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: 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: The Journal of Neuroscience

Article Title: The Small G-Protein Rac1 in the Dorsomedial Striatum Promotes Alcohol-Dependent Structural Plasticity and Goal-Directed Learning in Mice

doi: 10.1523/jneurosci.1644-23.2024

Figure Lengend Snippet: Figure 1. Alcohol activates Rac1 specifically in the DMS of male mice. A, Intermittent access to 20% alcohol in two-bottle choice (IA20%2BC) experimental timeline. B–D, The NAc (B), the DLS (C), and the DMS (D) were harvested 4 h into the last drinking session (binge, B) or after 24 h of abstinence (withdrawal, WD). Rac1-GTP pulldown assay was conducted on cell lysates. Levels of GTP-bound Rac1 were normalized to total Rac1 and presented as percentage of the average of the water values. E, After 4 weeks of IA20%2BC, the DMS was harvested, and Rac1-GTP pulldown assay was conducted on cell lysates. Levels of GTP-bound Rac1 were normalized to total Rac1 and presented as the percentage of the average of the water values. F, Female mice underwent IA20%2BC before the DMS was harvested and the percentage of Rac1-GTP was calculated. RhoA-GTP (G) or Cdc42-GTP (H) pulldown assay was conducted on cell lysates after IA20%2BC and quantified using the Western blot. Levels of GTP-bound RhoA or Cdc42 were normalized to total respective protein and presented as the percentage of average of the water values. I, Mice underwent 21 sessions of 10%CA, and the DMS was harvested. Rac1-GTP pulldown assay was conducted on cell lysates and quantified using the Western blot. Rac1-GTP was normalized to total Rac1 and presented as the percentage of average of water values. J, Mice underwent intermittent access to 1% sucrose for 2 weeks, and the DMS was harvested. Rac1-GTP pulldown assay was performed on cell lysates, and Rac1-GTP was normalized to total Rac1 and presented as average of water values. K, Mice underwent 7 weeks of IA20%2BC, and LIMK and cofilin phosphorylation in the DMS were examined using Western blot analysis. The levels of phospho-LIMK (pLIMK) and phospho-cofilin (pCofilin) were normalized to total respective protein and quantified as a percentage of the average of the water values. Data are represented as mean ± SEM and analyzed using one-way ANOVA (C, D, F, K) with post hoc Šidák's multiple-comparison test, Kruskal–Wallis test (B, E, G), or unpaired two-tailed t test (H, I, J). *p < 0.05, **p < 0.01, ***p < 0.001; ns, nonsignificant. n = 4–7 per group.

Article Snippet: The plasmid containing the T17N Rac1 mutation [plasmid 22784, pCyPet-Rac1(T17N)] and AAV-GFP (pAAV.CMV.PI.EGFP.WPRE. bGH; 1.6 × 1013 vg/ml) were purchased from Addgene.

Techniques: Western Blot, Phospho-proteomics, Comparison, Two Tailed Test

Journal: The Journal of Neuroscience

Article Title: The Small G-Protein Rac1 in the Dorsomedial Striatum Promotes Alcohol-Dependent Structural Plasticity and Goal-Directed Learning in Mice

doi: 10.1523/jneurosci.1644-23.2024

Figure Lengend Snippet: Figure 2. Alcohol activation of the LIMK/cofilin signaling pathway and subsequent F-actin formation in the DMS depends on Rac1. A, Rac1-DN mechanism of action. Rac1-DN has a mutation in its P-loop and does not bind GTP. GEFs bind to Rac1-DN but are unable to exchange GDP for GTP. The GEFs remain bound to Rac1-DN and ignore endogenous Rac1. B, HEK293 cells infected with AAV-Rac1-DN imaged in both bright-field and GFP to detect viral infection (2× magnification). Western blot analysis of Rac1 protein, phospho-LIMK, and phosphorylation of ERK1/2 in non- infected cells and cells infected with AAV-Rac1-DN after stimulation with 10% FBS. C, Confirmation of viral overexpression during dissection on the DMS of a mouse infected with AAV-Rac1-DN. A 2× image from EVOS FL tabletop fluorescent microscope. D, E, Left images (4× magnification) depict the specificity of the infection site for AAV-GFP (D) and AAV-Rac1-DN (E); scale bar, 500 μm. Right images (20× magnification) depict solely neurons infected in the DMS by both AAV-GFP (D) and AAV-Rac1-DN (E); scale bar, 100 μm. Each slice is stained with anti-GFP (green) and anti-NeuN (red) antibodies, along with DAPI (magenta). F, Experimental timeline. Mice received bilateral infusion of AAV-Rac1-DN or AAV-GFP in the DMS and were allowed 3 weeks for overexpression. After IA20%2BC or water only for 7 weeks, the DMS was harvested. G, Mice were infected with AAV-GFP or AAV-Rac1-DN before undergoing IA20%2BC. The DMS was harvested, and the total Rac1 protein level was examined using a Western blot and normalized to GAPDH. Data are presented as mean ± SEM and analyzed using two-way ANOVA with post hoc Šidák's multiple-comparison test. ***p < 0.001 ****p < 0.0001; ns, nonsignificant. n = 6 per group. H–J, Phosphorylation of LIMK and cofilin were examined using Western blot analysis.

Article Snippet: The plasmid containing the T17N Rac1 mutation [plasmid 22784, pCyPet-Rac1(T17N)] and AAV-GFP (pAAV.CMV.PI.EGFP.WPRE. bGH; 1.6 × 1013 vg/ml) were purchased from Addgene.

Techniques: Activation Assay, Mutagenesis, Infection, Western Blot, Phospho-proteomics, Over Expression, Dissection, Microscopy, Staining, Comparison

Journal: The Journal of Neuroscience

Article Title: The Small G-Protein Rac1 in the Dorsomedial Striatum Promotes Alcohol-Dependent Structural Plasticity and Goal-Directed Learning in Mice

doi: 10.1523/jneurosci.1644-23.2024

Figure Lengend Snippet: Figure 3. Rac1 promotes remodeling of dendritic arbors in the DMS. Low titer of AAV-GFP or AAV-Rac1-DN (7 × 109–3.5 × 1010 vg/ml) was infused bilaterally into the DMS. After 1 week of recovery, mice underwent 7 weeks of IA20%2BC. Mice consuming water only were used as the control. Twenty-four hours after the last drinking session, mice were perfused, and brains were sliced into 100 μm sections before MSN morphology was analyzed. A, A sample image (40× magnification) of a GFP-positive DMS MSN. Scale bar, 50 μm. B, An example of the reconstruction of the GFP-positive neuron. C, Scheme of the morphological parameters measured in each neuron. Dendritic order increased after each branching point, defined as a dendritic intersection. D, E, Analysis of neuronal dendritic arborization. Sholl analysis was performed on reconstructed neurons. The number of intersections was analyzed for each point (D) and the AUC (E) was calculated. F, Measurement of total dendritic length. G, The number of branching points. H, The number of ending points. I, The number of branches per dendritic order. Data are represented as mean ± SEM and analyzed by two-way ANOVA. Main effect of virus is represented graphically. *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001. Water + AAV-GFP, 19 neurons from 6 mice; alcohol + AAV-GFP, 23 neurons from seven mice; water + AAV-Rac1-DN, 15 neurons from six mice; alcohol + AAV-Rac1-DN, 10 neurons from three mice.

Article Snippet: The plasmid containing the T17N Rac1 mutation [plasmid 22784, pCyPet-Rac1(T17N)] and AAV-GFP (pAAV.CMV.PI.EGFP.WPRE. bGH; 1.6 × 1013 vg/ml) were purchased from Addgene.

Techniques: Control, Virus

Journal: The Journal of Neuroscience

Article Title: The Small G-Protein Rac1 in the Dorsomedial Striatum Promotes Alcohol-Dependent Structural Plasticity and Goal-Directed Learning in Mice

doi: 10.1523/jneurosci.1644-23.2024

Figure Lengend Snippet: Figure 4. Rac1 in the DMS is required for the alcohol-mediated morphological changes in dendritic spines. Low titer of AAV-GFP or AAV-Rac1-DN (7 × 109–3.5 × 1010 vg/ml) was infused bilaterally into the DMS. After 1 week of recovery, mice underwent 7 weeks of IA20%-2BC. Mice consuming water only were used as the control. Twenty-four hours after the last drinking session, mice were perfused, and MSN dendritic spine morphology was analyzed. A, A sample image of a GFP-positive DMS MSN (100× magnification) from each group. Scale bar, 10 μm. B, Average dendritic spine density (number of spines/10 µm). C, Average dendritic spine area (µm2). D, Average dendritic spine volume (µm3). E, Average dendritic spine length (µm). F, Average diameter of dendritic spine heads (µm). G, Dendritic spine length-to-width ratio. H, The percentage of filopodia, thin, stubby, and mushroom dendritic spines for water- and alcohol-drinking mice infected with either AAV-GFP or AAV-Rac1-DN. Data are represented as mean ± SEM and analyzed using two-way ANOVA (B–G) or three-way ANOVA (H) and Šidák's post hoc multiple-comparison test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, ns: non-significant. Water + AAV-GFP: 14 neurons from five mice; alcohol + AAV-GFP, 13 neurons from five mice; water + AAV-Rac1-DN: 14 neurons from five mice; alcohol + AAV-Rac1-DN, 11 neurons from four mice.

Article Snippet: The plasmid containing the T17N Rac1 mutation [plasmid 22784, pCyPet-Rac1(T17N)] and AAV-GFP (pAAV.CMV.PI.EGFP.WPRE. bGH; 1.6 × 1013 vg/ml) were purchased from Addgene.

Techniques: Control, Infection, Comparison

Journal: The Journal of Neuroscience

Article Title: The Small G-Protein Rac1 in the Dorsomedial Striatum Promotes Alcohol-Dependent Structural Plasticity and Goal-Directed Learning in Mice

doi: 10.1523/jneurosci.1644-23.2024

Figure Lengend Snippet: Figure 5. Rac1 does not play a role in the development and maintenance of voluntary alcohol intake. A, Development: surgery and IA20%2BC timeline. Mice received a bilateral, dual infusion of AAV-Rac1-DN or AAV-GFP as a control. After 3 weeks of recovery, IA20%2BC began and continued for 7 weeks. B, Weekly average alcohol consumption (g/kg/24 h) of development drinking mice. C, Water consumption averages for the IA20%2BC development drinking groups. D, Preference for alcohol during the development IA20%2BC drinking, with an index above 50 indicating a preference for alcohol. E, Maintenance: surgery and IA20%2BC timeline. Mice first underwent IA20%2BC for 7 weeks. Groups were balanced based on average daily alcohol consumption in the last six drinking sessions. Mice received a bilateral, dual infusion of AAV-Rac1-DN or AAV-GFP as a control and were allowed 1 full week of recovery. IA20%2BC resumed for 4 weeks. F, Weekly average consumption (g/kg/24 h) for maintenance-drinking mice. G, Water consumption average for maintenance-drinking mice. H, Preference for alcohol for maintenance-drinking mice. All data are represented as mean ± SEM and analyzed with two-way ANOVA with repeated measures. n = 9–10.

Article Snippet: The plasmid containing the T17N Rac1 mutation [plasmid 22784, pCyPet-Rac1(T17N)] and AAV-GFP (pAAV.CMV.PI.EGFP.WPRE. bGH; 1.6 × 1013 vg/ml) were purchased from Addgene.

Techniques: Control

Journal: The Journal of Neuroscience

Article Title: The Small G-Protein Rac1 in the Dorsomedial Striatum Promotes Alcohol-Dependent Structural Plasticity and Goal-Directed Learning in Mice

doi: 10.1523/jneurosci.1644-23.2024

Figure Lengend Snippet: Figure 6. Rac1 in the DMS is required for alcohol-associated goal–directed learning. A, OSA of 20% alcohol paradigm. Mice underwent surgery to overexpress AAV-Rac1-DN or AAV-GFP in the DMS. After 3 weeks of recovery, mice underwent 7 weeks of IA20%2BC to develop a preference for alcohol before transitioning to 20% alcohol OSA for 1 week of training and 4 experimental weeks. B, C, The group average of active (B) and inactive (C) lever presses during the 2 h sessions. D, Discrimination index for the active lever. Calculated as proportion of active lever presses compared with total lever presses. E, Proportion of rewarded lever presses. Calculated as rewarded lever presses compared with total lever presses. F, Alcohol consumption during each session.

Article Snippet: The plasmid containing the T17N Rac1 mutation [plasmid 22784, pCyPet-Rac1(T17N)] and AAV-GFP (pAAV.CMV.PI.EGFP.WPRE. bGH; 1.6 × 1013 vg/ml) were purchased from Addgene.

Techniques:

Journal: The Journal of Neuroscience

Article Title: The Small G-Protein Rac1 in the Dorsomedial Striatum Promotes Alcohol-Dependent Structural Plasticity and Goal-Directed Learning in Mice

doi: 10.1523/jneurosci.1644-23.2024

Figure Lengend Snippet: Figure 7. Rac1 in the DMS is required for alcohol action–outcome associations. A, Contingency degradation paradigm. Mice received a dual infusion of AAV-GFP or AAV-Rac1-DN into the DMS. After a recovery period, mice underwent IA20%2BC for 7 weeks before being trained to operant self-administer alcohol. Mice were first trained on an FR1 schedule before progressing to a RR schedule of reinforcement. Under RR, mice received a reward after a random number of active lever presses within a range (e.g., RR2 1–3 presses, RR3 2–4 presses, RR4 3–5 presses). Mice were trained for 5 sessions at each reinforcement step and spent 10 sessions at RR4 before testing. During the contingency degradation test, there were two types of sessions: ND and D. During nondegraded sessions, rewards were delivered on an RR4 schedule. During degraded sessions, rewards were delivered at a rate equal to the average of the last week of training, but active lever presses had no effect. Testing consisted of a ND session followed by a D session, repeated three times. B, Average active lever presses during ND and D testing sessions for AAV-GFP and AAV-Rac1-DN mice. C, Average port entries during ND and D testing sessions for AAV-GFP and AAV-Rac1-DN mice. Data are represented as mean ± SEM and analyzed using two-way ANOVA with Šidák's post hoc multiple-comparison test. ***p < 0.001, ns: non-significant. n = 7 per group.

Article Snippet: The plasmid containing the T17N Rac1 mutation [plasmid 22784, pCyPet-Rac1(T17N)] and AAV-GFP (pAAV.CMV.PI.EGFP.WPRE. bGH; 1.6 × 1013 vg/ml) were purchased from Addgene.

Techniques: Comparison

Journal: The Journal of Neuroscience

Article Title: The Small G-Protein Rac1 in the Dorsomedial Striatum Promotes Alcohol-Dependent Structural Plasticity and Goal-Directed Learning in Mice

doi: 10.1523/jneurosci.1644-23.2024

Figure Lengend Snippet: Figure 8. Rac1 in the DMS is not required for sucrose goal-directed learning. A, OSA of 1% sucrose paradigm. Mice underwent surgery to overexpress AAV-Rac1-DN or AAV-GFP in the DMS. After 3 weeks of recovery, mice underwent IA20%2BC for 7 weeks. One percent sucrose OSA began with 1 week of training and 4 weeks of experimental tracking. Variables tracked during the session include (B) active lever presses and (C) inactive lever presses. D, Discrimination and (E) proportion of rewarded lever presses were calculated. Data are represented as mean ± SEM and analyzed using RM two-way ANOVA. n = 7, 6.

Article Snippet: The plasmid containing the T17N Rac1 mutation [plasmid 22784, pCyPet-Rac1(T17N)] and AAV-GFP (pAAV.CMV.PI.EGFP.WPRE. bGH; 1.6 × 1013 vg/ml) were purchased from Addgene.

Techniques: