A total of 400?mL PBS and 1?L PI (50?g/mL) were added to each sample before measurements. carcinoma cells were decided using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, 3??103 cells were plated in 96-well plates NUN82647 and allowed to attach overnight. Five impartial wells were repeated for each experiment. A total of 20?mL MTT (Sigma) solution (5?mg/mL in PBS) was added to each well and the plates were incubated for additional 4?h at 37; 150?l dimethyl sulfoxide was added to each well before measurement at absorbance of 490?nm. Transwell migration assay Transwell migration assay was NUN82647 performed to test the migratory ability of RCC cells following Gal-3 knockdown. The same numbers of cells in serum-free Dulbecco’s altered eagle medium in the upper chamber of 12-well plates were allowed to migrate for 8?h toward Dulbecco’s modified eagle medium containing 10% FBS in the lower chamber. After migration, cells in the lower surface of the membrane were fixed and stained with Giemsa. Images were acquired with a bright field microscope at 20??magnification. To quantify migratory cells, four impartial fields were analyzed by using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The percentage of cells that migrated was normalized to control group. Annexin V-Fluorescein isothiocyanate/propidium iodide Apoptosis was decided using Annexin V-Fluorescein isothiocyanate/propidium iodide (PI) double staining. Briefly, 200?mL cells (1??106 cells per mL) were centrifuged at 1000?rpm for 5?min (4). Cells were washed with pre-cooled PBS and resuspended in 100?L binding buffer with 2?L Annexin V-Fluorescein isothiocyanate (20?g/mL). The mixed samples were kept in dark for 15?min. A total of NUN82647 400?mL PBS and 1?L PI (50?g/mL) were added to each sample before measurements. The value was read using flow cytometer (Beckman Coulter EPICS XL). Cells without staining were used as unfavorable control. PI staining Twenty-four hours after shRNA transfection, cells were NUN82647 harvested and fixed in pre-colded ethanol NUN82647 overnight. PI (50?g/mL) was added to each sample before measurements. Cell cycle distribution was analyzed using flow cytometer (Beckman Coulter EPICS XL). Western blotting Cells were produced to subconfluency on 6-well plates and exposed to Gal-3 knockdown. Twenty-four hours later, cells were collected, washed with PBS, and resuspended in hypotonic buffer (20?mM Mouse monoclonal to ERBB3 HEPES, 10?mM KCl, 1?mM MgCl2, 0.5?mM DTT, 0.1% Triton X-100, 20% glycerol, 5?g leupeptin per mL, 10?g aprotinin per mL, and 500?M phenyl-methylsulfonyl fluoride). The cells were centrifuged at 850?for 5?min. The supernatant was used as the cytoplasmic fraction. Protein concentrations were measured using BCA Protein Assay Kit (Beyotime Biotechnology, China). Total proteins were separated by SDS-PAGE (12.5% polyacrylamide) by the method of Laemmli and transferred to a PVDF membrane. The presence of proteins was detected using human anti-Gal-3 antibody H-160 (1:500) (Santa Cruz, USA), anti-Actin antibody (1:2000) (Sigma, USA), anti-p27 antibody (1:1000) (Santa Cruz, USA), anti-p21 (1:1000) (Santa Cruz, USA), and anti-Cyclin D1 antibody (1:1000) (Santa Cruz, USA). Expression levels of proteins were detected using the enhanced chemiluminescence system. For densitometric analysis of western blots, Image J software was used. Quantification of Caspase-3 activity Caspase-3 activity was decided using Caspase-3 assay kit (Beyotime Institute of Biotechnology, China) by detecting the color reporter molecule, p-nitroanilide (pNA), after cleavage from the substrate of Ac-DEVD-pNA.15 Cells were seeded at 4??105 cells per well in a 6-well plate and incubated overnight. Twenty-four hours after shRNA Gal-3 inhibition, cell lysates were assayed for Caspase-3 activity. Ten microliters of cell lysate was mixed with pre-cooled substrate answer Ac-DEVD-pNA (0.2?mM) and incubated at 37 for 60C120?min. The mixture without cell lysate was used as blank control. Relative fluorescent value was acquired at 405?nm wavelength. The activity of.