SUMOylation is dispensable whereas the BB2 motif and amino acids mutated in ATO-resistant APL patients are required for effective CyPN accumulation. (A) Schematic overview of PML structural organization and mutations used in this study. Mutations identified in PML/RARA transcripts isolated from ATO-resistant APL patients are shown in red. (B) IF showing nucleoporins (red) together with WT or mutated EYFP-PML1 (green) stably expressed in HaCaT/shPML cells. Representative images of untreated cells and cells treated with 0.5μM ATO are shown. Images are projections of 5 to 6 confocal z-scans. Scale bar, 10 μm. (C) Quantification of CyPNs regenerated by WT or mutated EYFP-PML1 in HaCaT/shPML cells incubated in the presence or absence of 0.5μM ATO for 24 hours. For each sample more than 80 cells were analyzed. Data represent average of 3 independent experiments ± SD (*P < .05, ***P < .001). (D) IF showing nucleoporins (red) together with WT EYFP-PML1 or EYFP-PML1 containing the patient derived mutations A216V and L218P (green) stably expressed in HaCaT/shPML cells. Representative images showing untreated cells or cells treated with 0.5μM ATO are shown. Images display projections of 5 to 6 confocal z-scans. Scale bar, 20 μm. (E) Quantification of CyPNs regenerated by WT or mutated EYFP-PML1 in HaCaT/shPML cells incubated in the presence or absence of 0.5μM ATO for 24 hours. For each sample more than 100 cells were analyzed. Data represent average of 3 independent experiments ± SD (***P < .001).