Effects of HDAC inhibitor trichostatin A (TSA) and DNMT inhibitor 5-Aza-2′-deoxycytidine (5-Aza-dC) on RGS10 transcript expression and cell viabilities.

<p>Total RNA was isolated from untreated control cells and TSA or 5-Aza-dC treated cells. The relative expression of RGS10 mRNA was quantified by qRT-PCR and normalized to GAPDH transcript expression * p<0.05; ** p<0.005. <b>A</b>) HDAC inhibition increases RGS10 expression in chemoresistant A2780-AD cells. The cells were plated in a 10 cm² plate and incubated for 24 hours. The following day, cells were treated with 500 nM TSA and were incubated for an additional 48 hours. <b>B</b>) DNMT inhibitor 5-Aza-dC enhances RGS10 expression in chemoresistant cells. Two million A2780-AD cells were seeded in 10 cm² plates and were incubated for 24 hours. The following day, cells were treated with 20 µM 5-Aza-dC. Media and drug were refreshed every 24 hours. After indicated incubation time (3, 5, and 7 days), cells were harvested, mRNA was isolated, and cDNA was generated and quantified using qRT-PCR with specific primers and probe. <b>C</b>) A2780-AD cells were plated in 96-well plates and treated with 5 µM 5-Aza-dC for 5 days, 500 nM TSA for 36 hours, a combination of 5 µM 5-Aza-dC for 5 days and 500 nM TSA for the final 36 hours or DMSO. Gene expression was assessed using qRT-PCR as described, and normalized to RPL13A gene expression. The arrow indicates the expression level predicted by an additive effect of TSA and 5-Aza-dC. <b>D</b>) In a parallel experiment, A2780-AD cells were treated under the same conditions as 4C with or without 30 µM cisplatin for the final 12 hours. Cell survival was assessed using CellTiter-Blue fluorimetric viability assays. ***: p<0.001 comparing epigenetic drug to DMSO control in the absence of cisplatin. ###: p<0.001 comparing vehicle versus cisplatin treatment within epigenetic drug treatment groups. The dotted box indicates the cell viability predicted by an additive effect of TSA and 5-Aza-dC. <b>E</b>) A2780/AD cells (5000 cells/well) were plated in 96-well plate and transfected with negative control or RGS10 siRNA duplexes (Ambion Grand Island, NY) as per the manufacturer's protocol using Dharmafect1 transfection reagent (Dharmacon). Cells were dosed with a combination of 5 µM 5-Aza-dC for 3 days and 500 nM TSA for the last 36 h or DMSO. 30 µM cisplatin or vehicle was added for the last 12 h. Cell survival was assessed using CellTiter-Blue fluorimetric viability assays.