br Matrigel transmembrane invasion assay br Cell
2.10. Matrigel transmembrane invasion assay
Cell migration across the matrigel barrier was determined by BD BioCoat Matrigel invasion chambers (BD Bioscience, Bedford, MA). The matrigel coated inserts were re-hydrated with serum free DMEM for 30 min at 37 °C in 5% CO2 followed by the washing of inserts with serum free media. MCF-7 and MDA MB-231 were seeded at the seeding density of 1 × 104 cells/well with or without treatment of HC9 extract into the invasion chambers and 10% DMEM medium was added as a chemo attractant to the lower chambers. The chambers were incubated at 37 °C in 5% CO2 for 24 h. The non-invading cells in the upper chambers were removed by gently wiping with the wet cotton swabs. Invading cells that were transversed to the matrigel and attached to the bottom of the membrane were fixed with 10% formalin and stained with 0.5% crystal violet. The membranes were removed from the inserts and examined under microscope and the numbers of invading cells were counted. Journal of Ethnopharmacology 242 (2019) 112022
2.11. Reverse transcription PCR analysis
The total cellular RNA was extracted from control as well as cells treated with diﬀerent concentrations of HC9 (10–80 μg/ml) by a one-step Tirapazamine guanidine isothiocyanate-phenol method using TRI reagent (Invitrogen) from both the cell lines. RNA was precipitated with iso-propanol and the concentration was quantified by using Nanodrop in-strument (Eppendorﬀ BioPhotometer plus). Total RNA was extracted and reverse transcribed. The expression of HIF-1α was examined by amplifying cDNA template by using gene-specific primers by PCR method. The primers used were β-actin-F: 5′-taccactggcatcgtgatg-gact-3'; β-actin-R: 5′-tttctgcatcctgtcggaaat-3'; Hif-1α (F): 5′-gtcgga-cagcctcaccaaacagagc-3'; Hif-1α (R): 3′-gttaacttgatccaaagctctgag-5'. PCR was run as follows: 25–30 cycles at 95 °C for 5 min, 95 °C for 1 min, annealing at 42 °C for 1 min and extension at 72 °C for 1 min and a final extension. RT-PCR products were then separated on a 1.2% agarose gel and visualized by staining with ethidium bromide. The intensities of the bands corresponding to the RT-PCR products were quantified by using phosphor imager (Alpha Imager, Alpha Innotech) and normalized with respect to β-actin product.
2.12. Gelatin zymography
The serum free conditioned medium collected from control as well as HC9 treated MCF-7 (10–40 μg/ml) and MDA MB-231 (10–80 μg/ml) cells was analyzed for pro-matrix metalloproteinase-9 (pro-MMP-9) and pro-matrix metalloproteinase-2 (pro-MMP-2) activity by gelatin zymo-graphy as described previously. Briefly, MCF-7 and MDA MB-231 were seeded at 5 × 105/ml seeding density in 6 well plates. The cells were treated with HC9 (0–80 μg/ml) in serum free media for 24 h. The conditioned media from control as well as treated cells were collected and concentrated in Centricon YM-30 tubes (Millipore, MA). The sam-ples were subjected to 7.5% SDS-polyacrylamide gel containing gelatin (0.5 mg/ml) under non-reducing conditions. The gel was washed with 0.25% Triton X-100 followed by overnight incubation at 37 °C in in-cubation buﬀer containing 150 mM NaCl, 100 mM CaCl2, 50 mM Tris-HCl pH 7.5, 1% Triton X-100 and 0.02% NaN3. The gel was stained with 0.1% Coomassie Brilliant blue R-250 in 40% isopropanol and destained in 7% acetic acid, examined and photographed. Gelatinase activation was visualized as unstained clear bands against the blue-stained gelatin background. The quantification of bands in control and treated samples was performed by densitometric analysis on Alpha Imager using Alpha Ease FC software, Alpha Innotech.
For western blotting, the control and HC9 treated MCF-7 (10–40 μg/ ml) and MDA MB-231 (10–80 μg/ml) cells were harvested, washed with 1X PBS and lysed by using lysis buﬀer as previously described (18).The protein was estimated using Bradford reagent (Biorad Laboratories Inc, CA, USA). Equal amount of protein was loaded on to 10% SDS-poly-acrylamide gel and transferred electrophoretically to Amersham Hybond-P PVDF membrane (GE Healthcare, UK) in sodium phosphate buﬀer (pH 6.8). The membrane was blocked in 5% BSA in TBST and incubated at room temperature for 3 h or overnight at 4 °C with mouse monoclonal antibodies against p53, SMAR1, p16, MMP-2 and tubulin (Santacruz, CA, USA) as well as with rabbit polyclonal antibodies against CDP/Cux, p21, Rb, phospo-Rb, VEGF, NFқB and goat polyclonal antibody against COX-2 (Santacruz, CA, USA) at 1:2000 dilution re-spectively. The membrane was washed in TBST and incubated with secondary IgG HRP conjugate at 1:5000 dilution. Proteins were visua-lized by using a chemiluminescence kit (Amersham ECL Advance western blotting detection kit, GE Healthcare, UK) and densitometric analysis of X-ray films was performed by Image J gel analysis software.