ATO-induced SUMOylation is counteracted by cell division. (A) IF showing HaCaT cells treated with the indicated combinations of 1.0μM ATO and 1.0mM TMD. Cells were pretreated for 12 hours with TMD before administration of ATO. Incubation was then continued with both drugs present for an additional 24 hours. DAPI (blue) and PML (red) is shown. Scale bar, 20 μm. (B-D) Quantification of CyPNs in HaCaT (B), NB4 (C), and T2 (D) cells after treatment with 1.0μM ATO and/or 1.0mM TMD as aforementioned in panel A. More than 200 cells were analyzed per sample. Data represent average of 2 independent experiments ± SD (***P < .001). (E-G) Western blot showing PML expression in HaCaT (E), NB4 (F), and T2 (G) cells treated with 1.0μM ATO and/or 1.0mM TMD as aforementioned in panel A. β-actin was used as loading control. (H) HaCaT cells stably expressing FLAG-PML1 were treated with 1.0μM ATO and harvested for analysis by Western blotting at indicated time points using urea or Triton X-100–based extraction buffers. β-actin was used as loading control. (I) HaCaT cells expressing FLAG-PML1 were treated with 1.0μM ATO and/or 1.0mM TMD as in panel A, and subsequently harvested for analysis by Western blotting at the indicated time points. β-actin was used as loading control. (J) IF analysis of HaCaT cells treated with 1.0μM ATO as indicated using antibodies against PML (red) and SUMO1 (green). Yellow rectangles highlight nuclear PML bodies (PML/SUMO1 colocalization), whereas green rectangles highlight CyPNs (absence of PML/SUMO1 colocalization). DAPI staining is shown in blue. Scale bar, 10 μm.