gfp rac1 t17n  (Addgene inc)

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: 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: 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: 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: GFP- Rac1 WT, GFP- Rac1 Q61L, GFP- Rac1 T17N were obtained from Addgene.

Techniques: Sequencing, Activation Assay, Activity Assay, Migration

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: 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: 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: 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: GFP- Rac1 WT, GFP- Rac1 Q61L, GFP- Rac1 T17N were obtained from Addgene.

Techniques: Sequencing, Activation Assay, Activity Assay, Migration

Journal: PLoS Pathogens

Article Title: Autocrine STAT3 activation in HPV positive cervical cancer through a virus-driven Rac1—NFκB—IL-6 signalling axis

doi: 10.1371/journal.ppat.1007835

Figure Lengend Snippet: A) Representative luciferase reporter assay from C33A cells co-transfected with GFP tagged E6 and a ConA reporter containing tandem κB binding sites . Promoter activity was measured using a dual-luciferase system. Data are presented as relative to the GFP transfected control. B) Representative western blot of C33A and normal human keratinocytes (NHK) cells transiently transfected with GFP or GFP tagged HPV18 E6 and analysed for phosphorylated and total p65 expression. Expression of HPV E6 was confirmed using a GFP antibody in the C33A cells. In NHK cells an antibody directly detecting HPV18 E6 was used to confirm expression of the GFP E6 fusion protein. GAPDH served as a loading control. C) Representative western blot of HeLa cells transfected with a pool of two specific siRNAs targeting HPV18 E6 and analysed for the expression of phosphorylated and total p65. Knockdown of HPV18 E6 was confirmed using an antibody against HPV18 E6 and p53. GAPDH served as a loading control. D-F) C33A cells were co-transfected with GFP, GFP tagged HPV18 E6 or GFP tagged HPV18 E6 and mutant IκBα (IκBm). Cells were then either left untreated or treated with IKK inhibitor VII (IKKi). D) Total RNA was extracted for RT-qPCR analysis of IL-6 expression. Samples were normalized against U6 mRNA levels. Representative data are presented relative to the GFP control. E) Cell lysates were analysed for the expression of phosphorylated and total p65 and IL-6. Expression of HPV E6 was confirmed using a GFP antibody and GAPDH served as a loading control. F) The culture medium was analysed for IL-6 protein by ELISA. G-I) HeLa and CaSKi cells were transfected with mutant IκBα (IκBm) or treated with IKK inhibitor VII (IKKi). G) Total RNA was extracted for RT-qPCR analysis of IL-6 expression. Samples were normalized against U6 mRNA levels. Representative data are presented relative to the DMSO or pcDNA control. H) Cell lysates were analysed for the expression of phosphorylated and total p65 and IL-6. GAPDH served as a loading control. I) The culture medium was analysed for IL-6 protein by ELISA. Bars represent the means ± standard deviation from at least three independent biological repeats. *P<0.05, **P<0.01, ***P<0.001 (Student’s t-test).

Article Snippet: The IκBα S33/36A mutant was a kind gift from Prof Ronald Hay (University of Dundee). pLNCX myr HA AKT1 K179M (AKT-DN) vector was purchased from Addgene (Addgene, 9006) and murine retrovirus envelope and GAG/polymerase plasmids were kindly provided by Professor Greg Towers (University College London). pcDNA-GFP-Rac1-T17N (Rac1-DN) was kindly provided by Professor Adrian Whitehouse (University of Leeds).

Techniques: Luciferase, Reporter Assay, Transfection, Binding Assay, Activity Assay, Western Blot, Expressing, Mutagenesis, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: PLoS Pathogens

Article Title: Autocrine STAT3 activation in HPV positive cervical cancer through a virus-driven Rac1—NFκB—IL-6 signalling axis

doi: 10.1371/journal.ppat.1007835

Figure Lengend Snippet: A) C33A cells were co-transfected with GFP, GFP tagged HPV18 E6 or GFP tagged HPV18 E6 and mutant IκBα (IκBm). Cells were then either left untreated or treated with IKK inhibitor VII (IKKi). Cell lysates were analysed for phosphorylated and total p65, phosphorylated and total STAT3 expression. Expression of HPV E6 was confirmed using a GFP antibody and GAPDH served as a loading control. B) Representative western blot of HeLa cells treated with increasing doses of the IKKα/β inhibitor IKK inhibitor VII (IKKi). Cell lysates were analysed for the expression of phosphorylated and total p65, phosphorylated and total STAT3 and IL-6 expression. GAPDH served as a loading control. C) Representative western blot of HeLa cells transfected with mutant IκBα (IκBm). Cell lysates were analysed as in B). D) C33A cells were serum starved for 24 hours. Cells were then treated with HeLa conditioned media for 2 hours from HeLa cells treated with DMSO or IKKi or transfected with pcDNA or IκBm. Cell lysates were analysed for phosphorylated and total STAT3 expression. GAPDH served as a loading control. E) C33A cells were serum starved for 24 hours. Cells were then treated with HeLa condition media for 2 hours from HeLa cells treated with DMSO or IKKi or transfected with pcDNA or IκBm. Cells were analysed by immunofluorescence staining for total STAT3 (green) and counterstained with DAPI to highlight the nuclei (blue in the merged panels). Scale bar 20 μm. F) C33A were treated with DMSO or IKKi or transfected with pcDNA or IκBm before treatment with 20 ng/mL recombinant human IL-6 for 30 mins. Cells were analysed by immunofluorescence staining for total STAT3 (green) and counterstained with DAPI to highlight the nuclei (blue in the merged panels). Scale bar 20 μm.

Article Snippet: The IκBα S33/36A mutant was a kind gift from Prof Ronald Hay (University of Dundee). pLNCX myr HA AKT1 K179M (AKT-DN) vector was purchased from Addgene (Addgene, 9006) and murine retrovirus envelope and GAG/polymerase plasmids were kindly provided by Professor Greg Towers (University College London). pcDNA-GFP-Rac1-T17N (Rac1-DN) was kindly provided by Professor Adrian Whitehouse (University of Leeds).

Techniques: Transfection, Mutagenesis, Expressing, Western Blot, Immunofluorescence, Staining, Recombinant

Journal: PLoS Pathogens

Article Title: Autocrine STAT3 activation in HPV positive cervical cancer through a virus-driven Rac1—NFκB—IL-6 signalling axis

doi: 10.1371/journal.ppat.1007835

Figure Lengend Snippet: A) C33A cells were co-transfected with GFP, GFP tagged HPV18 E6 or GFP tagged HPV18 E6 and dominant negative AKT (AKT-DN). Cells were then either left untreated or treated with AKT inhibitor VIII (AKTi). Cell lysates were analysed for phosphorylated and total AKT, and phosphorylated and total STAT3 expression. Expression of HPV E6 was confirmed using a GFP antibody and GAPDH served as a loading control. B) Representative western blot of HeLa cells treated with increasing doses of the AKTi. Cell lysates were analysed for the expression of phosphorylated and total AKT, phosphorylated and total STAT3 and IL-6 expression. GAPDH served as a loading control. C) Representative western blot of HeLa cells transfected with dominant negative AKT (AKT-DN). Cell lysates were analysed as in B). D) C33A cells were serum starved for 24 hours. Cells were then treated with HeLa conditioned media for 2 hours from HeLa cells treated with DMSO or AKTi or transfected with pcDNA or AKT-DN. Cell lysates were analysed for phosphorylated and total STAT3 expression. GAPDH served as a loading control. E) C33A cells were serum starved for 24 hours. Cells were then treated with HeLa condition media for 2 hours from HeLa cells treated with DMSO or AKTi or transfected with pcDNA or AKT-DN. Cells were analysed by immunofluorescence staining for total STAT3 (green) and counterstained with DAPI to highlight the nuclei (blue in the merged panels). Scale bar 20 μm. F) C33A were treated with DMSO or AKTi or transfected with pcDNA or AKT-DN before treatment with 20 ng/mL recombinant human IL-6 for 30 mins. Cells were analysed by immunofluorescence staining for total STAT3 (green) and counterstained with DAPI to highlight the nuclei (blue in the merged panels). Scale bar 20 μm.

Article Snippet: The IκBα S33/36A mutant was a kind gift from Prof Ronald Hay (University of Dundee). pLNCX myr HA AKT1 K179M (AKT-DN) vector was purchased from Addgene (Addgene, 9006) and murine retrovirus envelope and GAG/polymerase plasmids were kindly provided by Professor Greg Towers (University College London). pcDNA-GFP-Rac1-T17N (Rac1-DN) was kindly provided by Professor Adrian Whitehouse (University of Leeds).

Techniques: Transfection, Dominant Negative Mutation, Expressing, Western Blot, Immunofluorescence, Staining, Recombinant

Journal: PLoS Pathogens

Article Title: Autocrine STAT3 activation in HPV positive cervical cancer through a virus-driven Rac1—NFκB—IL-6 signalling axis

doi: 10.1371/journal.ppat.1007835

Figure Lengend Snippet: A) Representative western blot of C33A cells transiently transfected with GFP or GFP tagged HPV18 E6 and analysed for active Rac1 using a Rac1 activity assay. Expression of HPV E6 was confirmed using a GFP antibody. GAPDH served as a loading control. B) Representative western blot of HeLa cells transfected with a pool of two siRNAs targeting HPV18 E6 and analysed for active Rac1 using a Rac1 activity assay. Knockdown of HPV18 E6 was confirmed using an antibody against HPV18 E6 and p53. GAPDH served as a loading control. C-E) C33A cells were co-transfected with GFP, HA tagged HPV18 E6 alone or co-transfected with Rac1 N17 (Rac1-DN). Cells were then either left untreated or treated with the Rac1 inhibitor NSC23866 (NSC). C) Total RNA was extracted for RT-qPCR analysis of IL-6 expression. Samples were normalized against U6 mRNA levels. Representative data are presented relative to the GFP control. D) Cell lysates were analysed for the expression of phosphorylated and total p65, phosphorylated and total AKT and IL-6 protein. Expression of HPV E6 was confirmed using a HA antibody, Rac1-DN expression was confirmed using a GFP antibody and GAPDH served as a loading control. E) The culture medium was analysed for IL-6 protein by ELISA. F-H) HeLa and CaSki cells transfected with Rac1-DN or treated with NSC. F) Total RNA was extracted for RT-qPCR analysis of IL-6 expression. Samples were normalized against U6 mRNA levels. Representative data are presented relative to the GFP control. G) Cell lysates were analysed for the expression of phosphorylated and total p65, phosphorylated and total AKT and IL-6. Expression of Rac1-DN was confirmed using a GFP antibody and GAPDH served as a loading control. H) The culture medium was analysed for IL-6 protein by ELISA. Bars represent the means ± standard deviation from at least three independent biological repeats. *P<0.05, **P<0.01, ***P<0.001 (Student’s t-test).

Article Snippet: The IκBα S33/36A mutant was a kind gift from Prof Ronald Hay (University of Dundee). pLNCX myr HA AKT1 K179M (AKT-DN) vector was purchased from Addgene (Addgene, 9006) and murine retrovirus envelope and GAG/polymerase plasmids were kindly provided by Professor Greg Towers (University College London). pcDNA-GFP-Rac1-T17N (Rac1-DN) was kindly provided by Professor Adrian Whitehouse (University of Leeds).

Techniques: Western Blot, Transfection, Activity Assay, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: PLoS Pathogens

Article Title: Autocrine STAT3 activation in HPV positive cervical cancer through a virus-driven Rac1—NFκB—IL-6 signalling axis

doi: 10.1371/journal.ppat.1007835

Figure Lengend Snippet: A) C33A cells were co-transfected with GFP, HA tagged HPV18 E6 or HA tagged HPV18 E6 and Rac1-DN. Cells were then either left untreated or treated with the Rac1 inhibitor NSC23866 (NSC). Cell lysates were then analysed for phosphorylated and total STAT3 expression. Expression of HPV E6 was confirmed using a HA antibody, Rac1-DN expression was confirmed using a GFP antibody and GAPDH served as a loading control. B) Representative western blot of HeLa cells treated with increasing doses of the NSC. Cell lysates were analysed for the expression of phosphorylated and total STAT3 and IL-6 expression. GAPDH served as a loading control. C) Representative western blot of HeLa cells transfected with Rac1-DN. Cell lysates were analysed as in B). D) C33A cells were serum starved for 24 hours. Cells were then treated with HeLa conditioned media for 2 hours from HeLa cells treated with DMSO or NSC or transfected with GFP or Rac1-DN. Cell lysates were analysed for phosphorylated and total STAT3 expression. GAPDH served as a loading control. E) C33A cells were serum starved for 24 hours. Cells were then treated with HeLa condition media for 2 hours from HeLa cells treated with DMSO or NSC or transfected with GFP or Rac1-DN. Cells were analysed by immunofluorescence staining for total STAT3 (green) and counterstained with DAPI to highlight the nuclei (blue in the merged panels). Scale bar 20 μm. F) C33A were treated with DMSO or NSC or transfected with GFP or Rac1-DN before treatment with 20 ng/mL recombinant human IL-6 for 30 mins. Cells were analysed by immunofluorescence staining for total STAT3 (green) and counterstained with DAPI to highlight the nuclei (blue in the merged panels). Scale bar 20 μm.

Article Snippet: The IκBα S33/36A mutant was a kind gift from Prof Ronald Hay (University of Dundee). pLNCX myr HA AKT1 K179M (AKT-DN) vector was purchased from Addgene (Addgene, 9006) and murine retrovirus envelope and GAG/polymerase plasmids were kindly provided by Professor Greg Towers (University College London). pcDNA-GFP-Rac1-T17N (Rac1-DN) was kindly provided by Professor Adrian Whitehouse (University of Leeds).

Techniques: Transfection, Expressing, Western Blot, Immunofluorescence, Staining, Recombinant

Journal: PLoS Pathogens

Article Title: Autocrine STAT3 activation in HPV positive cervical cancer through a virus-driven Rac1—NFκB—IL-6 signalling axis

doi: 10.1371/journal.ppat.1007835

Figure Lengend Snippet: Schematic diagram of E6 mediated activation of STAT3 signalling in HPV+ cervical cancer cells. Through an undetermined mechanism E6 activates a pathway containing the Rac1 GTPase and its substrate AKT resulting in NFκB phosphorylation and IL-6 transcription. IL-6 functions in an autocrine and paracrine manner to induce STAT3 phosphorylation and nuclear translocation. Active STAT3 drives a gene expression programme required for cervical cancer cell proliferation and survival.

Article Snippet: The IκBα S33/36A mutant was a kind gift from Prof Ronald Hay (University of Dundee). pLNCX myr HA AKT1 K179M (AKT-DN) vector was purchased from Addgene (Addgene, 9006) and murine retrovirus envelope and GAG/polymerase plasmids were kindly provided by Professor Greg Towers (University College London). pcDNA-GFP-Rac1-T17N (Rac1-DN) was kindly provided by Professor Adrian Whitehouse (University of Leeds).

Techniques: Activation Assay, Translocation Assay, Expressing

Journal: PLoS ONE

Article Title: Involvement of Local Lamellipodia in Endothelial Barrier Function

doi: 10.1371/journal.pone.0117970

Figure Lengend Snippet: A . Impact of thrombin on GTP-bound RhoA, Rac1, and Cdc42 in cultured HUVEC. Untreated cells served as control. B . Impact of S1P on GTP-bound RhoA, Rac1, and Cdc42 in cultured HUVEC. Untreated cells served as control, and vehicle controls were also tested at the 0.5-min and 10-min time points. The numbers in parentheses indicate the number of replicates for each group. *P<0.05 compared to control (no treatment).

Article Snippet: The pcDNA3-GFP-Rac1 (wild type) and pcDNA3-GFP-Rac1 T17N (dominant negative) plasmids were obtained from Cell Biolabs (San Diego, CA).

Techniques: Cell Culture

Journal: PLoS ONE

Article Title: Involvement of Local Lamellipodia in Endothelial Barrier Function

doi: 10.1371/journal.pone.0117970

Figure Lengend Snippet: A . Rac1-GTP levels in control HUVEC and cells treated with 50 μM NSC23766 for 30 min. B . Time course of mean changes in TER caused by 50 μM NSC23766 (N = 8), compared to control (N = 8). C . Time course of changes in lamellipodia protrusion frequency after the addition of 50 μM NSC23766 (N = 9 cells studied. *P<0.05 versus baseline (BL). D . Time course of changes in the permeability of isolated rat mesenteric venules to albumin in response to 50 μM NSC23766 (N = 4) compared to control (N = 4). *P<0.05 versus control, same time point.

Article Snippet: The pcDNA3-GFP-Rac1 (wild type) and pcDNA3-GFP-Rac1 T17N (dominant negative) plasmids were obtained from Cell Biolabs (San Diego, CA).

Techniques: Permeability, Isolation

Journal: PLoS ONE

Article Title: Involvement of Local Lamellipodia in Endothelial Barrier Function

doi: 10.1371/journal.pone.0117970

Figure Lengend Snippet: A . P s albumin of HUVEC monolayers expressing GFP, GFP-Rac1-WT, or GFP-Rac1-DN (N = 4 for each group) ~16 h after transfection. Panels B , C , and D show expression of each construct, also shown in . These images were obtained ~16 h after transfection. The small arrows indicate lamellipodia, while the arrows with wider arrowheads show filopodia that were prevalent in cells expressing GFP-Rac1-DN. Lamellipodia parameters were also evaluated over a 10-min period: E . Protrusion frequency, F . Protrusion distance, G . Withdrawal Time, H . %Protrusions with a withdrawal time > 5 min. *P<0.05 between the indicated groups. N = 9 cells studied in each group.

Article Snippet: The pcDNA3-GFP-Rac1 (wild type) and pcDNA3-GFP-Rac1 T17N (dominant negative) plasmids were obtained from Cell Biolabs (San Diego, CA).

Techniques: Expressing, Transfection, Construct