Journal: Frontiers in Oncology

Article Title: TUFT1 Promotes Triple Negative Breast Cancer Metastasis, Stemness, and Chemoresistance by Up-Regulating the Rac1/β-Catenin Pathway

doi: 10.3389/fonc.2019.00617

Figure Lengend Snippet: Role of TUFT1 in TNBC migration and invasiveness by promoting the Rac1/β-catenin signaling pathway. (A) Down-regulation of TUFT1 by shRNA dramatically decreased the protein level of Rac1-GTP in MDA-MB-231 cells ( n = 3). (B) Overexpression of TUFT1 increased the protein level of Rac1-GTP in HCC1937 cells ( n = 3). (C) Western blot showed effect of Rac1 inhibitor NSC23766 on Rac1-GTP in MDA-MB-231 cells ( n = 3). (D) Down-regulation of Rac1-GTP treated with NSC23766 decreased expression of β-catenin in TUFT1—MDA-MB-231 and TUFT1—HCC1937 cells ( n = 3). Down-regulation of Rac1-GTP treated with NSC23766 decreased cell invasiveness (E) and migration in (F) in TUFT1—MDA-MB-231 and TUFT1—HCC1937 cells ( n = 3). Results are presented as means ± SD. The statistical significance was assessed by student's t -test; * p < 0.05, ** p < 0.01.

Article Snippet: NSC23766 (obtained from Tocris Bioscience) was used to inhibit Rac1 activation.

Techniques: Migration, shRNA, Over Expression, Western Blot, Expressing

Journal: Frontiers in Oncology

Article Title: TUFT1 Promotes Triple Negative Breast Cancer Metastasis, Stemness, and Chemoresistance by Up-Regulating the Rac1/β-Catenin Pathway

doi: 10.3389/fonc.2019.00617

Figure Lengend Snippet: Role of TUFT1 in TNBC stemness by promoting the Rac1 signaling pathway. (A) Down-regulation of Rac1-GTP treated with NSC23766 decreased expression of Nanog, Sox2, and Oct4 levels in TUFT1—MDA-MB-231 and TUFT1—HCC1937 cells ( n = 3). (B) Down-regulation of Rac1-GTP treated with NSC23766 decreased number of mammary spheres formed by TUFT1 - MDA-MB-231 cells ( n = 3). Results are presented as means ± SD. The statistical significance was assessed by student's t -test; * p < 0.05, ** p < 0.01.

Article Snippet: NSC23766 (obtained from Tocris Bioscience) was used to inhibit Rac1 activation.

Techniques: Expressing

Journal: The Journal of pathology

Article Title: Serotonin uptake is required for Rac1 activation in Kras-induced acinar-to-ductal metaplasia in the pancreas.

doi: 10.1002/path.5147

Figure Lengend Snippet: Figure 1. Inhibition of 5-HT uptake prevents Rac1 activation in pancreatic acinar cells. (A) Molecular modeling of serotonylated Q61 predicts a low probability of water molecule (H2O) access (arrowhead) to GTP for hydrolysis and consequent lock of Rac1 in the GTP-bound state. 5-HT (carbon atoms in purple) was modeled by a covalent bond to the side chain of Q61 according to the crystal structure of wild-type Rac1 [green ribbon; Protein Data Bank (PDB) code 3TH5]. The GTP-mimic inhibitor (non-hydrolysable GNP, phosphoaminophosphonic acid-guanylate ester) is shown (carbon atoms in cyan) together with the magnesium ion (marine blue sphere). The leucine side chain of the Q61L mutant is also shown (gray; PDB code 4GZL). Nitrogen, oxygen and phosphorus atoms are in blue, red, and orange, respectively. Left panel: full view of the protein. Right panel: enlarged view of the catalytic site. Illustration prepared with PyMol (Pymol.org by Schroedinger). (B) Rac1 G-LISA showed lack of Rac1 activation 60 min after cerulein (Cer) injection in Tph1−/−mice lacking peripheral 5-HT. (C) Liquid scintillation counting showed that 20 μM fluoxetine (FL) prevented [3H]5-HT uptake in both freshly isolated pancreatic acini and AR42J acinar cells. Micrographs of cells are shown. (D) Quantification of [3H]5-HT by liquid scintillation counting in total cell lysate and trichloroacetic acid-precipitated proteins of AR42J cells. (E) Schematic representation of the FRET sensor used. FRET was performed with the donor (CFP) recovery after acceptor (YFP) photobleaching method. Lower panels: confocal images of Cer-stimulated AR42J cells before and after photobleaching. (F) Time-course quantification of Rac1 activation by FRET assay in Cer-stimulated AR42J cells in the presence of 20 μM FL. (G) Immunoblotting showed lack of Pak1 phosphorylation in Cer-stimulated AR42J cells pretreated with 20 μM FL. Mean ± SEM (n ≥3), *p < 0.01. Ctrl, control; NS, not significant; OD, optical density; pPak, phosphorylated Pak. Scale bars: 100 μm [except in (E): 10 μm].

Article Snippet: Biochemical reagents Stock solutions for fluoxetine (Tocris, Bristol, UK), the Rac1 inhibitor NSC23766 (Tocris) and cerulein (Sigma, Buchs, Switzerland) were made in 0.9% NaCl.

Techniques: Inhibition, Activation Assay, Mutagenesis, Injection, Isolation, Western Blot, Phospho-proteomics, Control

Journal: The Journal of pathology

Article Title: Serotonin uptake is required for Rac1 activation in Kras-induced acinar-to-ductal metaplasia in the pancreas.

doi: 10.1002/path.5147

Figure Lengend Snippet: Figure 2. Fluoxetine (FL) treatment reduces ADM lesion formation in a 3D culture system. (A) Freshly isolated KrasG12D-derived acinar cells (day 0) transdifferentiated into ADM during a period of 6 days after collagen embedding, as demonstrated by structural rearrangements. (B and C) RT-qPCR revealed that ADM transdifferentiation was accompanied by downregulation of adult acinar cell markers (B) and upregulation of ADM markers (C). (D) RT-qPCR of Rac1 signaling components. (E) RT-qPCR of 5-HT signaling-related genes. (F) RT-qPCR for Tph1 expression in KrasG12D as compared with WT acini. (G) Schematic representation of in vitro drug treatment: 20 μM FL, 100 μM iRac1 and 1 mM Cys were applied daily on collagen-embedded acini according to a preventive (from day 0) or therapeutic (from day 4) regimen. (H) Representative micrographs of ADM structures following 4 days of preventive pharmacological treatment. (I) Quantification of newly formed ADMs at the indicated time points following preventive pharmacological treatment. Note the complete abolition of ADM upon FL treatment (red columns). (J) Actin (phalloidin, green) and nuclear [4′,6-diamidino-2-phenylindole (DAPI), blue] staining showing structural destabilization of ADM after 4 days of preventive FL treatment. (K) Left panels: representative micrographs of ADM upon therapeutic FL treatment. Right panel: quantification of ADM diameter. (L) Quantification of newly formed ADMs at the indicated time points following treatment with 10 μM 5-HT2B inhibitor SB204741 (SB). Insets: representative micrographs of ADM structures following 4 days of pharmacological treatment. Mean ± SEM (n = 3), *p < 0.05. Scale bars: 25 μm. Ctrl, control.

Article Snippet: Biochemical reagents Stock solutions for fluoxetine (Tocris, Bristol, UK), the Rac1 inhibitor NSC23766 (Tocris) and cerulein (Sigma, Buchs, Switzerland) were made in 0.9% NaCl.

Techniques: Isolation, Derivative Assay, Quantitative RT-PCR, Expressing, In Vitro, Staining, Control

Journal: The Journal of pathology

Article Title: Serotonin uptake is required for Rac1 activation in Kras-induced acinar-to-ductal metaplasia in the pancreas.

doi: 10.1002/path.5147

Figure Lengend Snippet: Figure 3. Fluoxetine (FL) treatment reduces the formation of premalignant lesions in vivo. (A) Hematoxylin and eosin (HE) staining showing premalignant lesions in KrasG12D mice at the ages of 5 weeks (early lesions) and 10 weeks (established lesions) as compared with WT mice. (B) RT-qPCR of Tiam1 [Rac1 upstream guanine nucleotide exchange factors (GEF)] and Pak1 (Rac1 downstream effector) in pancreata of 5-week-old and 10-week-old WT and KrasG12D mice. (C) Rac1 immunofluorescence staining (red). Nuclei are stained with 4′,6-diamidino-2-phenylindole (blue). Note Rac1 localization on apical membranes in WT acini (arrows) and re-localization to the basolateral membranes in KrasG12D-driven ADM lesions (arrowheads). (D) Expression of 5-HT signaling-related genes in pancreata of 5-week-old and 10-week-old WT and KrasG12D mice. (E) HE and mucin staining were utilized to measure the area occupied by abnormal tissue and PanIN type I lesions, respectively, upon FL treatment in KrasG12D mice. Lesion area is expressed as percentage of total tissue area. (F) α-SMA and collagen staining showed reduced stromal reaction upon FL treatment. (G) F4/80 and CD3 staining showed reduced inflammatory reaction upon FL treatment. (H) Von Willebrand factor (vWF) staining and positive vessel quantification in control and FL-treated KrasG12D mice. Mean ± SEM (n ≥4), *p < 0.05. Scale bars: 50 μm.

Article Snippet: Biochemical reagents Stock solutions for fluoxetine (Tocris, Bristol, UK), the Rac1 inhibitor NSC23766 (Tocris) and cerulein (Sigma, Buchs, Switzerland) were made in 0.9% NaCl.

Techniques: In Vivo, Staining, Quantitative RT-PCR, Immunofluorescence, Expressing, Control

Journal: Oncotarget

Article Title: Hypoxia/reoxygenation-experienced cancer cell migration and metastasis are regulated by Rap1- and Rac1-GTPase activation via the expression of thymosin beta-4

doi:

Figure Lengend Snippet: (A) HeLa cells were transfected with Rap1N17 or control plasmid (Mock) vectors for 24 h. Rac1 and Rap1 activity were detected by GST-pulldown and western blotting. (B) HeLa cells were treated with 50 μM CPT, and GST-pulldown was used to detect Rap1 and Rac1 activation. (C) HeLa cells were treated with a Rac1-GTPase inhibitor, NSC23766 (40 or 60 μM), or DMSO for 12 h. Rac1 and Rap1 activities were detected by GST-pulldown and western blotting. (D–F) HeLa cells were transfected with His-Rac1 V12, His-Rac1 N17, or pCDNA3.1 control (Mock) plasmid vectors for 24 h, and assayed for Rac1 and Rap1 activities by GST-pulldown and western blotting (D). Tβ4 transcript levels were measured by RT-PCR (E, left) or realtime PCR (E, right). Tβ4 protein levels were detected by western blotting (F). (G) Cells were transfected with siRNA-Tβ4 or scrambled control siRNA for 24 h, and incubated in the presence or absence of NSC23766; Rac1 and Rap1 activities were detected by GST-pulldown and western blotting. Data shown are representative of three independent experiments (A–G). Band intensities were normalized relative to controls using NIH image analysis software (Image J, version 1.62). Fold changes relative to the control are indicated under each band (A–D, F, and G). Data in bar graph are presented as means ± SD (E). * p < 0.05; ** p < 0.01 relative to the control (A–G).

Article Snippet: NSC23766 trihydrochloride was purchased from Axon Medchem (Groningen, Netherlands), and dissolved in dimethyl sulfoxide (DMSO) with the final concentration not exceeding 0.05%.

Techniques: Transfection, Control, Plasmid Preparation, Activity Assay, Western Blot, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Incubation, Software

Journal: Oncotarget

Article Title: Hypoxia/reoxygenation-experienced cancer cell migration and metastasis are regulated by Rap1- and Rac1-GTPase activation via the expression of thymosin beta-4

doi:

Figure Lengend Snippet: (A–D) B16F10 cells were cultivated in vitro to log phase. Five wild-type C57BL/6 mice were inoculated with 2 × 10 5 B16F10 cells by tail vein injection. NSC23766 was then injected intraperitoneally at a dose of 2.5 mg/kg every 12 h for 12 d. Mice were then sacrificed on day 19, and examined for lung metastasis (A). The number of lung metastases was assessed by counting tumor colonies under a light dissection microscope (B). Rap1 activity in NSC23766-treated and control B16F10 tumor-bearing mouse lung tissues was detected by GST-pulldown and western blotting. Band intensities were normalized relative to controls using NIH image analysis software (Image J, version 1.62). Fold increases relative to the control are indicated under each band (C). RNA was isolated from control and NSC23766-administered mouse lung tissues. Tβ4 transcript levels were measured by RT-PCR (D, top) or realtime PCR (D, bottom). Data in bar graph are presented as means ± SD (B and D). * p < 0.05; ** p < 0.01 relative to saline control (B–D). (E–H) B16F10 cells were transfected with Rac1N17 (E and F), or treated with NSC23766 (G and H) for 24 h. Five wild-type C57BL/6 mice were inoculated with 2 × 10 5 Rac1N17-transfected (E and F) or NSC23766-treated (G and H) B16F10 cells by tail-vein injection. Mice were then sacrificed on day 14, and examined for lung metastasis. (E and G). The number of lung metastases was assessed by counting tumor colonies under a light dissection microscope. Data in bar graph are presented as means ± SD (F and H). ** p < 0.01 relative to mock-treated (F) or B16F10 cell-injected controls (H). Data shown are representative of three independent experiments (A-H).

Article Snippet: NSC23766 trihydrochloride was purchased from Axon Medchem (Groningen, Netherlands), and dissolved in dimethyl sulfoxide (DMSO) with the final concentration not exceeding 0.05%.

Techniques: In Vitro, Injection, Dissection, Microscopy, Activity Assay, Control, Western Blot, Software, Isolation, Reverse Transcription Polymerase Chain Reaction, Saline, Transfection

Journal: Oncotarget

Article Title: Hypoxia/reoxygenation-experienced cancer cell migration and metastasis are regulated by Rap1- and Rac1-GTPase activation via the expression of thymosin beta-4

doi:

Figure Lengend Snippet: (A–E) HeLa cells were treated with NSC23766 (40 μM) or DMSO, and incubated under normoxic or hypoxic conditions for 45 min, followed by reoxygenation for 60 min. Rac1 activity was detected by GST-pulldown and western blotting (A). A confluent monolayer of HeLa cells was then scratched with a sterile pipet tip, and incubated for 18 h under normoxic conditions. Migration of cells into the space left by the scratch was photographed using a phase contrast microscope at 200× magnification (B). The empty area remaining at 18 h was quantified using NIH image analysis software (Image J, version 1.62), and compared to that of the 0-h time point (C). RNA was isolated from cells, and Tβ4 transcript levels measured by RT-PCR (D, top) or realtime PCR (D, bottom). Rap1 activity was detected by GST-pulldown and western blotting (E). Band intensities were normalized relative to controls using NIH image analysis software (Image J, version 1.62). Fold changes relative to the control are indicated under each band A, (E). Data in bar graph are presented as means ± SD (C and D). * p < 0.05; ** p < 0.01 relative to NSC23766-untreated normoxia control. # p < 0.05; ## p < 0.01 relative to NSC23766-untreated hypoxia-reoxygenation group (A–E). (F) NSC23766-treated HeLa cells were subjected to a cell migration assay using a Boyden chamber. Migrated cells were counted at 6, 12, and 18 h after incubation. Data are presented as means ± SD. ** p < 0.01 vs. control at the 0-h time point. ## p < 0.01 relative to NSC23766-untreated group at each time point. (G and H) HeLa cells were then transfected with Rac1N17 or control plasmid vectors for 6 h. A confluent monolayer of HeLa cells was scratched with a sterile pipet tip and incubated for 45 min under normoxia or hypoxia, followed by reoxygenation for 18 h. Migration of cells into the space left by the scratch was photographed using a phase contrast microscope at 200× magnification (G) The empty area remaining at 18 h was quantified using NIH image analysis software (Image J, version 1.62), and compared to that of the 0-h time point. Data in bar graph are presented as means ± SD. ** p < 0.01 relative to NSC23766-untreated normoxia control. & p < 0.05 relative to Rac1N17-untreated hypoxia-reoxygenation group (H). Data shown are representative of three independent experiments (A–H).

Article Snippet: NSC23766 trihydrochloride was purchased from Axon Medchem (Groningen, Netherlands), and dissolved in dimethyl sulfoxide (DMSO) with the final concentration not exceeding 0.05%.

Techniques: Incubation, Activity Assay, Western Blot, Sterility, Migration, Microscopy, Software, Isolation, Reverse Transcription Polymerase Chain Reaction, Control, Cell Migration Assay, Transfection, Plasmid Preparation

Journal: Cellular and Molecular Immunology

Article Title: DOCK2 regulates antifungal immunity by regulating RAC GTPase activity

doi: 10.1038/s41423-021-00835-0

Figure Lengend Snippet: DOCK2 is required for RAC1 activation and C. albicans clearance. WT THP-1 cells and DOCK2-KO THP-1 cells were stimulated with heat-killed C. albicans (MOI = 2) ( A , left) or Mannan (100 μg/ml) ( B , left) for the indicated times, followed by a RAC1 activity assay as described in the Methods section. Densitometric analysis of data from three independent experiments was performed for heat-killed C. albicans (MOI = 2) ( A , right) and α-Mannan (100 μg/mL) ( B , right) stimulation. C , D WT THP-1 cells and DOCK2-KO THP-1 cells were mixed with equal amounts of live C. albicans (MOI = 1) and cocultured for 24 h, followed by crystal violet staining of C. albicans ( C ). Scale bar = 5 μm. The fungal burden ( D ) was determined as described in the Methods section. E WT THP-1 cells overexpressing empty vector or Flag-DOCK2 were mixed with equal amounts of live C. albicans (MOI = 1) and cocultured for 24 h, and the fungal burden was determined as described in the Methods section. F DOCK2-KO THP-1 cells reconstituted with empty vector, WT DOCK2 or DOCK2 V1538A were mixed with equal amounts of live C. albicans (MOI = 1) and cocultured for 24 h, and the fungal burden was determined as described in the Methods section. BMDMs from WT mice were pretreated with NSC23766 (10 μM) for 1 h, followed by stimulation with Curdlan (100 μg/mL) ( G ), Mannan (100 μg/mL) ( H ) or heat-killed C. albicans (MOI = 2) ( I ) for the indicated times. Cell lysates were analyzed by western blotting for the indicated proteins. BMDMs from WT mice were pretreated with NSC23766 (10 μM) for 1 h and were then stimulated with Curdlan (100 μg/mL) ( J ) or Mannan (100 μg/mL) ( K ) for the indicated times, followed by real-time PCR to analyze the expression of the indicated genes. L BMDMs from WT mice were pretreated with NSC23766 (10 μM) for 1 h and were then stimulated with HKCA (MOI = 2), Curdlan (100 μg/mL) or α-Mannan (100 μg/mL) for 24 h, followed by ELISA to measure the production of the indicated proteins. M BMDMs from WT mice were pretreated with NSC23766 (10 μM) for 1 h prior to stimulation with heat-killed C. albicans (MOI = 2) for the indicated times, and cell survival was examined by an MTT assay. * P < 0.05; ** P < 0.01; *** P < 0.001 based on a two-tailed unpaired t test ( A , B , D , E , F , J – M ). All error bars indicate the S.E.M of biological replicates. The data are representative of three independent experiments

Article Snippet: NSC23766 and NAC were purchased from Topscience (cat nos.

Techniques: Activation Assay, Activity Assay, Staining, Plasmid Preparation, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, MTT Assay, Two Tailed Test

Journal: Cellular and Molecular Immunology

Article Title: DOCK2 regulates antifungal immunity by regulating RAC GTPase activity

doi: 10.1038/s41423-021-00835-0

Figure Lengend Snippet: ROS play a critical role in antifungal signaling activation. WT THP-1 cells and DOCK2-KO THP-1 cells were stimulated with Curdlan (100 μg/mL) ( A ), Mannan (100 μg/mL) ( B ) or heat-killed C. albicans (MOI = 2) ( C ) for the indicated times, followed by measurement of ROS production. D WT THP-1 cells were pretreated with NSC23766 (10 μM) for 1 h and were then stimulated with Curdlan (100 μg/mL), α-Mannan (100 μg/mL) or heat-killed C. albicans (MOI = 2) for 30 min, followed by measurement of ROS production via a fluorescent ROS probe. E DOCK2-KO THP-1 cells reconstituted with vector, WT DOCK2 or DOCK2 V1538A were stimulated with Curdlan (100 μg/mL), α-Mannan (100 μg/mL) or heat-killed C. albicans (MOI = 2) for 30 min, followed by measurement of ROS production via a fluorescent ROS probe. F BMDMs from WT mice were pretreated with NAC (10 μM) for 1 h, followed by stimulation with Curdlan (100 μg/mL), Mannan (100 μg/mL) or heat-killed C. albicans (MOI = 2) for the indicated times. Cell lysates were analyzed by western blotting for the indicated proteins. BMDMs from WT mice were pretreated with NAC (10 μM) for 1 h and were then stimulated with Curdlan (100 μg/mL) ( G ), Mannan (100 μg/mL) ( H ) or heat-killed C. albicans (MOI = 2) ( I ) for the indicated times, followed by real-time PCR to analyze the expression of the indicated genes. J BMDMs from WT mice were pretreated with NAC (10 μM) for 1 h and were then stimulated with Curdlan (100 μg/mL), α-Mannan (100 μg/mL) or heat-killed C. albicans (MOI = 2) for 24 h, followed by ELISA of the indicated proteins. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 based on a two-tailed unpaired t test ( D , E , G – J ) or two-way ANOVA ( A–C ). All error bars indicate the S.E.M of biological replicates. The data are representative of three independent experiments

Article Snippet: NSC23766 and NAC were purchased from Topscience (cat nos.

Techniques: Activation Assay, Plasmid Preparation, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test