Fig 2: Nfats are regulated by Sox10 in oligodendroglia. a QrtPCR-determined Rplp0-normalized Nfat expression in mouse oligodendrocytes after 6 (open bars, set to 1), 24 (light grey bars) or 48 h (dark grey bars) differentiation (n = 3) (Nfatc1: 24 h 1.4 ± 0.1, 48 h 2.2 ± 0.5; Nfatc2: 24 h 2.8 ± 0.1, 48 h 2.0 ± 0.2; Nfatc3: 24 h 1.6 ± 0.2, 48 h 2.2 ± 0.2; Nfatc4: not detected). b Immunocytochemistry on differentiating DAPI-counterstained (blue) mouse oligodendrocytes after 48 h with anti-Nfat antibodies (green). c Nuclear versus cytoplasmic Nfatc2 signal intensity in rat oligodendroglia treated with ionomycin (grey bars, iono) or DMSO (open bars, Ctr) (n = 3, 30 cells analyzed per experiment) (values: 0.60 ± 0.07 for Ctr, 0.97 ± 0.04 for iono). d Nuclear fraction of Nfatc2-eYFP in transfected mouse oligodendroglial cells before and after treatment with ionomycin or DMSO (n = 4 with ~40 cells analysed per time point and condition) (values before and after 5, 10, and 30 min treatment: 9.7 ± 3.0%, 17.1 ± 3.9%, 21.6 ± 5.8%, and 17.4 ± 5.8% for Ctr; 9.9 ± 3.6%, 25.5 ± 2.8%, 43.0 ± 4.2%, and 37.9 ± 8.4% for ionomycin). e Gapdh-normalized Nfatc2 quantification by qrtPCR in wildtype (Sox10endog +, set to 100%) or Sox10-deficient variant (Sox10endog -) Oln93 cells, before and after transduction with a control (LVempty) or Sox10-expressing (LVSox10) lentivirus (n = 3) (wildtype: 100.0 ± 6.1; variant: 3.1 ± 0.8; LVempty-infected variant: 1.5 ± 0.4; LVSox10-infected variant: 40.3 ± 9.6). f Sox10-dependent (+, light grey bars = low amounts; ++, dark grey bars = high amounts) fold inductions ± SEM of luciferase reporters carrying Nfatc2 distal promoter (dProm) or ECR87 in transfected N2a cells after 48 h (n = 3) (dProm: 3.4 ± 0.6 and 6.8 ± 1.4; ECR87: 19.7 ± 0.9 and 21.4 ± 1.3). g Mouse Nfatc2 locus with exons (brown boxes), transcription start sites (arrows), and Sox10 ChIP-Seq peaks (blue boxes). h Putative (light blue) and EMSA-confirmed (dark blue) Sox10 binding sites in ECR87. i Sox10-dependent (+, grey bars) fold inductions ± SEM of wildtype or mutant ECR87 luciferase reporters with inactivated Sox10 binding sites (S2m, S3m, S2/3m) in transfected N2a cells after 48 h (n = 3) (WT: 28.1 ± 1.0; S2m: 11.3 ± 0.5; S3m 8.4 ± 0.9; S2/3m 5.9 ± 0.6). j Sox10 immunohistochemistry (green) on cortical slices 7 days after transduction with a retroviral ECR87-tdTomato reporter (red). DAPI counterstain in blue. Scale bars, 50 µm. Statistical significance was determined by Bonferroni-corrected one-way (a) or two-way ANOVA (d) and two-tailed Student’s t-test (c, e, f, i) (*P = 0.05; **P = 0.01; ***P = 0.001)

Fig 3: NFATs are relevant in human oligodendrocytes. a NFAT expression in human iOLs (grey bars) and NPCs (open bars) by qrtPCR after normalization to GAPDH. Average NFAT levels in NPCs were set to 1 (NFATc1: 1.4 ± 0.3; NFATc2: 14.6 ± 1.9; NFATc3: 1.6 ± 0.3 and NFATc4: 0.9 ± 0.2). b Immunocytochemistry of MBP-positive (red) iOL with antibodies directed against NFATs (white in upper row, green in lower row); counterstaining with Hoechst dye (blue). c–g Analysis of differentiating iOL cultured 21 (c, e, h) or 35 days (d, f, g, i, j) in absence (Ctr, open bars) or presence of 0.1 (light grey bars) to 0.5 µM (dark grey bars) VIVIT (c–g, n = 4) or 1 (light grey bars) to 10 µM (dark grey bars) FK506 (h–j, n = 3–4). VIVIT/FK506 incubation was continuous (c–f, h, i) or from days 21–35 (g, j). Cultures were stained with anti-O4 (green, c) and anti-MBP antibodies (green, d). From staining and FACS analysis (see Suppl. Fig. 7c, d) the fraction of O4-positive cells after 21 days (e, h) and MBP-positive cells after 35 days (f, i) was determined. For treatment during days 21–35, the fraction of FACS-sorted O4-positive cells was determined that had reached a MBP-positive stage (g, j). The relative number of marker-positive cells under control conditions was set to 1 and used to normalize in pairwise fashion (values: 1 for control, 0.97 ± 0.01 for 21 days 0.1 µM VIVIT, 0.68 ± 0.06 for 21 days 0.5 µM VIVIT, 0.86 ± 0.10 for 35 days 0.1 µM VIVIT, 0.60 ± 0.10 for 35 days 0.5 µM VIVIT, 0.82 ± 0.05 for 0.1 µM VIVIT from days 21–35, 0.86 ± 0.04 for 0.5 µM VIVIT from days 21–35, 0.99 ± 0.21 for 21 days 1 µM FK506, 0.39 ± 0.17 for 21 days 10 µM FK506, 1.12 ± 0.36 for 35 days 1 µM FK506, 0.55 ± 0.09 for 35 days 10 µM FK506, 0.83 ± 0.17 for 1 µM FK506 from days 21–35 and 0.86 ± 0.10 for 10 µM FK506 from days 21–35). k–n NFAT immunohistochemistry in human brain tissue. Antibodies directed against NFATc3 and NFATc4 (red in k and l, black in m) were combined with anti-NOGOA antibodies (green in k and l, red in m). Arrows mark double positive cells, arrowheads NOGOA-positive oligodendrocytes without NFATc4. n Percentage of NOGOA-positive oligodendrocytes with nuclear NFATc4 in periplaque white matter (86 ± 4%), active and demyelinating lesions (41 ± 8%), active and post-demyelinating lesions (41 ± 8%), active and remyelinating plaques (59 ± 19%), and control CNS tissue (99 ± 1%). o Model of oligodendroglial Nfat action. Statistical significance was determined by two-tailed Student’s t-test (a) or Bonferroni-corrected one-way ANOVA (e–j, n) (*P = 0.05; **P = 0.01; ***P = 0.001). Scale bars, 50 µm

Fig 4: Sox10-dependent enhancer activation is stimulated by Nfatc2 and inhibited by Olig2 and Nkx2.2. a Transient transfections of N2a cells with luciferase reporters carrying Nkx2.2 ECR19 in the absence (-) or presence (+) of Sox10 and constitutively active Nfatc2 as effectors (n = 3). Activation of reporter gene expression was determined in extracts 48 h post transfection and is presented as fold inductions ± SEM with transfections in the absence of effectors arbitrarily set to 1 (58.5 ± 4.0 for Sox10, 0.3 ± 0.0 for Nfatc2, and 130.5 ± 11.6 for Sox10 in combination with Nfatc2). b, c Neural tube electroporations in HH11-stage chicken embryos (n = 3). The relative number of electroporated cells, in which Nkx2.2 expression was induced 48 h after neural tube electroporation of Sox10 and Nfatc2, was quantified (b: 0 for control, 19.5 ± 4.9 for Sox10, 25.9 ± 4.1 for Nfatc2, and 39.0 ± 4.6 for Sox10 in combination with Nfatc2). Transverse sections were used for quantifications (c). Sections were probed for the occurrence of Nkx2.2 (white in upper row, red in lower row). Electroporated cells on the left side all express GFP (green) and to variable extent Nkx2.2 (white in upper row, red in lower row). Nuclei are counterstained by DAPI (blue). Scale bar, 50 µm. d, e Transient transfections of N2a cells with luciferase reporters carrying ECR19 (d) or OLEa (e) in the absence of added transcription factors (-) or in the presence (+) of Sox10, Olig2, Nkx2.2, and combinations thereof (n = 3). Activation of reporter gene expression was determined in extracts 48 h post transfection and is presented as fold inductions ± SEM with transfections in the absence of added transcription factors being arbitrarily set to 1 (ECR19: 55.3 ± 7.7 in the presence of Sox10, 0.7 ± 0.1 in the presence of Olig2 and 13.1 ± 2.0 in the presence of Sox10 and Olig2; OLEa: 21.6 ± 1.3 in the presence of Sox10, 2.2 ± 0.1 in the presence of Nkx2.2 and 8.8 ± 1.2 in the presence of Sox10 and Nkx2.2). Statistical significance was determined by two-tailed Student’s t-test (*P = 0.05; **P = 0.01; ***P = 0.001)

Fig 5: Knockdown expression of CnB, NFAT1 or NFAT2 impairs in vivo tumor growth. (a) Immunohistochemical analysis of NFAT1 expression and subcellular localization in control (LKO), CnB1-silenced (shCnB) and NFAT1-silenced (shNFAT1) 4T1 tumors. A lymph node section of control mice (left panel) has been used as positive control for NFAT1 expression and nuclear localization. Arrows point to NFAT1-expressing lymphoid infiltrating cells, whereas stars point to nuclear NFAT1 in mammary tumor cells. (b) 4T1 cells (5 × 105; n=4 for each 4T1 cell derivatives) were inoculated in the inguinal mammary fat pad of syngeneic Balb/c mice. Tumor size was measured every 3 days with a caliper and the tumor volume calculated according to the equation V= (L × W2)/2. Data are represented as mean±S.E.M. (c) Tumor volume at the time of killing, 3–5 weeks after inoculation. Control pLKO tumors (white dots, n=21), shCnB1 tumors (light gray, n=21), shNFAT1 tumors (black dots, n=21), shNFAT2 tumors (gray dots, n=16). Data are represented as mean±S.E.M. (d) Quantification of the number of proliferative cells (Ki67+, upper panel) and apoptotic cells (CC3+, lower panel) in tumors (n=3) 15 days after inoculation. Numbers are the results of counting five consecutive fields at the × 20 magnification. Data are represented as mean±S.E.M. (e) Tumors excised 15 days after orthotopic injection were fixed, paraffin embedded and further analyzed for the expression of cleaved caspase 3 (CC3) by immunohistochemistry. The right panels represent a higher magnification of the insets shown in respective left panels. A representative picture of each tumor is shown. Arrows point to CC3-positive cells. Note the unspecific brown staining of necrotic areas observed at the × 1 magnification