H3k27ac

ChIP assays were performed as reported previously [60 (link)]. Briefly, 2 wells of differentiating cells from a P-24 well plate were pooled after fixing with 1% formaldehyde for 10 min. The cells were washed and lysed in cell lysis buffer. The chromatin was sheared by sonication in diagenode bioruptor for 10 min at 30 sec on/off cycles. The lysate was immunoprecipitated using ChIP grade antibodies to H3Ac (Millipore, Cat. No.: 06-599), PCAF (Cell Signaling Technology, Cat. No.: 3378) and H3K27Ac (ABclonal, Cat. No.: A7253). ChIP with normal IgG (Millipore, Cat # PP64B) was used as the negative control. ChIP’d DNA was recovered and analyzed by RT-qPCR using SYBR® Green Real-Time PCR Master Mix (ThermoFisher Scientific) in a 10 μl reaction volume. Primer sequences used to amplify promoter regions are provided in Supplementary Table 1.

For Co-IP assay, cells were collected and lysed on ice with lysis buffer containing 0.5% NP40. The lysates were pre-cleared by incubation with protein A beads. The protein complex was then precipitated by a specific antibody together with protein A beads followed by extensive washing. The resulting materials were analyzed by western blotting as previously described^{81 (link)}. For IF analysis, PDLSCs were fixed using 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 and blocked with 4% BSA/PBS. Then the cells were treated with primary antibodies followed by incubation with FITC or TRITC–conjugated secondary antibodies. Coverslips were mounted in a glycerol/PBS solution containing DAPI. Confocal images were obtained using a Zeiss LSM confocal laser-scanning microscope. Antibodies against EZH2 (#5246), E2F1 (#3742), lamin A/C (#4777), H3K27me3 (#9733), H3K9me2 (#4685), H3K9me3 (#13969), RB and phospho-RB (S780, S795, and S807/811) (RB Antibody Sampler Kit #9969) were purchased from Cell Signaling Technology (CST). Antibodies against GAPDH (AC002) and α-Tubulin (AC007) were purchased from Abclonal. Antibody against CEMP-1 (ab134231) was from Abcam. Antibody against CAP (sc-53947) was from Santa Cruz. Antibodies used in IF were: EZH2 (612667, BD Pharmingen), H3K27ac (A7253, Abclonal), MED1 (A300-793A, Bethyl) and VAV2 (YT4864, Immunoway).

We ground the tissue with a tissue grinder (Shanghai Jingxin Industrial Development Co., Ltd., China). Then, the tissue was fixed with 1% formaldehyde and incubated at room temperature for 10 min to make DNA protein cross-links. Then, glycine was added to stop the cross-linking and incubated at room temperature for 5 min. One milliliter tissue lysis containing protease inhibitors (MCE, USA) was added to suspend tissue, and then, tissue was sonicated using EPISONIC (USA) to get 200–300 bp of chromatin fragments. Immunoprecipitation was performed with STAT1 (1:1000, Cell Signaling Technology, 14994S), p-STAT1 (1:1000, Cell Signaling Technology, 9167S), and H3K27ac (1:50, ABclonal Technology, A7253). The chromatin DNA was extracted using DNA purification kit (TIANGEN, China), and the specific primers of TNFAIP2 and LCP2 enhancer were used for PCR. The primer sequences were as follows: TNFAIP2-F5′-GTGCCTTCCAGTCAGAGGAG-3′, R5′-GCATCATAGGGAGGTC.
AGGA-3′, LCP2-F5′-GGGGTTTGTGCAGAGAGAGA-3′, R5′-CTTTGCCCAGACCTACCAAG-3′.