Muc1 c

Human tissue of patient with NSCLCwere collected from 2014 to 2017 in Taihe Hospital. Immunohistochemistry analyses were performed using an DAKO EnVision system as described previously. The following antibodies were used: MUC1-C from Cell Signaling (catalog no. #16564). This study about patients with NSCLC was approved by the ethics committee of Hubei Taihe Hospital.

Chromatin immunoprecipitation (ChIP) was performed on cells crosslinked with 1% formaldehyde for 5 minutes at 37°C, quenched with 2 mol/L glycine and washed with PBS, and then sonicated in a Covaris E220 sonicator to generate 300–600 bp DNA fragments. Immunoprecipitation was performed using a control IgG (Santa Cruz Biotechnology) and antibodies against MUC1-C (Cell Signaling Technology, catalog no. 16564, RRID:AB_2798765), JUN (Abcam, catalog no. ab32137, RRID:AB_731608), ARID1A (Cell Signaling Technology, catalog no. 12354, RRID: AB_263710), PBRM1 (Cell Signaling Technology, catalog no. E6N2K), EP300 (Cell Signaling Technology, catalog no. D2X6N), H3K27ac (Abcam, catalog no. ab4729, RRID:AB_2118291), H3K4me1 (Abcam, catalog no. ab8895, RRID:AB_306847), and H3K4me3 (Abcam, catalog no. ab8580; RRID:AB_306649). Precipitated DNAs were detected by PCR using primers listed in Supplementary Table S3. Quantitation was performed on immunoprecipitated DNA using SYBR-green and the CFX384 Real-Time PCR Machine (Bio-Rad). Data are reported as fold enrichment relative to IgG (8 (link)).

Protein extraction, two-dimensional polyacrylamide gel electrophoresis, and transfer to nitrocellulose membrane were carried out as previously described [11 (link),12 (link),15 (link)]. The membrane was incubated with the following antibodies: cleaved CASP3, cleaved PARP, PARP, p-EGFR, EGFR, p-AKT, AKT, p-ERK, ERK, p-STAT3, STAT3, eIF4A3, p-4E-BP1, mTOR, p-P70-S6, MUC1, and MUC1-C, purchased from Cell Signaling Technology (Danvers, MA, USA). The antibody to glyceraldehyde-3-phosphate dehydrogenase was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).