dock1 sirna  (Santa Cruz Biotechnology)

Journal: Current Cancer Drug Targets

Article Title: TBOPP, a DOCK1 Inhibitor, Potentiates Cisplatin Efficacy in Breast Cancer by Regulating Twist-mediated EMT

doi: 10.2174/0115680096281231240202073558

Figure Lengend Snippet: DOCK1 knockdown enhances breast cancer cell sensitivity to cisplatin. ( A ) Validation of DOCK1 knockdown efficiency following the transfection of breast cancer cells with DOCK1 siRNA or negative control, respectively. ( B and C ) Cisplatin sensitivity of breast cancer cells transfected with DOCK1 siRNA or negative control, as measured by a CCK-8 assay. The IC 50 value of the cells was calculated following DOCK1 inhibition. * P < 0.05; ** P < 0.01. ( D ) Cellular proliferation was measured using EdU staining and the EdU-positive cell ratio was calculated. (Scale bar: 100 μm). * P < 0.05; ** P < 0.01. ( E ) A Western blot analysis was performed to evaluate the level of DOCK1 expression in cisplatin-treated breast cancer cells. * P < 0.05. ( F and G ) The level of DOCK1 protein and mRNA expression was detected in breast cancer cells via Western blot and qRT-PCR. ( H ) The IC 50 of cisplatin was positively correlated with the level of DOCK1 mRNA in three breast cells.

Article Snippet: For the cell transfection assay, DOCK1 siRNA, Twist siRNA, or negative siRNA were synthesized by Santa Cruz Biotechnology (CA, USA).

Techniques: Knockdown, Biomarker Discovery, Transfection, Negative Control, CCK-8 Assay, Inhibition, Staining, Western Blot, Expressing, Quantitative RT-PCR

Journal: Current Cancer Drug Targets

Article Title: TBOPP, a DOCK1 Inhibitor, Potentiates Cisplatin Efficacy in Breast Cancer by Regulating Twist-mediated EMT

doi: 10.2174/0115680096281231240202073558

Figure Lengend Snippet: TBOPP can effectively enhance the effect of cisplatin on BC in vivo . ( A ) Representative images of xenograft tumors in BALB/c nude mice after different groups of treatment (Normal group, TBOPP group, Cisplatin group or TBOPP+ Cisplatin group). ( B ) The Growth curves of xenograft tumors in each group was statistically analyzed in 0 days to 14 days. ( C and D ) The Ki-67 expression and cell apoptosis in each group was detected by immunohistochemistry and a TUNEL assay, respectively. * P < 0.05. ( E ) The expression of DOCK1, Twist and Vimentin was measured by qRT-PCR in Normal group, TBOPP group, Cisplatin group or TBOPP+ Cisplatin group. * P < 0.05; ** P < 0.01.

Article Snippet: For the cell transfection assay, DOCK1 siRNA, Twist siRNA, or negative siRNA were synthesized by Santa Cruz Biotechnology (CA, USA).

Techniques: In Vivo, Expressing, Immunohistochemistry, TUNEL Assay, Quantitative RT-PCR

Journal: Current Cancer Drug Targets

Article Title: TBOPP, a DOCK1 Inhibitor, Potentiates Cisplatin Efficacy in Breast Cancer by Regulating Twist-mediated EMT

doi: 10.2174/0115680096281231240202073558

Figure Lengend Snippet: The effect of DOCK1 on EMT. ( A and B ) The level of E-cadherin and Vimentin protein expression was detected in BC cells via Western blot. ( C ) DOCK1 was negatively correlated with the level of E-cadherin mRNA and was positively correlated with the level of Vimentin mRNA in BC cells. ( D and E ) Western blot analyses were performed to evaluate the level of DOCK1, Vimentin, and E-cadherin expression in BC cells transfected with DOCK1 siRNA or NC, respectively. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: For the cell transfection assay, DOCK1 siRNA, Twist siRNA, or negative siRNA were synthesized by Santa Cruz Biotechnology (CA, USA).

Techniques: Expressing, Western Blot, Transfection

Journal: Current Cancer Drug Targets

Article Title: TBOPP, a DOCK1 Inhibitor, Potentiates Cisplatin Efficacy in Breast Cancer by Regulating Twist-mediated EMT

doi: 10.2174/0115680096281231240202073558

Figure Lengend Snippet: The effect of DOCK1 knockdown on EMT in the cisplatin-treated breast cancer cells. ( A-D ) A Western blot was used to detect the level of DOCK1, E-cadherin, and vimentin expression. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: For the cell transfection assay, DOCK1 siRNA, Twist siRNA, or negative siRNA were synthesized by Santa Cruz Biotechnology (CA, USA).

Techniques: Knockdown, Western Blot, Expressing

Journal: Current Cancer Drug Targets

Article Title: TBOPP, a DOCK1 Inhibitor, Potentiates Cisplatin Efficacy in Breast Cancer by Regulating Twist-mediated EMT

doi: 10.2174/0115680096281231240202073558

Figure Lengend Snippet: DOCK1 knockdown promotes cell sensitivity to cisplatin via Twist. ( A ) Heatmap of EMT-associated genes in response to DOCK1 siRNA. ( B ) A CCK-8 assay was performed to determine the viability of three breast cancer cell lines transfected with Twist siRNA or in combination with DOCK1 siRNA. ( C and D ) Western blotting was used to measure the level of DOCK1, Twist, E-cadherin, and vimentin protein expression in three breast cancer cell lines. ** P < 0.01; *** P < 0.001. ( E ) A CCK-8 assay was performed to determine the viability of three breast cancer cell lines transfected with the Twist plasmid or in combination with DOCK1 siRNA. ( F and G ) Western blotting was used to measure the level of DOCK1, Twist, E-cadherin, and vimentin protein expression in three breast cancer cell lines.

Article Snippet: For the cell transfection assay, DOCK1 siRNA, Twist siRNA, or negative siRNA were synthesized by Santa Cruz Biotechnology (CA, USA).

Techniques: Knockdown, CCK-8 Assay, Transfection, Western Blot, Expressing, Plasmid Preparation

Journal: Oncology Letters

Article Title: Downregulation of Dock1 and Elmo1 suppresses the migration and invasion of triple-negative breast cancer epithelial cells through the RhoA/Rac1 pathway

doi: 10.3892/ol.2018.9077

Figure Lengend Snippet: Interaction between Dock1 and Elmo1. (A) Reverse transcription-quantitative polymerase chain reaction and (B) western blot analysis were performed on the expression levels of Dock1 and Elmo1 in MDA-MB-231 cells, MDA-MB-231 cells transfected with an empty vector, MDA-MB-231 cells transfected with si-Dock1 and MDA-MB-231 cells transfected with si-Elmo1. *P<0.05 and **P<0.01 vs. NC. Dock1, dedicator of cytokinesis 1; Elmo1, engulfment and cell motility 1; si, small interfering RNA; NC, negative control.

Article Snippet: Dock1 siRNA (targeting sequence: 5′-GGCCTACACTTTGCTTCTGC-3′), Elmo1 siRNA (targeting sequence: 5′-CGACAAUGUAACUCUGCAA-3′) and unspecific scrambled siRNA (50 nM) (targeting sequence: 5′-ACGUGACACGUUCGGAGAATT-3′) vectors (Invitrogen; Thermo Fisher Scientific, Inc.) were transfected into MDA-MB-231 cells using Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 48 h. Subsequent experiments were performed 48 h after transfection.

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Transfection, Plasmid Preparation, Small Interfering RNA, Negative Control

Journal: Oncology Letters

Article Title: Downregulation of Dock1 and Elmo1 suppresses the migration and invasion of triple-negative breast cancer epithelial cells through the RhoA/Rac1 pathway

doi: 10.3892/ol.2018.9077

Figure Lengend Snippet: Downregulation of Dock1 and Elmo1 inhibits the viability of MDA-MB-231 cells. (A) Cell Counting kit-8 and (B) plate colony formation assays were performed to measure the viability of MDA-MB-231 cells, MDA-MB-231 cells transfected with an empty vector, MDA-MB-231 cells transfected with si-Dock1 and MDA-MB-231 cells transfected with si-Elmo1. *P<0.05 and **P<0.01 vs. NC. Dock1, dedicator of cytokinesis 1; Elmo1, engulfment and cell motility 1; si, small interfering RNA; NC, negative control.

Article Snippet: Dock1 siRNA (targeting sequence: 5′-GGCCTACACTTTGCTTCTGC-3′), Elmo1 siRNA (targeting sequence: 5′-CGACAAUGUAACUCUGCAA-3′) and unspecific scrambled siRNA (50 nM) (targeting sequence: 5′-ACGUGACACGUUCGGAGAATT-3′) vectors (Invitrogen; Thermo Fisher Scientific, Inc.) were transfected into MDA-MB-231 cells using Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 48 h. Subsequent experiments were performed 48 h after transfection.

Techniques: Cell Counting, Transfection, Plasmid Preparation, Small Interfering RNA, Negative Control

Journal: Oncology Letters

Article Title: Downregulation of Dock1 and Elmo1 suppresses the migration and invasion of triple-negative breast cancer epithelial cells through the RhoA/Rac1 pathway

doi: 10.3892/ol.2018.9077

Figure Lengend Snippet: Downregulation of Dock1 and Elmo1 suppresses the migration and invasion abilities of MDA-MB-231 cells. (A) Migration and (B) invasion assays were performed to evaluate the migration and invasion abilities of MDA-MB-231 cells, MDA-MB-231 cells transfected with an empty vector, MDA-MB-231 cells transfected with si-Dock1 and MDA-MB-231 cells transfected with si-Elmo1. *P<0.05, **P<0.01 and ***P<0.001 vs. NC. Dock1, dedicator of cytokinesis 1; Elmo1, engulfment and cell motility 1; si, small interfering RNA; NC, negative control.

Article Snippet: Dock1 siRNA (targeting sequence: 5′-GGCCTACACTTTGCTTCTGC-3′), Elmo1 siRNA (targeting sequence: 5′-CGACAAUGUAACUCUGCAA-3′) and unspecific scrambled siRNA (50 nM) (targeting sequence: 5′-ACGUGACACGUUCGGAGAATT-3′) vectors (Invitrogen; Thermo Fisher Scientific, Inc.) were transfected into MDA-MB-231 cells using Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 48 h. Subsequent experiments were performed 48 h after transfection.

Techniques: Migration, Transfection, Plasmid Preparation, Small Interfering RNA, Negative Control

Journal: Oncology Letters

Article Title: Downregulation of Dock1 and Elmo1 suppresses the migration and invasion of triple-negative breast cancer epithelial cells through the RhoA/Rac1 pathway

doi: 10.3892/ol.2018.9077

Figure Lengend Snippet: Downregulation of Dock1 and Elmo1 reduces Rac1 activity and the expression of migration-associated proteins in MDA-MB-231 cells. (A) A Rac activity assay was performed to evaluate the Rac1 activity of MDA-MB-231 cells. (B) Reverse transcription-quantitative polymerase chain reaction and (C) western blot analysis assays were performed to measure the expression levels of FAK, Talin and Vinculin in MDA-MB-231 cells, MDA-MB-231 cells transfected with an empty vector, MDA-MB-231 cells transfected with si-Dock1 and MDA-MB-231 cells transfected with si-Elmo1. *P<0.05, **P<0.01 and ***P<0.001 vs. NC. Dock1, dedicator of cytokinesis 1; Elmo1, engulfment and cell motility 1; Rac1, Ras-related C3 botulinum toxin substrate 1; FAK, focal adhesion kinase; si, small interfering RNA; NC, negative control.

Article Snippet: Dock1 siRNA (targeting sequence: 5′-GGCCTACACTTTGCTTCTGC-3′), Elmo1 siRNA (targeting sequence: 5′-CGACAAUGUAACUCUGCAA-3′) and unspecific scrambled siRNA (50 nM) (targeting sequence: 5′-ACGUGACACGUUCGGAGAATT-3′) vectors (Invitrogen; Thermo Fisher Scientific, Inc.) were transfected into MDA-MB-231 cells using Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 48 h. Subsequent experiments were performed 48 h after transfection.

Techniques: Activity Assay, Expressing, Migration, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Plasmid Preparation, Small Interfering RNA, Negative Control

Journal: Oncology Letters

Article Title: Downregulation of Dock1 and Elmo1 suppresses the migration and invasion of triple-negative breast cancer epithelial cells through the RhoA/Rac1 pathway

doi: 10.3892/ol.2018.9077

Figure Lengend Snippet: Downregulation of Dock1 and Elmo1 affected the RhoA/Rac1 pathway. Western blot analyses were performed to evaluate the expression levels of (A) RhoA QTPase and RhoA total, and (B) Rac1 GTPase and Rac1 total in MDA-MB-231 cells, MDA-MB-231 cells transfected with an empty vector, MDA-MB-231 cells transfected with si-Dock1 and MDA-MB-231 cells transfected with si-Elmo1. **P<0.01 and ***P<0.001 vs. NC. Dock1, dedicator of cytokinesis 1; Elmo1, engulfment and cell motility 1; RhoA, Ras homolog gene family, member A; Rac1, Ras-related C3 botulinum toxin substrate 1; si, small interfering RNA; NC, negative control.

Article Snippet: Dock1 siRNA (targeting sequence: 5′-GGCCTACACTTTGCTTCTGC-3′), Elmo1 siRNA (targeting sequence: 5′-CGACAAUGUAACUCUGCAA-3′) and unspecific scrambled siRNA (50 nM) (targeting sequence: 5′-ACGUGACACGUUCGGAGAATT-3′) vectors (Invitrogen; Thermo Fisher Scientific, Inc.) were transfected into MDA-MB-231 cells using Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 48 h. Subsequent experiments were performed 48 h after transfection.

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Small Interfering RNA, Negative Control

Journal: Scientific Reports

Article Title: Profiling of miRNAs and target genes related to cystogenesis in ADPKD mouse models

doi: 10.1038/s41598-017-14083-8

Figure Lengend Snippet: Expression validation of key miRNAs and target mRNAs in kidney tissues of Pkd1 conditional knockout mice. ( A ) The expression level of 13 key miRNAs and ( C ) candidate target mRNAs of miR-182-5p were confirmed in kidneys of Pkd1 f/f :HoxB7-Cre at P1, P3, and P7 using quantitative real-time RT-PCR. Target mRNAs included genes related with cystogenesis and the actin cytoskeleton ( Wasf2 , Dock1 , Itga4 ). RNU6 and 18 s rRNA were used as internal controls for miRNA and mRNA, respectively. The experiment was performed in triplicate. n ≥ 3 for each time point. ( B ) Alteration of miR-182-5p expression in Pkd1 f/f :HoxB7-Cre mouse kidney at P7. At P7, the expression pattern of miR-182-5p is detected by section in situ hybridization (ISH) in Pkd1 f/f and Pkd1 f/f :HoxB7-Cre mice. M, medulla; C, cortex; G, glomerulus; Cy, cyst; black arrow, miR-182-expressing glomerulus; black dotted box, high magnification region; red dotted line, cyst lining epithelial cells. scale bars indicate left 200 µm; middle 100 µm; right 50 µm. ( D ) Relative 3′UTR luciferase activity of Wasf2 , Dock1 , and Itga4 genes upon transfecting mIMCD cells with Negative Control mimic (NC mimic) or miR-182-5p mimic. Mutating the seed sequence of miR-182-5p induced rescued luciferase activity of the psiCHECK-2 vector. WT (wild type), MT (mutant type). Data are presented as mean ± SD of three independent experiment in triplicate. *** P < 0.001; ** P < 0.01.

Article Snippet: Small interfering RNA (siRNA) targeting mouse Wasf2 , Dock1 , or Itga4 (Bioneer, Korea) genes were transfected into mIMCD cells at 20 nM using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions for 24 to 48 h. miRNA mimics and inhibitors for mmu-miR-182-5p (Ambion) were reverse-transfected into mIMCD cells at 10 nM using siPORT NeoFX transfection reagent (Ambion) for 24 to 48 h. The control experiments were transfected with scramble miRNA (Pre-miR miRNA Precursor and miR-Vana miRNA inhibitor Molecules, Negative Control #1, Ambion).

Techniques: Expressing, Biomarker Discovery, Knock-Out, Quantitative RT-PCR, In Situ Hybridization, Luciferase, Activity Assay, Negative Control, Sequencing, Plasmid Preparation, Mutagenesis

Journal: Scientific Reports

Article Title: Profiling of miRNAs and target genes related to cystogenesis in ADPKD mouse models

doi: 10.1038/s41598-017-14083-8

Figure Lengend Snippet: Defects in the actin cytoskeleton promote cyst development and growth. ( A , D ) Pkd1, Pkd2, Wasf2 , Dock1 , and Itga4 siRNA-transfected mIMCD cells showed formed larger cysts than control siRNA-transfected mIMCD cells did. After 6 days of mIMCD culture in matrigel, representative images were obtained and cyst lumen sizes were randomly measured in mIMCD spheroids. Each spheroid was stained with rhodamine-labeled phalloidin (red) for F-actin. Nuclei were counterstained with DAPI (blue). ( B ) Expression of Wasf2, Dock1, and Itga4 protein (red) was analyzed by immunofluorescence in kidneys of Pkd1 f/f and Pkd1 f/f :HoxB7-Cre mice. These target proteins were significantly repressed in collecting duct-specific cyst lining epithelial cells (green), but not in normal tubules. Cy, cyst. Scale bar indicates 20 μm. ( C ) Five hours after scratching, mIMCD cells transfected with siRNAs for Wasf2 , Dock1 , or Itga4 formed fewer lamellipodia and reduced expression of actin cytoskeleton proteins on lamellipodia at the leading edge compared with either control siRNA- or negative control mimic-transfected cells. Scale bars represent 25 μm (in the smaller square) and 75 μm. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Small interfering RNA (siRNA) targeting mouse Wasf2 , Dock1 , or Itga4 (Bioneer, Korea) genes were transfected into mIMCD cells at 20 nM using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions for 24 to 48 h. miRNA mimics and inhibitors for mmu-miR-182-5p (Ambion) were reverse-transfected into mIMCD cells at 10 nM using siPORT NeoFX transfection reagent (Ambion) for 24 to 48 h. The control experiments were transfected with scramble miRNA (Pre-miR miRNA Precursor and miR-Vana miRNA inhibitor Molecules, Negative Control #1, Ambion).

Techniques: Transfection, Control, Staining, Labeling, Expressing, Immunofluorescence, Negative Control

Journal: Scientific Reports

Article Title: Profiling of miRNAs and target genes related to cystogenesis in ADPKD mouse models

doi: 10.1038/s41598-017-14083-8

Figure Lengend Snippet: Role of miR-182-5p in renal cystogenesis linked to defects in the actin cytoskeleton. ( A ) Altered protein expression levels observed upon transfection of mIMCD cells with miR-182-5p mimic or miR-182-5p inhibitor were confirmed by western blot analysis. β-Actin was used as an internal control. Each experiment was performed in triplicate. ( B ) Pictures were taken five hours after scratching. Ectopic expression of miR-182-5p prevented the reorganization of actin filaments in mIMCD cells. Scale bars represent 25 μm (in the smaller square), and 75 μm. ( C ) miR-182-5p precursors or inhibitors (20 nM) were transfected into mIMCD cells, and then cells were observed in 3D culture conditions. The cysts in miR-182-5p precursor-treated cells were bigger than cysts in NC treated cells. miR-182-5p inhibitor-transfected mIMCD cells showed reduced of cyst lumen size. Immunostaining with Wasf2, Dock1, and Itga4 (green); F-actin (red); DAPI (blue). *** P < 0.001.

Article Snippet: Small interfering RNA (siRNA) targeting mouse Wasf2 , Dock1 , or Itga4 (Bioneer, Korea) genes were transfected into mIMCD cells at 20 nM using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions for 24 to 48 h. miRNA mimics and inhibitors for mmu-miR-182-5p (Ambion) were reverse-transfected into mIMCD cells at 10 nM using siPORT NeoFX transfection reagent (Ambion) for 24 to 48 h. The control experiments were transfected with scramble miRNA (Pre-miR miRNA Precursor and miR-Vana miRNA inhibitor Molecules, Negative Control #1, Ambion).

Techniques: Expressing, Transfection, Western Blot, Control, Immunostaining

Journal: Scientific Reports

Article Title: Profiling of miRNAs and target genes related to cystogenesis in ADPKD mouse models

doi: 10.1038/s41598-017-14083-8

Figure Lengend Snippet: The effect of miR-182-5p inhibition in attenuating cyst growth in vivo . ( A ) The schedule for the injection study. ( B ) Immunofluorescence staining of kidney sections from control lentivirus- (left) or inhibitory miR-182-5p lentivirus- (right) injected Pkd1 f/f :Aqp2-Cre mice. Red indicates collecting duct, green indicates proximal tubule, and blue indicates the nucleus. ( C ) Serum BUN levels, ( D ) kidney weight-to-body weight ratio, ( E ) expression of miR-182-5p and ( F ) expression of Wasf2, Dock1 , and Itga4 . n = 5, control lentivirus- and n = 6, inhibitory miR-182-5p lentivirus-injected mice, respectively * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Small interfering RNA (siRNA) targeting mouse Wasf2 , Dock1 , or Itga4 (Bioneer, Korea) genes were transfected into mIMCD cells at 20 nM using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions for 24 to 48 h. miRNA mimics and inhibitors for mmu-miR-182-5p (Ambion) were reverse-transfected into mIMCD cells at 10 nM using siPORT NeoFX transfection reagent (Ambion) for 24 to 48 h. The control experiments were transfected with scramble miRNA (Pre-miR miRNA Precursor and miR-Vana miRNA inhibitor Molecules, Negative Control #1, Ambion).

Techniques: Inhibition, In Vivo, Injection, Immunofluorescence, Staining, Control, Expressing

Journal: Oncogene

Article Title: The focal adhesion-associated proteins DOCK5 and GIT2 comprise a rheostat in control of epithelial invasion

doi: 10.1038/onc.2016.345

Figure Lengend Snippet: GIT2 represses DOCK5-regulated and Rac1-dependent cell spreading, as well as FA turnover. HeLa cells were transfected with the indicated shRNA expression vectors and selected in 2.5 μg/ml of puromycin for 72 h. Cells were then cultured for an additional 24 h in the absence of puromycin. ( a ) Phase contrast images of control, GIT2-kd, DOCK1-kd, DOCK5-kd and GIT2-kd+DOCK5-kd cells cultured on collagen. ( b ) Quantification of cell area occupied by control, GIT2-kd, DOCK5-kd and GIT2-kd+DOCK5-kd HeLa cells 1 h after plating on collagen. ( c ) Control and DOCK5-kd cells were plated on collagen and assayed for adhesion after 1 h. All values are means±s.d. ( n =3). ( d ) Quantification of the distribution of areas occupied by control, GIT2-kd, GIT2-kd+Rac1-kd and GIT2-kd+Rac3-kd HeLa cells 1 h after plating on collagen. ( e ) Specificity of NewEast Biosciences antibody for Rac-GTP. Recombinant Rac1 was loaded with either GDP or GTPγS followed by immunoprecipitation with monoclonal antibody to Rac1-GTP and western blotting. ( f ) Localization of Rac-GTP in control, GIT2-kd, DOCK5-kd and GIT2-kd+DOCK5-kd cells 1 h after plating on collagen. ( g ) FA turnover in live HeLa cells expressing paxillin-GFP. Representative confocal images illustrate FA formation over times indicated in control, GIT2-kd, DOCK5-kd and GIT2+DOCK5-kd cells grown on glass. ( h ) Rate (min −1 ) of assembly, disassembly and turnover of FAs as measured by change in GFP fluorescence over time for control, GIT2-kd, DOCK5-kd and GIT2+DOCK5-kd Hela cells, *** P< 0.001, ** P< 0.002, analysis of variance with Tukey–Kramer post hoc testing.

Article Snippet: To determine the requirement for DOCK1 and DOCK5 in GIT2-regulated cell spreading , HeLa cells were transfected with 50 nM of siRNA duplexes targeting DOCK1 (Ambion ID# 145822; GE Healthcare Life Sciences) or DOCK5 (Ambion ID# 130801) using LipofectAmine 2000.

Techniques: Transfection, shRNA, Expressing, Cell Culture, Recombinant, Immunoprecipitation, Western Blot, Fluorescence

Journal: Oncogene

Article Title: The focal adhesion-associated proteins DOCK5 and GIT2 comprise a rheostat in control of epithelial invasion

doi: 10.1038/onc.2016.345

Figure Lengend Snippet: GIT2 modulates a Crk-p130Cas-DOCK5 signaling pathway required cell spreading. ( a ) Puromycin selected control, GIT2-kd, DOCK5-kd or DOCK5 and GIT2-kd cells were plated on collagen-coated plates for 2.5 h. Next, whole-cell lysates were processed for western blotting to detect GIT2, DOCK1, DOCK5 and ERK as loading control, as well as phosphorylated forms and total levels of p130Cas, FAK and paxillin. ( b ) Quantification of phospho-p130Cas, -paxillin, and -FAK levels from a . All values are means±s.d. ( n =3). * P< 0.05; ** P< 0.02; *** P< 0.01 by unpaired two-tailed Student's t -test. ( c ) DOCK5 binds to Crk in a GIT2-regulated manner. HeLa cells with or without GIT2 depletion were plated on collagen-coated plates for 2.5 h. The cells were then lysed and the samples processed for immunoprecipitation with anti-Crk antibody and western blotting to detect Crk, p130Cas and Crk in immunoprecipitates and whole-cell lysates. ( d ) Quantification of the amount of DOCK5 associated with Crk in control and GIT2-kd cells. Values are means±s.d. ( n =3). *** P< 0.01 by unpaired two-tailed Student's t -test. ( e ) Cells with or without knockdown of GIT2 were transfected overnight with a Myc-tagged form of the CBD of DOCK5 comprises amino acids 1712–1871. Transfected cells were plated on collagen for 1 h and labeled to detect Myc (green) and Arp3 (red). ( f ) Quantification of cell area from e . All values are means±s.d. ( n =3). *** P< 0.01 by unpaired two-tailed Student's t -test.

Article Snippet: To determine the requirement for DOCK1 and DOCK5 in GIT2-regulated cell spreading , HeLa cells were transfected with 50 nM of siRNA duplexes targeting DOCK1 (Ambion ID# 145822; GE Healthcare Life Sciences) or DOCK5 (Ambion ID# 130801) using LipofectAmine 2000.

Techniques: Western Blot, Two Tailed Test, Immunoprecipitation, Transfection, Labeling

Journal: Oncogene

Article Title: The focal adhesion-associated proteins DOCK5 and GIT2 comprise a rheostat in control of epithelial invasion

doi: 10.1038/onc.2016.345

Figure Lengend Snippet: DOCK5 localization is controlled by GIT2. ( a ) Localization of DOCK1 (green) or DOCK5 (green) together with paxillin (red) in HeLa cells transfected with DOCK1 or DOCK5 expression constructs. ( b ) Localization of DOCK5 (green) and paxillin (red) in control and GIT2-depleted cells transfected with DOCK5 expression construct and plated on collagen for 1 h. ( c ) HeLa cells were transfected with control, GIT2, and DOCK5 shRNA expression vectors and selected in puromycin as described in . Pooled knockdown cells were plated on collagen for 1 h and labeled to detect DOCK5 (green) and paxillin (red). ( d ) Pooled knockdown cells plated on collagen for 1 h were labeled to detect DOCK5 (green) and Arp3 (red). All scale bars represent 10 μm each.

Article Snippet: To determine the requirement for DOCK1 and DOCK5 in GIT2-regulated cell spreading , HeLa cells were transfected with 50 nM of siRNA duplexes targeting DOCK1 (Ambion ID# 145822; GE Healthcare Life Sciences) or DOCK5 (Ambion ID# 130801) using LipofectAmine 2000.

Techniques: Transfection, Expressing, Construct, shRNA, Labeling

Journal: Oncogene

Article Title: The focal adhesion-associated proteins DOCK5 and GIT2 comprise a rheostat in control of epithelial invasion

doi: 10.1038/onc.2016.345

Figure Lengend Snippet: DOCK5 is required for mammary epithelial cell invasion and wound repair. ( a ) MCF10A cells were cultured on fibronectin. Cell monolayers were labeled to detect DOCK5 (green) and Arp3, cortactin or paxillin (red) 6 h after wounding. Scale bar indicates 20 μm. ( b ) MCF10A cells selected to conditionally express control, GIT2, or GIT2 and DOCK5 shRNA were treated with doxcycline for 72 h. Lysates were immunoblotted to detect GIT2, DOCK5, DOCK1 and ERK. ( c ) To quantify changes in MCF10A invasion, control (black), GIT2-kd (red), or GIT2+DOCK5-kd (blue) cells were harvested and added to the top chamber of Matrigel-coated transwell filters and incubated for 12 h in the presence of 10 ng/ml of EGF as a chemoattractant. Cells that migrated to the bottom chamber were quantified. Values are means±s.d. ( n =3). ( d ) MDA-MB-231 cells transiently transfected with siRNA targeting DOCK5 and DOCK1 were lysed and immunoblotted. ( e ) siRNA control (black), DOCK5-kd (red) and DOCK1-kd (blue) MDA-MB-231 cells were assayed for invasion through Matrigel-coated filters in the presence of 10% serum as a chemoattractant. Values are means ±s.d. ( n =3). ( f ) Immunolocalization of DOCK5 (green) and Arp3 (red) in MDA-MB-231 control and DOCK5-kd cells. Scale bar represents 10 μM. ( g ) Whole-cell lysates of MDA-MB-231 cells were processed for western blotting to detect GIT2, DOCK1, DOCK5 and ERK as loading control, as well at phosphorylated forms and total levels of p190Cas, FAK and paxillin. ( h ) Quantification of phospho-p130Cas, -paxillin and -FAK levels from g . ( i ) MDA-MB-231 cells stably transduced with empty lentiviral vector (control), lentiviral expression constructs encoding wild-type DOCK5 or catalytically inactive DOCK5(ISP) were assayed for invasion through Matrigel-coated filters in the presence of 10% serum as a chemoattractant. All values in this figure are means±s.d. from a minimum of n =3 or more experiments. * P< 0.05; ** P< 0.02; *** P< 0.01 by unpaired two-tailed Student's t -test.

Article Snippet: To determine the requirement for DOCK1 and DOCK5 in GIT2-regulated cell spreading , HeLa cells were transfected with 50 nM of siRNA duplexes targeting DOCK1 (Ambion ID# 145822; GE Healthcare Life Sciences) or DOCK5 (Ambion ID# 130801) using LipofectAmine 2000.

Techniques: Cell Culture, Labeling, shRNA, Incubation, Transfection, Western Blot, Stable Transfection, Transduction, Plasmid Preparation, Expressing, Construct, Two Tailed Test