Figure 1.
Figure 1. Effect of VHL loss and hypoxia on VCAM-1 levels in ccRCC cell lines. (A) VCAM-1 mRNA levels were determined by quantitative RT-PCR in 786-O and RCC4 transfected with empty vector pRv (786-O-pRv and RCC4-pRv) or with pRv-VHL (786-O-VHL and RCC4-VHL), Caki-1, and Caki-2 and exposed to normoxia (Nx) or hypoxia 0.5% O2 (Hp) for 24 h. mRNA levels are expressed as fold-change and normalized over the levels of VHL-positive cells in normoxic conditions and controlled with β-Actin as the housekeeping gene. Statistical analysis was done using two-way ANOVA followed by Bonferroni’s posthoc test. n = 6 experiments. (B) VCAM-1 and VHL protein levels were analyzed in total cell lysates from 786-O-pRv, 786-O-VHL, RCC4-pRv, RCC4-VHL, Caki-1, and Caki-2 cell lines exposed to normoxia or hypoxia 0.5% O2 for 24 h. A representative Western blot (n = 6) is shown. As a loading control, α-tubulin was used. (C) 786-O-VHL cells were treated with 2 µg/ml actinomycin D and then cultured under normoxia or hypoxia for the indicated times. Gene expression is represented as fold-change and normalized over the levels of VHL-positive cells in normoxic conditions (considered 100%) and controlled with β-Actin gene expression levels. n = 4 experiments. (D) VCAM-1 mRNA levels were determined by quantitative RT-PCR before splicing (measuring nuclear heterogeneous RNA) in 786-O-pRv, 786-O-VHL, RCC4-pRv, and RCC4-VHL exposed to normoxia or hypoxia 0.5% O2 for 24 h. Gene expression is represented as fold-change over the levels of VHL-positive cells in normoxic conditions and controlled with β-Actin as the housekeeping gene. Statistical analysis between different conditions was done using two-way ANOVA followed by Bonferroni’s posthoc test. n = 4 experiments. (E) VCAM-1 levels at the cell membrane were determined by flow cytometry in 786-O-VHL and 786-O-pRv cells exposed to normoxia or hypoxia 0.5% O2 for 24 h. Quantification of the fluorescence mean in each condition is shown. Statistical analysis was done using two-way ANOVA followed by Bonferroni’s posthoc test. n = 4 experiments. The values represent the mean ± SEM. ***, P < 0.001 and ****, P < 0.0001 were considered significant.

Effect of VHL loss and hypoxia on VCAM-1 levels in ccRCC cell lines. (A) VCAM-1 mRNA levels were determined by quantitative RT-PCR in 786-O and RCC4 transfected with empty vector pRv (786-O-pRv and RCC4-pRv) or with pRv-VHL (786-O-VHL and RCC4-VHL), Caki-1, and Caki-2 and exposed to normoxia (Nx) or hypoxia 0.5% O2 (Hp) for 24 h. mRNA levels are expressed as fold-change and normalized over the levels of VHL-positive cells in normoxic conditions and controlled with β-Actin as the housekeeping gene. Statistical analysis was done using two-way ANOVA followed by Bonferroni’s posthoc test. n = 6 experiments. (B) VCAM-1 and VHL protein levels were analyzed in total cell lysates from 786-O-pRv, 786-O-VHL, RCC4-pRv, RCC4-VHL, Caki-1, and Caki-2 cell lines exposed to normoxia or hypoxia 0.5% O2 for 24 h. A representative Western blot (n = 6) is shown. As a loading control, α-tubulin was used. (C) 786-O-VHL cells were treated with 2 µg/ml actinomycin D and then cultured under normoxia or hypoxia for the indicated times. Gene expression is represented as fold-change and normalized over the levels of VHL-positive cells in normoxic conditions (considered 100%) and controlled with β-Actin gene expression levels. n = 4 experiments. (D) VCAM-1 mRNA levels were determined by quantitative RT-PCR before splicing (measuring nuclear heterogeneous RNA) in 786-O-pRv, 786-O-VHL, RCC4-pRv, and RCC4-VHL exposed to normoxia or hypoxia 0.5% O2 for 24 h. Gene expression is represented as fold-change over the levels of VHL-positive cells in normoxic conditions and controlled with β-Actin as the housekeeping gene. Statistical analysis between different conditions was done using two-way ANOVA followed by Bonferroni’s posthoc test. n = 4 experiments. (E) VCAM-1 levels at the cell membrane were determined by flow cytometry in 786-O-VHL and 786-O-pRv cells exposed to normoxia or hypoxia 0.5% O2 for 24 h. Quantification of the fluorescence mean in each condition is shown. Statistical analysis was done using two-way ANOVA followed by Bonferroni’s posthoc test. n = 4 experiments. The values represent the mean ± SEM. ***, P < 0.001 and ****, P < 0.0001 were considered significant.

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