Confocal microscope

HCT116 p53^+/+ and HCT116 p53^−/− cells were stably transfected with KDEL-DsRed2 (Clontech: pDsRed-ER vector), and treated cells were fixed in 4% paraformaldehyde for 15 min at room temperature. Cells were then extensively washed to remove any debris. All images were acquired using an Olympus confocal microscope and processed using Adobe Photoshop software.

Cells for immunofluorescence were fixed in ice-cold methanol for 15 min at -20 °C and further treated with 0.1% Triton X-100 for 30 min. Cells were then blocked by 5% BSA for 30 min at room temperature. Primary antibodies were diluted in universal antibody diluent (NCM Biotech) and incubated with prostate cells overnight at 4 °C. The cells were then washed three times with 0.02% TBST, and were incubated with secondary antibodies for 50 min at room temperature followed by wash with 0.02% TBST twice. DAPI was incubated with cells at room temperature for 5 min. Images were acquired with a STEDYCON confocal microscope and processed using Image J and Adobe Photoshop. Quantification of the m⁶A, RBM15, IGF2BP1/2/3 and S9.6 signal intensities was performed according to the previously described method [7 (link)].