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. 2021 Apr 9;10(4):860. doi: 10.3390/cells10040860

Figure 3.

Figure 3

Overexpression of MECP2 phosphorylation-deficient variants increases the expression of pro-inflammatory cytokines in BV2 cells upon LPS/IFNγ-induced inflammation. BV2 cells were transduced with lentivirus vectors encoding MECP2-wild type (WT)-Myc, MECP2-S80A-Myc, MECP-S423A-Myc, or plasmid backbone only (Control). The vector backbone contained the elongation factor-1 alpha (EF1α) promoter and green fluorescent protein (ZsGreen1) coding sequence separated by an internal ribosome entry site (IRES). Inflammation was induced by treatment with LPS (200 ng/mL) and IFNγ (20 ng/mL) for 24 h. (a) β-actin-normalized MECP2-WT, MECP2-S423A and MECP2-S80A mRNA expression in vehicle and LPS/IFNγ-treated BV2 cells. Data are shown as mean + SEM of n = 4. Kruskal–Wallis followed by Dunn’s multiple comparisons test, ** p < 0.01, * p < 0.05 (b) Representative blot image showing MECP2-WT, MECP2-S423A, and MECP2-S80A overexpression in vehicle and LPS/IFNγ-treated BV2 cells (left). Quantification of β-actin normalized exogenous MECP2 protein levels (right). Data are shown as mean + SEM of n = 4. Independent samples t-test; **** p < 0.0001. (c) β-actin-normalized mRNA expression of the pro-inflammatory cytokines Il6 and Tnf upon LPS/IFNγ-induced inflammation in BV2 cells expressing MECP2-WT, MECP2-S80A, MECP2-S423A, or Control vector. Data are shown as mean + SEM of n = 4. One-way ANOVA, post-hoc LSD. For Treatment effect: Independent-samples t-test or independent samples Mann–Whitney U test was used; **** p < 0.0001, *** p < 0.001, ** p < 0.01. (d) Homeostatic (P2ry12) and disease-associated microglia (DAM; Trem2/Cst7) RNA signature upon LPS/IFNγ-induced inflammation in BV2 cells. Data are shown as mean + SEM of n = 4. One-way ANOVA, post-hoc LSD. For Treatment effect: Independent-samples t-test or independent samples Mann–Whitney U test was used; ** p < 0.01, * p < 0.05.