FIG. 2.

Silencing of TRPC1 inhibits EPC proliferation and migration. (A) TRPC1 mRNA expression levels were determined using real-time RT-PCR. Parallel amplification of the rat housekeeping β-actin gene was used as an internal control. Transduction of EPCs with siRNA targeting TRPC1 (siRNA-TRPC1) or shRNA targeting TRPC1 (shRNA-TRPC1) greatly decreased TRPC1 mRNA expression at 48 h post-transduction. The results are expressed as the mean±SEM of 3 experiments. n=3, ^#P<0.05. (B) Top: TRPC1 protein levels were examined by western blot analysis. Equal loading was confirmed by staining for β-actin. Transduction of EPCs with siRNA-TRPC1 or shRNA-TRPC1 clearly decreased TRPC1 protein levels at 48 h post-transduction. The data shown are representative of 3 different experiments. Bottom: Densitometric analysis of TRPC1 protein expression levels, normalized to expression levels of the housekeeping β-actin gene, was determined using the Quantity One program. The results are expressed as the mean±SEM (n=3), ^#P<0.05. (C) Effects of TRPC1 knockdown on EPC proliferation. A [³H]-thymidine incorporation assay was used to examine EPC proliferation. Transfection of EPCs with siRNA-TRPC1 or shRNA-TRPC1 significantly decreased the uptake of [³H]-thymidine by EPCs at 48 h after infection. The data are presented as the mean±SD (n=9), ^#P<0.05. (D) Transfection of EPCs with siRNA-TRPC1 or shRNA-TRPC1 clearly inhibited the proliferation of EPCs at 48 h after infection. The data are presented as the mean±SEM (n=3), ^#P<0.05 versus siRNA-control, *P<0.05 versus shRNA-control. (E) Silencing of TRPC1 inhibited EPC migration, as analyzed by a modified Boyden-chamber assay. Transfection of EPCs with siRNA-TRPC1 or shRNA-TRPC1 notably decreased the number of migrating EPCs. The data are presented as the mean±SEM (n=5), ^#P<0.05. (F) The SOC-mediated influx of Ca²⁺ was examined under stimulation with 1 μM thapsigargin during the change from Ca²⁺-free conditions to 2 mM Ca²⁺. Left: The maximum amplitude of [Ca²⁺]i induced by SOCE significantly decreased after transfection with siRNA-TRPC1 or shRNA-TRPC1 for 48 h. Right: Statistical analysis of SOCE among different groups. The data are presented as the mean±SEM of 3 different experiments, n=6, ^#P<0.05 versus shRNA-control, *P<0.05 versus siRNA-control. RT-PCT, reverse transcription–polymerase chain reaction; SEM, standard error mean; SD, standard deviation; SOCs, store-operated calcium channels; SOCE, store-operated Ca²⁺ entry.