Fig. 6: Cit-k KO dorsal OPCs are more vulnerable to oxidative stress compared to Cit-k KO ventral OPCs. | Nature Communications

Fig. 6: Cit-k KO dorsal OPCs are more vulnerable to oxidative stress compared to Cit-k KO ventral OPCs.

From: Molecular and functional heterogeneity in dorsal and ventral oligodendrocyte progenitor cells of the mouse forebrain in response to DNA damage

Fig. 6

a γH2AX expression in acutely MACS-isolated OPCs of P10 Cit-k KO mice. b Number of γH2AX+ foci in dOPCs vs. vOPCs (n = 3 each, two-tailed Mann–Whitney U-test, n.s.). c DHE labeling in acutely MACS-isolated OPCs of P10 Cit-k KO mice. d DHE integrated density in dOPCs vs. vOPCs (n = 5 each, Two-tailed Paired t-test, **P = 0.0013, t(4) = 8.043). e NBT reduction assay in Cit-k KO dOPCs vs. vOPCs (normalized to dOPC mean value. n = 4/group, Two-way Anova: Genotype: P < 0.0001, F(1,21) = 92.83; Region: P = 0.0067, F(1,21) = 9.045; Genotype × Region: P = 0.0204, F(1,21) = 6.288). Orange and blue lines represent mean (solid line) ± SE (dotted lines) of WT dOPCs and WT vOPCs, respectively. f, g Western blotting (f) and densitometric analysis (g) of γH2AX and NRF2 protein expression in P10 Cit-k KO dOPCs vs. vOPCs (n = 4/group; γH2AX: n.s.; NRF2: P = 0.0155, t(3) = 4.984, Two-tailed Paired t-test). h NRF2 (green) expression in MACSorted Cit-k KO AN2+ (red) dOPCs and vOPCs in vitro. DAPI (blue) counterstains cell nuclei. i NRF2 staining integrated density in MACSorted Cit-k KO dOPCs vs. vOPCs (n = 4/group; P = 0.0087, t(3) = 6.129, Two-tailed Paired t-test). j Heatmap of the qRT-PCR analysis of the mRNAs of Nrf2, Keap1, and NRF2-target genes in dOPCs and vOPCs MACSorted from P10 WT and Cit-k KO mice (Two-way Anova followed by Bonferroni’s Multiple Comparison Test; n, P, and F values in Supplementary Table 1). Dot plots are included in Supplementary Fig 7. k Log(inhibitor) vs. response curve (Non-linear regression inhibition curve) representing percentages of viable OPCs after an acute H2O2 treatment (0, 100, 500, 5000 µM). LC50 KO dOPCs = 38.96 µM, R2 = 0.885; LC50 KO vOPCs = 1481 µM, R2 = 0.859; LC50 WT dOPCs = 1015 µM, R2 = 0.895; LC50 WT vOPCs = 2046 µM, R2 = 0.907). l Cit-k KO dOPC density at t0 (i.e., 30 min after plating) and at t = 2 DIV in Ctrl conditions vs. in presence of NAC or DMF (n = 5/group; P < 0.0001, F(3) = 53.86, One-way Anova Repeated Measures). In l data are mean ± SE. In b, d, e, g, i lines connect paired samples (i.e., dOPC and vOPC samples from the same mouse). Scale bars: 5 µm in a, c, h; 10 µm in l. DHE dihydroethidium, DIV days in vitro, DMF dimethyl fumarate, dOPC dorsal oligodendrocyte progenitor cell, H2O2 hydrogen peroxide, LC50 inhibitory concentration 50—concentration that produces 50% decrease in viable cells, NAC N-acetyl-L-cysteine, NBT Nitroblue Tetrazolium, vOPC ventral oligodendrocyte progenitor cell, WT wild-type; P postnatal day, γH2AX phosphorylated histone H2AX. *P < 0.05; **P < 0.01; ***P < 0.001. Source data are provided as a Source Data file.

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