Figure 8

hTid-1 knockdown inhibits Met activation and HGF-mediated migration. (a) Left panel, 786-0 RCC cells that had been stably infected with either a control lentivirus or a si-hTid-1 expressing lentivirus were serum-starved overnight and treated with HGF for the indicated time points. Whole-cell lysates were collected and resolved by SDS–PAGE. Western blotting was used to analyze phospho-Met (Tyr1234/Tyr1235, pMet), total Met and hTid-1 levels. Right panel, densitometric analysis was used to determine the degree of Met phosphorylation relative to total Met levels and is shown as the relative pixel density of pMet:Met compared with untreated cells. (b) Left panel, 786-0 cells that have been stably infected with either a control lentivirus or a si-hTid-1 expressing lentivirus were serum-starved overnight and used in a transwell migration assay. A total of 7.5 × 10⁵ cells were plated in the upper chamber with HGF in the lower chamber. Cells were allowed to migrate for 24 h, and representative pictures for each filter is shown. Right panel, The number of cells that migrated was counted in five individual fields/filter and the average number of cells/field was calculated, and is shown as a ratio to the number of cells that migrated in the control condition. The data shown represent pooled data from three independent experiments. An unpaired t-test was used to determine significance. *, a P-value of <0.05 was considered significant. (c) hTid-1 knockdown inhibits signaling through Erk1/2 and STAT3. 786-0 RCC cells that had been stably infected with either a control lentivirus or a si-hTid-1 expressing lentivirus were serum-starved overnight and treated with 10 ng/ml HGF for the indicated time points. Whole-cell lysates were prepared and subjected to immunoblot analysis using antibodies specific for phospho-Met (Tyr1234/1235, pMet), total Met, pSTAT3, total STAT3, phospho-Erk1/2 (pErk1/2), hTid-1 and β-actin.