H3k27me3

Terminal tumor tissue and whole lungs were stored in 10% neutral buffered formalin for fixation. Formalin-fixed, paraffin-embedded tissues were sectioned and stained with hematoxylin (Vector Laboratories, H-3401) and eosin (Harleco, 200-12) to evaluate tissue morphology. Trichrome staining was performed at the University of Iowa Comparative Pathology Laboratory. For IHC, sections were deparaffinized in xylene and rehydrated through a series of ethanol washes. Antigen retrieval was performed in citrate-based buffer (Vector Laboratories). After washing, samples were stained using VECTASTAIN Elite ABC-HRP kit, per the manufacturer’s instructions (Vector Laboratories). Antibodies included H3K27me3 (Cell Signaling Technology; H3K27me3 clones C36B11, 9733S, used 1:200). Slides were counterstained using IHC-optimized hematoxylin (Vector Laboratories) and mounted with Permount (Fisher Chemical, SP15-500). Lung metastases were quantified from H&E-stained lung sections by evaluating 4 independent sections at 100 μm intervals through the lung. Images were taken using an EVOS light imaging system at 10×, 20×, or 40× original magnification, with figure scale bars at 200 μm.

Total protein was measured using a modified Lowry method. Protein (7 µg for cells and 5 µg for tissues) was run on the automated Western blotting system, WES [78 (link)] (Cat No: 004-600, Protein Simple, San Jose, CA, USA). The primary anti-rabbit antibodies for EZH2 (1:50, Cat No: 5426S), FOXP3 (1:25, Cat No: 12632S), JARID2 (1:50, Cat No: 13594S), and H3K27me3 (1:25, Cat No: 9733S) (Cell Signaling, Danvers, MA, USA), anti-rabbit β-actin (1:100, Cat no: 4970S, Cell Signaling, Danvers, MA, USA), and anti-rabbit H3 (1:100, Cat No: 39451, Active Motif, Carlsbad, CA, USA) were used to measure expression levels within the samples. HRP-conjugated rabbit secondary antibody provided in the WES kit was used. Plates (12-230 kDa and 25 capillary) were run using default settings and results analyzed using the Compass for WES software (Version 5.0.1). Band area given by the software was used and normalized to β-actin or H3. Results were expressed as a ratio in which media alone (for cell treatments) or EuN (for patient tissues) was considered to be 1. It is important to note that proteins examined using the automated Western Blotting system, WES, will have different expected molecular weights compared to traditional Western blotting, due to differences in technology.

Antibodies to H3K27me3, JMJD3, p-Smad3, Smad3, p-AKT, AKT, p-ERK1/2, ERK1/2, Notch1, Jagged-1, UTX and DNMT1 were purchased from Cell Signaling Technology (MA, USA). Antibodies to fibronectin, α-SMA, collagen III, JMJD3, TGF-β1, Notch3 were purchased from Abcam (MA, USA). An antibody to Smad7 was obtained from Santa Cruz Biotechnology (CA, USA). An antibody to β-actin, PTEN and FBXW7 were obtained from proteintech (Wuhan, China). β-tubulin was purchased from Sigma-Aldrich (MO, USA). GSKJ4 was purchased from Selleck (Houston, USA). DMSO and other chemicals were obtained from Beyotime (Shanghai, China). Lipofectamine 3000 was obtained from Invitrogen-Thermo Fisher Scientific (CA, USA).

Cell lysates were collected by boiling pellets in hot 1% SDS lysis buffer (1% SDS, 100 mM NaCl, 10 mM Tris (pH 7.5)) for 5 minutes. Proteins were resolved by SDS-PAGE gels and transferred to PVDF membranes before blocking with 5% milk in TBST for 1 hour. Membranes were incubated with primary antibody overnight at 4°C and appropriate HRP-conjugated secondary antibody after washing with TBST. HRP signal was detected using film. Antibodies were purchased from Cell Signaling Technologies for H3K27me3 (cat# 9733S, RRID AB_2616029), H3K27ac (cat# 4353S, RRID AB_10545273), H3 (cat# 4499S, RRID AB_10544537), GAPDH (cat# 2118S, RRID AB_ 561053), SUZ12 (cat# 3737S, RRID AB_2196850), ATF3 (cat# 18665S, RRID AB_2827506), pAMPKα (cat# 2535S, RRID AB_331250), and AMPKα (cat# 2532S, RRID AB_330331), or from BD BioSciences for EZH2 (#612666, RRID AB_2102429).