Analyses of the protein localization of PML, PR-WT, and PR-B/L-mut using IF staining. (A) HeLa cells were incubated with or without As2O3 (10μM) for 8 hours, and endogenous PML was detected with an anti-PML antibody. PML nuclear bodies were detected in green. The nuclei were stained with 4,6 diamidino-2-phenylindole (DAPI; blue). Note that the microgranular pattern of NBs without As2O3 (i and iii) was altered to become a macrogranular pattern with As2O3 (iv and vi). (B) Flag-tagged PR-WT was used for the same assay as described in panel A. Anti-FLAG and anti-PML antibodies were used to detect PR-WT and endogenous PML, respectively. PML bodies were confirmed in the cytoplasm with a microgranular pattern without As2O3 (i,ii, and iv) and a macrogranular pattern with As2O3 (v,vi, and viii). (C) When using Flag-tagged PR-B/L-mut, localization showed a diffuse pattern mostly in the cytoplasm with and without As2O3. (D) Flag-tagged PR-WT or PR-B/L-mut was overexpressed in U937 cells without As2O3, and the same IF staining was performed. Note that PR-B/L-mut localization was confirmed in the cytoplasm as a diffuse pattern. Magnification is 800×.
