Fig 7.

Arkadia is involved in arsenic-induced degradation of sumoylated PML. (A) Depletion of Arkadia inhibits arsenic-induced degradation of sumoylated PML. HT1080-GFP-PML cells were transfected with a nontargeting siRNA (siNT) and siRNA SMARTpool targeting Arkadia (siArk) or RNF4 (siRNF4). Cells were exposed to arsenic at the indicated times. Whole-cell lysates were analyzed by Western blotting with anti-Ark, anti-RNF4, anti-GFP, and antiactin antibodies. A high exposure (exp.) and a low exposure of the same anti-GFP Western blot are shown. (B) Depletion of Arkadia inhibits arsenic-induced degradation of PML nuclear bodies. HT1080-GFP-PML cells were transfected with siNT, siArk, or siRNF4. Cells were exposed to arsenic at the indicated times before fixation, permeabilization, and DAPI staining. GFP-PML (green) expression and cellular localization were then analyzed by GFP-based fluorescence with the same laser intensity for each condition and are represented alone or merged with DAPI (blue). Bars, 20 μm. (C) Quantification of the number of PML nuclear bodies visualized with anti-PML staining in 30 cells under the indicated conditions, as described for panel B. (D and E) The same experiments described for panels A and B, respectively, performed with two individual siRNAs targeting Arkadia, siArk(1), and siArk(2). Numbers to the left of the gels are molecular masses (in kDa).