Fig 1: nCGA altered the migration-related proteins in OH cultured VSMCs. Cells were co-treated with OH, MLE, and nCGA (40 or 120 μM) for 24 h and then subjected to immunodetection conducted with antibodies against by Western blot analyses. (A) Densitometry was used to quantify p-FAK relative to total FAK, p-Src relative to total Src, and Integrin β3 relative to β-actin. (B) The gelatin zymography was then performed to analyze the activities of MMP-9 and MMP-2 as described in the Materials and Methods. (C) Densitometry was used to quantify NF-κB, p-IκB relative to total IκB, Rac1, Rho A, and Cdc42 relative to β-actin. The quantitative data are presented as the mean ± SD from a minimum of three independent experiments. # p < 0.05 as compared to the C (control). * p < 0.05 as compared to the OH group.
Fig 2: PPARβ/´ upregulates pro-tumoural marker gene expression and laminin 332 deposition in actinic keratosis, enhancing SCC progression.A mRNA expression of epithelial-to-mesenchymal transition (EMT) markers in 20 actinic keratoses with moderate atypia (grade II) of Pparβ/δ+/+ and Pparβ/δ−/− mice by real-time RT-PCR. Means ± SEM are presented; p-values are **p = 0.0077 (Tgfβ1), ***p = 0.0009 (Krt13), *p = 0.046 (Vim), *p = 0.045 (Snai1), *p = 0.032 (Snai2), *p = 0.028 (Gsc), **p = 0.009 (Itgαv), *p = 0.025 (Itgα6), *p = 0.022 (Itgβ1), *p = 0.013 (Itgβ6), *p = 0.035 (Cadh12), *p = 0.020 (Col7α1), *p = 0.035 (Mmp19), *p = 0.025 (Lamα3), *p = 0.043 (Mmp2) calculated by two-tailed Student's t-test. The full gene names are given in supplementary Table S2.B Representative immunofluorescence staining of PLA experiments for laminin 332/β4 integrin (red spots) in actinic keratosis with moderate atypia (grade II) sections from Pparβ/δ+/+ and Pparβ/δ−/− mice. DAPI, blue. Scale bars, 50 μm.C Representative immunofluorescence staining of PLA experiments for β4 integrin/Rac1 (red spots) and laminin 332 in actinic keratosis with moderate atypia (grade II) sections from Pparβ/δ+/+ and Pparβ/δ−/− mice. DAPI, blue. Scale bars, 50 μm.D Quantification of PLA staining. Average number of PLA signals (red spots) per nucleus from n = 10 actinic keratoses with moderate atypia sections/genotype (*p = 0.024, t-test).E Actinic keratosis (AK) with grading of cellular atypia (mild, grade I; moderate, grade II; severe, grade III) and SCC distribution in 25 tumours collected from each Pparβ/δ+/+ and Pparβ/δ−/− mouse graded histologically in a blinded manner according to Rowert-Huber et al and to the Broders' classification, respectively.
Fig 3: 6-TG reduced HSV-1 replication by suppressing Rac1 activity. (a) Rac1 siRNA effectively silenced Rac1 protein in HCECs as shown by Western blotting. (b) Rac1 siRNA-treated HCECs reduced HSV-1 replication compared to that of the nonspecific siRNA-treated HCECs during HSV-1 infection. (c) NSC23766 significantly suppressed HSV-1 replication in a dose-dependent manner. In-Cell Western blotting analysis of HSV-1 gD and DRAQ5 from HSV-infected HCECs in the presence of NSC23766 (3, 10, and 30 μM) at 24 h. In addition, Western blotting analysis of HSV-1 gD and GAPDH from HSV-infected HCECs in the presence of NSC23766 (3, 10, and 30 μM) at 24 h. Significance between untreated and NSC23766-treated and HSV-1-infected cells was determined by one-way ANOVA with Tukey’s multiple comparison test. (d) HCECs were infected with HSV-1 at a low MOI (MOI = 0.1) and at a high MOI (MOI = 1) for 24 h, respectively. Rac1 activity was detected by GST pulldown assay, and total Rac1 protein was measured by Western blotting assay. (e) HCECs were treated with 6-TG at 5 μM or 10 μM for 24 h, respectively. The amount of active, GTP-loaded Rac1 was determined by using a GST pulldown assay. Western blotting analysis of Rac1 protein and gD-1 from untreated and 6-TG-treated cells. (f) HCECs were treated with 6-TG at 5 μM and 10 μM for 2 h followed by infection with HSV-1 for 24 h. The amount of active, GTP-loaded Rac1was determined by using a GST pulldown assay. Western blotting analysis of Rac1 protein and GAPDH from untreated and 6-TG-treated cells. The numbers represent the relative density of the band in comparison to the corresponding untreated control normalized to GAPDH. The value of untreated HCECs is set at 1.00 (100%). All data are representative of three independent experiments.
Fig 4: PKCɛ is involved in sortilin-induced vascular damage.(A) Representative immunoblots and densitometric analyses of 3 independent experiments evaluating protein levels of phospho-PYK2, phospho-PKCɛ, and Rac1-GTP in WT, S1P3–/–, and gp91phox–/– vessels exposed to vehicle or sortilin for 60 minutes. (B) Acetylcholine-evoked vasorelaxation in WT mesenteric arteries exposed to vehicle or pretransfected with either siRNA silencing PKCɛ or a scrambled siRNA and then exposed to sortilin for 60 minutes (n = 6). (C) Representative immunoblots and densitometric analyses of 3 independent experiments evaluating phospho-PYK2, and Rac1-GTP levels in WT mesenteric arteries pretransfected with either siRNA silencing PKCɛ or a scrambled siRNA and then exposed to sortilin; the effectiveness of PKCɛ silencing was determined by Western blotting (n = 3). Data are represented as mean ± SD. One-way ANOVA (A and C) or 2-way ANOVA (B) followed by Bonferroni’s post hoc test was used.
Fig 5: Phosphorylation of PKCɛ and PYK2 is required for Rac1-dependent NOX2 activation.(A) Representative confocal immunofluorescence staining of Rac1 in HUVECs treated with vehicle (Ctrl) or sortilin alone or pretreated with the S1P3 inhibitor TY52156. Arrows indicate membrane translocation of Rac1. Scale bar: 10 μm. Insets show higher magnification, zoom ×4.7. (B) Acetylcholine-evoked vasorelaxation in WT mesenteric arteries exposed to vehicle, sortilin alone, or sortilin in the presence of NSC23766 (n = 3). (C) Acetylcholine-evoked vasorelaxation in WT mesenteric arteries treated with vehicle or sortilin alone or pretreated with either ML171 or GSK2795039 before sortilin stimulation (n = 3). (D) NOX activity in HUVECs treated with vehicle or sortilin alone or preincubated with ML171 or GSK2795039 before sortilin (n = 4–5 replicates from 3 independent experiments). (E) NOX activity in WT and gp91phox–/– mesenteric arteries exposed to vehicle or sortilin (n = 3 replicates from 3 independent experiments). Data are expressed as increase of chemiluminescence per minute in arbitrary units. (F) Acetylcholine-evoked vasorelaxation in mesenteric arteries from WT and gp91phox–/– mice exposed to vehicle or sortilin (n = 5). (G) Representative immunoblots and densitometric analyses of 3 independent experiments evaluating protein levels of phospho-Tyr579-PYK2, phospho-Ser729-PKCɛ, phospho-Thr497-PKCα, PKC, S1P3, and Rac1-GTP in HUVECs treated with vehicle or sortilin in the presence or absence of TY52156 or GSK2795039. Data are represented as mean ± SD. One-way ANOVA (D, E, and G) or 2-way ANOVA (B, C, and F) followed by Bonferroni’s post hoc test was used. (C) *P < 0.0001 versus vehicle or GSK2795039 plus sortilin at the same acetylcholine concentration (as indicated by color code).
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