Fig 1: Phenotypic analysis of neural progenitors and differentiating cells upon overexpression of uc.478 and uc.479(A) The diameter of neurospheres was measured for WT or MeCP2 KO cells upon overexpression of uc.478 or uc.479. Graphs represent the median and interquartile range (a two-tailed t test was used; *p < 0.05, **p < 0.01, ***p < 0.001). Images correspond to representative spheres of each condition. Scale bar in the sphere, 100 µm. (B) Control WT neural progenitors or cells overexpressing uc.478 or uc.479, as indicated, were driven toward glutamatergic differentiation for 7 days, stained with MAP2, and reconstructed in silico from confocal images with NeuronStudio software. One representative picture for each condition is shown. (C) Automatic analysis of the cells in (B) allowed total branchpoints and spine count per condition (n = 20 neurons, one-way ANOVA was used; *p < 0.05, **p < 0.01). (D) For the same cells as in (B), the abundance of each spine subtype was determined with NeuronStudio software (n = 20 neurons, one-way ANOVA was used; *p < 0.05, **p < 0.01). (E–G) the same analysis as in (B)–(D) was performed in MeCP2 KO cells. (H) Expression levels of VGlut1 and VGlut2 mRNA were analyzed by qRT-PCR in WT or MeCP2 KO cells overexpressing uc.478 or uc.479 and driven toward glutamatergic differentiation for 7 or 14 days, as indicated. The graph represents the mean ± SD of three independent replicas (unpaired t test was used; *p < 0.05, **p < 0.01, ***p < 0.001). (I) Western blot analysis of VGlut1 and VGlut2 protein levels from the same MeCP2 KO cells as in (H).
Fig 2: MeCP2 ablation causes a defect in cell proliferation with a delay in cell-cycle progression. (A) PC-3, (B) LNCaP and (C) NIH-3 T3 cells were transfected with siRNA MeCP2 or non-targeting siRNA CTRL oligos; MeCP2 ablation was checked at 5 and 7 days after transfection and PC-3, LNCaP and NIH-3 T3 cells, and cell proliferation was analysed (data are mean ± SD bars calculated from three independent experiments). (D) A representative FACS analysis of PC-3 treated with control siRNA (siRNA CTRL: plots 1, 2 and 3) or with siRNA against MeCP2 (siRNA MeCP2: plots 4, 5 and 6) that shows a delay in cell-cycle progression. (E) After 7 days of MeCP2 silencing was observed an enrichment of cells in sub-G0/G1 phase (M1 bar), a representative FACS analysis is shown in E1. The synthesis of three independent FACS experiments after 7 days of MeCP2 silencing is represented in bar chart E2 (statistics were performed using Student’s test. *: significant difference between siRNA CTRL and siRNA MeCP2 PC-3 cells, P < 0.005). (F) A representative bi-parametric BrdU/PI FACS analysis of PC-3 cells after 7 days of MeCP2 silencing is shown in F1; the synthesis of three independent bi-parametric BrdU/PI FACS analysis of PC-3 cells after 7 days of MeCP2 silencing is represented in bar chart F2 (statistics were performed using ANOVA. *: significant difference in cell cycle phases between siRNA CTRL and siRNA MeCP2 PC-3 cells, P < 0.05).
Fig 3: Tubacin rescues ciliogenesis in MeCP2‐deficient cells AWT and Mecp2 null MEF cells were starved for 48 h and then treated with the HDAC6 inhibitor tubacin (TUB) (1 μM) for 48 h or with DMSO (VEH) or left untreated (UT). Cells were fixed and stained for anti‐acetylated α‐tubulin, and cilia were counted. The graph shows the percentage of ciliated cells out of three independent experiments (mean ± SE, *P < 0.05; **P < 0.01, two‐way ANOVA followed by Bonferroni post hoc test). Two‐way ANOVA reported a significant genotype effect (F (1, 10) = 17.94, P < 0.01), a significant tubacin treatment (F (2, 10) = 4.425, P < 0.05), and a significant interaction (F (2, 10) = 7.266, P < 0.05).BPrimary cortical neurons were treated at DIV5 with DMSO (VEH) or tubacin (TUB, 1 μM) for 48 h, or left untreated (UT), fixed at DIV7, and stained with anti‐AC3 antibody. The graph shows the percentage of ciliated cells obtained from three independent experiments counting n = 300 UT cells, n = 216 VEH‐treated cells, and n = 267 TUB‐treated cells for each genotype (mean ± SE, *P < 0.05; **P < 0.01, two‐way ANOVA followed by Bonferroni post hoc test). Two‐way ANOVA reported a significant genotype effect (F (1, 15) = 23.42, P < 0.001), a significant treatment effect (F (2, 15) = 11.25, P < 0.01), and a significant interaction (F (2, 15) = 4.853, P < 0.05).C MECP2‐silenced or control hTERT‐RPE‐1 cells were starved for 48 h, treated with tubacin (TUB, 1 μM) for 24 h, and then exposed to 200 nM SAG for 24 h in the presence of tubacin. Cells were stained with anti‐Smo and anti‐acetylated α‐tubulin antibodies, and the percentages of Smo‐positive cilia were calculated. The graph shows the averages of three independent experiments (mean ± SE, **P < 0.01, three‐way ANOVA followed by Tukey's post hoc test). Statistical analysis reported a significant difference between genotype (F (1, 2) = 21.47, P < 0.001), a significant SAG effect (F (2, 2) = 154.5, P < 0.0001), and a significant tubacin effect (F (1, 2) = 25.18, P < 0.001). In detail, a significant interaction between genotype and SAG treatment (F (2, 2) = 5.413, P < 0.05) and between SAG and tubacin treatment (F (2, 2) = 6.939, P < 0.01) was reported, demonstrating respectively a difference between CTRL‐ and MeCP2‐depleted cells in response to SAG and a different SAG effect when cells were treated with tubacin.DRepresentative immunostaining of Smo (green) and acetylated α‐tubulin (red) in control (CTRL) and silenced (siMeCP2) hTERT‐RPE‐1 cells, after stimulation with 200 nM SAG in the presence or absence of the HDAC6 inhibitor tubacin (TUB). Scale bar = 50 μm, and 20 μm in the enlarged image. Source data are available online for this figure.
Fig 4: Pol III co-occupies methylated SINEs with MBPs.(a) Semiquantitative ChIP assay in A31 fibroblasts showing specific binding of TFIIIB, TFIIIC and pol III to B1 and B2 loci, as well as 7SL, but not the Apo-E gene. Histone H3 and TAFI48 provide positive and negative controls, respectively. (b) Semiquantitative ChIP assay in HeLa cells showing occupancy of pol III, TFIIIB and TFIIIC at Alu loci from chromosomes 6, 10, 19 and 22, as well as 7SL and Apo-E genes. ChIPs for histone H3 and TAFI48 provide positive and negative controls, respectively. No antibody was used for the mock sample. (c) Mean±s.e.m. of the percentage input bound in three independent ChIP–quantitative PCR (qPCR) assays in HeLa cells, of the indicated proteins at individual Alu loci from chromosomes 6, 10, 19 and 22, as well as 7SL and Apo-E loci and Alu PV subfamily consensus. ChIPs for histone H3 and TAFI48 provide positive and negative controls, respectively. No antibody was used for the mock samples. P values are calculated by t-test. (d) Mean±s.e.m. of four independent sequential ChIP–qPCR assays in which DNA immunoprecipitated from HeLa cells using pol III antibody was reprecipitated using antibodies against pol III, TFIIIB, TAFI48 (negative control), MBD1, MBD2 and MeCP2, as indicated. No TAFI48 signal was detected on Alu(c6).
Fig 5: HDAC6 inhibition restores neuronal defects associated with the lack of Mecp2HDAC6 inhibition restores neuronal defects associated with the lack of Mecp2 ARepresentative images of MAP2‐positive WT and Mecp2 null neurons (DIV7). At DIV5, neurons were treated with tubacin (TUB, 1 μM for 48 h) and Aurora A inhibitor TC‐S 7010 (TC‐S, 7 nM for 48 h) or left untreated and morphologically analyzed by Sholl analysis plug‐in. Scale bar = 20 μm.BThe graph reports the mean ± SE of the number of intersections measured 85–145 nm from the soma calculated by Sholl analysis for WT (n = 39 neurons) and Mecp2 null neurons, when left untreated (n = 57 neurons) or treated with tubacin (n = 53 neurons) or TC‐S 7010 (n = 38 neurons). Data from WT and null neurons were compared by Mann–Whitney test (**P < 0.01); drug effects on null cells were analyzed by one‐way ANOVA followed by Dunn's post hoc test (*P < 0.05; **P < 0.01).CRepresentative images of WT and Mecp2 null neurons (DIV14) immunostained for MAP2 (white), Synapsin1/2 (green), and Shank2 (red). Mecp2 null cells were treated at DIV12 with tubacin (TUB, 1 μM for 48 h) or Aurora A inhibitor TC‐S 7010 (TC‐S, 7 nM for 48 h). Scale bar = 15 μm, and 5 μm in the enlarged image.D, EThe graphs represent the mean ± SE of the Synapsin1/2 and the Shank2 puncta density in WT (n = 67 neurons) and Mecp2 null neurons, when untreated (n = 51 neurons) or treated with tubacin (n = 59 neurons) or TC‐S 7010 (n = 55 neurons). Neurons derived from at least 6 different biological replicates. Data from WT and null neurons were compared by Mann–Whitney test (**P < 0.01; ***P < 0.001); drug effects on null cells were analyzed by one‐way ANOVA followed by Dunn's post hoc test (*P < 0.05; ***P < 0.001).FThe graph depicts the mean ± SE of the number of co‐localized pre‐ and post‐synaptic puncta (n = 20 neurons deriving from three different biological samples). Data from WT and null neurons were compared by Mann–Whitney test (***P < 0.001); drug effects on null cells were analyzed by one‐way ANOVA followed by Dunn's post hoc test (*P < 0.05). Source data are available online for this figure.
Supplier Page from MilliporeSigma for Anti-MeCP2 antibody produced in rabbit