Journal: Cancer gene therapy

Article Title: Pals1 functions in redundancy with SMAP1 to inhibit Arf6 in order to prevent Rac1-dependent colorectal cancer cell migration and invasion.

doi: 10.1038/s41417-022-00570-2

Figure Lengend Snippet: Fig. 1 Loss of Pals1 in a colorectal cancer cell line HCT116 results in TJ defects, enhanced migration and invasion. A Immunostaining of confluent HCT116 and HCT116ΔPals1 cells with indicated antibodies, B Activation of Rac1 in wild type and Pals1-deficient HCT116 was quantified using G-LISA assay. C Representative images and quantification of the FRET signal of a biosensor targeting active Rac1, transfected in HCT116 and HCT116ΔPals1 cells. Results are representative of 4 experiments. D Quantification of Rac1 biosensor FRET signals at the cell body and the cell cortex in HCT116 and HCT116ΔPals1 cells. Scale bars are 20 µm in A, C.

Article Snippet: The Rac1 G-LISA activity assay (#BK128, Cytoskeleton Inc.) was performed according to manufacturer’s instructions, using 0.5 mg/ml protein concentration of cell lysates.

Techniques: Migration, Immunostaining, Activation Assay, Transfection

Journal: Cancer gene therapy

Article Title: Pals1 functions in redundancy with SMAP1 to inhibit Arf6 in order to prevent Rac1-dependent colorectal cancer cell migration and invasion.

doi: 10.1038/s41417-022-00570-2

Figure Lengend Snippet: Fig. 2 Deletion of Pals1 in Caco-2 cells does not result in enhanced migration and invasion or upregulation of active Arf6 or Rac1. A Immunostaining of confluent Caco-2 and Caco-2ΔPals1 cells with the indicated antibodies. B Representative images from wound healing assays of Caco-2 and Caco-2ΔPals1 cells and the corresponding quantification (N = 3). C Representative images and quantification of transwell matrigel invasions assays of Caco-2 and Caco-2ΔPals1 cells (N = 5). D Western blot and CBB-stained gel of pulldown experiments to detect active Arf6 from lysates of Caco-2 and Caco-2ΔPals1 cells (N = 6). E Western blot and CBB-stained gel of pulldown experiments to detect active Rac1 from lysates of Caco-2 and Caco-2ΔPals1 cells (N = 3). F Representative images and quantification of the FRET signal of a biosensor targeting active Rac1, transfected in Caco-2 and Caco-2ΔPals1 cells. Results are representative of 4 experiments. Scale bars are 20 µm in A and F, 100 µm in B.

Article Snippet: The Rac1 G-LISA activity assay (#BK128, Cytoskeleton Inc.) was performed according to manufacturer’s instructions, using 0.5 mg/ml protein concentration of cell lysates.

Techniques: Migration, Immunostaining, Western Blot, Staining, Transfection

Journal: Cancer gene therapy

Article Title: Pals1 functions in redundancy with SMAP1 to inhibit Arf6 in order to prevent Rac1-dependent colorectal cancer cell migration and invasion.

doi: 10.1038/s41417-022-00570-2

Figure Lengend Snippet: Fig. 3 Pals1-deficient DLD1 do not exhibit increased Arf6/Rac1 activity or enhanced cell migration/invasion. A Immunostaining of confluent DLD1 and DLD1ΔPals1 cells with the indicated antibodies. B Representative images from wound healing assays of DLD1 and DLD1ΔPals1 cells and the corresponding quantification (N = 3). C Representative images and quantification of transwell matrigel invasion assays of DLD1 and DLD1ΔPals1 cells (N = 3). D Western blot and CBB-stained gel of pulldown experiments to detect active Arf6 from cell lysates of DLD1 and DLD1ΔPals1 cells (N = 8). E Western blot and CBB-stained gel of pulldown experiments to detect active Rac1 from cell lysates of DLD1 and DLD1ΔPals1 cells (N = 3). Scale bars are 20 µm in A and 100 µm in B.

Article Snippet: The Rac1 G-LISA activity assay (#BK128, Cytoskeleton Inc.) was performed according to manufacturer’s instructions, using 0.5 mg/ml protein concentration of cell lysates.

Techniques: Activity Assay, Migration, Immunostaining, Western Blot, Staining

Journal: Cancer gene therapy

Article Title: Pals1 functions in redundancy with SMAP1 to inhibit Arf6 in order to prevent Rac1-dependent colorectal cancer cell migration and invasion.

doi: 10.1038/s41417-022-00570-2

Figure Lengend Snippet: Fig. 4 Knockout of Pals1 in mesenchymal-like RKO cells does not affect cell motility. A Western blot analysis of the expression of E-Cadherin in different colorectal cancer cell lines. B Representative images from wound healing assays of RKO and RKOΔPals1 cells and the corresponding quantification (N = 6). C Representative images and quantification of transwell matrigel invasion assays of RKO and RKOΔPals1 cells (N = 4). D Western blot and CBB-stained gel of pulldown experiments to detect active Arf6 from cell lysates of RKO and RKOΔPals1 cells (N = 6). E Western blot and CBB-stained gel of pulldown experiments to detect active Rac1 from cell lysates of RKO and RKOΔPals1 cells (N = 3). Scale bars are 100 µm in B.

Article Snippet: The Rac1 G-LISA activity assay (#BK128, Cytoskeleton Inc.) was performed according to manufacturer’s instructions, using 0.5 mg/ml protein concentration of cell lysates.

Techniques: Knock-Out, Western Blot, Expressing, Staining

Journal: Cancer gene therapy

Article Title: Pals1 functions in redundancy with SMAP1 to inhibit Arf6 in order to prevent Rac1-dependent colorectal cancer cell migration and invasion.

doi: 10.1038/s41417-022-00570-2

Figure Lengend Snippet: Fig. 6 SW48ΔPals1 cells display enhanced Arf6/Rac1 activation and increased cell migration, which can be rescued by SMAP1 transfection. A Immunostaining of confluent SW48 and SW48ΔPals1 cells with the indicated antibodies. B Representative images and quantification of the FRET signal of a biosensor targeting active Rac1, transfected in SW48 and SW48ΔPals1 cells. Results are representative of 3 experiments. C Western blot of cell lines with and without SMAP1 overexpression. Empty vector was used as negative control. D, E Quantification of cell migration (scratch assay, D) and invasions assay (E) of the indicated cell lines. F Rac1 activation of the indicated cell lines quantified by G-LISA. G Western blot and CBB-stained gel of pulldown experiments to detect active Arf6 from cell lysates of the indicated cell lines (N = 3). H Survival probability of colorectal cancer patients with only low Pals1 expression, only low SMAP1 expression or low Pals1 and low SMAP1 expression. Scale bars are 20 µm in A and B.

Article Snippet: The Rac1 G-LISA activity assay (#BK128, Cytoskeleton Inc.) was performed according to manufacturer’s instructions, using 0.5 mg/ml protein concentration of cell lysates.

Techniques: Activation Assay, Migration, Transfection, Immunostaining, Western Blot, Over Expression, Plasmid Preparation, Negative Control, Wound Healing Assay, Staining, Expressing

Journal: Cell reports

Article Title: Optimizing metastatic-cascade-dependent Rac1 targeting in breast cancer: Guidance using optical window intravital FRET imaging.

doi: 10.1016/j.celrep.2021.109689

Figure Lengend Snippet: Figure 1. Rac1 signaling is increased in MMTV-PyMT-driven metastatic breast cancer (A) Schematic of Rac1-FRET biosensor in FRET conformation upon GTP loading and Rac1 activation in cells crossed to the MMTV-PyMT-driven breast cancer model. (B) Representative images and quantification of upregulated Rac1 activity in primary MMTV-PyMT-driven mammary tumors compared to WT mammary glands (n = 7 mice per condition, 991 cells in total). (C) Spatiotemporal monitoring of live Rac1 activity in the context of the native tumor microenvironment showing the local vasculature (Qdot655) and second harmonic generation (SHG) imaging visualizing the local ECM monitored by optical window imaging in primary tumors (i). Longitudinal imaging can be achieved using these windows and further improved by image stabilization, allowing for the assessment of Rac1 activity in relation to tumor microenvironment (ii). (D) Tracking of Rac1 activity in relation to the proximity of cells to local tumor vasculature, with example excerpts showing cells proximal and distal to the local vasculature (n = 5 mice, 166 cells). (E) Upregulation of Rac1 activity at the invasive border of primary tumors as quantified through an optical window (n = 5 mice, 180 cells). Columns show averages, and error bars represent SEM; scale bars, 50 mm. Unpaired Welch’s t test, ****p < 0.0001 and *p < 0.05 (B and E); one-way ANOVA, *p < 0.05 (D).

Article Snippet: Reagent or Resource Source Identifier Antibodies Mouse monoclonal IgG1 anti-Rac1 Cytoskeleton Cat# ARC03, RRID:AB_10709099 Rabbit poly-clonal IgG anti-GFP Abcam Cat# ab290, RRID:AB_303395 Rabbit monoclonal IgG1 anti-GAPDH Cell Signaling Cat# 2118, RRID:AB_561053 Sheep-anti-Mouse IgG HRP-linked GE healthcare Cat# NA931, RRID:AB_772210 Donkey-anti-Rabbit IgG HRP-linked GE healthcare Cat# NA934, RRID:AB_772206 Rabbit monoclonal IgG1 anti-Ki67 ThermoFisher Scientific Cat# RM-9106-S1, RRID:AB_149792 Rabbit polyclonal anti-cleaved Caspase-3 Cell Signaling Cat# 9661, RRID:AB_2341188 Rabbit monoclonal anti-Cyclin B1 (EPR17060) Abcam Cat# ab181593, RRID:AB_2820245 Rabbit polyclonal anti- Cyclin A2 (C-19) Santa Cruz Biotechnology Cat# sc-596 Rabbit polyclonal anti-PAK1 Cell Signaling Cat# 2602, RRID:AB_330222 Rabbit polyclonal anti-PAK1 (Ser199/204)/PAK2 (Ser192/197) Cell Signaling Cat# 2605, RRID:AB_2160222 Mouse Monoclonal anti-beta-actin (clone AC-15) Merck (SigmaAldrich) Cat# A5441, RRID:AB_476744 Bacterial and virus strains E. coli DH5a ThermoFisher Scientific Cat# 18265017 Chemicals, peptides and recombinant proteins Qtracker 655 Vascular Label ThermoFisher Scientific Cat# Q21021MP AnnexinV-Cy5 BioVision Cat# 1013 Propidium iodide Merck, Sigma-Aldrich Cat# P4170 SalI Promega Cat# R6051 NdeI New England Biolabs Cat# R0111S Rac1 Pull-down Activation Assay Biochem Kit CytoSkeleton Inc. Cat# BK035 Experimental models: Cell lines PyMT 20065 (from mouse 20065, female) SEARCHBreast https://searchbreast.org/ MMTV-PyMT;Rac1-FRET (from mouse 8625, female) This study N/A MMTV-PyMT;Rac1-FRET;P-Rex1 ± (from mouse 138509, female) This study N/A Experimental models: Organisms/strains Rac1-FRET mice (ubiquitously expressed, ‘‘ON’’) Prof. Heidi CE Welch, Babraham Institute, UK, Johnsson et al. 2014 N/A MMTV-Her2 mice The Jackson Laboratory Stock# 002376 MMTV-PyMT mice The Jackson Laboratory Stock# 002374 K14-CreER mice The Jackson Laboratory Stock# 005107 LSL-K-Ras G12D mice The Jackson Laboratory Stock# 008179 Rac1-flox mice Prof L. Machesky, Prof O. Sansom, CRUK Beatson Institute, University of Glasgow N/A P-Rex1 KO mice Prof. Heidi CE Welch, Babraham Institute, UK, Welch et al. 2005 N/A FVB/NJ (Ausb) mice The Jackson Laboratory Stock# 001800 NOD.Cg-Prkdc < scid > IL2rg < tm1Wjl > /SzJ (Ausb) mice The Jackson Laboratory Stock# 005557 BALB/c-Fox1nu (Ausb) mice Charles River Strain# 194 (Continued on next page) Cell Reports 36, 109689, September 14, 2021 e1

Techniques: Activation Assay, Activity Assay, Imaging

Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 1. Loss of NF1 reduces RAC1-driven melanoblast migration. A. Scratch-like migration assay representing the percentage of cell coverage after 6 h, 9 h and 12 h using either WT or NF1+/−melanoblasts (MB) in the presence of a RAC1 activator (CN04). B. RAC1 activity was measured by G-lisa in WT and NF1+/−melanoblasts (MB). C. Scratch-like migration assay after 3 h, 6 h, 9 h and 12 h in NF1+/−melanoblasts 48 h-post transfection with either a scramble siRNA (SCR) or with an NF1-specific siRNA (siNF1). D. Expression status of NF1 and expression of phosphorylated and non-phosphorylated ERK and AKT in NF1+/−melanoblasts by western blot. α-actinin was used as a loading control. GTP-RAC1 pulldown and total lysates were blotted with α-RAC1 antibody. E. Scratch-like migration assay representing the percentage of cell coverage after 9 h and 12 h in NF1+/−melanoblasts 48 h-post transfection with either a scramble siRNA (SCR) or with an NF1-specific siRNA (siNF1) and in the presence or absence of a RAC1 activator (CN04). *: SCR vs. siNF1, #: -CN04 vs. +CN04. F. GTP-RAC1 pulldown and total lysates were blotted with α-RAC1 antibody in the presence or absence of a RAC1 activator (CN04). **P < 0.01, *P < 0.05, ns: not significant (unpaired Student's t-test). All error bars represent the SEM of at least three independent experiments.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Migration, Activity Assay, Transfection, Expressing, Western Blot, Control

Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 2. Loss of NF1 increases melanoma migration and is associated with increased PREX1 expression. A. NF1 mRNA expression under NF1 silencing with two siRNAs (NF1.6 and NF1.11) in SK-mel-23, Mel501, and SK-mel-103 melanoma cell lines. B. PREX1 mRNA expression under NF1 silencing with two siRNAs in SK-mel-23, Mel501, and SK- mel-103 cell lines. C. Scratch-like migration assay representing the percentage of cell coverage after 6 h, 12 h and 24 h under NF1 silencing in SK-mel-23, Mel501, and SK-mel- 103 cell lines. D. Scratch-like migration assay as in C, after additional transfection with siRNA control (scramble) or with PREX1 siRNA (siPREX1). E. Scratch-like migration assay as in C. in the absence (control) or presence (RAC1 inhibitor) of a RAC1 inhibitor. ***P < 0.001, **P < 0.01, *P < 0.05 (unpaired Student's t-test). All error bars rep- resent the SEM of at least three independent experiments.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Migration, Expressing, Transfection, Control

Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 4. PREX is upregulated in low NF1 expressing melanoma metastases. A. Representative microphotographs of Tissue Microarray (TMA) containing primary and metastatic melanoma samples analysed by immunohistochemistry using a specific antibody against NF1, RAC1 and PREX1. Bar, 100 μm. B. Scoring of the immunohistochemistry staining was performed according to our previously described protocol [24]. Duplicates of valid punch samples are represented for each condition. Significance was tested using two-tailed t-test with *P < 0.05 and ns: not significant.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Expressing, Microarray, Immunohistochemistry, Staining, Two Tailed Test

Journal: Molecular Microbiology

Article Title: Enzymatic activities and functional interdependencies of Bacillus subtilis lipoteichoic acid synthesis enzymes

doi: 10.1111/j.1365-2958.2010.07472.x

Figure Lengend Snippet: In vitro activity of B. subtilis LtaS-type enzymes. A. Chemical structures of fluorescently labelled NBD-PG and NBD-DAG lipids with known S. aureus LtaS and B. cereus PLC cleavage site indicated by an arrow. B. Coomassie stained gel of purified B. subtilis LtaS-like proteins. Extracellular enzymatic domains of B. subtilis LtaS BS , YfnI, YqgS and YvgJ were purified as N-terminal His-tag fusion proteins and 10 µg purified protein separated on a 10% SDS-PAGE gel and visualized by staining with Coomassie brilliant blue. C. TLC analysis of B. subtilis LtaS BS , YfnI, YvgJ and YqgS in vitro reaction products. The NBD-PG lipid substrate was incubated with eLtaS BS , eYfnI, eYvgJ or eYqgS enzyme. Subsequently, lipids were extracted and separated by TLC and fluorescent lipid bands visualized by scanning plates with a fluorescence imager. As negative and positive controls, reactions were set up without enzyme or with the B. cereus PLC enzyme respectively. Note that only 10% of the PLC reaction was run on the TLC plate. Positions of NBD-PG and presumed NBD-DAG reaction product are indicated on the left and proteins added to each reaction are shown on the top of the panel.

Article Snippet: Briefly, 1.8 ml of 10 mM sodium succinate buffer, pH 6.0, ionic strength (µ) = 50 mM (adjusted with NaCl) was added to 25 µg of TLC-purified NBD-PG lipid (Avanti; order number 810163).

Techniques: In Vitro, Activity Assay, Staining, Purification, SDS Page, Incubation, Fluorescence

Journal: Molecular Microbiology

Article Title: Enzymatic activities and functional interdependencies of Bacillus subtilis lipoteichoic acid synthesis enzymes

doi: 10.1111/j.1365-2958.2010.07472.x

Figure Lengend Snippet: Kinetic measurements for recombinant LtaS BS , YfnI, YqgS and YvgJ enzymes. A. Time-course experiment. Enzyme reactions were set up as described under Experimental procedures , aliquots removed at the indicated time points and reactions stopped by the addition of chloroform and methanol. Lipids were separated on TLC plates and the NBD-DAG reaction product quantified. For each time point and enzyme the average value and standard deviation of three values is plotted. Three independent experiments were performed and a representative graph is shown. B. Maximal enzyme activity of B. subtilis LtaS BS , YfnI, YqgS and YvgJ. The slope of the linear fit through the first three data points of the curve shown in (A) was used to calculate the maximal enzyme activity for each B. subtilis LtaS orthologue. Three independent time-course experiments were used to determine an average value and standard deviation for the maximal enzyme activity and these values are plotted.

Article Snippet: Briefly, 1.8 ml of 10 mM sodium succinate buffer, pH 6.0, ionic strength (µ) = 50 mM (adjusted with NaCl) was added to 25 µg of TLC-purified NBD-PG lipid (Avanti; order number 810163).

Techniques: Recombinant, Standard Deviation, Activity Assay

Journal: Molecular Microbiology

Article Title: Enzymatic activities and functional interdependencies of Bacillus subtilis lipoteichoic acid synthesis enzymes

doi: 10.1111/j.1365-2958.2010.07472.x

Figure Lengend Snippet: Metal and substrate specificity of recombinant B. subtilis LtaS-type enzymes. A. B. subtilis LtaS-type enzymes require Mn 2+ for activity. In vitro enzyme assays were set up with NBD-PG lipid as the substrate in the presence of 10 mM MgCl 2 , MnCl 2 , CaCl 2 or ZnCl 2 and reactions were initiated by the addition of eLtaS BS . As controls, reactions were set up without enzyme or without metal ion added. Samples were incubated for 3 h at 37°C, lipids extracted and separated by TLC. Plates were scanned and signals of the reaction product quantified. Reactions were set up in triplicate and the average value and standard deviation plotted. The average fluorescence reading for the reactions set up with MnCl 2 was set to 1 and other values were adjusted accordingly. Similar results were obtained for B. subtilis YfnI, YqgS and YvgJ (see ). B. NBD-PG is the sole lipid substrate for B. subtilis LtaS BS , YfnI, YqgS and YvgJ. Standard enzyme reactions were set up using NBD-PG, NBD-PS, NBD-PE or NBD-PC as substrate (indicated on the left of the panel) and reactions were initiated by the addition of the different B. subtilis enzymes. As a negative control, lipid substrates were incubated without enzyme (no enz.) and as a positive control, a PLC reaction using NBD-PG as substrate was run alongside on each TLC plate in order to determine the mobility of the reaction product. Three independent experiments were performed and a representative result is shown. Note that only the upper part of the TLC plates is shown with the area of the reaction product.

Article Snippet: Briefly, 1.8 ml of 10 mM sodium succinate buffer, pH 6.0, ionic strength (µ) = 50 mM (adjusted with NaCl) was added to 25 µg of TLC-purified NBD-PG lipid (Avanti; order number 810163).

Techniques: Recombinant, Activity Assay, In Vitro, Incubation, Standard Deviation, Fluorescence, Negative Control, Positive Control

Journal: Communications Biology

Article Title: Extracellular osmolarity regulates osteoblast migration through the TRPV4-Rho/ROCK signaling

doi: 10.1038/s42003-025-07946-8

Figure Lengend Snippet:

Article Snippet: Rac1/Cdc42 antibody , CST, America.

Techniques: Saline, Lysis, Staining

Journal: PLoS ONE

Article Title: Type VI collagen promotes lung epithelial cell spreading and wound-closure

doi: 10.1371/journal.pone.0209095

Figure Lengend Snippet: Quantification of 10hr 16HBE wound-width relative to 0hr controls on COL6, COL1, Matrigel coated wells, and uncoated tissue-culture wells after treatment with inhibitors of PI3K (LY294002, 5 μM), CDC42 (ZCL 278, 55 μM), RHOA (CCG 1423, 3 μM), FAK (PF 573228, 40 nM), RAC (EHT 1864, 600 nM), and ERK (FR 180204, 3 μM). (a) Heat map indicating the effect of inhibitor relative to control for cells on each matrix. A lighter color represents more wound closure relative to 0hr. A yellow circle represents p<0.05. Plots present wound-healing rate of cells on COL6, Matrigel, COL1, and plastic after treatment with (b) PI3K inhibitor, (c) CDC42 inhibitor, and (d) RAC1 inhibitor. ** p<0.01, *** p<0.001. n = 6.

Article Snippet: Plasmids containing GFPmPA-GFP-N1 (a gift from Michael Davidson, Addgene plasmid # 54712), and constitutively active FAK (pGFP FAK Y397F, a gift from Kenneth Yamada, Addgene plasmid # 50516), CDC42 (a gift from Joan Brugge, Addgene plasmid #14568), and RAC1 (pcDNA3-EGFP-RAC1-Q61L, a gift from Gary Bokoch, Addgene plasmid #12981) all contain GFP and were transfected individually [ , ].

Techniques: Control

Journal: PLoS ONE

Article Title: Type VI collagen promotes lung epithelial cell spreading and wound-closure

doi: 10.1371/journal.pone.0209095

Figure Lengend Snippet: (a) Representative images of GFP-only, Rac1, Cdc42 and Pi3k-overexpressing cell spreading on Matrigel. Quantification of GFP-positive 16HBE spreading 3 hours after plating on (b) Matrigel, (c) COL1, (d) uncoated tissue-culture wells, and (e) COL6 after transfection with constitutively active signaling constructs for FAK, RAC1, ERK, RHOA, CDC42, or PI3K. Black lines represent spreading of GFP-transfected cells on each matrix, respectively. Grey lines represent spreading of GFP transfected cells on COL6. N = 6. * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: Plasmids containing GFPmPA-GFP-N1 (a gift from Michael Davidson, Addgene plasmid # 54712), and constitutively active FAK (pGFP FAK Y397F, a gift from Kenneth Yamada, Addgene plasmid # 50516), CDC42 (a gift from Joan Brugge, Addgene plasmid #14568), and RAC1 (pcDNA3-EGFP-RAC1-Q61L, a gift from Gary Bokoch, Addgene plasmid #12981) all contain GFP and were transfected individually [ , ].

Techniques: Transfection, Construct