Journal: Oncogene

Article Title: Upregulation of PIP3-Dependent Rac Exchanger 1 (P-Rex1) Promotes Prostate Cancer Metastasis

doi: 10.1038/onc.2009.30

Figure Lengend Snippet: P-Rex1 promotes prostate cancer cell migration and invasion via Rac activation. (a) CWR22Rv1 cells (left panel) or PC3-LN4 cells (right panel) were transfected with control GFP, GFP-tagged wild-type P-Rex1 or P-Rex1(-DH) mutant for 48 h, and then subjected to Transwell migration assay for 12 h (CWR22Rv1 cells) or 5 h (PC3-LN4) toward NIH3T3 CM. Transfected PC3-LN4 cells were selected by flow cytometry cell sorting based on GFP fluorescence prior to migration assays due to a low transfection efficiency (~25%). Bars show mean ± S.E. with *p<0.05 compared to control set at 100% (n=3). Inset: Expression of GFP-tagged P-Rex1 and P-Rex1(-DH) protein detected by western blot analysis using anti-GFP antibody. (b) CWR22Rv1 cells stably expressing wild-type P-Rex1, P-Rex1 “GEF-dead” mutant or control vector were subjected to migration and invasion assays for 12 h. Cells stably expressing wild-type P-Rex1 were pretreated without (none) or with 50 μM NSC for 15 min. Bars show mean ± S.E. with *p<0.01 compared to control (n=3). Inset: Western blot analysis of expression levels of P-Rex1 and its mutant. (c) P-Rex1 activates Rac1 in prostate cancer cells. The amount of activated GTP-bound Rac1 was measured by a Rac activation assay. The image shown is representative of 3 separate experiments. (d) P-Rex1 increases lamellipodia formation. Serum-starved CWR22Rv1 cells or PC-3 cells (positive control) were incubated with NIH3T3 CM for 5 h and then stained with rhodamine-conjugated phalloidin (red) for detection of F-actin.

Article Snippet: Additional reagents were NSC23766, Y27632 and anti-GFP antibody (EMD Biosciences); cell permeable C3 transferase and F-actin visualization kit (Cytoskeleton); Rac activation assay kit, Rho assay reagent, anti-Rac1 antibody (clone 23A8), and Immobilon-FL (Millipore); mouse anti-RhoA (26C4), and rabbit anti-HA and anti-β-actin antibodies (Santa Cruz); FBS (Hyclone); Lipofectamine TM 2000, AlexaF488-labeled secondary antibody, RPMI-1640 and DMEM (Invitrogen); EGF, Matrigel and fibronectin (BD Biosciences); CXCL12 (R&D Systems); and IRDye-labeled secondary antibodies (LI-COR Biosciences).

Techniques: Migration, Activation Assay, Transfection, Control, Mutagenesis, Transwell Migration Assay, Flow Cytometry, FACS, Fluorescence, Expressing, Western Blot, Stable Transfection, Plasmid Preparation, Positive Control, Incubation, Staining

Journal: Current Biology

Article Title: WASP Restricts Active Rac to Maintain Cells’ Front-Rear Polarization

doi: 10.1016/j.cub.2019.10.036

Figure Lengend Snippet:

Article Snippet: Active Rac pull-down assay , Cytoskeleton , No. #BK035.

Techniques: Recombinant, Pull Down Assay, Plasmid Preparation, Expressing, Software

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Hyperinsulinemia stimulates angiogenesis of human fetoplacental endothelial cells: a possible role of insulin in placental hypervascularization in diabetes mellitus.

doi: 10.1210/jc.2013-1210

Figure Lengend Snippet: Figure 4. Pharmacological inhibitors delineated the signaling pathways involved in insulin- induced angiogenesis. The inhibitory effects of L-NAME (400 M) and NSC23766 (200 M) on insulin (10 nM) stimulated eNOS- and Rac1-activity in feto-placental EC were tested by NO bioavailability assay (A) and immunoblotting for Rac1-GTP (B), respectively. The blocking effect of wortmannin on Akt activation in feto-placental EC was shown previously (8). EC were pretreated for 1h either with an inhibitor of (C) PI3K (wortmannin; 100 nM), (D) eNOS (L-NAME) or (E) Rac1 activation (NSC23766; 200 M). Cells were then seeded in growth factor-reduced Matrigel and treated with insulin (INS, 10 nM) or VEGF (650 pM) as positive control. Network formation was monitored in a cell observer and total tube length was quantified 6h after seeding. Data of the NO bioavailability assay are means SD of 3 different cell isolations each measured in quadruplicates. One representative immunoblot out of 4 is shown for Rac1-GTP. Data of in vitro network formation assays are means SD of 3 different cell isolations each measured in triplicates. * P .05 vs control, § P .05 vs insulin.

Article Snippet: Rac1 activation by GTP was measured using the luminescence based G-LISATM Rac1 Activation Assay Biochem KitTM (Cytoskeleton, Denver, CO) according to the manufacturers instruction.

Techniques: Protein-Protein interactions, Activity Assay, Western Blot, Blocking Assay, Activation Assay, Positive Control, In Vitro, Control

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Hyperinsulinemia stimulates angiogenesis of human fetoplacental endothelial cells: a possible role of insulin in placental hypervascularization in diabetes mellitus.

doi: 10.1210/jc.2013-1210

Figure Lengend Snippet: Figure 5. Effect of insulin on Rac1 activity and actin cytoskeleton arrangement of human feto-placental EC. A) Rac1 activity was measured in control cells (C) and after 30min insulin treatment (INS; 10 nM). Data are a mean of two experiments with different EC isolations, each run in triplicates. B-J) The effect on actin arrangement was further determined by phalloidin staining. Cells were seeded in gelatin- coated glass chamber slides. Starved cells (S) were cultured 24h in EBM containing 0.5% FCS and another 24h in EBM without FCS, and nonstarved cells (NS) were cultured under normal (5% FCS) conditions. Starved cells were either pretreated with wortmannin (WORT, 100 nM), NSC23766 (NSC, 200 M) or UO126 (10 M) for 1h (E-J). Cells were then stimulated with insulin (INS, 10 nM) or HBSS as vehicle for 30min and stained with phalloidin texas-red. B) Nonstarved cells show organized parallel F-actin stress fibers (arrows) with very intense phalloidin staining. C) Starved cells show membrane ruffle formation (filled arrows), less actin staining and altered cell shape. D) After insulin stimulation, starved cells restored actin organization to the cell shape observed in the nonstarved cells. H-I) The insulin effect was abolished with pretreatment of starved cells with wortmannin or NSC23766. J) Pretreatment with UO126 did not inhibit the insulin effect. Cells pretreated with inhibitors alone are shown in E (wortmannin), F (NSC23766) and G (UO126). Representative images of two experiments with different EC isolations, each run in triplicates.

Article Snippet: Rac1 activation by GTP was measured using the luminescence based G-LISATM Rac1 Activation Assay Biochem KitTM (Cytoskeleton, Denver, CO) according to the manufacturers instruction.

Techniques: Activity Assay, Control, Staining, Cell Culture, Membrane

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Hyperinsulinemia stimulates angiogenesis of human fetoplacental endothelial cells: a possible role of insulin in placental hypervascularization in diabetes mellitus.

doi: 10.1210/jc.2013-1210

Figure Lengend Snippet: Figure 6. Scheme of proposed signaling pathways by which insulin stimulates angiogenesis in human feto-placental EC. Signaling proteins phosphorylated, or activated through binding to GTP after insulin stimulation identified here are highlighted in bold circles: Insulin binding to IR leads to its autophosphorylation and subsequent IRS1 phosphorylation and PI3K activation. Active PI3K generates PIP3, which activates Akt or downstream guanidine exchange factors (GEFs) which can activate Rac1 which binds GTP in its activated form. Active Akt phosphorylates and, consequently, inactivates GSK3, leading to cell cycle progression. Akt also phosphorylates eNOS leading to NO production. NO generation and Rac1 activation will contribute to the insulin-induced angiogenesis in placental EC. The pharmacological inhibitors wortmannin, L-NAME and NSC23766 confirmed the role of insulin on actin reorganization and its effects on stimulation of angiogenesis.

Article Snippet: Rac1 activation by GTP was measured using the luminescence based G-LISATM Rac1 Activation Assay Biochem KitTM (Cytoskeleton, Denver, CO) according to the manufacturers instruction.

Techniques: Protein-Protein interactions, Binding Assay, Phospho-proteomics, Activation Assay

Journal: PLoS ONE

Article Title: CD148 Tyrosine Phosphatase Promotes Cadherin Cell Adhesion

doi: 10.1371/journal.pone.0112753

Figure Lengend Snippet: A) Effects of CD148 in E-cadherin contact formation were examined by a calcium-switch assay and immunofluorescence staining. CD148 WT, but not CS, promotes E-cadherin contact formation in A431D/E-cadherin WT cells, as evidenced by the expanded and intense E-cadherin distribution (left panels), while CD148 WT shows no effects in A431D/E-cadherin 764 AAA cells (right panels). B) Activities of Rac1, Cdc42, and RhoA were measured in the condition of calcium switch assay. Active and total levels of Rac1, Cdc42, and RhoA proteins were assessed by the pull-down assays and/or immunoblotting described in the “ ” (left panels). The activity was normalized to total amount of protein using densitometry and expressed as fold change (right panels). The data show means ± SEM of quadruplicate determinations. **P<0.05 vs. CD148 (−) cells.

Article Snippet: RhoA, Rac1, and Cdc42 activities were assessed using the Rac1 and RhoA pull-down activation assay kit (Cytoskeleton, Inc., Denver, CO) according to the manufacturer’s instruction.

Techniques: Immunofluorescence, Staining, Western Blot, Activity Assay

Journal: PLoS ONE

Article Title: CD148 Tyrosine Phosphatase Promotes Cadherin Cell Adhesion

doi: 10.1371/journal.pone.0112753

Figure Lengend Snippet: A and B) CD148-introduced or CD148-negative A431D/E-cadherin WT (panel A) and A431D/E-cadherin 764 AAA (panel B) cells were subjected to a hanging drop assay. Rac1, Cdc42, and RhoA activities were assessed at the indicated time points. Active and total levels of Rac1, Cdc42, and RhoA proteins were assessed by pull-down assays and/or immunoblot analysis (left panels). The relative levels of active versus total Rac1, Cdc42, and RhoA were quantified by densitometric analysis (right panels). The data show means ± SEM of quadruplicate determinations. **P<0.05 vs. CD148 (−) cells. CD148 WT, but not CS, increases Rac1 activity in A431D/E-cadherin WT cells, while this effect is not observed in A431D/E-cadherin 764 AAA cells. C) Effects of CD148 WT in cell-cell adhesion were assessed by a hanging drop assay in the presence or absence of Rac1 inhibitor NSC 23766 (75 µM). CD148 increase of cell-cell adhesion is largely diminished by Rac1 inhibition.

Article Snippet: RhoA, Rac1, and Cdc42 activities were assessed using the Rac1 and RhoA pull-down activation assay kit (Cytoskeleton, Inc., Denver, CO) according to the manufacturer’s instruction.

Techniques: Western Blot, Activity Assay, Inhibition

Journal: Experimental neurology

Article Title: RvD1binding with FPR2 attenuates inflammation via Rac1/NOX2 pathway after neonatal hypoxic-ischemic injury in rats

doi: 10.1016/j.expneurol.2019.112982

Figure Lengend Snippet: Effects of FPR2 siRNA and Rac1 activation CRISPR on infarct area and geotaxis reflex at 24 h post hypoxia-ischemia (HI). (A and B)The infarction area was significantly enlarged with FPR2 siRNA/Rac1 activation CRISPR interventions when compared with respective controls. (C) Interventions with FPR2 siRNA or Rac1 activation CRISPR significantly worsened geotaxis reflex compared with respective controls. ANOVA followed by Tukey test was used for analysis. Data were shown as mean ± SD (n=6 per group). * P < 0.05 vs. sham group; # P < 0.05 vs. HI+vehicle group, & P < 0.05 vs. HI+RvD1+scramble siRNA group, @ P < 0.05 vs. control CRISPR. RvD1, resolvin D1.

Article Snippet: 1 μg of Rac1 CRISPR activation plasmid (Santa Cruz, USA) or control CRISPR Activation Plasmid (Santa Cruz, USA) were given via ICV injection as described above at 48 h before HI.

Techniques: Activation Assay, CRISPR, Control

Journal: Experimental neurology

Article Title: RvD1binding with FPR2 attenuates inflammation via Rac1/NOX2 pathway after neonatal hypoxic-ischemic injury in rats

doi: 10.1016/j.expneurol.2019.112982

Figure Lengend Snippet: Activation of Rac1 decreased the anti-inflammation function of RvD1 at 24 h post hypoxia-ischemia (HI). (A) Representative Western blot bands. (B-G) Quantification of Western blot bands showed that Rac1 activation CRISPR inhibited the effect of RvD1 on decreasing the expression of GTP-Rac1 (C), total Rac1 (D), NOX2 (E), IL-1β (F), and TNF-α(G) compared with HI+RvD1+control CRISPR. ANOVA followed by Tukey test was used for analysis. Data were shown as mean ± SD (n=6 per group). * P < 0.05 vs. sham group; # P < 0.05 vs. HI+vehicle group; & P < 0.05 vs. HI+RvD1+control CRISPR group. RvD1, resolvin D1.

Article Snippet: 1 μg of Rac1 CRISPR activation plasmid (Santa Cruz, USA) or control CRISPR Activation Plasmid (Santa Cruz, USA) were given via ICV injection as described above at 48 h before HI.

Techniques: Activation Assay, Western Blot, CRISPR, Expressing, Control

Journal: Experimental neurology

Article Title: RvD1binding with FPR2 attenuates inflammation via Rac1/NOX2 pathway after neonatal hypoxic-ischemic injury in rats

doi: 10.1016/j.expneurol.2019.112982

Figure Lengend Snippet: Knockdown of FPR2 reduced the anti-inflammation effect of RvD1 at 24 h post hypoxia-ischemia (HI). (A) Representative Western blot bands. (B-G) Quantification of Western blot bands showed that RvD1 significantly increased the expression of FPR2 (B), whereas decreased the expression of GTP-Rac1 (C), total Rac1(D), NOX2(E), IL-1β (F), and TNF-α(G) compared with HI+vehicle group. However, FPR2 siRNA reversed the effect of RvD1 compared with HI+RvD1+scramble siRNA group. ANOVA followed by Tukey test was used for analysis. Data were shown as mean ± SD (n=6 per group). * P < 0.05 vs. sham group; # P < 0.05 vs. HI+vehicle group; & P < 0.05 vs. HI+RvD1+scramble siRNA group. RvD1, resolvin D1.

Article Snippet: 1 μg of Rac1 CRISPR activation plasmid (Santa Cruz, USA) or control CRISPR Activation Plasmid (Santa Cruz, USA) were given via ICV injection as described above at 48 h before HI.

Techniques: Knockdown, Western Blot, Expressing

Journal: Cells

Article Title: Enhanced Migration of Fuchs Corneal Endothelial Cells by Rho Kinase Inhibition: A Novel Ex Vivo Descemet's Stripping Only Model.

doi: 10.3390/cells13141218

Figure Lengend Snippet: Figure 5. Activation of Rac1 driving lamellipodia formation at the leading edge enhancing cell migration. Rac1 activation in normal and FECD cells at (A) baseline and (B) after the treatment with ripasudil compared to baseline. (C) The expression of ARPC2 at the leading edge in normal and FECD cells with and without ripasudil. Pearson’s correlation between normal and FECD cells at (D) baseline and (E) after the treatment with ripasudil compared to baseline. * p < 0.05; ** p < 0.01; *** p < 0.001. The data are expressed as mean ± SEM. Blue: nucleus; Red: actin; Green: ARPC2; Yellow: merging of actin and ARPC2. Scale: 10 µm.

Article Snippet: All of the cell lines were plated on a 96-well plate, treated with 1 μM of ripasudil or left untreated as controls, and incubated for 24 h before the GTPase assay was performed using the GTPase-Glo Assay kit (Promega, Madison, WI, USA) and Active Rac1 kit (SignalChem, Richmond, BC, Canada).

Techniques: Activation Assay, Migration, Expressing

Journal: PLoS ONE

Article Title: K-Ras Mediated Murine Epidermal Tumorigenesis Is Dependent upon and Associated with Elevated Rac1 Activity

doi: 10.1371/journal.pone.0017143

Figure Lengend Snippet: ( A ) Macroscopic view of oral papillomas from LSL-K-Ras G12D ;K14-Cre:ER (upper panel) and LSL-K-Ras G12D ;K14-Cre:ER;Rac1 Wt/- mice (lower panel). Deficiency for one Rac1 allele dramatically reduced papilloma growth. ( B ) Kaplan-Meier survival curve of Rac Wt/- (n = 20), LSL-K-Ras G12D ;K14-Cre:ER (n = 78) and LSL-K-Ras G12D ;K14-Cre:ER;Rac1 Wt/- (n = 22) mice shows the dramatic difference between the two mouse strains. Median survival for LSL-K-Ras G12D ;K14-Cre:ER;Rac1 Wt/- mice was determined to be 75 days, which is 50% greater than the 51 day survival time of LSL-K-Ras G12D ;K14-Cre:ER mice. ( C ) Staining for the marker of cell proliferation Ki-67 revealed markedly lower levels in tumors from LSL-K-Ras G12D ;K14-Cre:ER;Rac1 Wt/- mice relative to tumors from LSL-K-Ras G12D ;K14-Cre:ER + mice.

Article Snippet: Antibodies used were against active Rac1-GTP (Neweast Biosciences cat. no.: 26903), Rac1 (Santa Cruz Biotechnology Inc. cat. no.: sc-217).

Techniques: Staining, Marker

Journal: PLoS ONE

Article Title: K-Ras Mediated Murine Epidermal Tumorigenesis Is Dependent upon and Associated with Elevated Rac1 Activity

doi: 10.1371/journal.pone.0017143

Figure Lengend Snippet: ( A ) Active Rac1 was immunoprecipitated from lysates derived from NIH 3T3 cells that had been treated for the indicated time periods with PDGF (10 ng/ml). Western analysis for Rac1 was carried out on immunoprecipitated protein as well as the original lysate as indicated. ( B ) Histogram showing active Rac1 immunofluorescence intensity averaged over 5 fields of NIH 3T3 cells increasing over the indicated time periods of PDGF treatment. ( C ) Staining for actin with Texas-red conjugated phalloidin (red) and co-immunofluorescence for active Rac1 (green) in representative fields of NIH 3T3 cells used in the quantification shown in panel B, demonstrating increased active Rac1 and changes to the actin cytoskeleton over the indicated time periods of PDGF treatment. Boxed areas indicated by numbers 1-3 are shown enlarged. ( D ) Immunohistochemical analysis for Rac1 and active Rac1 of oral mucosa from LSL-K-Ras G12D , LSL-K-Ras G12D ; K14-Cre:ER and Rac1 Wt/- ; LSL-K-Ras G12D ; K14-Cre:ER showing dramatic increase in active Rac1 in LSL-K-Ras G12D ; K14-Cre:ER tissue, which is reduced when one Rac1 allele is lacking. Histogram shows results of positive pixel analysis for active Rac1staining.

Article Snippet: Antibodies used were against active Rac1-GTP (Neweast Biosciences cat. no.: 26903), Rac1 (Santa Cruz Biotechnology Inc. cat. no.: sc-217).

Techniques: Immunoprecipitation, Derivative Assay, Western Blot, Immunofluorescence, Staining, Immunohistochemical staining