(A) The 786-0 and Caki-1 cells, and (B) the Neo and wt-VHL cells were transfected with the 2.6-kb VEGF promoter-luciferase construct, and cultured for 12 hours. The cells were then treated overnight (12 hours) either with CsA (5.0 μg/ml) or with the vehicle alone. Following CsA treatment, the cells were harvested and fold increase in luciferase activity was calculated as the relative luciferase counts from the CsA-treated group of cells compared with cells treated with the vehicle alone. The data reflect three independent experiments. Columns, average of triplicate readings of two different samples; bars, +/- SD. *, p<0.05 compared with respective vehicle-treated controls, and **, p<0.05 compared with either CsA-treated 786-0 or CsA-treated Neo cells. In the lower panel of (B), the expression of pVHL in wt-VHL cells was confirmed by Western blot analysis. (C) The Neo and wt-VHL cells were treated either with CsA (5.0 μg/ml) or the vehicle alone for 2 hours. The cells were lysed and the extracts were immunoprecipitated with anti-Sp1. Immunoprecipitates (IP) were captured by protein A-sepharose beads, and were separated by SDS-PAGE. Western blot (Blot) was performed with anti-PKCζ or anti-Sp1. The bottom panel represents the total lysate input of PKCζ. Representative of three independent experiments with similar findings.

(A) The 786-0 cells were treated with different concentrations (1.0 and 5.0 μg/ml) of CsA or with the vehicle alone (control) for 24 hours. Whole cell lysates were prepared, and Western blot analysis was performed using anti-VEGF to quantitate VEGF protein expression (upper panel). The expression of β-actin in these cells was analyzed by Western blot analysis using anti-β-actin (lower panel). The bar graph below the Western blot illustrates the relative expression VEGF by densitometry, where the signals were standardized to the expression of the internal control β-actin. Representative of three independent experiments. Columns, average of relative intensity of VEGF expression from three different blots; bars, +/- SD. (B) The 786-0 and TEC cells were transfected with the full-length (2.6-kb) VEGF promoter-luciferase construct (1.0 μg), cultured for 12 hours, and then treated overnight (12 hours) either with different concentrations (1.0 and 5.0 μg/ml) of CsA or with the vehicle alone (control). (C) The 786-0 cells were pretreated with the pharmacological kinase inhibitors PD98059 (10 μM), LY294002 (10 μM), or Calphostin C (250 nM). Control cells were pretreated with the vehicle alone. (D) The 786-0 cells were pretreated either with different concentrations (50-250 nM) of Calphostin C or with the vehicle alone. In (C) and (D), the pretreated cells were transfected with the 2.6-kb VEGF promoter-luciferase construct (1.0 μg), cultured for 12 hours, and then treated overnight (12 hours) either with CsA (5.0 μg/ml) or with the vehicle alone (control), in absence/presence of the kinase inhibitors. In (B), (C), and (D), the cells were harvested after 12 hours of CsA treatment, and fold increase in luciferase activity was calculated as the relative luciferase counts from the CsA-treated or pharmacological inhibitor-treated group of cells compared with that of cells treated with the vehicle alone. The data reflect three independent experiments. Columns, average of triplicate readings of two different samples; bars, +/- SD. In (A-D), *, p<0.05 compared with vehicle-treated control; and in (C) and (D), **, p<0.05 compared with CsA-treated but pharmacological inhibitor-untreated group.

(A) The 786-0 cells were treated either with different concentrations (1.0 and 5.0 μg/ml) of CsA or the vehicle alone for 2 hours. The cells were lysed and the extracts were immunoprecipitated with anti-Sp1. Immunoprecipitates (IP) were captured by protein A-sepharose beads, and were boiled in SDS buffer and separated by SDS-PAGE. Western blot (Blot) was performed with anti-PKCζ, anti-PKCδ or anti-Sp1. The bottom panels represent the total lysate inputs of PKCζ and PKCδ. Representative of three independent experiments with similar findings. (B) The 786-0 cells were treated either with different concentrations (1.0 and 5.0 μg/ml) of CsA or the vehicle alone for 2 hours. Nuclear extracts were prepared from these cells. EMSA was performed with the nuclear extracts of 786-0 cells using a chemiluminescent kit as described in methods. The probe used was a biotin-labeled oligonucleotide duplex containing an Sp1 binding site. The Sp1 antibody (1.0 μg) or the control IgG, or the unlabeled Sp1 consensus oligonucleotide (10-fold molar excess) was added to the binding reaction mixture of the indicated samples to either supershift or compete away the CsA (5.0 μg/ml)-induced specific protein-DNA complex formation (right panel). Representative of three independent experiments with similar findings. (C) The 786-0 cells were transfected with the 2.6-kb, 0.35-kb, or 0.07-kb VEGF promoter-luciferase construct, and cultured for 12 hours. (D) The 786-0 cells were transfected either with HIF-2α siRNA (25 nM) or control siRNA for 48 hours. The cells were then transfected with the 2.6-kb VEGF promoter-luciferase construct, and cultured for 12 hours. In (C) and (D), the cells were then treated overnight (12 hours) either with CsA (5.0 μg/ml) or with the vehicle alone. Following CsA treatment, the cells were harvested and the luciferase activity of each group of cells was calculated as described in methods. The data reflect three independent experiments. Columns, average of triplicate readings of two different samples; bars, +/- SD. *, p<0.01 compared with vehicle-treated controls of the respective promoter constructs. In (D), the knock down of HIF-2α was confirmed by Western blot analysis following 48 hours of siRNA transfection (upper right corner).

(A) The 786-0 cells were serum starved, and then treated with different concentrations (1.0 and 5.0 μg/ml) of CsA or with the vehicle alone (control) for 24 hours. CsA-induced VEGF release in cell culture medium was measured by ELISA as described in methods. The data reflect three independent experiments. Columns, average value of VEGF release from triplicate readings of two different samples; bars, +/- SD. *, p<0.01 compared with vehicle-treated controls. (B) 1.0 × 10⁶ human renal tumor cells (786-0) were injected subcutaneously in nude mice, and they (n=5 in each group) were treated either with CsA (10 mg/kg/day) or with vehicle as a control. Treatment was continued for 25 days, and tumor volume was monitored at regular intervals using a digital caliper. Tumors were harvested 25 days after tumor injection. The data reflect three independent experiments. Points, average value of tumor volume; bars, +/- SD. The CsA-treated group had average tumor volumes that were significantly different than the vehicle-treated group (p<0.05). (C) Total RNA was isolated from harvested tumor tissues (day 25) and reverse transcribed. Fold change in VEGF mRNA expression was measured by real time PCR using gene-specific primers for human VEGF. The data reflect three independent experiments, resulting from duplicate readings of two different samples. Columns, average value of VEGF mRNA expression; bars, +/- SD. *, p<0.05 compared with vehicle-treated controls. (D) Representative photomicrographs showing the immunohistochemical expression of CD31 in harvested tumor tissues (day 25) (magnification: 400X). The right panel represents the mean vessel density calculated by standard grid counting of CD31-positive vessels at 400X magnification. The data reflect three independent experiments, in which three to four non-overlaping fields of each specimen were analyzed in a blinded manner. Columns, average value of vessel counts; bars, +/- SD. *, p<0.05 compared with vehicle-treated controls.

(A) The 786-0 cells were pretreated either with different concentrations (250-1000 nM) of Go6976 or with the vehicle alone. The pretreated cells were transfected with the 2.6-kb VEGF promoter-luciferase construct (1.0 μg), cultured for 12 hours, and then treated overnight (12 hours) either with CsA (5.0 μg/ml) or with the vehicle alone (control), in absence/presence of the kinase inhibitors. Following CsA treatment, the cells were harvested and fold increase in luciferase activity was calculated as the relative luciferase counts from the CsA-treated group of cells compared with that of cells treated with the vehicle alone. The data reflect three independent experiments. Columns, average of triplicate readings of two different samples; bars, +/- SD. *, p<0.05 compared with vehicle-treated control. (B) The 786-0 cells were co-transfected with the 2.6 kb VEGF promoter-luciferase construct, and a dominant-negative (DN) mutant of either PKCζ or PKCδ. Control cells were co-transfected with the VEGF promoter-luciferase construct and the empty expression vector. Following transfection, the cells were cultured for 12 hours, and then treated overnight (12 hours) either with CsA (5.0 μg/ml) or with the vehicle alone (control). Following CsA treatment, the cells were harvested and fold increase in luciferase activity was calculated as the relative luciferase counts from the CsA-treated group of cells compared with that of cells transfected with the empty vector, and treated with the vehicle alone. The data reflect three independent experiments. Columns, average of triplicate readings of two different samples; bars, +/- SD. *, p<0.01 compared with vehicle-treated control, and **, p<0.01 compared with empty vector-transfected and CsA-treated group. (C) and (D) The 786-0 cells were treated either with different concentrations (1.0 and 5.0 μg/ml) of CsA or with the vehicle alone for 2 hours. In (C), the cells were lysed, and Western blot analysis using anti-phospho-PKCζ or anti-phospho-PKCδ was performed to quantitate the expression of phospho-PKCζ and phospho-PKCδ respectively (first and third panels). The expression of total cellular PKCζ or PKCδ was analyzed by Western blot analysis using anti-PKCζ or anti-PKCδ (second and fourth panels). Representative of three independent experiments with similar findings. In Supplementary Figure-3, a bar graph illustrates the relative expression phospho-PKCζ and phospho-PKCδ by densitometry, where the signals were standardized to the expression of total PKCζ and total PKCδ (from three different experiments). Although not shown, a similar level of phosphorylation of both PKCζ and PKCδ was observed even at later time points of CsA treatment (up to 12 hours). In (D), the cells were lysed, and PKCζ was immunoprecipitated from cell lysate using anti-PKCζ. Using immunoprecipitated PKCζ, a kinase assay was performed as described in the methods. The data for the PKCζ kinase activity are expressed as fold induction with respect to the activity in vehicle-treated control cells. The data reflect three independent experiments, resulting from duplicate readings of two different samples. Columns, average value of PKCζ kinase activity; bars, +/- SD. *, p<0.05 compared with vehicle-treated control.

(A) 0.5 × 10⁶ syngenic tumor cells (CT26) were injected subcutaneously in BALB/c mice, and fully MHC mismatched cardiac transplants were performed in these animals 7 days later. Following cardiac transplantation, the mice (n=5 in each group) were treated either with CsA (10 mg/kg/day) or with vehicle as a control. Treatment was continued for 14 days (i.e. up to 22 days following tumor injection), and tumor volume was monitored using digital caliper on alternate days. Tumors were harvested after 22 days of tumor injection. The data reflect three independent experiments. Columns, average value of tumor volume; bars, +/- SD. *, p<0.01 compared with vehicle-treated control. (B) Total RNA was isolated from harvested tumor tissues (day 22), and reverse transcribed. Fold change in VEGF mRNA expression was measured by real time PCR using gene-specific primers for murine VEGF. The data reflect three independent experiments, resulting from duplicate readings of two different samples. Columns, average value of VEGF mRNA expression; bars, +/- SD. *, p<0.01 compared with vehicle-treated control. (C) Representative photomicrographs showing the immunohistochemical expression of CD31 in harvested tumor tissues (day 22) (magnification: 400X). The right panel represents the mean vessel density calculated by standard grid counting of CD31-positive vessels at 400X magnification. The data reflect three independent experiments, in which three to four non-overlaping fields of each specimen were analyzed in a blinded manner. Columns, average value of vessel counts; bars, +/- SD. *, p<0.05 compared with vehicle-treated control. (D) 0.5 × 10⁶ syngenic tumor cells (CT26) were injected subcutaneously in BALB/c mice, and fully MHC mismatched cardiac transplants were performed in these animals 7 days later. Following cardiac transplantation, the mice (n=5 in each group) were treated either with i) CsA (10 mg/kg/day) + control IgG, ii) CsA (10 mg/kg/day) + anti-VEGF and iii) vehicle. Treatments were continued until the mice were sacrificed (up to 30 days following tumor injection). Tumor volume was monitored on alternate days. Points, average value of tumor volume; bars, +/- SD. On days 22-30, the CsA + control IgG-treated group had average tumor volumes that were significantly higher than the vehicle-treated group (p<0.05), and the CsA + anti-VEGF-treated group had average tumor volumes that were significantly lower than the CsA + control IgG-treated group (p<0.05).