Jmjd3

AR (Millipore Cat# 06-680, RRID:AB_310214), ARv7 (Precision antibody Cat# AG10008, RRID:AB_2631057), Bcl-2 (R&D Systems Cat# AF810, RRID:AB_355621; Cat# MAB8272, RRID:AB_10890789; Dako clone 124 Cat# M0887, RRID:AB_2064429), β4 integrin (Abcam, Cambridge, UK, #ab133682, RRID:AB_2923284, and 450-11 A, RRID:AB_396065, as in [24 (link)], β-actin (Abcam Cat# ab8227, RRID:AB_2305186), E-cadherin (GeneTex Cat# GTX100443, RRID:AB_10729586 and Abcam, #ab231303, RRID:AB_2923285), Cytokeratin (Agilent Cat# GA053, RRID:AB_2892089 and Santa Cruz Biotechnology Cat# sc-81,714, RRID:AB_2191222), Goat-anti-Mouse Alexa Fluor 546 (Thermo Fisher Scientific Cat# A-11,003, RRID:AB_2534071), Goat anti-Mouse IgG HRP (Bio-Rad Cat# 170–6516, RRID:AB_11125547), Goat-anti-Rabbit IgG Alexa Fluor 546 (Thermo Fisher Scientific Cat# A-11,010, RRID:AB_2534077), Goat-anti-Rabbit IgG HRP (SeraCare KPL Cat# 5220 − 0336, RRID:AB_2857917), IgG (Bethyl Cat# P120-101, RRID:AB_479829), JMJD3 (Abcam Cat# ab38113, RRID:AB_943898), H3K27me3 (Active Motif Cat# 39,155, RRID:AB_2561020), HSP90 (Cell Signaling Technology Cat# 4877, RRID:AB_2233307), UTX (Abcam Cat# ab36938, RRID:AB_883400).

786-0 and ACHN cells were washed with PBS buffer twice and then homogenized in 200μl radioimmuno-precipitation assay (RIPA) buffer containing the protease inhibitors cocktail(1 mmol/L) and phenylmethylsulfonyl fluoride (100μg/mL). Homogenates were centrifuged and supernatants were collected. A total of 50 μg of protein separated by 10% sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membrane were saturated with 5% skim milk in TBST (50 mM Tris–HCl, 150 mM NaCl, 0.1% Tween-20) for 2h and then incubated with primary antibodies at 4°C overnight. The primary antibodies used in this study included rabbit polyclonal antibodies to UTX (1:1,000, Abcam, Hong Kong, China), JMJD3 (1:1500, Abcam), EZH2 (1:500, Santa Cruz Biotechnology, Hong Kong, China), H3K27me3 (1:1,500, Epigentek, Brooklyn, USA), H3 (1:2,000, Sigma-Aldrich, St Louis, USA) and actin (1:2,500, Sigma, St Louis, USA). The membranes were incubated with HRP-conjugated goat anti-rabbit antibody (1:5,000, Sigma) for 1 h at room temperature and then exposed to enhanced chemiluminescence substrate (Millipore, Rockford, USA), and detection was performed using a film.

Total cell proteins were extracted using radio-immunoprecipitation assay (RIPA) lysis buffer (Cat# R0020, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Protein concentration was quantified using a bicinchoninic acid (BCA) protein assay kit (Cat# ab102536, Abcam Inc., Cambridge, UK) and adjusted to the same concentration. Proteins were loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After separation, proteins were transferred to a nitrocellulose membrane, followed by blocking with 5% skim milk for 1–2 h. Diluted primary antibodies JMJD3 (Abcam, Cambridge, UK; 1: 1000), H3K27me3 (Abcam, Cambridge, UK; 1: 2000), H3 (Cell Signaling Technologies, Danvers, MA, USA; 1: 1000), and β-actin (Santa Cruz, CA, USA; 1: 2000) were added and incubated with NC membranes overnight at 37 °C. After washed three times with Tris-buffered saline Tween-20 (TBST), the membranes were incubated with corresponding horseradish peroxidase (HRP)-labeled secondary rabbit anti-mouse IgG (West Grove, PA, USA; 1: 10000) for 2 h. The immunocomplexes on the membrane were visualized using enhanced chemiluminescence (ECL) solution (Thermo Fisher Scientific Inc., Waltham, MA, USA), and band intensities were quantified using ImageJ software. The experiment was repeated 3 times independently.

Sample collection and protein extraction were performed in a manner similar to ELISA. Protein concentrations were mixed with a 5 × Laemmli loading buffer and then were heated at 100°C for 5 min. Subsequently, 20 μg of the protein sample was loaded in a prepared sodium dodecyl sulfate-polyacrylamide gel (5% stacking gel and 8 or 10% resolving gel, according to the molecular weight of the protein) and was separated by electrophoresis. The protein was electro-transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, CA, USA). The PVDF membranes were blocked with 5% defatted milk in a Tris buffered saline with 0.1% Tween-20 (TBST) for 90 min at room temperature and were incubated with primary antibodies against iNOS (1:500, Abcam, USA), Jmjd3 (1:800, Abcam, USA), H3K27me3 (1:1000, Biogot Technology, Co., Ltd), and β-actin (1:5000, Biogot Technology, Co., Ltd) at 4°C overnight. The secondary antibodies (1:10000, ZSGB-BIO, China) were incubated for 60 min at room temperature. After washing thrice with TBST, the PVDF membranes were incubated with a prepared enhanced chemiluminescence mixture (Millipore Corp, Billerica, MA, USA) for 1 min and were visualized on film in the dark. The gray value of the bands was quantified with the Image J 14.0 software.

ChIP assays were performed using a ChIP Assay Kit (Millipore, Billerica, MA, USA) according to the manufacturer's instructions. Briefly, CD4⁺ T cells were crosslinked with 1% formaldehyde and sonicated to shear the DNA to 500 ∼ 1,000-bp fragments. Protein/DNA complexes were precipitated with histone-specific antibodies. The H3K27me3 and JMJD3 antibodies were purchased from Abcam (MA, USA). Protein-DNA crosslinks were reversed during incubation at 65°C, and precipitated DNA was then extracted with phenol / chloroform and further purified using ethanol prior to the amplification of the target DNA by real-time quantitative PCR (RT-qPCR). The following primer pairs were used: CD11a, forward 5′-AAATGGAAGGACCCTGATGCTC-3′ and reverse 5′-TGTAGCGGATGATGTCTTTGGC-3′.

The cells were washed twice with PBS buffer and homogenized in 200μl radioimmuno-precipitation assay (RIPA) buffer containing the protease inhibitors cocktail(1 mmol/L) and phenylmethylsulfonyl fluoride (100μg/mL). Homogenates were centrifuged and supernatants were collected. A total of 50 μg of protein separated by 10% sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were saturated with 5% skim milk in TBST (50 mM Tris–HCl, 150 mM NaCl, 0.1% Tween-20) for 2h and then incubated with primary antibodies at 4°C overnight. The primary antibodies used in this study included rabbit polyclonal antibodies to JMJD3 (1:1500, Abcam, Shanghai, China), H3K27me3 (1: 1,500, Cell Signaling Technology, Massachusetts, USA) and H3 (1:2,000, Cell Signaling Technology). The membranes were incubated with HRP-conjugated goat anti-rabbit antibody (1:5,000, Sigma, St Louis, USA) for 1 h at room temperature and exposed to enhanced chemiluminescence substrate (Millipore, Rockford, USA). The detection was performed using a film.

The tissues and cells were added radioimmuno-precipitation assay(RIPA) buffer containing the protease inhibitors cocktail (1 mmol/L) and phenylmethylsulfonyl fluoride (100μg/mL), and sonicated to prepare homogenates. Homogenates were centrifuged and supernatants were collected. 50 μg protein was separated by 10% sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were saturated with 5% skim milk in TBST for 2h and then incubated with primary antibodies at 4°C overnight. The primary antibodies used in this study included rabbit polyclonal antibodies to JMJD3 (1:1500, Abcam, Hong Kong, China), H3K27me3 (1:1,500, Epigentek, Brooklyn, USA) and β-Actin (1:2,500, Sigma, St Louis, USA). The membranes were incubated with HRP-conjugated goat anti-rabbit antibody (1:5,000, Sigma) for 1 h at room temperature and then exposed to enhanced chemiluminescence substrate (Millipore, Rockford, USA), and detection was performed using a film.