Fig 2: Decreased MARCKS expression inhibits intimal hyperplasia formation in vivo. A, MARCKS protein is up-regulated during the formation of intimal hyperplasia in the murine carotid ligation model. Common carotid arteries in WT mice were ligated at the carotid bifurcation and harvested at days 0 (untreated), 7, 14, and 28. Total lysates from 3 arteries (6 µg of total protein) were used to assess phospho-MARCKS (Pi-MARCKS), MARCKS, a-SMA, and ß-actin expression at each time point with Western blot analysis. B, Protein expression was normalized to GAPDH. MARCKS expression increases early in the formation of intimal hyperplasia. Maximal MARCKS expression occurs at 14 days. C, The caroitd artery was examined with immunostaining of frozen sections at an early phase of intimal hyperplasia formation (day 7 postligation). Confocal microscopy was used to identify MARCKS (red), a-SMA (green), and the murine endothelial cell marker, CD31 (white). At day 7, the injured carotid arteries in WT mice had developed a significant hyperplastic lesion. The merged confocal images demonstrate that MARCKS was highly expressed in the injured arteries. MARCKS is localized in the polarized leading edge (white arrows) of invading VSMCs. Scale bars=20 µm. D, MARCKS expression increased in WT mice as a result of carotid ligation (day 14). MARCKS expression increased only slightly after carotid ligation in MARCKS+/- mice (M+/-). Rac1 and PAK1 activation were lower in the ligated carotid artery (day 14) of MARCKS+/- mice (M+/-) as compared to WT mice. E, Representative VVG-stained sections of ligated carotid artery are shown at day 14 in WT (left) and MARCKS +/- (right) mice. There was a significantly greater hyperplastic response observed in WT mice compared to MARCKS+/- mice (M +/-). Scale bars=200 µm. F, Intimal hyperplasia was quantified with intima area/media area ratio, n=3. CD indicates cluster of differentiation; MARCKS, myristoylated alanine-rich protein kinase substrate; PAK, p21-activated kinase; SM, smoothelin; SMA, smooth muscle actin; VSMC, vascular smooth muscle cell; VVG, Verhoeff-Van Gieson; WT, wild type. *P<0.0001.

Fig 3: PAP and its interaction with Pak1. A: Pak1 is divided into N-terminal and C-terminal halves. The N-terminal half contains p21 binding domain (PBD) followed by a kinase inhibitory domain, which overlap with each other. The proline rich motifs interact with different cellular proteins including Nck, Grb2 and Pix, etc. PAP is derived from the autoinhibitory domain linked to a TAT sequence. PAP binds to Pak1 and may activate Pak1 through attenuation of Pak1 autoinhibition in a similar way as Cdc42 and Rac1 do. B: We examined the effects of the PAP on Pak1 phosphorylation in cultured neonatal rat ventricular myocytes (NRVMs) that were treated with PAP (20 µg/ml) for 2 hours. Immunoblotting analyses of Pak1 phosphorylation indicate that Pak1 activation was induced by angiotensin II (Ang II), in NRVMs (n = 3 independent experiments).

Fig 4: Chemotactic extravasation and metastatic colony formation are regulated by PAK1. a Scratch wound migration assay. Cells were plated and grown to confluency. A wound was manually made and cell motility was monitored every 4 h. b Chemotactic cell migration. Cells were serum starved and subjected to transwell migration towards 25 ng/ml EGF or 50 µM GABA for 6 h. Cells that migrated to the underside of the membrane were counted and are represented as a percent of uninfected MDA-MB-231BR cell migration. c Cells were plated in a 96 well plate and proliferation was measured using a MTT proliferation assay for 3 consecutive days. d, e 50 ng/ml EGF or 100 µM GABA and control (water) in Matrigel were added on top of the CAM. MDA-MB-231 and MDA-MB-231BR control or PAK1 shRNA cells were i.v injected into the CAM vasculature and d counted at the location under the chemoattractant, or control Matrigel, re-counted 24 h later and is represented as a percentage of total cells at time zero. e Quantification of metastatic colonies 5 days post-injection. Metastatic colonies were visualized and counted in the CAM via fluorescent microscopy (f)

Fig 5: Insulin signaling following overnight fatty acid treatment in L6 myotubes. Insulin signaling in L6 myotubes following overnight treatment in no fatty acid control (CON), palmitate (PALM, 500 µmol/L) or oleate (OLEA, 500 µmol/L), under basal serum-free or 100 nmol/L insulin for 2, 5 or 10 min. (A) Rac1-GTP binding was assessed via immunosorbent assay. Western blotting was used to assess (B) phosphorylation of PAK 1 and PAK2 (pPAK1/2 Thr423/Thr402), and (C–D) phosphorylation of Akt at Thr308 (pAkt Thr308) and Ser473 (pAkt Ser473). (E) Representative western blot images. Insulin signaling outcomes were analyzed by two-way time × treatment ANOVA. A main effect of insulin was evaluated by comparing 2, 5, and 10 min of insulin stimulation with basal using Dunnett's analysis. Main effects of fatty acid treatments were evaluated by comparing palmitate and oleate with the no fatty acid control condition using Dunnett's analysis. Total protein abundance of insulin signaling proteins was not affected by insulin or fatty acid treatments. Main effect P-values for time and treatment, respectively, are provided for total Rac1 (P = 0.33, P = 0.31), total Akt, (P = 0.76, P = 0.17) and total PAK1, (P = 0.39, P = 0.95). Insulin signaling analysis reflects three independent experiments each consisting of n = 3 for a total sample size of n = 9 for all conditions. Data are presented as mean + SD.