Journal: Genes & Cancer

Article Title: Hax-1 is required for Rac1-Cortactin interaction and ovarian carcinoma cell migration

doi:

Figure Lengend Snippet: (A) Lysates (25 μg) from HOSE, SKOV3, HEYA8, OVCAR3, 2008, and OVCA429 ovarian cancer cells were collected, separated by 10% SDS-PAGE and subjected to immunoblot analysis with antibodies specific to Hax-1 or GAPDH (loading control). Expression levels of Hax-1 were quantified, normalized for the loading control (GAPDH), and the results were plotted as percent increase over the expressions levels seen in HOSE cells (mean ± SEM; n = 3). (B) Two transiently transfected shRNA constructs with Red fluorescent protein tag (RFP) for Hax 1(shHax #1, shHax #3) along with control cells that express non-specific shRNA with RFP (sh-NS) were assessed for Hax-1 expression using immunoblot analysis with antibodies to Hax-1. The blots were then stripped and reprobed with antibodies to GAPDH to monitor equal loading of protein. Expression of Hax-1 was quantified and presented as percent change over Hax-1 levels from control cells that express non-specific shRNA (mean ± SEM; n = 3). Statistical significance was assessed using One tailed t-test (* p <0.05 and ** p<0.001). (C) To determine the migratory potential, 1×10 6 cells, plated in 35 mm plates were allowed to adhere overnight, serum deprived for 16 hours, and treated with 0.5 μM Mitomycin-C to arrest cell division. A linear scratch wound was made across the cell monolayer and cells were stimulated with 10% FBS or 20 μM LPA. Fields of view (10X) were selected at random, photographed and marked for re-identification. The identical fields were re-imaged following 24 hours of incubation and the images presented are representative of three independent experiments, each performed with triplicate fields of view. The percentage of migration in the serum starved (0.2% BSA), 10% FBS stimulated and 20 μM LPA treated groups were calculated in comparison with the control (mean ± SEM; n = 3). Statistical significance was assessed using One tailed t-test. * p <0.05; ** p<0.001.

Article Snippet: pRFP-C-RS scrambled control shRNA construct (TR30015) and eight pRFP-C-RS shRNA constructs (TF312515) against HAX-1 with Chloramphenicol and Puromycin resistance markers were purchased from OriGene Technologies, Inc., Rockville, MD.

Techniques: SDS Page, Western Blot, Expressing, Transfection, shRNA, Construct, One-tailed Test, Incubation, Migration

Journal: Genes & Cancer

Article Title: Hax-1 is required for Rac1-Cortactin interaction and ovarian carcinoma cell migration

doi:

Figure Lengend Snippet: (A) Transient silencing of Hax-1 was carried out by transfecting SKOV3 cells with vectors encoding shRNA specific for Hax-1 (sh-Hax 1 & sh-Hax #3) or scrambled shRNA control for 48 hours (sh-NS). These transfectants were unstimulated (SS), stimulated with LPA (20 μM), or FBS (10%) and their migratory responses were monitored as described under Materials and Methods. At 24 hours following stimulation, images were obtained from random fields of view at 10X magnification. The images shown are representative of three independent experiments, each performed with triplicate fields of view. (B) Cell migration profiles were quantified by enumerating the migrated cells in a minimum of three different fields. Results are presented as the number of migrated cells per field and the bars represent mean ± SEM from there independent experiments. Silencing of endogenous Hax-1 was monitored by immunoblot analysis using antibodies to Hax-1 (inset). The blot was stripped and reprobed with antibodies to GAPDH to monitor equal loading of protein.

Article Snippet: pRFP-C-RS scrambled control shRNA construct (TR30015) and eight pRFP-C-RS shRNA constructs (TF312515) against HAX-1 with Chloramphenicol and Puromycin resistance markers were purchased from OriGene Technologies, Inc., Rockville, MD.

Techniques: shRNA, Migration, Western Blot

Journal: Genes & Cancer

Article Title: Hax-1 is required for Rac1-Cortactin interaction and ovarian carcinoma cell migration

doi:

Figure Lengend Snippet: (A) SKOV3 cells in which the expression of Hax-1 was silenced by shRNA to Hax-1 (sh-Hax #1 and sh-Hax #3) and the control cells expressing non-specific shRNA (shNS) were sequentially immunostained with primary mouse monoclonal Rac1 (1:200) antibody for 1h, washed, incubated with secondary Alexa 647-conjugated goat anti-mouse IgG (1:200, Red) for 1h washed and then stained with Alexa 488-conjugated anti-mouse Cortactin (Millipore, MA) antibody for 1h (1:200, Green). Fluorescent micrographs were collected with a Leica SP2 MP Confocal microscope using 63x Plan APO 1.4 NA oil immersion objective. Colocalization of Rac1 and cortactin (Yellow) were analyzed, on 3 images for each condition per experiment, using NIH ImageJ software. The scale bar 20 μm is common for all channel images. In order to determine the transfection efficiency/ silencing efficiency for Hax-1, the RFP-tagged shRNA transfected cells were fixed with 3% paraformaldehyde and the nuclei were DAPI stained. RFP and DAPI fluorescent micrographs were collected with a Nikon TE2000-E inverted microscope using 20x Plan Fluor NA 0.45 dry objective. (B) Using NIH ImageJ software, numbers of transfected and untransfected cells were counted. The transfection efficiency was calculated as the ratio of transfected to untransfected cells and the graph represents the percentage of transfected cells. (C) For the quantitative determination of the Rac1-cortactin colocalization, Pearson's correlation coefficient (PCC) calculated using JACoP (Just Another Colocalization Plugin) for each transfection condition was normalized to the vector control transfection. Average of the normalized PCC is represented as a graph and the statistical significance was assessed using one-tailed t-test (*p <0.1 and ** p<0.05). (D) SKOV3 cells (1×10 6 ) were transiently transfected with vectors encoding control shRNA (sh-NS) or shRNA specific to Hax-1 (sh-Hax #1 and Hax #3). At 48 hours following transfection, 1mg of the cellular proteins was immunoprecipitated with cortactin ( Left Panel ) or Rac1 ( Right Panel ) antibodies. Immunoprecipitates were resolved by SDS-PAGE and immunoblot analysis was carried out to detect the coimmunoprecipitation of Rac1 or cortactin respectively along with Hax-1. (E) 25 μg of the cell lysates served as input controls and were assessed for the expression levels of Rac1, cortactin, and Hax-1. Reprobing the blots with GAPDH antibody served to monitor equal loading of protein. The experiments were repeated at least three times and the presented results are from a typical experiment.

Article Snippet: pRFP-C-RS scrambled control shRNA construct (TR30015) and eight pRFP-C-RS shRNA constructs (TF312515) against HAX-1 with Chloramphenicol and Puromycin resistance markers were purchased from OriGene Technologies, Inc., Rockville, MD.

Techniques: Expressing, shRNA, Incubation, Staining, Microscopy, Software, Transfection, Inverted Microscopy, Plasmid Preparation, One-tailed Test, Immunoprecipitation, SDS Page, Western Blot

Journal: Genes & Cancer

Article Title: Hax-1 is required for Rac1-Cortactin interaction and ovarian carcinoma cell migration

doi:

Figure Lengend Snippet: (A) SKOV3 control cells transfected with the empty pcDNA3.1+ vector (VC) and SKOV3 cells transiently transfected with HA-epitope tagged domains of Hax-1, Hax-D1, Hax-D2, Hax-D3, Hax-D4, and Hax-D5 (denoted as D1, D2, D3, D4, and D5 respectively), were utilized to analyze the role of Hax-1 domains on cell migration. At 24 hours after transfection, 0.5 μM Mitomycin-C was added to the transfectants (5×10 5 /dish) to prevent cell division. Linear scratch wounds were made with 200 μl pipette tips across the monolayer of cells in the respective dishes to initiate the wound-healing assay. At 0 hour, fields of view (10X) were selected at random, photographed and marked for re-identification. The identical fields were re-imaged after 24 hours of incubation. The images presented are representative of three independent experiments, each performed with triplicate fields of view. (B) Percentage of wound closure and the migration inhibition percentage were calculated based on the migration of the transfectants expressing vector control and the respective domains. The statistical significance was assessed using students-t test. * p<0.05. Error bars are presented as mean ± SEM for triplicate experiments. (C) Expression of the respective Hax-1 domains was monitored by immunostaining with HA-epitope antibody. Three different fields were viewed and three independent experiments were performed. Fluorescent micrographs were collected with a Leica SP2 MP Confocal microscope using 63x Plan APO 1.4 NA oil immersion objective. The results presented here are from a typical experiment and the scale bar is 10 μm for all the images. (D) Expression of HA-tagged Hax-D1-D5 domains (labeled as D1, D2, D3, D4, D5) were monitored along with vector control (VC) by immunoblot analysis using lysates (50 μg) derived from the respective transfectants. Lysates were resolved in 15% SDS-PAGE gels and immunoblotted with an antibody against HA-epitope. The blot was stripped and reprobed with GAPDH antibody to monitor equal loading of protein. The HA-epitope reactive bands were quantified and plotted as percent expression over GAPDH levels (mean ± SEM; n = 3).

Article Snippet: pRFP-C-RS scrambled control shRNA construct (TR30015) and eight pRFP-C-RS shRNA constructs (TF312515) against HAX-1 with Chloramphenicol and Puromycin resistance markers were purchased from OriGene Technologies, Inc., Rockville, MD.

Techniques: Transfection, Plasmid Preparation, Migration, Transferring, Wound Healing Assay, Incubation, Inhibition, Expressing, Immunostaining, Microscopy, Labeling, Western Blot, Derivative Assay, SDS Page

Journal: Genes & Cancer

Article Title: Hax-1 is required for Rac1-Cortactin interaction and ovarian carcinoma cell migration

doi:

Figure Lengend Snippet: (A) pcDNA3-SKOV3 vector control cells (VC) and pcDNA3 Hax D1-D5 domain transfected cells (D1, D2, D3, D4, D5) were immunostained with primary mouse monoclonal Rac1 (1:200) antibody for 1h, washed, incubated with Alexa 568-conjugated goat anti-mouse IgG for 1h (Red), washed and then stained with Alexa 488-conjugated anti-mouse Cortactin (Millipore, MA) antibody for 1h (Green). Fluorescent micrographs were collected with a Leica SP2 MP Confocal microscope using 63x Plan APO 1.4 NA oil immersion objective. Colocalization of Rac and Cortactin (Yellow) were analyzed, on 3 images for each condition per experiment, using NIH ImageJ software. The scale bar 10 μm is common for all images. (B) For the quantification of Rac1-cortactin colocalization, Pearson's correlation coefficient (PCC) calculated using JACoP (Just Another Colocalization Plugin) for each transfection condition was normalized to the vector control transfection and the average of the normalized PCC is represented as a graph (Figure 6-side panel, lower figure). The statistical significance assessed using one-tailed t-test. ** p<0.05. (C) Lysates (25 μg) from cells transfected with vector-control or vectors encoding Hax-D1-D5 domains were subjected to immunoblot analysis to monitor the expression of HA-tagged Hax-1 domains (D1, D2, D3, D4, and D5), endogenous Rac1, cortactin using the respective antibodies. Equal loading of the lysates was monitored by reprobing the blots with GAPDH antibody. The experiments were repeated at least three times and the presented results are from a typical experiment.

Article Snippet: pRFP-C-RS scrambled control shRNA construct (TR30015) and eight pRFP-C-RS shRNA constructs (TF312515) against HAX-1 with Chloramphenicol and Puromycin resistance markers were purchased from OriGene Technologies, Inc., Rockville, MD.

Techniques: Plasmid Preparation, Transfection, Incubation, Staining, Microscopy, Software, One-tailed Test, Western Blot, Expressing

Journal: Genes & Cancer

Article Title: Hax-1 is required for Rac1-Cortactin interaction and ovarian carcinoma cell migration

doi:

Figure Lengend Snippet: LPA, upon binding to one or more LPA receptors, activates Gα 13 to stimulate the invasive migration of carcinoma cell lines [ , ]; Gα 13 in turn stimulates Hax-1 and Hax-1, thus stimulated by the upstream LPA-LPAR signaling promotes the interaction of Rac1 and cortactin through specific domains ( see text ). Hax-1 mediated interaction between Rac1 and cortactin is required for LPA-stimulated migration of the ovarian carcinoma cell line, SKOV3. The role of Hax-1 in subsequent molecular events during cell migration remains to be clarified.

Article Snippet: pRFP-C-RS scrambled control shRNA construct (TR30015) and eight pRFP-C-RS shRNA constructs (TF312515) against HAX-1 with Chloramphenicol and Puromycin resistance markers were purchased from OriGene Technologies, Inc., Rockville, MD.

Techniques: Binding Assay, Migration

Journal: F1000Research

Article Title: Immunoblotting validation of research antibodies generated against HS1-associated protein X-1 in the human neutrophil model cell line PLB-985.

doi: 10.12688/f1000research.6516.2

Figure Lengend Snippet: ( A ) Western blot analysis using rabbit and mouse pre-immune serum from 0.1 × 10 6 , 0.5 × 10 6 , and 1 × 10 6 differentiated PLB-985, control shRNA, and Hax1 shRNA cells. ( B ) Western blot analysis using goat anti-rabbit IgG 680LT only on cell lysates from 0.1 × 10 6 , 0.5 × 10 6 , and 1 × 10 6 differentiated PLB-985, control shRNA and Hax1 shRNA expressing PLB-985 cells. Two predominant background bands can be observed at a relative mobility of 60 and 70 kDa, and one band around 30 kDa. These background bands can also be seen in , , and . ( C ) Subsequent incubation with rabbit and mouse anti-Hax1 from Western blots shown in B demonstrate the appearance of the Hax1 band at the predicted 35 kDa size.

Article Snippet: A mouse Hax1 monoclonal antibody (BD Biosciences) that is routinely used in publications investigating Hax1 , , – directed against Hax1 amino acids 10–148, and a rabbit polyclonal antibody (Proteintech Group, Inc.) directed against the full length Hax1 protein .

Techniques: Western Blot, shRNA, Expressing, Incubation

Journal: F1000Research

Article Title: Immunoblotting validation of research antibodies generated against HS1-associated protein X-1 in the human neutrophil model cell line PLB-985.

doi: 10.12688/f1000research.6516.2

Figure Lengend Snippet: Details of Primary and Secondary Antibodies.

Article Snippet: A mouse Hax1 monoclonal antibody (BD Biosciences) that is routinely used in publications investigating Hax1 , , – directed against Hax1 amino acids 10–148, and a rabbit polyclonal antibody (Proteintech Group, Inc.) directed against the full length Hax1 protein .

Techniques: Concentration Assay

Journal: F1000Research

Article Title: Immunoblotting validation of research antibodies generated against HS1-associated protein X-1 in the human neutrophil model cell line PLB-985.

doi: 10.12688/f1000research.6516.2

Figure Lengend Snippet: Western blot analysis of differentiated PLB-985 cell lysates from 0.1 × 10 6 , 0.5 × 10 6 , and 1 × 10 6 cells from three independent replicates. Mouse anti-tubulin (beta-) is used as a loading control and can be seen present at a relative mobility of 55 kDa in the goat anti-mouse 800 channel. Mouse anti-Hax1 detects a band with a relative mobility of 35 kDa as predicted. Hax1 can be detected in densities of 0.5 × 10 6 and 1 × 10 6 cells.

Article Snippet: A mouse Hax1 monoclonal antibody (BD Biosciences) that is routinely used in publications investigating Hax1 , , – directed against Hax1 amino acids 10–148, and a rabbit polyclonal antibody (Proteintech Group, Inc.) directed against the full length Hax1 protein .

Techniques: Western Blot

Journal: F1000Research

Article Title: Immunoblotting validation of research antibodies generated against HS1-associated protein X-1 in the human neutrophil model cell line PLB-985.

doi: 10.12688/f1000research.6516.2

Figure Lengend Snippet: ( A – C ) Western blot analysis of differentiated PLB-985 cell lysates from 0.1 × 10 6 , 0.5 × 10 6 , and 1 × 10 6 cells expressing either control shRNA or Hax1 shRNA from three independent replicates. Mouse anti-tubulin (beta-) is used as a loading control and can be seen present at a relative mobility of 55 kDa. Both mouse and rabbit anti-Hax1 detects a band at a relative mobility of 35 kDa as predicted. ( D ) Quantification of the band intensities of tubulin and Hax1 relative to control shRNA from three independent Western blots. Error bars indicate standard error of the mean. p values were calculated using paired t-test to assess significance relative to control shRNA.

Article Snippet: A mouse Hax1 monoclonal antibody (BD Biosciences) that is routinely used in publications investigating Hax1 , , – directed against Hax1 amino acids 10–148, and a rabbit polyclonal antibody (Proteintech Group, Inc.) directed against the full length Hax1 protein .

Techniques: Western Blot, Expressing, shRNA

Journal: F1000Research

Article Title: Immunoblotting validation of research antibodies generated against HS1-associated protein X-1 in the human neutrophil model cell line PLB-985.

doi: 10.12688/f1000research.6516.2

Figure Lengend Snippet: Western blot analysis of differentiated PLB-985 cell lysates from 0.1 × 10 6 , 0.5 × 10 6 , and 1 × 10 6 cells from three independent replicates. Mouse anti-tubulin (beta-) is used as a loading control and can be seen present at a relative mobility of 55 kDa. Rabbit anti-Hax1 detects a band at a relative mobility of 35 kDa as predicted. Hax1 can be detected in densities as low as 0.1 × 10 6 cells ( C ).

Article Snippet: A mouse Hax1 monoclonal antibody (BD Biosciences) that is routinely used in publications investigating Hax1 , , – directed against Hax1 amino acids 10–148, and a rabbit polyclonal antibody (Proteintech Group, Inc.) directed against the full length Hax1 protein .

Techniques: Western Blot

Journal: F1000Research

Article Title: Immunoblotting validation of research antibodies generated against HS1-associated protein X-1 in the human neutrophil model cell line PLB-985.

doi: 10.12688/f1000research.6516.2

Figure Lengend Snippet: ( A ) Western blot analysis of differentiated PLB-985 cell lysates from 0.1 × 10 6 , 0.5 × 10 6 , and 1 × 10 6 cells comparing mouse and rabbit anti-Hax1 antibodies. Lysates from the same cell extractions were run on a single SDS-PAGE gel and blotted onto a single nitrocellulose membrane. After transfer, the membrane was divided and probed with either mouse anti-Hax1 or rabbit anti-Hax1. The membranes were imaged simultaneously. ( B ) Quantification of the ratios of Hax1 and tubulin band intensities from three independent blots were measured and plotted. Error bars indicate standard deviation.

Article Snippet: A mouse Hax1 monoclonal antibody (BD Biosciences) that is routinely used in publications investigating Hax1 , , – directed against Hax1 amino acids 10–148, and a rabbit polyclonal antibody (Proteintech Group, Inc.) directed against the full length Hax1 protein .

Techniques: Western Blot, SDS Page, Standard Deviation