Journal: Oncogene

Article Title: TIPE1 induces apoptosis by negatively regulating Rac1 activation in hepatocellular carcinoma cells.

doi: 10.1038/onc.2014.208

Figure Lengend Snippet: Figure 5. TIPE1 inhibits Rac1 activation, leading to cell growth repression. (a) Co-immunoprecipitation to test the interaction between TIPE1 and Rac1 in HEK293 cells. (b) Pull-down assay to detect the GTP-Rac1 level in HEK293 cells. (c) Western blot to detect protein levels of p-p65, p65, p-JNK and JNK in HEK293 cells 8 h after transfection with or without pRK5-myc-Rac1-Q61L and increasing amounts of pcTIPE1-HA plasmids. (d) Cytometry analysis of Bel7402 cells after co-transfected with TIPE1 and pRK5-myc-Rac1-Q61L plasmids for 24 h. (e) Rac1 knockdown destroys the increased cell growth induced by TIPE1 siRNA 313 in HepG2.2.15 cells. **Po0.01. TIPE1 and Rac1 levels were determined by reverse transcription–PCR. The experiments were repeated for three times and one representative result is shown.

Article Snippet: Wild-type Rac1 expression plasmid pRK5myc-Rac1-wt, activated Rac1 expression plasmid pRK5-myc-Rac1-Q61L and activated H-Ras expression plasmid pBabe H-Ras 12V were obtained from Addgene (Cambridge, MA, USA).

Techniques: Activation Assay, Immunoprecipitation, Pull Down Assay, Western Blot, Transfection, Cytometry, Knockdown, Reverse Transcription

Journal: Journal of leukocyte biology

Article Title: The trafficking protein JFC1 regulates Rac1-GTP localization at the uropod controlling neutrophil chemotaxis and in vivo migration

doi: 10.1002/JLB.1VMA0818-320R

Figure Lengend Snippet: (a) The distribution of Rac1-Q61L transfected into primary WT or JFC1−/− neutrophils was analyzed by TIRF microscopy. Upper panels, representative images of unstimulated neutrophils are shown and representative dynamic studies are presented in supplementary movies S5 and S6. Lower panels, representative images of fMLF-stimulated neutrophils are shown. The white arrows point to the uropods of these cells showing accumulation of Rac1-Q61L-GFP in JFC1−/− but not in wild type neutrophils. Scale bars: 5 μm. (b) Immunofluorescence analysis of endogenous JFC1 (red) and Rac1-GTP (green) in neutrophilic-differentiated HL60 cells. Arrowheads indicate examples of vesicles that are positive for both JFC1 and Rac1 at the uropod and back body. Scale bar, 5 μm. (c) Super-resolution microscopy analysis (STORM) of the localization of endogenous JFC1 and Rac1-GTP in neutrophilic-differentialted HL60 cells. JFC1 and Rac1-GTP are detected adjacent to each other (arrowheads <50 nm apart) compatible with putative, in situ, protein-protein interaction; Scale bar, 5 μm. (d) Quantification of the super-resolution microscopy analysis showing the distance between JFC1 and Rac1-GTP centroids was performed as described under “Materials and Methods” and results are expressed as a percentage of total pairs at <50nm distance for each cell. A total of at least 10,000 JFC1 and Rac1-GTP pairs were analyzed from at least 4 individual cells. Mean ± S.E.M, **, p < 0.01, ***, p<0.001.(e) Chemotactic responses of wild type (WT, black bars) or JFC1−/− (red bars) neutrophils in response to the CXC chemokines CXCL2 and KC. Mean ± SEM, n=6 mice analyzed independently in two independent experiments. NS, non-stimulated.

Article Snippet: Expression vectors: GFP- Rac1 WT, GFP- Rac1 Q61L, GFP- Rac1 T17N were obtained from Addgene.

Techniques: Transfection, Microscopy, Immunofluorescence, Super-Resolution Microscopy, In Situ

Journal: Journal of leukocyte biology

Article Title: The trafficking protein JFC1 regulates Rac1-GTP localization at the uropod controlling neutrophil chemotaxis and in vivo migration

doi: 10.1002/JLB.1VMA0818-320R

Figure Lengend Snippet: (a) Coimmunoprecipitation analysis of the JFC1-Rac1 interaction. Cells were transfected with myc-JFC1 and with either WT Rac1-GFP, the constitutively active Rac1 Q61L-GFP or the dominant negative Rac1 T17N-GFP. Cell lysates were used in pulldown assays, carried out using anti-myc antibodies and magnetic beads. Western blots are representative of at least three experiments with similar results. (b) Densitometric quantification of the immunoprecipitated bands from 3 independent experiments using the ImageJ software. The data is represented as mean ± SEM. ** p< 0.01. (c) Pulldown experiments were performed in cells transfected with GFP-Rac1Q61L with either wild type myc-JFC1 or with the point mutant myc-JFC1-W83S, which lacks binding to Rab27a. (d) Pulldown experiments were performed in cells transfected with EGFP-Rab27a with either myc-JFC1 WT or with the myc-JFC1-W83S mutant.

Article Snippet: Expression vectors: GFP- Rac1 WT, GFP- Rac1 Q61L, GFP- Rac1 T17N were obtained from Addgene.

Techniques: Transfection, Dominant Negative Mutation, Magnetic Beads, Western Blot, Immunoprecipitation, Software, Mutagenesis, Binding Assay

Journal: Journal of leukocyte biology

Article Title: The trafficking protein JFC1 regulates Rac1-GTP localization at the uropod controlling neutrophil chemotaxis and in vivo migration

doi: 10.1002/JLB.1VMA0818-320R

Figure Lengend Snippet: (a-e) Comparative quantitative analysis of the localization of endogenous active Rac1 in stimulated wild type and JFC1−/− neutrophils. (a) Quantitative analysis of total Rac1-GTP in neutrophils isolated from WT and JFC1−/− mice after stimulation with 100nM fMLF for 10 min at 37 °C. Active Rac1 was quantified using an antibody specific for the GTP bound form of Rac1 as described under “Material and Methods” and Rac1-GTP fluorescence intensity in the whole cell was analyzed using ImageJ. The data is indicated as mean± SEM of the arbitrary fluorescent units from an n=3 mice. (b) Representative images of WT and JFC1−/− cells showing Rac1-GTP accumulation in the tail upon fMLF stimulation for 10 min. (c) The length of Rac1-GTP uropods was analyzed using the ImageJ software. The data is expressed as mean ± SEM from a total of 180 cells from 3 independent experiments. (d) The % of total Rac1-GTP accumulated in the tail for each cell was calculated from the fluorescent intensity of Rac1-GTP in the uropod relative to the whole cell, measured using the ImageJ software. (e) The tail area was measured and Rac1 tail intensity per unit area was calculated using ImageJ. (c-e). WT (black circles); JFC1−/− (red triangles). Data are represented as mean ± SEM and were obtained from an n=4 mice. *** p<0.001, **** p<0.0001. (f-k) Effects of microtubule disruption (f-h) or ROCK inhibition (i-k) on the distribution of endogenous active Rac1 in WT neutrophils. Isolated neutrophils were seeded onto glass coverslips for 30 min, treated with the indicated inhibitors and stimulated with 100nM fMLF for 10 min at 37 °C. Active Rac1 was quantitatively analyzed by confocal microscopy as described above. (f) Representative image of WT and JFC1−/− cells showing Rac1-GTP accumulation in the tail upon treatment with 10μM Nocadozole followed by fMLF stimulation. (g) Percentage of total cells expressing Rac1-GTP-positive tails. (h) % of total (whole cell) Rac1-GTP accumulated in the tail for each Nocodazole- or vehicle-treated cell. (i) Representative images of WT and JFC1−/− cells showing Rac1-GTP accumulation in the tail upon treatment with 10μM Y27632 prior to fMLF stimulation. (j) The length of the Rac1-GTP tails in vehicle vs Y27632-treated cells. The data is expressed as mean ± SEM obtained from 3 independent experiments. (k) Percentage of total Rac1-GTP accumulated at the uropods of Y27632 or vehicle treated cells. n=3 mice. Mean ± SEM. (l) Quantitative analysis of RhoA activity in wild type (WT) and JFC1−/− neutrophils that have been left untreated (NS) or stimulated with fMLF for 30 seconds (30”) or for 3 minutes (3’). Mean ± SEM, n=3. *, p<0.05.

Article Snippet: Expression vectors: GFP- Rac1 WT, GFP- Rac1 Q61L, GFP- Rac1 T17N were obtained from Addgene.

Techniques: Isolation, Fluorescence, Software, Disruption, Inhibition, Confocal Microscopy, Expressing, Activity Assay

Journal: Journal of leukocyte biology

Article Title: The trafficking protein JFC1 regulates Rac1-GTP localization at the uropod controlling neutrophil chemotaxis and in vivo migration

doi: 10.1002/JLB.1VMA0818-320R

Figure Lengend Snippet: A schematic representation showing a proposed model for the sequence of events in a WT vs a JFC1−/− neutrophil. In a WT neutrophil the interaction of JFC1 with Rac1-GTP allows trafficking of Rac1-GTP, thus preventing its accumulation in the uropod and allowing for RhoA cyclic activation and subsequent tail retraction (left panel). In contrast, in a JFC1−/− neutrophil, Rac1-GTP, a proposed negative regulator of RhoA (52) but also suggested to positively regulate uropod-localized RhoA activity (13), accumulates in the uropod, affecting RhoA-activation-deactivation cycling, and causing increased uropod length and thus impaired directional migration (right panel).

Article Snippet: Expression vectors: GFP- Rac1 WT, GFP- Rac1 Q61L, GFP- Rac1 T17N were obtained from Addgene.

Techniques: Sequencing, Activation Assay, Activity Assay, Migration

Journal: Oncogene

Article Title: TIPE1 induces apoptosis by negatively regulating Rac1 activation in hepatocellular carcinoma cells.

doi: 10.1038/onc.2014.208

Figure Lengend Snippet: Figure 5. TIPE1 inhibits Rac1 activation, leading to cell growth repression. (a) Co-immunoprecipitation to test the interaction between TIPE1 and Rac1 in HEK293 cells. (b) Pull-down assay to detect the GTP-Rac1 level in HEK293 cells. (c) Western blot to detect protein levels of p-p65, p65, p-JNK and JNK in HEK293 cells 8 h after transfection with or without pRK5-myc-Rac1-Q61L and increasing amounts of pcTIPE1-HA plasmids. (d) Cytometry analysis of Bel7402 cells after co-transfected with TIPE1 and pRK5-myc-Rac1-Q61L plasmids for 24 h. (e) Rac1 knockdown destroys the increased cell growth induced by TIPE1 siRNA 313 in HepG2.2.15 cells. **Po0.01. TIPE1 and Rac1 levels were determined by reverse transcription–PCR. The experiments were repeated for three times and one representative result is shown.

Article Snippet: Wild-type Rac1 expression plasmid pRK5myc-Rac1-wt, activated Rac1 expression plasmid pRK5-myc-Rac1-Q61L and activated H-Ras expression plasmid pBabe H-Ras 12V were obtained from Addgene (Cambridge, MA, USA).

Techniques: Activation Assay, Immunoprecipitation, Pull Down Assay, Western Blot, Transfection, Cytometry, Knockdown, Reverse Transcription

Journal: Cancers

Article Title: RAC1B Regulation of TGFB1 Reveals an Unexpected Role of Autocrine TGFβ1 in the Suppression of Cell Motility

doi: 10.3390/cancers12123570

Figure Lengend Snippet: RAC1B depletion controls TGFβ1 gene expression and secretion in Panc1 and MDA-MB-231 cells. ( A ) Concentration of bioactive TGFβ1 in culture supernatants, as measured by ELISA, of Panc1 cells genetically engineered to lack exon 3b of RAC1 and hence expression of RAC1B (Panc1 RAC1BKO ) or empty lentiviral vector (LV) control cells (827.4 ± 166.9 vs. 35.2 ± 40.7, n = 4, p = 0.0012, left-hand graph), or Panc RAC1BKD and ctrl cells (393.3 ± 98.2 vs. 264.8.2 ± 98.3, n = 3, p = 0.0038) or Panc1 TGFB1KD and ctrl cells (393.3 ± 98.2 vs. 85.5 ± 38.9, n = 3, p = 0.029, right-hand graph). Cells were allowed to condition the media for 24 h. ( B ) As in ( A ), except that culture supernatants were retrieved from two individual clones (cl.) of MDA-MB-231 cells stably transfected with HA-RAC1B. Data shown are representative of three assays performed in total (means ± SD from triplicate samples). ( C ) Panc1 RAC1BKO cells were subjected to qPCR analyses of TGFB1 ( n = 5), TGFB2 ( n = 3), or TGFB3 ( n = 3), whereas Panc1 RAC1BKD cells underwent qPCR analysis of INHBA . ( D ) MDA-MB-231 RAC1BKD cells were subjected qPCR analysis of TGFB1 . Data in ( C , D ) are the mean ± SD of three different transfections. The asterisks indicate significant differences (* p < 0.05; ** p < 0.01; *** p < 0.001). Successful KO or KD of RAC1B or TGFB1 in Panc1 and MDA-MB-231 cells, and verification of ectopic overexpression of HA-RAC1B in MDA-MB-231 cells is shown in .

Article Snippet: An expression vector for full length human TGFβ1 (cat.# SC119746) was purchased from OriGene Technologies Inc. (Rockville, MD, USA), while a MYC-tagged version of constitutively active RAC1 (Q61L mutation) in the pRK5 vector was a kind gift of G.M.

Techniques: Gene Expression, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Plasmid Preparation, Control, Clone Assay, Stable Transfection, Transfection, Over Expression

Journal: Cancers

Article Title: RAC1B Regulation of TGFB1 Reveals an Unexpected Role of Autocrine TGFβ1 in the Suppression of Cell Motility

doi: 10.3390/cancers12123570

Figure Lengend Snippet: Effects of antibody-mediated neutralization of secreted TGFβ on cell motility. ( A ) MDA-MB-231 cells (10,000/well) were subjected to real-time cell migration assay in culture medium (50 µL) containing only 1% fetal bovine serum (FBS) and in the absence or presence of either vehicle, IgG1 isotype control antibody (Isotype ctrl, 5 µg/mL) or pan-anti-TGFβ1/2/3 antibody (αTGFβ, 5 µg/mL). The assays shown are representative of three assays performed in total. Data represent the mean ± SD of three parallel wells. Differences between IgG1 isotype-treated and αTGFβ-treated cells were first significant at 2:00 and remained so during the course of the assay. The assay shown is representative of three assays. Data represent the mean ± SD of triplicate wells. ( B ) After completion of the assay shown in ( A ), cells were retrieved from the wells and processed for RNA isolation and qPCR analysis of SNAI1 , SMAD3 or RAC1 . SNAI1 rather than SNAI2 was chosen because of its closer association with invasive vs. non-invasive breast carcinomas and the stronger efficiency and potency to bind to the CDH1 promoter and repress its transcription . ECAD expression remained unaltered. Data represent the mean ± SD of triplicate wells. The asterisks indicate statistical significance relative to isotype ctrl treated cells. * p < 0.05.

Article Snippet: An expression vector for full length human TGFβ1 (cat.# SC119746) was purchased from OriGene Technologies Inc. (Rockville, MD, USA), while a MYC-tagged version of constitutively active RAC1 (Q61L mutation) in the pRK5 vector was a kind gift of G.M.

Techniques: Neutralization, Cell Migration Assay, Control, Isolation, Expressing

Journal: Cancers

Article Title: RAC1B Regulation of TGFB1 Reveals an Unexpected Role of Autocrine TGFβ1 in the Suppression of Cell Motility

doi: 10.3390/cancers12123570

Figure Lengend Snippet: Effect of KD of TGFB1 on random cell migration in MDA-MB-231 and Panc1 cells. ( A ) MDA-MB-231 (left-hand graph) or Panc1 (right-hand graph) cells were transiently transfected twice with 50 nM each of irrelevant ctrl siRNA or TGFB1 siRNA and 48 h later subjected to real-time cell migration assays on an xCELLigence platform. The data shown are each representative of three assays. ( B ) Panc1 cells were transfected with 50 nM of ctrl siRNA, or 25 nM each of TGFB1 siRNA + ctrl siRNA, RAC1B siRNA + ctrl siRNA, or a combination of TGFB1 and RAC1B siRNA, and evaluated for migratory activity as in ( A ). Differences between curve B and curve C were first significant at 6:00 and all later time points. ( C ) Panc1 cells stably expressing a HA-tagged version of RAC1B (Panc1 HA-RAC1B ) or empty vector (V) control cells were transfected with TGFB1 or ctrl siRNA and subsequently subjected to impedance-based migration assay. Data in ( A – C ) are the means ± SD of 3–4 wells and are representative of at least 3 independent experiments. Verification of TGFβ1 KD by ELISA is shown in C and elevated TGFB1 expression in Panc1 HA-RAC1B cells in . KD of RAC1B or TGFB1, or overexpression of HA-RAC1B, was verified by immunoblotting (insets in panels A , B and C , respectively) and in . In the inset in panel B, all signals are from the same blot/exposure from which irrelevant lanes have been removed (indicated by thin vertical lines between lanes 2, 3 and 4, see uncropped blot in ). C, ctrl; R, RAC1B; T, TGFB1.

Article Snippet: An expression vector for full length human TGFβ1 (cat.# SC119746) was purchased from OriGene Technologies Inc. (Rockville, MD, USA), while a MYC-tagged version of constitutively active RAC1 (Q61L mutation) in the pRK5 vector was a kind gift of G.M.

Techniques: Migration, Transfection, Activity Assay, Stable Transfection, Expressing, Plasmid Preparation, Control, Enzyme-linked Immunosorbent Assay, Over Expression, Western Blot

Journal: Cancers

Article Title: RAC1B Regulation of TGFB1 Reveals an Unexpected Role of Autocrine TGFβ1 in the Suppression of Cell Motility

doi: 10.3390/cancers12123570

Figure Lengend Snippet: Effects of siRNA-mediated inhibition of TGFβ1 or RAC1B on genes involved in EMT and cell motility. ( A ) Panc1 or MDA-MB-231 cells were transiently transfected twice with 50 nM of either ctrl siRNA, TGFβ1 siRNA, RAC1B siRNA, or a combination of both, as indicated. Forty-eight h later, cells were processed for qPCR analysis of CDH1 and SNAI1 , and GAPDH as an internal control. ( B ) Panc1 TGFB1KD cells were subjected to immunoblot analysis of ECAD, SNAIL, RAC1B, and HSP90 as a loading control. ( C ) As in ( A ), except that Panc1 TGFB1KD cells were subjected to amplification of SMAD3 mRNA and Panc1 SMAD3KD cells subjected to amplification of TGFβ1 mRNA. ( D ) Immunoblot analysis of SMAD3 in Panc1 TGFB1KD cells. ( E ) Immunoblot analysis of ECAD in Panc1 SMAD3KD cells. Data in ( A , C ) represent the mean ± SD of GOI after normalization with the housekeeping genes (TBP or GAPDH) from three independent transfection experiments. The graphs below the blots in panels ( B , D , E ) depict data quantification based on densitometric readings from band intensities after normalization with those for HSP90 (mean ± SD; B , D : n = 5, E : n = 4). The asterisks indicate significant differences (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: An expression vector for full length human TGFβ1 (cat.# SC119746) was purchased from OriGene Technologies Inc. (Rockville, MD, USA), while a MYC-tagged version of constitutively active RAC1 (Q61L mutation) in the pRK5 vector was a kind gift of G.M.

Techniques: Inhibition, Transfection, Control, Western Blot, Amplification

Journal: Cancers

Article Title: RAC1B Regulation of TGFB1 Reveals an Unexpected Role of Autocrine TGFβ1 in the Suppression of Cell Motility

doi: 10.3390/cancers12123570

Figure Lengend Snippet: Effect of ectopic SMAD3 expression on cell migration following RNAi-mediated depletion of endogenous TGFβ1. Panc1 or MDA-MB-231 cells were transfected with 50 nM each of either ctrl siRNA or TGFB1 siRNA in combination with either empty pcDNA3.1 vector or pcDNA3.1 containing a SMAD3 cDNA. Forty-eight h after transfection cells were analyzed by real-time cell migration assay. Data are the means ± SD of parallel 3-4 wells and are representative of 3 independent experiments. The differences between curves D and B were first significant ( p < 0.05) at 3:30 (Panc1) or 3:00 (MDA-MB-231), and for Panc1 cells remained significant over the 24-h assay period. A transient effect of ectopic SMAD3 was also seen in control conditions (curves A vs. curves C, significant between time points 6:30 and 13:00 (Panc1) and 4:00 and 7:30 (MDA-MB-231) in accordance with previous findings ). KD of TGFB1 or overexpression of SMAD3 was verified by immunoblotting (insets), siCo, ctrl siRNA; siTB1, TGFB1 siRNA; V, empty vector; pS3, SMAD3 expression vector.

Article Snippet: An expression vector for full length human TGFβ1 (cat.# SC119746) was purchased from OriGene Technologies Inc. (Rockville, MD, USA), while a MYC-tagged version of constitutively active RAC1 (Q61L mutation) in the pRK5 vector was a kind gift of G.M.

Techniques: Expressing, Migration, Transfection, Plasmid Preparation, Cell Migration Assay, Control, Over Expression, Western Blot

Journal: Cancers

Article Title: RAC1B Regulation of TGFB1 Reveals an Unexpected Role of Autocrine TGFβ1 in the Suppression of Cell Motility

doi: 10.3390/cancers12123570

Figure Lengend Snippet: Cartoon illustrating the pathways through which RAC1B targets TGFβ and its receptors for inhibition in PDAC/TNBC-derived cells. Left, RAC1B promotes expression of SMAD7 and SMAD7-mediated degradation (DG) of ALK5. Right, RAC1B—in a RAC1-dependent or independent manner (see Discussion)—promotes expression and secretion of endogenous/autocrine (aTGFβ1). Its occupation of surface receptors induces their rapid internalization and because receptors are replenished only very slowly the cells adopt a refractory state (desensitization, DS) against acute stimulation with paracrine, stromal cell-derived TGFβ (sTGFβ). The sTGFβ activates ALK5 which phosphorylates SMAD2 and SMAD3 at their C-terminus (pSMAD2/3C), while aTGFβ1—in addition to its effects on receptor turnover—upregulates SMAD3 mRNA and protein abundance. Both pathways converge on the miR-200/ZEB1 autoregulatory loop via intermittent transcriptional activation of miR-200 family members to decrease (pSMAD2/3C) or increase (SMAD3) ECAD expression and to stimulate (pSMAD2/3C) or repress (SMAD3) EMT and cell migration (CM) (denoted by arrows or lines). Upper right, The RAC1B-aTGFβ1-SMAD3 pathway may also induce BGN, which prevents receptor activation by sequestration (SQ) of sTGFβ in the TME. Arrows indicate induction/activation and lines repression/inhibition. Stippled red lines denote a still hypothetical interaction. The bracketed numbers next to arrows/lines denote relevant references.

Article Snippet: An expression vector for full length human TGFβ1 (cat.# SC119746) was purchased from OriGene Technologies Inc. (Rockville, MD, USA), while a MYC-tagged version of constitutively active RAC1 (Q61L mutation) in the pRK5 vector was a kind gift of G.M.

Techniques: Inhibition, Derivative Assay, Expressing, Quantitative Proteomics, Activation Assay, Migration