Figure 7: PIB5PA is suppressed by HDAC2 and HDAC3 in melanoma cells.

(a) Total RNA from ME1007 and Mel-FH cells treated with 5-aza (5-aza-2′-deoxycytidine; 10 μM) for 96 h were subjected to quantitative PCR (qPCR) analysis (n=3, mean±s.e.m.). (b) Whole-cell lysates from HEMa-LP melanocytes, and ME1007 and Mel-FH melanoma cells treated with 5-aza (10 μM) for 96 h were subjected to western blot analysis. (c) Whole-cell lysates from ME1007 and Mel-FH cells treated with SAHA (4 μM) for indicated periods were subjected to western blot analysis. (d) Total RNA from ME1007 and Mel-FH cells treated with SAHA (4 μM) for indicated periods were subjected to qPCR analysis (n=3, mean±s.e.m.). (e) ME1007 and Mel-FH cells were transiently transfected with the pGL3-basic-based reporter constructs, pGL3-vector and pGL3-INPP5J core promoter (pGL3-INPP5J−2,016/+114), respectively. Twenty-four hours later, cells were treated with SAHA (4 μM) for a further 24 h, followed by measurement of the luciferase activity (n=3, mean±s.e.m.). (f) ME1007 and Mel-FH cells were transfected with the control or PIB5PA siRNA. Twenty-four hours later, total RNA was subjected qPCR analysis of PIB5PA mRNA expression (n=3, mean±s.e.m.). (g) ME1007 and Mel-FH cells were transfected with the control or PIB5PA siRNA. Twenty-four hours later, cells were treated with SAHA (4 μM) for a further 24 h. Whole-cell lysates were subjected to western blot analysis. (h) Quantification of pSer473-Akt normalized to total Akt levels as shown in g (n=3, mean±s.e.m.). (i) Total RNA from ME1007 and Mel-FH cells treated with MC1568 (5 μM), MS275 (5 μM) and SAHA (4 μM), respectively, for 24 h was subjected to qPCR analysis of PIB5PA mRNA. The relative abundance of PIB5PA mRNA in cells treated with MC1568, MS275 or SAHA was normalized to that in cells without treatment (n=3, mean±s.e.m.). (j) Whole-cell lysates from ME1007 and Mel-FH cells treated with MC1568 (5 μM), MS275 (5 μM) and SAHA (4 μM), respectively, for 24 h were subjected to western blot analysis. (k) Whole-cell lysates from ME1007 and Mel-FH cells transfected with the control, HDAC1, HDAC2 or HDAC3 siRNA were subjected to western blot analysis. (l) ME1007 cells were cotransfected with the control, HDAC2 or HDAC3 siRNA, and pGL3-INPP5J-(−2,016/+114). Luciferase activity was measured 24 h after transfection (n=3, mean±s.e.m.).