Immunofluorescence analysis of KDM4 proteins

1x105 HCC38 cells were grown on coverslips and treated with siRNAs as described previously up to 70%-80% confluence. Forty-eight hours post-treatment, the cells were pre-extracted with 0.2% PBS/Tween 100 and fixed in 3.7% formaldehyde for 15 min at room temperature. The fixed cells were permeabilized with 0.5% Triton X-100 in PBS and blocked with 3% bovine serum albumin in PBS for 1 hour. Next, the cells were incubated overnight at 4°C with anti-KDM4C (Abcam ab85454), anti-KDM4A (Abcam 24545) or anti-trimethyl H3K9 (Abcam ab10812) antibodies at 1:250 dilution followed by three washes with PBS containing 0.01% Triton X-100 and then incubation with Alexa Fluor 488 (1:500 dilution, Abcam 150077). The cells were washed twice and mounted in ProLong Diamond Antifade Mountant with DAPI (Life Technologies, ThermoFischer Scientific, Waltham, USA). Images were acquired on an Eclipse Ni-E microscope (Nikon, Melville, USA) and analyzed with ImageJ software (NIH) at 40X-100X by triplicate in three independent samples.^{22 (link)}