Fig. 1.

HR23B in CTCL cells. (A) Immunoblot showing protein levels of FLAG-HR23B in uninduced (−) and induced (+) U2OS-TET cells (#1 and #2) with actin as a loading control. Dox, doxycycline. (B) U2OS-TET cells expressing ectopic FLAG-HR23B were treated with doxycycline for 16 h before an additional 48-h treatment with SAHA (2, 5, 10, and 20 μM) or DMSO control (0). (i) Immunoblot showing protein levels of FLAG-HR23B in uninduced (−) and induced (+) U2OS-TET cells (#1), HR23B, and PARP cleavage. Actin was used as a loading control. (ii) Graph showing the percentage sub-G1 cells in induced (black bar) cell lines treated with SAHA compared with DMSO control (clear bar). (C) The levels of sub-G1 apoptotic cells determined by FACS analysis after treating the U2OS cell line (#1) expressing ectopic FLAG-HR23B. Cells were treated with doxycycline for 16 h before an additional 48-h treatment with SAHA (10 μM), PXD101 (4 μM), apicidin (4 μM), or DMSO control. The graph shows the percentage of sub-G1 cells in induced (black bar) cell lines treated with HDAC inhibitors compared with DMSO control (clear bar). (D) Immunoblot showing protein levels of HR23B in the CTCL cell lines MYLA, SeAx, and HUT78 treated with the indicated dose of SAHA (μM). Acetylated H3 and actin (loading control) are shown for comparison. (E) Level of sub-G1 apoptotic cells determined by FACS after treating MYLA, SeAx, or HUT78 with the HDAC inhibitors SAHA (S), TSA (T), PXD101 (P), and apicidin (A), all at 5 μM, and valproic acid (V) at 5 mM. (F) MYLA cells were treated with HR23B or nontargeting control (NT) siRNA for 48 h, followed by immunoblotting. Actin served as the loading control. (G) The levels of sub-G1 MYLA cells determined by FACS after treatment with HR23B siRNA or nontargeting (NT) control siRNA for 48 h followed by treatment with DMSO control (0) or increasing concentrations of SAHA, 3, 10, or 30 (μM), as indicated for 72 h (n = 4; error bars, SEM).