Table 1.
List of novel and known mediators of thrombin–induced endothelial response identified in the functional siRNA screen.
Fig 1.
RhoGTPases and thrombin stimulation.
A—Quantification of the area under the response curve (AUC) of HUVECs transfected with 22 different RhoGTPase-targeting siRNAs and stimulated with 1U/ml of thrombin for 3h at 72h post-transfection (n = 3±SD). In blue, left, NT control siRNA (n = 18, in triplicate), in red, siRhoD significantly different AUC compared to NT control siRNA and in green, two potentially relevant genes: siRhoT2 and siRND3. (Color code refers to the change in AUC, see S1A–S1C Fig) ***P<0.001 in two-tailed Student’s T-test and FDR<0.05; B-D—Normalized endothelial resistance of HUVECs transfected with indicated siRNAs and stimulated with 1U/ml of thrombin for 3h at 72h post-transfection (n = 3±SD). Arrow indicates the time point at which thrombin was added.
Fig 2.
Contribution of RhoGEFs and associated proteins to the response to thrombin.
A—Quantification of the area under the response curve (AUC) of HUVECs transfected with 99 different siRNAs targeting RhoGEFs (and associated genes) and stimulated with 1U/ml of thrombin for 3h at 72h post-transfection (n = 3±SD). In blue, on the left, NT control siRNA (n = 18, in triplicate), in red, RhoGEFs significantly different AUC compared to NT control siRNA and in green: siLARG, siGEF-H1, siGNA12 and siPLXNB3. (Color code refers to the change in AUC, see S1A–S1C Fig) ****P<0.0001 in two-tailed Student’s T-test and FDR<0.05; ***P<0.001 in two-tailed Student’s T-test and FDR<0.05; **P<0.01 in two-tailed Student’s T-test and FDR<0.05; *P<0.05 in two-tailed Student’s T-test and FDR<0.05; B-K, Normalized endothelial resistance of HUVECs transfected with the indicated siRNAs and stimulated with 1U/ml of thrombin for 3h at 72h post-transfection (n = 3±SD); Arrow indicates the time point at which thrombin was added.
Fig 3.
Contribution of RhoGAPs and associated proteins to the response to thrombin.
A—Quantification of the area under the response curve (AUC) of HUVECs transfected with 71 different siRNAs targeting RhoGAPs (and associated genes) and stimulated with 1U/ml of thrombin for 3h at 72h post-transfection (n = 3±SD). In blue, on the left, NT control siRNA (n = 18, in triplicate), in red, RhoGAPs significantly different AUC compared to NT control. (Color code refers to the change in AUC, see S1A–S1C Fig) ****P<0.0001 in two-tailed Student’s T-test and FDR<0.05; ***P<0.001 in two-tailed Student’s T-test and FDR<0.05; B-D—Normalized endothelial resistance of HUVECs transfected with the indicated siRNAs and stimulated with 1U/ml of thrombin for 3h at 72h post-transfection (n = 3±SD); Arrow indicates the time point at which thrombin was added.
Fig 4.
Contribution of Rho effector proteins and associated proteins to the response to thrombin.
A—Quantification of the area under the response curve (AUC) of HUVECs transfected with 80 different siRNAs targeting Rho-effector proteins (and associated genes) stimulated with 1U/ml of thrombin for 3h at 72h post-transfection (n = 3±SD). In blue, on the left, NT control siRNA (n = 18, in triplicate), in red Rho-effector proteins with a significantly different AUC compared to NT control siRNA and in green, siMLC1. (Color code refers to the change in AUC, see S1A–S1C Fig) ****P<0.0001 in two-tailed Student’s T-test and FDR<0.05; B-G—Normalized endothelial resistance of HUVECs transfected with the indicated siRNAs and stimulated with 1U/ml of thrombin for 3h at 72h post-transfection (n = 3±SD); Arrow indicates the moment at which thrombin was added.