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. Author manuscript; available in PMC: 2020 Sep 1.
Published in final edited form as: Cell Signal. 2019 May 1;61:120–129. doi: 10.1016/j.cellsig.2019.04.013

Figure 2. Silencing of Beclin1 attenuates thrombin-induced NF-κB activity and inflammatory gene expression in EC.

Figure 2.

(A) HPAEC were transfected with si-Con or si-Beclin1 using DharmaFect1. After 24 h, cells were again transfected with NF-κBLUC and Renilla-LUC constructs using DEAE-dextran as described in Materials and Methods. Cells were treated with thrombin for 6 h and the cell extracts were assayed for Firefly and Renilla luciferase activities. Renilla luciferase was used as an internal control for transfection efficiency. Bars indicate mean ± SEM (n = 4-6) for each condition, and were analyzed by ANOVA. (B) HPAEC were transfected with si-Con or si-Beclin1 for 48 h prior to treatment with thrombin for 6 h. Cells were lysed and Western blot analysis was performed to determine VCAM-1 levels. The levels of Actin were used to monitor protein loading. Bars indicate mean ± SEM (n = 4) for each condition, and were analyzed by ANOVA. (C) Cell supernatants were collected after 48 h transfection and 6 h thrombin treatment, and ELISA was performed to determine MCP-1 release. Bars indicate mean ± SEM (n = 4–6) for each condition, and data was analyzed by ANOVA.