Figure 1.

The absence of MECP2 leads to decreased cell viability and dysregulations in cell migration, integrin, and phagosome formation pathways in MGLs

(A) Representative images of CTRL, KO, and KO^R MGLs stained with CD68 and MeCP2 antibodies. Scale bar 10 μm.

(B) The percentage of cell viability was compared between different cell lines. Each data point shows the mean ± SEM, as indicated. Significance was tested using a two-way ANOVA with the Tukey’s multiple comparison test (^∗p = 0.0304 and ^∗p = 0.0247 for day 24 and 29, respectively) (three lines for CTRL MGLs and KO and two isogenic rescue lines for KO^R MGLs, n = 3 independent experiments for each sample at each time point).

(C) Bright-field images of CTRL, KO, and KO^R MGLs at day 7 of differentiation, scale bar 200 μm.

(D) Bright-field images of CTRL MGLs with (right) or without (left) masks that marked round (in green), elongated (in blue), or cell clumps (in red), scale bar 200 μm.

(E–H) Percentage of round cells, elongated cells, and cell clumps, respectively. (H) Area fold change compared to the control of CTRL, KO, and KO^R. One-way ANOVA with the Tukey’s multiple comparison test was performed (^∗p = 0.0323, ^∗∗p = 0.0022, and n.s. for E to G, respectively, for ^∗p = 0.0149 and ^∗∗p = 0.0021 for H (Each bar shows the mean ± SEM, each dot represents one sample, two different isogenic pairs, two different clones for CTRL with three independent experiments per sample).

(I) Heatmap showing the 39 differentially expressed genes (greater than 1.25-fold) between these two groups (KO n = 5 from two independent cell lines, two to three biological replicates, CTRL n = 5 from two independent cell lines, two to three biological replicates and KO^Rn = 3 from two independent cell lines, one to two independent experiments p < 0.05, data were analyzed for statistical significance using Rosalind onramp software, see supplemental information, methods' section). Increasing fold changes compared to CTRL are marked in red while decreasing ones are in blue.

(J) Top canonical pathways were obtained with 39 differentially expressed genes (DEGs) using Ingenuity Pathway Analysis (IPA), where significance was calculated by the right-tailed Fisher’s exact test (p < 0.05).

(K) The top network involving the majority of DEG obtained through IPA, genes that are downregulated are in green and upregulated are in red.

(L) Top 5 diseases and biological functions were obtained with IPA.