Role of VCAM-1/α4β1 binding in monocyte adhesion to ccRCC. (A) Analysis of VCAM-1 protein levels in 786-O-pRv or 786-O-VHL cells untreated or transfected with a scrambled siRNA (Scr) or siRNAs specific for VCAM-1. α-Tubulin was used as a loading control. A Western blot representative of three experiments is shown. (B) U937–calcein-AM–labeled cell adhesion on 786-O-pRv or 786-O-VHL cells untreated or transfected with a scrambled siRNA or siRNAs specific for VCAM-1. Cell adhesion was performed for 20 min at 37°C, and afterward, fluorescent cells were counted under the microscope. n = 5. (C) U937–calcein-AM–labeled adhesion experiment on 786-O-pRv or 786-O-VHL untreated (control [Ctr]) or treated with 10 µg/ml of blocking antibodies against β1, α4, or αL integrin subunits. Cell adhesion was performed for 20 min at 37°C, and afterward, fluorescent cells were counted under the microscope. n = 4. (B and C) Data are represented as number of cells ± SEM. 10 random fields were analyzed per condition. Statistical analysis was done using two-way ANOVA followed by Bonferroni’s posthoc test. (D) Effects of VHL loss in monocytic cell-mediated cytotoxicity against ccRCC cells. 786-O-pRv or 786-O-VHL target cells were seeded into 16-well sensor plates, and activated human monocytes treated with IFN-γ and LPS were directly added into wells at a 60:1 ratio, monocytes (Mono)/ccRCCs. Cell survival measurements were automatically collected every 5 min by the real-time cell electronic-sensing analyzer system (xCELLigence System) for up to 96 h. Cellular index results were expressed as cell survival percentage mean ± SEM at 96 h. Data are representative of three different experiments. Statistical analysis between different conditions was done using two-way ANOVA followed by Bonferroni’s posthoc test. **, P < 0.01 and ***, P < 0.001 were considered significant.