Id1 regulation of TSP1 in endothelial cells. (A) Relative expression of Id1 mRNA. HMVECs were treated with VEGF (100 pg/mL), VEGF, and 8CPT (100μM), or left untreated (control). Id1 mRNA expression was assessed by quantitative real-time PCR (qPCR). GADPH served as an internal control for each assay (data not shown). Relative expression was normalized to the untreated control and plotted as fold change. Bars represent mean ± SD (n = 3). (B) Down-regulation of TSP1 protein expression by Id1. HMVECs were transfected with vector or Id1 expression constructs and assayed 6 hours posttransfection. After Western blot analysis (left), quantitation of protein expression of TSP1 and Id1 relative to α-tubulin was performed using LI-COR Odyssey software (right). Bars represent means ± SD (n = 3). (C) mRNA expression of TSP1 in HMVECs after Id1 transfection. Samples were transfected and analyzed by qPCR, as in (B). Bars represent means ± SD (n = 3). (D) Up-regulation of TSP1 protein after depletion of Id1. HMVECs were transduced with lentivirus expressing Id1 shRNA or control lentivirus. After Western blot analysis (left), quantitation of protein expression of TSP1 and Id1 relative to α-tubulin was performed using LI-COR Odyssey software (right). Data shown are plotted as means ± SE (n = 3). An independent Id1 shRNA sequence gave similar results (data not shown).