Fig 2: Effect of simvastatin on the mevalonate pathway and the downstream targets. a Illustration of how simvastatin acts on the mevalonate pathway. b MOLM-13 and MV4-11 were exposed to 40 μM simvastatin for 48 h, and Western blotting analysis was then performed to determine the protein expression level of HMGCR, RAS, Rap1 and RhoB. c MOLM-13 and d MV4-11 cells were exposed to the designated concentrations of simvastatin for 48 h, and ERK, MEK and p38 and their phosphorylation levels were detected by Western blotting. HMGCR 3-hydroxy-3-methylglutaryl-CoA reductase, FPP farnesyl pyrophosphate, GGPP geranylgeranyl pyrophosphate, SIM simvastatin, GAPDH glyceraldehyde-3-phosphate dehydrogenase

Fig 3: Depletion of RhoB partially rescues the MLN7243-induced loss of endothelial integrity. A–E, HUVECs were transfected with nontargeting control siRNA (N.T.), siRhoA, and/or siRhoB and cultured for 72 h. A and B, normalized resistance of HUVECs with single knockdowns treated with 500 nM MLN7243. B, bar graphs represent mean + SD of normalized endothelial resistance after 5 h of four individual experiments relative to DMSO. C, Western blot analysis of RhoB and RhoA expression. Bar graphs show quantified RhoB and RhoA expression normalized to GAPDH and N.T. or N.T./N.T., respectively. Data are presented as mean + SD, n = 3. D and E, normalized endothelial resistance of HUVECs with double knockdowns of RhoA/B treated with 500 nM MLN7243. E, bar graphs represent mean + SD of normalized endothelial resistance after 5 h, relative to the DMSO control, of four individual experiments. ns, nonsignificant, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. HUVEC, human umbilical vein endothelial cell.

Fig 4: Model of differential regulation of endothelial integrity by RhoB and RhoA. A, GDI, GEF, and GAP proteins are primarily responsible to control RhoA activity conveying rapid responses to, mostly GPCR-binding, agonists. B, in contrast, the activity of RhoB is mainly regulated at the levels of its mRNA expression, protein synthesis, and ubiquitination-mediated degradation, which allows for a relatively stable level of constitutive signaling by RhoB. C, despite being differentially regulated, active RhoB and RhoA both signal toward their common effectors, including ROCK, driving the phosphorylation of MLC, which results in actomyosin contraction and an increase in endothelial permeability. GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; MLC, myosin light chain; ROCK, Rho-associated kinase.

Fig 5: Inhibition of ubiquitination by MLN7243 leads to increased RhoB expression and activity.A, Western blot analysis for pMLC of HUVECs treated with 500 nM MLN7243 for the indicated times. GAPDH was used as loading control. Bar graph shows quantification of pMLC expression normalized to GAPDH. Data are presented as mean + SD, n = 3. B and C, normalized resistance of HUVECs treated with 500 nM MLN7243, 10 μM Y27632, or both, and (C) relative drop in resistance after 4 h. Data are presented as mean + SD, n = 3. D, Western blot analysis and quantification of RhoA, RhoB, and Rac1 protein expression in HUVECs treated with 500 nM MLN7243. Bar graph shows quantification of RhoA/B, Rac1 expression, relative to GAPDH and DMSO. Data are presented as mean + SD, n = 3 to 4. E, immunofluorescent staining for RhoB (red) and counterstained with DAPI (blue) of HUVECs after treatment with 500 nM MLN7243. Scale bars represent 50 μm in overview images and 15 μm in zoomed images. Bar graph shows fluorescent intensity of RhoB staining. Data are represented as mean + SD of 10 regions of interest normalized to DMSO. F, Rhotekin pulldown of HUVECs treated with 500 nM MLN7243 followed by Western blot analysis for the GTP-bound, active forms of RhoA and RhoB. GAPDH was used as loading control. Bar graphs show quantification of RhoA/B.GTP normalized to GAPDH. Data are presented as mean + SD, n = 3. G, endogenous RhoB was immunoprecipitated from HUVECs treated with 500 nM MLN7243 followed by Western blot analysis of RhoB and ubiquitinated proteins (FK2). Input equals 8%. H, Western blot analysis of RhoB expression after treatment with 500 nM MLN7243 for the indicated times and with a medium refresh without MLN7243 after t = 7 h (right panel only). ns, nonsignificant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. HUVEC, human umbilical vein endothelial cell; MLC, myosin light chain.