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: Cancers

Article Title: Rac1 GTPase Regulates the βTrCP-Mediated Proteolysis of YAP Independently of the LATS1/2 Kinases

doi: 10.3390/cancers16213605

Figure Lengend Snippet: YAP levels are regulated by Rac1. ( A ) Impacts of oncogenic K-Ras G12D inhibition on levels of YAP in PC cells. HPAF/CD18 cells, known to express oncogenic K-Ras G12D , were treated with 50 nM MRTX1133 for the indicated times. The experiment was performed in medium supplemented with either 10% FBS or 0.3% FBS. Levels of pERK, YAP, and S127-phosphorylated YAP were measured by immunoblotting. Total ERK was used as a loading control. ( B ) YAP levels in PC cells treated with inhibitors of oncogenic Ras effectors. HPAF/CD18 cells were treated for 16 h with inhibitors of the MAPK pathway (50 μM U0126), PI3K kinase (20 μM LY294002), Rac1 GTPase (50 μM EHT-1864), and PAK1-3 kinases (5 μM FRAX597; 20 μM IPA-3). Levels of YAP and S127-phosphorylated YAP were measured by immunoblotting. Actin was used as a loading control. ( C – F ) YAP levels in a panel of human pancreatic cell lines treated with different Rac1 inhibitors. The indicated cell lines were treated with EHT-1864 (50 μM), NSC23766 (100 μM), and/or Ehop-016 (20 μM). Sixteen hours later, levels of YAP and S127-phosphorylated YAP were measured. Actin was used as an internal standard. ( G ) The siRNA-mediated knockdown of Rac1 reduces YAP levels in PC cells. In duplicate, HPAF/CD18 cells were transfected with Rac1 siRNA (Rac1) or with a non-targeting siRNA (NT). Two days later, levels of Rac1 and YAP were measured. GAPDH was used as an internal standard. Densitometry readings for the intensity ratios of Rac1/GAPDH and YAP/GAPDH, with the value of the first NT-transfected replicate arbitrarily set to 1. The dotted line indicates a lane that was spliced out of the raw imaging data. As shown in the supplement, the two sides of the dotted line are from the same exposure of the same blot. ( H ) Expression of dominant negative Rac1 T17N mutant reduces YAP levels in PC cells. HPAF/CD18 cells were infected for 24 h with adenoviral particles carrying no insert (Ad.CTR; 50 pfu/cell) or expressing Rac1 T17N (Ad.N17Rac1; 50 pfu/cell). Forty-eight hours post-infection, levels of YAP were quantified using western blotting. Actin was used as an internal standard. The right panel shows representative images of the infected cells at 48 h post-infection (100× magnification). The dotted line indicates a lane that was spliced out of the raw imaging data. As shown in the supplement, the two sides of the dotted line are from the same exposure of the same blot. For all panels, the relevant densitometry readings for the indicated intensity ratios (pYAP/ERK, YAP/ERK, pYAP/YAP, pYAP/Actin, YAP/Actin, Rac1/GAPDH, and YAP/GAPDH) are shown below the lanes of each western blot.

Article Snippet: The PCR product encoding myc-tagged Rac1 had been amplified from vector pRK5-myc-Rac1-wt (a gift from Gary Bokoch’s lab; purchased from Addgene cat# 12985) using forward primer 5′-TGGAAGATC TGGCCA CCATGGAGCA-GAAG-3′ (Msc1 site underlined) and reverse primer 5′-TGGC CTCGAG ATAAACAAGTTGGGCCATGGC-3′ (Xho1 site underlined).

Techniques: Inhibition, Western Blot, Control, Knockdown, Transfection, Imaging, Expressing, Dominant Negative Mutation, Mutagenesis, Infection

Journal: Cancers

Article Title: Rac1 GTPase Regulates the βTrCP-Mediated Proteolysis of YAP Independently of the LATS1/2 Kinases

doi: 10.3390/cancers16213605

Figure Lengend Snippet: Activated Rac1 mutant cooperates with E6 and E7 to up-regulate YAP levels. ( A ) Levels of YAP and Rac1 in a panel of PC cell lines and HPNE cells. Levels of the two proteins were quantified by immunoblotting in the indicated cell lines. GAPDH was used as an internal standard. The graph on the right correlates the protein levels of YAP and Rac1. The Spearman’s correlation coefficient (r s ) is shown. Cell lines with wild-type KRAS are labeled with a blue circle and those with oncogenic KRAS have the black circles. ( B ) Transduction of HPNE and HPNE/E6/E7 cells with activated forms of Rac1 and BRAF. HPNE cells (Puro R ) and HPNE/E6/E7 cells (Puro R , Neo R ) were infected with retroviral particles carrying no insert (pLXSH, Hygro R ) or expressing oncogenic mutants of either BRAF (pMSCV-BRAF V600E , Zeocin R ) or Rac1 (pLXSH-Myc-Rac1 G12V , Hygro R ). After 10 days of selection for viral integration, cells were examined for differences in YAP levels and markers of senescence. ( C ) Oncogenic BRAF triggers oncogene-induced senescence in HPNE cells. In triplicates, selected cells were plated at low density, histochemically stained to reveal SA-β-galactosidase activity, and then counter-stained with eosin. Representative images of the stained cells are shown on the left. Scale bars are 200 μm in length. The right graph shows the percentage of senescent cells in each population expressed as a mean +/− S.D. ( n = 3). * Statistically different from the other two populations in a Student’s t -test with p < 0.001. ( D ) Activated Rac1 cooperates with E6/E7 to elevate YAP levels in PC cells. Selected cell populations were analyzed by immunoblotting with the indicated antibodies. GAPDH was used as an internal standard. The dotted line indicates a lane that was spliced out of the raw imaging data. As shown in the supplement, the two sides of the dotted line are from the same exposure of the same blot. ( E ) YAP levels are slightly elevated in HPNE derivatives expressing oncogenic K-Ras G12D . HPNE/E6/E7 and HPNE/E6/E7/st cells and the same expressing oncogenic K-Ras G12D were analyzed for differences in levels of YAP and S127-phosphorylated YAP. Using a GST-PAK1 pulldown assay, levels of GTP-bound Rac1 (GTP-Rac1) were also measured, along with total Rac1 levels. Actin was used as an internal standard. For all panels, the relevant densitometry readings for the indicated intensity ratios (YAP/GAPDH, Rac1/GAPDH, Rac1/ERK, myc/ERK, p16/ERK, pYAP/ERK, YAP/ERK, pERK/ERK, pYAP/YAP, Rac1-GTP/Rac1, pYAP/Actin, and YAP/Actin) are shown below the lanes of each western blot.

Article Snippet: The PCR product encoding myc-tagged Rac1 had been amplified from vector pRK5-myc-Rac1-wt (a gift from Gary Bokoch’s lab; purchased from Addgene cat# 12985) using forward primer 5′-TGGAAGATC TGGCCA CCATGGAGCA-GAAG-3′ (Msc1 site underlined) and reverse primer 5′-TGGC CTCGAG ATAAACAAGTTGGGCCATGGC-3′ (Xho1 site underlined).

Techniques: Mutagenesis, Western Blot, Labeling, Transduction, Infection, Retroviral, Expressing, Selection, Staining, Activity Assay, Imaging

Journal: Cancers

Article Title: Rac1 GTPase Regulates the βTrCP-Mediated Proteolysis of YAP Independently of the LATS1/2 Kinases

doi: 10.3390/cancers16213605

Figure Lengend Snippet: YAP degradation after Rac1 inhibition requires the SCF βTrCP E3 ubiquitin ligase. ( A ) Time course of YAP decline after Rac1 inhibition. HPAF/CD18 cells were harvested at the indicated time point after the addition of EHT-1864 (50 μM). Levels of YAP and S127-phosphorylated YAP were quantified by immunoblotting. GAPDH was used as an internal standard. ( B ) MG132 blocks the degradation of YAP elicited by the inhibition of Rac1. After 12 h of exposure to EHT-1864 (50 μM; +) or vehicle control (−), HPAF/CD18 cells were exposed or not to MG132 (20 μg/mL) for 4 h prior to harvesting. ( C ) Schematic description of YAP structure showing the position and sequence of critical degrons and phosphorylation sites. Upper drawing shows the structure of YAP, including its heterodimerization domain (TEAD), WD40 domains (WW), transactivation domain (TAD), putative degrons (βTrCP1/2, FBXW7), and LATS1/2 phosphorylation sites (S61, S109, S127, S128, S131, S163, S164, and S381). Lower left panel: Sequence of the putative FBXW7 phosphodegron highlighting its consensus and its potentially required phosphoserine group (S351). Changes introduced by the S351A/P352A mutation are also shown. Lower right panel: Sequence of the βTrCP degron of YAP highlighting its consensus and its required CK1 (S384, S387) and LATS1/2 (S381) phosphorylation sites. Changes introduced by the D383A/S384A mutation are also shown. ( D ) Detection of the Flag-YAP proteins in retrovirally infected Panc1 cells. Panc1 cells infected with pLXSH viruses carrying no insert (Empty), Flag-tagged YAP (WT), or its various mutant versions (5SA, D383A/S384A, S351A/P352A) were analyzed for the presence of Flag-YAP. GAPDH was used as an internal control. ( E ) The βTrCP degron is needed for YAP degradation after Rac1 inhibition, but not the S381 LATS1/2 phosphorylation site. In duplicate, Panc1 cells expressing the different mutants of Flag-YAP were exposed to EHT-1864 (50 μM). Sixteen hours later, Flag-tagged proteins were quantified using western blotting. Again, GAPDH was used as an internal control. ( F ) The siRNA-mediated knockdown of Skp1 blocks YAP degradation after Rac1 inhibition. Panc1 cells were transfected with skp1 siRNA or with a non-targeting siRNA. Forty-eight hours later, transfected cells were treated with EHT-1864 (50 μM; +) or vehicle control (−) for 16 h prior to western blot analysis. ( G ) The siRNA-mediated knockdown of the βTrCP1/2 proteins blocks YAP degradation after Rac1 inhibition. Panc1 cells were transfected with a non-targeting siRNA or with siRNA against βTrCP1, βTrCP2, or both proteins. Forty-eight hours later, transfected cells were treated with EHT-1864 (50 μM; +) or vehicle control (−) for 16 h prior to western blot analysis. GAPDH was again used as an internal standard. For all panels, densitometry readings for the indicated intensity ratios (pYAP/GAPDH, YAP/GAPDH, Skp1/GAPDH, and FLAG/GAPDH) are shown below the lanes of each western blot.

Article Snippet: The PCR product encoding myc-tagged Rac1 had been amplified from vector pRK5-myc-Rac1-wt (a gift from Gary Bokoch’s lab; purchased from Addgene cat# 12985) using forward primer 5′-TGGAAGATC TGGCCA CCATGGAGCA-GAAG-3′ (Msc1 site underlined) and reverse primer 5′-TGGC CTCGAG ATAAACAAGTTGGGCCATGGC-3′ (Xho1 site underlined).

Techniques: Inhibition, Ubiquitin Proteomics, Western Blot, Control, Sequencing, Phospho-proteomics, Mutagenesis, Infection, Expressing, Knockdown, Transfection

Journal: Cancers

Article Title: Rac1 GTPase Regulates the βTrCP-Mediated Proteolysis of YAP Independently of the LATS1/2 Kinases

doi: 10.3390/cancers16213605

Figure Lengend Snippet: YAP degradation after Rac1 inhibition is LATS1/2-independent but requires CK1. ( A ) The silencing of LATS1/2 fails to prevent YAP degradation after Rac1 inhibition. Panc1 cells were transfected with a non-targeting siRNA or with siRNA against LATS1, LATS2, or both proteins. Forty-eight hours later, transfected cells were treated with EHT-1864 (50 μM; +) or the vehicle control (−) for 16 h prior to western blot analysis. GAPDH was again used as an internal standard. ( B ) Detection of LATS1 and LATS2 in the LATS1/2-proficient and -deficient HeLa cells. Cells were probed with the indicated antibodies. ( C , D ) LATS1/2 are not needed for YAP degradation after Rac1 inhibition. LATS1/2-proficient and -deficient cells HeLa cells were exposed to 50 μM EHT-1864 ( C ) or the DMSO vehicle ( D ) for 16 h, after which YAP levels were measured. ( E , F ) Panc1 cells expressing the Flag-YAP protein ( E ) or its 5SA mutant ( F ) were exposed or not to CK1 inhibitor IC-261 (10 μM), after which YAP levels were measured. GAPDH was used as an internal standard. For all panels, densitometry readings for the indicated intensity ratios (pYAP/GAPDH, YAP/GAPDH, LATS1/GAPDH, and FLAG/GAPDH) are shown below the lanes of each western blot.

Article Snippet: The PCR product encoding myc-tagged Rac1 had been amplified from vector pRK5-myc-Rac1-wt (a gift from Gary Bokoch’s lab; purchased from Addgene cat# 12985) using forward primer 5′-TGGAAGATC TGGCCA CCATGGAGCA-GAAG-3′ (Msc1 site underlined) and reverse primer 5′-TGGC CTCGAG ATAAACAAGTTGGGCCATGGC-3′ (Xho1 site underlined).

Techniques: Inhibition, Transfection, Control, Western Blot, Expressing, Mutagenesis

Journal: Cancers

Article Title: Rac1 GTPase Regulates the βTrCP-Mediated Proteolysis of YAP Independently of the LATS1/2 Kinases

doi: 10.3390/cancers16213605

Figure Lengend Snippet: A model for the regulation of YAP stability by Rac1 and Ras. Previous studies have revealed complex interactions between the Hippo and Ras pathways, including both positive and negative regulations of MST1/2 and LATS1/2 by the Ras oncogenes. Downstream of Ras, YAP can be activated by the AKT phosphorylation and inhibition of MST2 (Arrow #1) [ , ] or the MAPK-mediated phosphorylation of Ajuba, resulting in LATS1/2 inhibition (Arrow #2) . However, oncogenic Ras can also cause the RASSF1A-dependent activation of the MST2-LATS1 complex, leading to YAP phosphorylation and inhibition (Arrow #3) [ , , ]. These interactions are expected to impact the S381-phosphorylation of YAP, both positively and negatively. This S381 phosphorylation creates a priming site that allows the S384/S387 phosphorylation of YAP (in green) by casein kinase 1 (CK1). This phosphorylation activates a βTrCP degron (boxed) that promotes the polyubiquitination of YAP by the SCF βTrCP ligase and its degradation by the 26S proteasome. In this article, we describe a novel Rac1-regulated pathway that controls the stability of YAP (Arrow #4). This pathway operated outside of the Hippo pathway and did not require the LATS1/2 kinases or the phosphorylation of S381 to regulate YAP stability. Like the Hippo pathway, this other pathway regulated YAP stability in manners that were still dependent on both the integrity of the βTrCP degron and the kinase activity of CK1. We propose that this new pathway activates the βTrCP degron by the direct phosphorylation of S384 by a so-called “DSG” kinase. At the actin cytoskeleton (gray shaded area), the activity of this “DSG” kinase would normally be inhibited by Rac1 and its downstream effectors. Upon Rac1 inhibition, this kinase would rapidly phosphorylate S384, resulting in the CK1 phosphorylation of S387 and full activation of the degron. The existence of a so-called “DSG” kinase has been proposed before to explain the βTrCP-dependent proteolysis of Claspin [ , ].

Article Snippet: The PCR product encoding myc-tagged Rac1 had been amplified from vector pRK5-myc-Rac1-wt (a gift from Gary Bokoch’s lab; purchased from Addgene cat# 12985) using forward primer 5′-TGGAAGATC TGGCCA CCATGGAGCA-GAAG-3′ (Msc1 site underlined) and reverse primer 5′-TGGC CTCGAG ATAAACAAGTTGGGCCATGGC-3′ (Xho1 site underlined).

Techniques: Phospho-proteomics, Inhibition, Activation Assay, Activity Assay

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: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, 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, 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, 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, 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, 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, Activation Assay, Migration