Scratch wounds of equal size were introduced into the mono layer by a sterile pipette tip and cell debris generated from the scratch was washed away with 1x phosphate buffered saline. Cells were treated with vehicle or IC20 ACA in serum free medium for 24 h and microscopic images describing speed of wound closure was documented at various time intervals using an truly inverted fluorescence microscope, Nikon Eclipse TS 100 and analyzed using TScratch software, Version 1. 0. PARP cleavage assay The occurrence of apoptosis was assessed based on the proteolytic cleavage of PARP by caspase 3. Briefly, cells were treated with ACA and total proteins were extracted using the NE PERW nuclear and cytoplasmic extraction kit according to manufacturers protocol. Fractionation was done using SDS PAGE and electro transferred onto nitrocellulose membranes.
Total proteins were incubated with rabbit anti PARP antibodies and detected using an enhanced chemiluminescence reagent using x ray films. Apoptosis was represented by cleavage of 116 kDa PARP into an 85 kDa product. DNA fragmentation assay Cells were treated with ACA for 12 h and 24 h before harvesting, and total DNA was extracted from both untreated and treated cells using the Suicide TrackTM DNA Ladder isolation kit according to the manufacturers protocol. Extracted DNA was analysed on a 1. 0% agarose gel electrophoresis and stained with ethidium bromide. Fragmentation of DNA was observed under UV illumination and visualized using a gel documentation system.
Microarray global gene expression analysis To investigate changes brought upon by ACA in global gene expression, the Affymetrix GeneChipW Human Gene 1. 0 Sense Target Array was used according to manufacturers protocol. Briefly, total RNA from HSC 4 cells treated with ACA for 60 min and 120 min were extracted using the RNeasyW Plus Mini Kit according to manufacturers protocol and analyzed under the Agilent 2100 Bioanaly zer. RNA samples were then reverse transcribed, labelled and hybridized onto Affymetrix chips containing 764,885 probes repre senting and spanning across 28,869 human genes. Scan ning of all arrays was done using the Affymetrix GeneChipW Scanner. Statistical and gene expression analysis of triplicate arrays were done using the GeneSpringW GX version 10. 0 software employing principle component analysis plots, p value and fold change thresholds.
Western blot analysis To determine levels of protein expression, cytoplasmic and nuclear extracts from HSC 4 cells treated with ACA at IC50 concentrations for 2 h and 4 h were prepared using the NE PERW nuclear and cytoplasmic extraction kit according to manufacturers protocol. Protein concen tration was quantified and normalized Entinostat using the Quick Start Bradford protein assay kit 2 accord ing to manufacturers protocol. Fractionation of proteins were done using a 12.