After 30 min incubation at room temperature, 5 μl of propidium iodide was added in each well (1 μg/ml). Cellular DNA content was assessed by capillary cytometry (Guava EasyCyte 96 Plus). Data were analyzed on the Guava CytoSoft™ Express Pro software (Merck/Milli pore/Guava Tech). CytoSoft Express Pro was used to identify the three cell cycle phases and calculate relevant statistics, including population percentages (subG1, G0/G1, S and G2/M phases). Quantification of DNA methylation HeLa cells were treated with G extract (200 μg/ml) or luteolin (25 μM) for 48 hours. DNA was purified using QIAamp® DNA Kit. The content of methylated
DNA was determined Selleckchem AZD0530 using 200 ng of DNA from untreated cells, treated cells with G extract or luteolin, as described by the manufacturer; Sigma’s Imprint® Methylated DNA Quantification Kit. Western blot analysis HeLa cells (6 × 105) were seeded into 6-well cell culture plates and grown for 24 hours. Cells were treated with different
selleck chemical concentrations of G extract or luteolin for 24 and 48 hours. The cells were then harvested, centrifuged to discard the DMEM medium, washed with cold PBS (phosphate buffered saline), resuspended in RIPA buffer (25 mM Tris, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% SDS; Sigma–Aldrich, USA) containing protease inhibitors. Equal amounts of total protein were separated on 10–12% polyacrylamide gel and electrophoretically transferred to a nitrocellulose membrane. After blocking with 5% non-fat dry milk or 3% BSA (Bovine Serum Albumin) and tween 20 in GSK1120212 supplier selleck products PBS, the nitrocellulose membranes were incubated with either a mouse monoclonal anti-UHRF1 antibody (Proteogenix, Oberhausbergen, France), a mouse monoclonal anti-DNMT1 (clone 60B1220.1,
Proteogenix), and a rabbit polyclonal anti-p16INK4A antibody (DeltaBiolabs, Gilroy, CA) according to the manufacturer’s instructions (4°C, overnight). Membranes were thereafter incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (diluted to 1:10,000 for anti-mouse antibodies and 2: 10,000 for anti-rabbit antibody) at room temperature for 45 minutes. The membranes were then washed with TPBS five times. Signals were detected by chemiluminescence using the ECL Plus detection system (Amersham, GE Healthcare UK Limited). Statistical analysis Data were analyzed with student’s t-test and presented as mean value ± S.E.M of three independent measurements in separate experiments. Results Aqueous gall extract content Aqueous gall extract from L. guyonianum was the subject of a chemical study with the aim of having a global idea in their composition. The metabolites contents of the tested extract are presented in Table 1. Quantitative phytochemical analysis showed that the extract contained an important quantity of flavonoids, polyphenols, and tannins. In fact, 1 mg of G extract was equivalent to 85 μg of gallic acid and 460 μg of quercetin.