ATO stimulates increased accumulation of PML/RARA within CyPNs. (A) Western blot showing total RARA and PML/RARA expression in NB4 and T2 cells treated with or without 0.5μM ATO for 24 hours. Blots were probed using an anti-RARA antibody. β-actin was used as loading control. (B) Quantification of RARA-positive CyPNs in NB4 and T2 cells treated with or without 0.5μM ATO for 24 hours. For each sample more than 500 cells were analyzed. Data represent average of 3 independent experiments ± SD (***P < .001). (C) IF showing the localization of RARA (red) and nucleoporins (Nups; green) in NB4 and T2 cells treated with or without 0.5μM ATO for 24 hours. RARA-positive CyPNs are detected in NB4 cells, but not in T2 cells. Images represent projections of 5 to 6 confocal z-scans. Scale bar, 10 μm. (D) Western blot showing stable expression of WT and mutated (A216V and L218P) PML/RARA in HaCaT cells. Cells were treated with or without 0.5μM ATO for 24 hours and subsequently lysed in a urea-based extraction buffer. Blots were probed using an anti-RARA antibody. β-actin was used as loading control. (E) Quantitation of CyPNs in HaCaT cells stably expressing WT or mutated PML/RARA and treated with or without 0.5μM ATO for 24 hours. For each sample more than 100 cells were analyzed. Data represent average of 3 independent experiments ± SD (*P < .05, ***P < .001). (F) IF showing RARA (red) and nucleoporins (Nups; green) in HaCaT and HaCaT cells expressing WT or mutated PML/RARA (P/R). Selected foci containing Nups and RARA are highlighted. Scale bar, 20 μm.