Qcmtm 24 well fluorimetric cell invasion assay kit

Cell invasion experiments were performed using the QCMTM 24-well Fluorimetric Cell Invasion Assay kit (Chemicon, International, MI, USA) according to the manufacturer′s instructions. The kit uses an insert polycarbonate membrane with an 8-μm pore size. The insert was coated with a thin layer of EC Matrix that occluded the membrane pores and blocked migration of non-invasive cells. Culture medium (500 μl) supplemented with 10% FBS was used as chemoattractant. Cells that migrated and invaded the underside of the membrane were xed in 4% paraformaldehyde. The invading cells were stained by Calcein-AM, and the number was then determined by uorescence and reported as relative uorescence units (RFUs).
Effect of CR594175 silencing on the protein levels of CTNNB1, E-cadherin, C-myc, CyclinD1 and MMP-9 HepG2 cells were divided into three groups: a control group, Lv-NC group and Lv-shRNA-CR594175. Cells in logarithmic phase were seeded to 6-well plates at 5x10 5 cells/well. One day later, viral solution was added and the infection e ciency was evaluated by observing and analyzing the uorescent mark 72 hours after infection. Protein were isolated and subjected to western blotting for CTNNB1, E-cadherin, Cmyc, CyclinD1 and MMP-9 protein, respectively.

HepG2 cells infected with recombinant lentiviruses 72 hours, trypsinized and used for invasion assay. Cell invasion experiments were performed using the QCMTM 24-well Fluorimetric Cell Invasion Assay kit (ECM554, Chemicon International, USA) according to the manufacturer′s instructions. The kit uses an insert polycarbonate membrane with an 8-μm pore size. The insert was coated with a thin layer of EC Matrix TM that occluded the membrane pores and blocked migration of non-invasive cells. Culture medium (500 μl) supplemented with 10% FBS was used as chemoattractant. Cells that migrated and invaded the underside of the membrane were xed in 4% paraformaldehyde. The invading cells were stained by Calcein-AM, and the number was then determined by uorescence and reported as relative uorescence units (RFUs). The grouping and cell treatment were the same as those for cell viability assay. Seventy-two hours after lentiviral infection, cells were trypsinized and the viable cells were counted by using trypan blue staining, and seeded into the upper chamber of transwell at a density of 5 × 10 5 cells/well, and incubated under normal conditions for 48 hours, and then stained and counted. The grouping was the same as in the proliferation assay.

Cell invasion experiments were performed using the QCMTM 24-well Fluorimetric Cell Invasion Assay kit (Chemicon, International, MI, USA) according to the manufacturer′s instructions. The kit uses an insert polycarbonate membrane with an 8-μm pore size. The insert was coated with a thin layer of EC Matrix that occluded the membrane pores and blocked migration of non-invasive cells. Culture medium (500 μl) supplemented with 10% FBS was used as chemoattractant. Cells that migrated and invaded the underside of the membrane were xed in 4% paraformaldehyde. The invading cells were stained by Calcein-AM, and the number was then determined by uorescence and reported as relative uorescence units (RFUs).
Effect of lncRNA-CR594175 silencing on the protein levels of CTNNB1, E-cadherin, C-myc, CyclinD1 and MMP-9 HepG2 cells were divided into three groups: a control group, Lv-NC group and Lv-shRNA-CR594175. Cells in logarithmic phase were seeded to 6-well plates at 5x10 5 cells/well. One day later, viral solution was added and the infection e ciency was evaluated by observing and analyzing the uorescent mark 72 hours after infection. Proteins were isolated and subjected to western blotting for CTNNB1, E-cadherin, Cmyc, CyclinD1 and MMP-9 protein, respectively.

LNCaP and PC3 cells were used for cell viability assay by using a cell counting kit-8 assay kit (CCK-8, Dojindo, Japan). LNCaP cells were used for invasion assay by using the QCMTM 24-well Fluorimetric Cell Invasion Assay kit (Millipore) according to the manufacturer's instructions. Migration ability of PC3 cells was assessed using wound healing assay. The cells used in the experiments were infected with the lentiviruses (Lv-NC or Lv-miR-137 or Lv-shRNA-PBC11 or Lv-miR-137+ Lv-shRNA-PBC11) for 72huors,then EMT model was established by adding TGF-β1(5ng/ml) for 48 hours.