GSM9363255: DU145, GC7 3; Homo sapiens; RNA-Seq
DU145 Cells were seeded at 106 cells/10cm dish, treated with 40µM CG7 48h later (D0) during 24h. At the end of this treatment, cells were treated 10min with emetin (Sigma-Aldrich) 25µg/ml, washed twice with PBS, and harvested by trypsination. Cytoplasmic lysates were prepared by incubation of cells 10min in hypotonic buffer (10mM Tris-HCl pH 7.4, 0.5mM MgCl2, 10mM KCl, 1X CompleteTM EDTA free protease inhibitor (Roche) and 10U/mL RNAseOutTM (Invitrogen). Cells are disrupted by shaking in presence of ceramic beads (10sec at 4500rpm in a Precellys Evolution homogenizer). Nuclei were pelleted by centrifugation at 750 g for 5min at 4°C, and mitochondria were pelleted by centrifugation at 12,000 g for 10min at 4°C. The protein content of the cytosolic extract was measured by Bradford assay and samples containing 2mg protein were loaded on top of 15–50% sucrose gradients poured using the Gradient Master (Serlabo Technologies). After ultracentrifugation at 250 000g during 105min at 4°C on a SW41 Beckman rotor, fractions of 700µl each were collected from each gradient while 254nm absorbance profiles were generated using an ISCO UA-6 detector. the human genome (GRCh38) using STAR (v2.7.8a)