximate size in kDa. For quantification (H), band intensities in proliferating cells were set to 1 after normalization to GAPDH levels, and values for differentiating cells expressed relative to it (n = 3). Statistical significance was determined by two-tailed Student's t test (F) and one way Anova (H) (*P = 0.05; **P = 0.01; ***P = 0.001).

Fig 4: Occurrence and localization of KLF9 and KLF13 during oligodendroglial development. (A–D) Detection and quantification of KLF9 (A, B) and KLF13 (C, D) in oligodendroglial cells at P0, P7 and P21 following co-immunohistochemistry of Klf-specific antibodies (in green) with antibodies against SOX10, PDGFRA and MYRF (all in red). Exemplary pictures (A, C) were taken from the ventrolateral white matter of spinal cord tissue at P7. Quantification of the fraction of SOX10-positive oligodendroglial cells and MYRF-positive oligodendrocytes in the white matter that co-expressed KLF9 (B) or KLF13 (D). (E) Subcellular localization of KLF9 and KLF13 proteins by microscopy following co-immunohistochemistry of KLF-specific antibodies (in green) with antibodies against PDGFRA (red) and MBP (yellow), combined with a DAPI nuclear counterstain (magenta) in primary oligodendroglial cells cultured under proliferating (upper row) or differentiating (lower row) conditions. Scale bar: 50 µm (A, C), 25 µm (E).

Fig 5: Ep400 occurrence in the mouse spinal cord and dispensability for OPC specification. (A–I) Localization of Ep400 (red) determined by co-immunohistochemistry on spinal cord tissue at postnatal day 1 (P1) with the following CNS cell type markers (green): NeuN (A), Nfia (B), Gfap (C), Iba1 (D), Sox10 (E), Olig2 (F), Pdgfra (G), Nkx2.2 (H), Mbp (I). (J–O) Ep400 deletion in Ep400fl/fl Nestin::Cre (Ep400?N, K,M,O) embryos as determined by Ep400 immunohistochemistry (J,K,N,O) at E12.5 relative to age-matched controls (J,L,N). Nuclei were counterstained with Dapi (L–O). Shown is the ventral spinal cord surrounding the ventricular zone (P–W) Immunohistochemistry with antibodies directed against Olig2 (P–S) and Sox10 (T–W) on transverse spinal cord sections of control and Ep400?N embryos at E12.5 (P,Q,T,U) and E14.5 (R,S,V,W). Shown is the region around the pMN domain (P, Q, T, U) and ventral mantle zone (R,S,V,W). Scale bars: 30 µm (A) and 50 µm (K, P, R). (X, Y) Quantifications of Olig2 and Sox10 positive cells in spinal cord of control and Ep400?N embryos at E12.5 (X) and E14.5 (Y) (n = 3 embryos per genotype, counting three separate sections each). Mean absolute cell numbers per section (X) or relative numbers normalized to DAPI (Y) ± SEM are presented. No statistically significant difference between the genotypes was detected.