Mecp2

The frozen tissue sections were washed 3 times with 1 × PBS. After that, the tissue was blocked with 1 × PBS, 10% goat serum, and 0.3% Triton at 37 °C for 2 h. Subsequently, the tissue was incubated with primary antibodies (Sirt1, 1:400, rabbit anti-mouse, Cell Signaling Technology; BDNF, 1:750, rabbit anti-mouse, Abcam Plc.; MeCP2, 1:200, rabbit anti-mouse, Cell Signaling Technology) at 4 °C overnight. On the next day, the tissue slides were washed 3 times with 1 × PBS. Then the tissue was incubated with secondary antibodies (1:1000; goat anti-rabbit IgG H&L, Abcam Plc.) in PBS in the dark for 1 h. 1 × PBS washing was performed again. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI), followed by 1 × PBS washing. Quenched fluorogens were added, and the slides were sealed. Images were captured under a fluorescence microscope, and the quantification of immuno-positive cells was analyzed using the Image-Pro Plus software version 6.0.

Nuclei were obtained from neurospheres derived from each hiPSC line. ChIP was performed with antibodies against MeCP2 (Cell Signaling Technology), as described previously [53 (link)], with the following modifications. Neurospheres were crosslinked with 1% formaldehyde for 10 min to yield crosslinked chromatin. They were then incubated with glycine at a final concentration of 200 mM for another 5 min, and stored at −80°C until use. The lysed nuclear pellets were sonicated six times with a 30-sec ON, 60-sec OFF cycle by using a Bioruptor® Sonicator (Diagenode, Inc.). The crosslinked chromatin was subsequently eluted from magnetic beads (Dynabeads® M-280 Sheep anti-Rabbit IgG; Life Technologies). Co-immunoprecipitated DNA was detected via qPCR by using the following primers: STAT3 binding site-forward (5’-TCATGCCCAGTGAATGACTC-3’) and STAT3 binding site-reverse (5’-AGATGCCAGGCTGTCAGG-3’); and hGFAP exon1-forward (5’-AGAGCAGGATGGAGAGGAGA-3’) and hGFAP exon1-reverse (5’- CCTTGAAGCCAGCATTGAGT-3’).

After extracting the protein, its concentration was measured by the bicinchoninic acid protein (BCA) method (Beyotime, Shanghai, China). The protein in each sample was denatured by boiling with a loading buffer (1 μL buffer per 4 μL protein sample) at 100 °C for 5 min, then transferred to a Polyvinylidene Fluoride (PVDF) membrane, with 5% skim milk covered to block unspecific bindings, on a shaker at room temperature. After 2 h, the membrane was washed with Tris-buffered saline with Tween 20 (TBST). Primary rabbit anti-mouse antibodies targeting β-actin (1:1000; Cell Signaling Technology, Danvers, MA, USA), Sirt1 (1:1000; Cell Signaling Technology), BDNF (1:1000, Abcam, Cambridge County, UK), and MeCP2 (1:1000; Cell Signaling Technology) were added at 4 °C for overnight incubation. On the following day, after TBST washing, alkaline phosphatase (AP) -coupled goat anti-rabbit secondary antibodies (1:10,000; Haimingrui Biotech, Beijing, China) were added at room temperature, and incubation was completed on a shaker for 2 h. The TBST washing procedure was repeated. ECL substrate (Haimingrui Biotech, Beijing, China) was employed to develop protein bands. Protein expression was qualitatively analyzed with the β-actin as the internal reference to indicate the relative expression. The protein bands were analyzed using the Image-Pro Plus software to calculate optical density (OD).

Cells were fixed with PBS containing 4% paraformaldehyde for 30 min at room temperature and incubated with primary antibodies against the following proteins: MeCP2 (1:200, Cell Signaling Technology), phalloidin (1:2000, Dyomics), NANOG (1:500, CosmoBio), OCT4 (1:200, Santa Cruz Biotechnology, Inc.), TRA-1-60 (1:1000, Millipore), TRA-1-81 (1:1000, Millipore), βIII-tubulin (1:2000, Sigma Chemical Co.), MAP2 (1:250, Sigma Chemical Co.), and GFAP (1:1000, Invitrogen). They were then washed with PBS and incubated with an Alexa Fluor 488-, Alexa Fluor 555-, or Alexa Fluor 647-conjugated secondary antibody (1:500, Invitrogen), as appropriate. Images were obtained using an Axioplan 2 microscope (Carl Zeiss). The number of GFAP-positive cells was counted among 100 Hoechst-positive cells for each experiment (n = 5).

About 1 × 10⁵ cells were plated on coverslips 48 h prior and they were washed with PBS and then fixed with 4% paraformaldehyde for 15 min. After washing with PBS they were then permeabilized with 0.2% Triton X-100 for 20 min and blocked with 5% BSA for 30 min. Following that, cells were incubated with primary antibodies MeCP2 (Cell Signaling, 3456S), or HA (Cell Signaling, 3724S) for 1 h at room temperature, washed with PBS three times and then incubated in the dark with Phalloidin 568 and secondary antibodies ALEXA-488 goat anti rabbit conjugate for 1 h at room temperature. After three PBS washes the coverslips were mounted with ProLong^® Gold Antifade Mountant with DAPI (Thermo Fisher Scientific), the slides were allowed to cure for 48 h and then examined under the Nikon T-1E scanning confocal microscope, with a 60× objective, and analyzed with NIS software.