ASHD alters the expression of apoptotic proteins in Reh and K562 cells.

<p><b>A–C</b>. Cell lysates were prepared from Reh cells after incubating with ASHD (100 µM) for 24, 48 and 72 h. DMSO treated cell lysate was used as control. Approximately, 40 µg of protein per sample was resolved on SDS-PAGE and transferred to a PVDF membrane. The membrane was probed for the expression of BAD, BCL2 (<b>A</b>), PARP (<b>B</b>) and caspase 3, caspase 9 and caspase 8 (<b>C</b>) with specific primary antibodies and appropriate secondary antibodies. <b>D–E</b>. Cell lysates were prepared from K562 cells after incubating with ASHD (30 µM) for 48 h and used for western blotting. The proteins studied are BAD, BAX, PARP (<b>D</b>), and caspase 8, caspase 9 and cytochrome C (<b>E</b>). The α-tubulin was used as an internal loading control in all the panels. <b>F–G</b>. Effect of pancaspase inhibitor (z-VAD-FMK) on Reh cells treated with ASHD. Approximately 0.75×10⁵ cells/ml were cultured and incubated with 30 µM ASHD, with or without 50 µM z-VAD-FMK. DMSO treated cells were used as vehicle control. <b>F</b>. MTT assay showing effect of ASHD on cell proliferation following treatment with z-VAD-FMK at 24 and 48 h. <b>G</b>. Trypan blue assay showing cell viability. For other details refer Fig. 1 legend. Error bars in panels, <b>F</b> and <b>G</b> are based on three independent batches of experiments.