Barx1

Tissue and cell lysates were extracted using RIPA lysis buffer with the protease inhibitor phenylmethane sulfonyl fluoride (Merck Millipore, USA). Protein concentration was measured using the BCA protein assay kit (Beyotime Biotechnology) according to the manufacturer's instructions. Equivalent amounts of protein (30μg) were separated on 10% SDS PAGE gel and then transferred onto 0.45μm PVDF membranes (Millipore, Billeria, MA) according to the standard protocols. Membranes were blocked in 5% milk in TBST buffer for 1 hr at room temperature, followed by incubation with primary antibodies at 4°C overnight. After incubation with a secondary antibody for 1 hr at room temperature, proteins were detected using ECL regent (Millipore, Billeria, MA). Primary antibodies were as follows: Barx1 (Santa Cruz, sc-81956), β-actin (sc-47778), MGAT5 (Abcam, ab87977), and MMP9 (Cell Signaling, #13667).

A549 cells were cross-linked with 1% formaldehyde at RT for 10 min. The cross-linked chromatin was sonicated to generate DNA fragments averaging 100–200 bp in length by Bioruptor plus. Chromatin fragments were immunoprecipitated with antibodies against mouse normal IgG (4 μg, Santa Cruz), BARX1 (4 μg, Santa Cruz) and protein A-Sepharose beads. After washing and reversing the cross-links, the enriched DNA was purified and then examined by qRT-PCR. The primer sequences are listed in Supplementary Table 2.