Gsk j4 hcl s7070

HeLa (C01-CA) human cervical adenocarcinoma cells and SiHa (C01-FH) human squamous carcinoma cells were obtained from Novobio (Shanghai, China) and cultured in DMEM and RPMI-1640 medium containing 10% fetal bovine serum (FBS, EVERY GREEN, Zhejiang, China), respectively. The cells were then incubated in a humidified atmosphere at 37 °C with 5% CO₂. Selenium dioxide (Aldrich-200107) and GSK-J4 Hcl (S7070, a specific inhibitor of JMJD3/UTX), were purchased from Selleck (Houston, USA).

Antibodies used were anti: JMJD3^{28 (link)} (raised in the laboratory using amino acids 798–1095, dilution 1:200 for immunofluorescence (IF), 1:1000 for western blot (WB); and Abcam, ab38113, dilution 1:250 for IF), DAPI (ThermoFisher, D1306, dilution 1:500 for IF), β-TUBULIN (Millipore, MAB3408, dilution 1:5000 for WB), HA tag (Abcam, ab20084, dilution 1:5000 for WB), H3K9me3 (Abcam, ab8898, dilution 1:250 for IF), H3K27me3 (Millipore, 07449, dilution 1:500) and Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 488 (Invitrogen, A32731, dilution 1:1000 for IF), Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 555 (Invitrogen, A32727, dilution 1:1000 for IF). TGFβ and doxycycline hyclate were acquired from Millipore (GF111 and 324385 respectively). The doxycycline was used at a concentration of 1 ug/ml for 24 h. GSK-J4 inhibitor was acquired from Selleckchem (GSKJ4 HCl S7070) and used at a concentration of 1 uM for 6 h.