Suv39h1

ChIP assays were performed as previously described24 (link). Briefly, protein-DNA complexes were cross-linked by 1% formaldehyde for 10 min at 37 °C. Cells were resuspended in 200 μl of SDS lysis buffer and subjected to three cycles of sonication on ice with 10-S pulses. Then, sonicated samples were centrifuged to spin down cell debris, and the soluble chromatin solutions were immunoprecipitated using antibodies specific for p65(Bethyl Lab., Montgomery, TX, USA), HP1(GeneTex, San Antonio, TX, USA), SUV39H1, H3K9me3, and RNA polymerase II(Millipore, Billerica, MA, USA). The final immunoprecipitated DNA(IP DNA) were resolved in nuclease-free H₂O and the DNA solutions were quantified by quantitative PCR. Primers amplifying irrelevant segments of DNA in the IL-8 gene, the 3′ UTR sites, were used as site-specific binding controls.

Human MSCs grown on coverslips were fixed in 1% PFA (Polyformaldehyde). Human MSC spheroids were fixed in 3% PFA, embedded in OCT and cryosectioned (6–8 μm thickness). After permeabilization with 0.1% Triton X‐100, samples were incubated with desired primary antibodies, overnight at 4°C, followed by detection with a fluorescence‐conjugated secondary antibody. Nuclei were stained with 4, 6‐diamidino‐2‐phenylindole (DAPI). After mounting, samples were visualized under confocal microscope (FV1000; Olympus, Tokyo, Japan). Nuclear actin was observed directly. Confocal z‐scans were used and display an overlay of several picture 23.
F‐actin was labelled with Alexa Flura488‐phalloin (1:400; Life Technologies, Carlsbad, CA, USA); G‐ actin was labelled with Rhodamine‐DNase I (1:1000; Life Technologies); other antibody used in this study were α‐tubulin (1:400; Millipore, America); β‐actin (1:100; Santa Cruz, America); Nanog (1:200; ThermoFisher, America); Suv39h1 (1:200; Millipore, America); H3K9me3 (1:500; Abcam); Integrin β1 (1:100; Millipore); Vinculin (1:200; Santa Cruz); N‐cadherin (1:400; Cell Signaling Technology); β‐catenin (1:400; Sigma‐Aldrich); focal adhesion kinase (FAK, 1:1000; Millipore); phosphorylated FAK (p‐FAK, 1:1000; Millipore, America); E‐cadherin (1:2000; Life Technologies, America).

The antibodies employed in this assay included 5 μg antibody to Suv39h1 (Millipore, Billerica, MA, USA), H3K9me3 (Abcam Inc., Cambridge, UK), MyoD (Santa Cruz Biotechnology, CA, USA) or isoform IgG (Santa Cruz Biotechnology, CA, USA). IgG antibody was regarded as the NC. The pulled-down DNA was resuspended in 20 μL water and 1 μL immunoprecipitation samples were subjected to RT-qPCR. The content of amplified DNA was used to present the relative enrichment with input and values captured after normal IgG immunoprecipitation as the controls. The primer sequences of ChIP-PCR were shown in Table 2.