abi1 1b9  (MBL Life science)

Journal: bioRxiv

Article Title: Nance-Horan Syndrome-like 1 protein negatively regulates Scar/WAVE-Arp2/3 activity and inhibits lamellipodia stability and cell migration

doi: 10.1101/2020.05.11.083030

Figure Lengend Snippet: (A) Still images from live cell imaging showing NHSL1-EGFP co-expressed with mScarlet-I-tagged Abi1 and (B) Nap1 in B16-F1 cells. Representative images shown from three independent experiments. Scale bar represent 20 µm. Inset is a magnified view of the white box. Scale bar in inset (A) applies to both insets (A,B): 1.25 μm. See also related video S6. (C-E) Endogenous NHSL1 co-localises with Abi1 (C, NHSL1 pAb) Scar/WAVE1 (D, NHSL1 pAb ) , and Scar/WAVE2 (E, NHSL1 mAb ) at the very edge of lamellipodia in B16-F1 mouse melanoma cells. Scale bar in (C) applies also to (D,E): 20 µm. Inset represents a magnified view of the white box. Scale bar in inset in (C) applies also to inset for (D,E): 20 μm. Representative images shown from three independent experiments. See Fig. S8 for line scans of co-localisations in (C) and (E).

Article Snippet: Commercial primary antibodies: EGFP (Roche), Myc (9E10, Sigma), MBP (New England Biolabs), Abi1 (MBL, clone 1B9, D147-3), Abi2 (Epitomics, 3189-1), Scar/WAVE1 (BD 612276), Scar/WAVE2 rabbit mAb (D2C8, CST 3659), ARPC2 (07-227-I-100UG, Millipore).

Techniques: Live Cell Imaging

Journal: bioRxiv

Article Title: Nance-Horan Syndrome-like 1 protein negatively regulates Scar/WAVE-Arp2/3 activity and inhibits lamellipodia stability and cell migration

doi: 10.1101/2020.05.11.083030

Figure Lengend Snippet: (A) The Scar/WAVE complex co-immunoprecipitates with NHSL1. HEK cells were transfected with EGFP-NHSL1, and Myc-Pir121, -Nap-1, -WAVE2, -Abi2. NHSL-1 was immunoprecipitated (pAb 4457) from lysates and co-immunoprecipitation tested on a western blot with Myc and EGFP antibodies. Representative blots from three independent experiments (see Fig. S9A,B for full western blots). (B) and (C) Western blot showing endogenous NHSL1 pulled down with polyclonal NHSL1 (4457) antibody followed by western blotting with (B) Abi1 and (C) Scar/WAVE2 antibodies detecting endogenous co-immunoprecipitation of these proteins in MCF10A (Abi1) and B16-F1 (Scar/WAVE2) cell lysates. Immunoprecipitation using non-immune rabbit IgG served as a negative control. Representative blots from three independent experiments. (D) GST-pull downs using purified Glutathione-sepharose coupled GST-fusion proteins of Abi1 and c-Abl SH3 domains or GST alone from HEK cell lysates that were transfected with EGFP-NHSL1. Following GST-pulldown EGFP-NHSL1 was detected in a western blot with anti-EGFP antibodies. Ponceau staining of membrane reveals GST or GST-tagged Abi- or Abl-SH3 domains used. Representative blots from three independent experiments. (E) Western blot showing immunoprecipitation using NHSL1 polyclonal (4457) antibody or non-immune rabbit IgG control from HEK cell lysates expressing Myc-NHSL1 and all Myc-tagged components of the Scar/WAVE complex, including Myc-Abi1-full-length (E, left), Myc-Abi1-delta-SH3 (E, right). The western blot shows co-immunoprecipitation between NHSL1 and all components of the Scar/WAVE complex only when the Abi SH3 domain is present (Myc-HSPC300 is not shown). Representative blots from three independent experiments. (F) Quantification of band intensity from chemiluminescence from (E) imaged with a CCD camera. Co-immunoprecipitation is reduced by >80%. Bars indicate mean ± SEM (error bars); n = 3; One-way ANOVA: p=0.0002; F(7,16)=8.855; Sidak’s multiple comparisons test: PIR121 **, p=0.0093; Nap1 **, p=0.0028; WAVE2 **, p=0.0074; Abi1 **, p=0.0030. (G) HEK cells were transfected with EGFP-tagged NHSL1 or NHSL1-SH3 binding mutants or EGFP only as negative control and Myc-Abi1. After GFP-trap pulldown of wild type (WT) NHSL1 or different NHSL1-SH3 binding mutants co-precipitation was detected in a western blot with Myc antibody. Representative blots from five independent experiments. Source data are provided as a Source Data file.

Article Snippet: Commercial primary antibodies: EGFP (Roche), Myc (9E10, Sigma), MBP (New England Biolabs), Abi1 (MBL, clone 1B9, D147-3), Abi2 (Epitomics, 3189-1), Scar/WAVE1 (BD 612276), Scar/WAVE2 rabbit mAb (D2C8, CST 3659), ARPC2 (07-227-I-100UG, Millipore).

Techniques: Transfection, Immunoprecipitation, Western Blot, Negative Control, Purification, Staining, Expressing, Binding Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Abi1 loss drives prostate tumorigenesis through activation of EMT and non-canonical WNT signaling

doi: 10.1186/s12964-019-0410-y

Figure Lengend Snippet: ABI1 expression is downregulated during prostate tumor progression. Prostate cancer tissue microarray ( n = 505 patients) was stained for ABI1 to evaluate its expression during prostate tumor progression. ( a ) Representative ABI1 expression staining in Gleason 3, 4, and 5 patterns, as indicated; Benign indicates normal prostate epithelium. ( b ) Comparison of ABI1 expression among Gleason patterns, 3, 4 and 5 indicating significant downregulation of ABI1 in Gleason 5 vs. 3, ( p = 0.0012); or 4 ( p = 0.0025) (one-way ANOVA, multiple comparison). ( c ) Analysis of ABI1 expression in the tissue microarray indicating an association between low ABI1 expression and clinically significant events, such as biochemical recurrence, a metastatic event, and death; p = 0.038; 99% confidence interval: 0.03308–0.4292; Linear-by-Linear association exact test (Sytel Studio-9). ( d ) Representative Western blots showing reduced levels of ABI1 in different patient-derived organoid cell lines (left) and immortalized prostate cancer cell lines (right) compared with non-tumor 26Nb and RWPE-1 cells. Numbers next to bands in the RWPE-1 lane and on the right side of the panel indicate ABI1 isoform designation: top band, isoform 2; middle band, isoform 3; and bottom band, isoform 5 [ , ]; #, indicates the mutant ABI1 lacking exon 6 in LNCaP . GAPDH was used as the loading control. ( e ) Identification of mutations in the cDNA of ABI1 from organoid cultures of human metastatic prostate cancer cell lines. (A. and B.) Schematic of full-length ABI1 cDNA (isoform-1) with coding regions of consecutive exons (Ex1-Ex12) indicated (not drawn to scale). The ATG start codon (corresponding to nucleotide 1), TAA stop codon (corresponding to nucleotide 1527), and the T15, E3, and T33 primer sites (filled-in black arrows) are labeled. The T15, E3, and T33 primers were used to selectively amplify ABI1 cDNA via PCR from the MSK-PCa1 and MSK-PCa2 organoid cell lines. PCR products were subcloned into a pCR-Blunt II TOPO vector and sequenced. A. Schematic of mutation identified in cDNA clone-19 sequence from MSK-PCa1 cells. Sequencing of cDNA clone-19 revealed a 248-bp deletion spanning nucleotides 717 to 964 (from the 3′ end of Exon6, at the junction between Exon 6 and Exon 7, to the middle of Exon 9) that is predicted to result in a frame shift and subsequent stop codon at nucleotide 1170. B. Schematic of mutation identified in cDNA clone-9 from MSK-PCa2 cells. Sequencing of cDNA clone-9 revealed a 44-bp deletion spanning nucleotides 955 to 998 in the middle of Exon 9 that is predicted to result in a frame shift and subsequent stop codon at nucleotide 1047

Article Snippet: The resulting lysate was immunoprecipitated using anti-Abi1 antibody (clone 1B9, MBL) at an antibody:lysate ratio of 1:100.

Techniques: Expressing, Microarray, Staining, Western Blot, Derivative Assay, Mutagenesis, Labeling, Plasmid Preparation, Sequencing

Journal: Cell Communication and Signaling : CCS

Article Title: Abi1 loss drives prostate tumorigenesis through activation of EMT and non-canonical WNT signaling

doi: 10.1186/s12964-019-0410-y

Figure Lengend Snippet: ABI1 KO RWPE-1 cells exhibit reduced WAVE complex levels, which are rescued upon ABI1 re-expression. ( a ) Representative western blots showing reduced levels of different WAVE complex members in the absence of ABI1 in RWPE-1 cells; β-actin was used as the loading control. ( c ) The mRNA levels of all the WAVE complex genes (except ABI1) remained unchanged. The western blots from 3 independent experiments were quantified via densitometry analysis ( b - j ). The graphs were generated in Prism, and the data are represented as the mean ± SEM, unpaired Student’s t-test ( p < 0.05)

Article Snippet: The resulting lysate was immunoprecipitated using anti-Abi1 antibody (clone 1B9, MBL) at an antibody:lysate ratio of 1:100.

Techniques: Expressing, Western Blot, Generated

Journal: Cell Communication and Signaling : CCS

Article Title: Abi1 loss drives prostate tumorigenesis through activation of EMT and non-canonical WNT signaling

doi: 10.1186/s12964-019-0410-y

Figure Lengend Snippet: Loss of membrane localization of adherens junction proteins in ABI1 KO cells. ( a ) Immunostaining images showing localization of the adherens junction proteins (left) E-cadherin and (right) β-catenin. ( b ) Quantification of the number of cells with positive junctional staining per 100 cells is shown. Quantification was performed in 6 independent fields per cell line. The graph was generated with Prism software, and the results are represented as the mean ± SEM, unpaired Student’s t-test ( p < 0.05). ( c ) Western blot analysis of the same proteins showed a modest decrease or no change upon loss of Abi1

Article Snippet: The resulting lysate was immunoprecipitated using anti-Abi1 antibody (clone 1B9, MBL) at an antibody:lysate ratio of 1:100.

Techniques: Immunostaining, Staining, Generated, Software, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Abi1 loss drives prostate tumorigenesis through activation of EMT and non-canonical WNT signaling

doi: 10.1186/s12964-019-0410-y

Figure Lengend Snippet: ABI1 KO cells have increased migratory ability in 2D and 3D cultures. ( a ) Representative images from wound healing (scratch-wound) assays at 0 days and 4 days post-scratch demonstrating increased migration of the ABI1 KO cells. Scale bars represent 200 μm. ( b ) The covered area in images was quantified using ImageJ. The graph represents data from n = 3 experiments (with 3 independent fields per cell line per experiment) normalized to the parental RWPE-1 cell data. The graph was generated with Prism software, and the results are represented as the mean ± SEM, unpaired Student’s t-test; * ( p < 0.05); ** ( p < 0.01). ( c ) Representative images demonstrating altered growth characteristics evidenced by the appearance of the cells in a Matrigel overlay assay. Cells were plated on a chamber slide and overlaid with Matrigel the next day. Images were taken 4 days post-overlay. ABI1 KO cells grew in a 2D culture-like manner (second column), while control RWPE-1 (first column) and ABI1 overexpression cells (third column) primarily grew into spheroids. ( d ) When plated in regular 3D cultures, the ABI1 KO organoids showed invasive/migratory abilities. The images show parental cells, two failed CRISPR control clones (top row) and three different ABI1 KO clones (bottom row) at 4 days post-culture. Scale bars represent 100 μm

Article Snippet: The resulting lysate was immunoprecipitated using anti-Abi1 antibody (clone 1B9, MBL) at an antibody:lysate ratio of 1:100.

Techniques: Migration, Generated, Software, Overlay Assay, Over Expression, CRISPR, Clone Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Abi1 loss drives prostate tumorigenesis through activation of EMT and non-canonical WNT signaling

doi: 10.1186/s12964-019-0410-y

Figure Lengend Snippet: RWPE-1 ABI1 KO spheroids exhibit invasive migratory phenotype in 3D Matrigel cultures. Time-lapse images acquired overnight of representative spheroids showing key features of the spheroids. Cells were plated in 50% Matrigel and allowed to grow for 6 days before live overnight imaging (every 5 min for 14 h). ( a ) RWPE-1 parental, ( b ) Control clone 16, ( c ) ABI1 KO clone 12, ( d ) ABI1 KO clone 35, ( e ) ABI1 rescue Isoform 2, ( f ) ABI1 rescue Isoform 3 cells. ( g - h ) Analysis of the morphology and behavior of the spheroids was performed by quantifying the percentage of organoids exhibiting the following characteristics: spinning, spherical shape, grape-like morphology, projections and cell budding/migration from the spheroid. The control spheroids show a spherical shape, while the KO spheroids do not (black outline). KO spheroids feature projections (black filled arrows) and cells trying to escape from the organoid (white arrows). The data represent n = 3 experiments (18–25 spheroids per cell line per experiment) and were normalized to the parental RWPE-1 cell data. The graph was generated with Prism software, and the results are presented as the mean ± SEM, unpaired Student’s t-test with p -values * ( p < 0.05), ** ( p < 0.01), *** ( p < .001), and **** ( p < 0.0001)

Article Snippet: The resulting lysate was immunoprecipitated using anti-Abi1 antibody (clone 1B9, MBL) at an antibody:lysate ratio of 1:100.

Techniques: Imaging, Migration, Generated, Software

Journal: Cell Communication and Signaling : CCS

Article Title: Abi1 loss drives prostate tumorigenesis through activation of EMT and non-canonical WNT signaling

doi: 10.1186/s12964-019-0410-y

Figure Lengend Snippet: Differential gene expression pattern is correlated with EMT pathways and WNT signaling. The DEGs determined by RNA sequencing demonstrated ( a ) upregulation of EMT pathways and Wnt signaling. Individual genes, fold changes and p-values are listed. Right panel shows representative western blots of several EMT markers and different integrin proteins validate the RNA sequencing findings. ( b ) Western blots showing upregulation of STAT3 phosphorylation (Y705 and S727) and FYN activation. ( c ) ABI1 interacts with STAT3 and FYN in RWPE-1 cells. Western blotting results indicating that FYN (top panel) and STAT3 (middle panel) co-immunoprecipitated with Abi1. Input, RWPE-1 cell lysate; Flow-Through, unbound fraction of the lysate; IgG, control immunoprecipitation lacking anti-Abi1 antibody; IP, immunoprecipitation including the anti-Abi1 antibody. Asterisk in top panel indicates the corresponding bands in fractions. ( d - f ) ABI1 loss promotes nuclear localization of activated STAT3 as determined by pY705 antibody. ( d ) Western blotting analysis of p-STAT3 Y705 expression in cytoplasmic (left panel) or, nuclear fraction (right panel) of RWPE ABI1 KO, control and ABI1-rescued clones. ( e ) Immunofluorescence analysis of p-STAT3 Y705 levels in RWPE ABI1 KO clones. ( f ) Quantification of nuclear p-STAT3 Y705 levels, n = 3, p < 0.001. ( g ) An example of inverse correlation of ABI1 and pSTAT3 Y705 expression levels in a prostate tumor. Serial tumor sections were immunostained with antibodies to either ABI1 (left panel), or pSTAT3 Y705 (right panel). Arrows depict two example areas of correlation of low ABI1 and high pSTAT. ( h ) Schematic depicting the proposed mechanism of how Abi1 loss promotes the invasive potential of prostate epithelial cells. Center, ABI1 ( ABI1 ) is critical for cell junction maintenance through WAVE complex-mediated actin polymerization; disruption of ABI1 leads to loss of WAVE complex (WAVE complex) stability, resulting in lower levels of E-cadherin (CDH1) at cell-cell contacts and downregulation of cell-cell adhesion. Right, ABI1 pY421 binds with high affinity to FYN-SH2 domain. Disruption of ABI1 leads to constitutive activation of the SRC family kinase FYN (FYN) to stimulate STAT3 activation and promote its nuclear localization. Nuclear STAT3 promotes transcription of the epithelial-to-mesenchymal transition (EMT) gene program, including extracellular matrix metalloproteinases that promote migration through matrix degradation in the absence of proper cell-cell adhesion. Left, In the absence of ABI1, activated FYN acts on FAK kinase (FAK) to promote integrin signaling, which also supports cell migratory activity. Activated FAK may also directly bind and activate pSTAT3, thus providing another possibility for EMT pathway activation

Article Snippet: The resulting lysate was immunoprecipitated using anti-Abi1 antibody (clone 1B9, MBL) at an antibody:lysate ratio of 1:100.

Techniques: Expressing, RNA Sequencing Assay, Western Blot, Activation Assay, Immunoprecipitation, Clone Assay, Immunofluorescence, Migration, Activity Assay

Journal: Oncogenesis

Article Title: Disruption of Abi1/Hssh3bp1 expression induces prostatic intraepithelial neoplasia in the conditional Abi1/Hssh3bp1 KO mice

doi: 10.1038/oncsis.2012.28

Figure Lengend Snippet: Identification of Abi1 mutations in primary prostate tissue. ( a ) Diagram of ABI1/SSH3BP1 gene with identified prostate tumor mutations. Boxes indicate coding region of consecutive exons (1–12 as indicated) of ABI1/SSH3BP1 gene with exonic mutation listed above boxes. Deletions encompassing entire exons (exon 6 and exon 10) are indicated by lines. Numbers below boxes depict the last amino acid encoded by the specific exon or encoded by a shared codon from two consecutive exons. Lines with arrows indicate approximate locations of Abi1/Hssh3bp1 structural domains and identified binding regions for listed proteins or their domains. WAVE-BD, WAVE domain-binding region; , HHR, homeo-domain homologous region; Abl-SH3-SH2, c-Abl kinase SH3-SH2 domain-binding region PXXP-, Ser- or Pro-rich, sequences rich in SH3 domain-binding consensus PXXP, serine or proline residues, respectively; SH3, Src homology 3 domain. Indicated binding sites for: Spectrin-SH3, Spectrin Src Homology 3 domain; Eps8-SH3, Eps8 Src Homology 3 domain; p85, p85 regulatory subunit of phosphoinositide-3 kinase; p85-SH2, p85-Src Homology 2 domain; Abl-PRL, c-Abl kinase proline-rich region, N-WASP and mDiaLNCaP Δex6, indicates exon 6 deletion; ATG, start codon. ( b ) DNA sequence chromatograms demonstrating recurring ABI1 mutations. Chromatograms from tumor tissue and tissue adjacent to tumor or archived lymph node biopsy are shown. The text above chromatograms indicates location and primary consequence of mutations. Left panel demonstrates A363S and A363D mutations; middle panel, deletion following codon G331 (#G331); right panel demonstrates complete deletion of exon 10.

Article Snippet: For evaluating protein levels, the following antibodies were used: Abi1 (clone 1B9, MBL International, Woburn, MA, USA); Abi2 (p-20, #sc-20327, Santa Cruz Biotechnology); E-Cadherin (Cat. No. 4065) and phospho-Akt S473 (Cat. No. #9271) (Cell Signaling Technology, Inc., Danvers, MA, USA); B-catenin (Cat. No. 610154, BD Biosciences, San Jose, CA, USA); Ki-67 (Cat. No. VP-K451, Vector Laboratories); WAVE2 (H-110, #sc-33548; #07-410, Santa Cruz Biotechnology; rabbit polyclonal (#1735) and NAP1 were kind gifts from Theresia Stradal).

Techniques: Mutagenesis, Binding Assay, Sequencing

Journal: Oncogenesis

Article Title: Disruption of Abi1/Hssh3bp1 expression induces prostatic intraepithelial neoplasia in the conditional Abi1/Hssh3bp1 KO mice

doi: 10.1038/oncsis.2012.28

Figure Lengend Snippet: ABI1 mutations in primary prostate tumors

Article Snippet: For evaluating protein levels, the following antibodies were used: Abi1 (clone 1B9, MBL International, Woburn, MA, USA); Abi2 (p-20, #sc-20327, Santa Cruz Biotechnology); E-Cadherin (Cat. No. 4065) and phospho-Akt S473 (Cat. No. #9271) (Cell Signaling Technology, Inc., Danvers, MA, USA); B-catenin (Cat. No. 610154, BD Biosciences, San Jose, CA, USA); Ki-67 (Cat. No. VP-K451, Vector Laboratories); WAVE2 (H-110, #sc-33548; #07-410, Santa Cruz Biotechnology; rabbit polyclonal (#1735) and NAP1 were kind gifts from Theresia Stradal).

Techniques: Mutagenesis, Functional Assay, Phospho-proteomics

Journal: Oncogenesis

Article Title: Disruption of Abi1/Hssh3bp1 expression induces prostatic intraepithelial neoplasia in the conditional Abi1/Hssh3bp1 KO mice

doi: 10.1038/oncsis.2012.28

Figure Lengend Snippet: Expression of Abi1 in LNCaP cell line inhibits cellular growth, anchorage-independent growth and proliferation in vitro and in xenograft model. ( a ) Inhibition of cell growth, and ( b ) cell proliferation of LNCAP cells expressing Abi1. LNCaP cells transfected with Abi1 proliferate slower than mock-transfected cells. Cell proliferation was evaluated by 5-bromo-2-deoxyuridine incorporation as described in Materials and methods. The data indicate that the percentage of cells in S phase was significantly lower among cells transfected with Abi1 isoform 2 (Abi1) than among cells transfected with isoform 2-lacking exon 10 (Abi1 ΔEx10), or mock transfected, ( P <0.001, χ 2 ). ( c ) Expression of Abi1 but not the mutant Abi1 ΔEx10 promotes cell-to-cell adhesion. Cell aggregation assay was performed as described in Materials and methods. Left panel , quantification of cell-to-cell contacts; *** P <0.001, n =3. Right panel , representative images from indicated cell lines: cells with three or more contacts are indicated by asterisks. ( d ) Abi1 mutant A363S does not inhibit colony formation as well as wt Abi1. LNCaP cell lines expressing Abi1 or Abi1 containing a known prostate cancer mutation in exon 10 (A363S) were plated in soft agar and evaluated for colony growth 4 weeks later. Numbers of colonies over a defined size (>0.5 cm in images taken from agar plates) were scored positive and counted per region of interest (ROI), n =3. A363S cell line showed enhanced growth vs the Abi1 isoform 2 expressing cell line, ** P <0.01; * P <0.05. ( e ) Abi1 inhibits of tumor growth in LNCaP xenograft assay. Growth of tumors was monitored by prostate-specific antigen (PSA) as described in Materials and methods. Expression of Abi1 inhibits secretion of serum PSA. Some animals did not develop tumors hence no secretion of PSA was observed. Abi1, Abi1 isoform 2 expressing cell line; mock, cell line expressing empty plasmid. Comparison between the cell lines indicates statistical significance between the groups ( n =0.0374, two-way analysis of variance).

Article Snippet: For evaluating protein levels, the following antibodies were used: Abi1 (clone 1B9, MBL International, Woburn, MA, USA); Abi2 (p-20, #sc-20327, Santa Cruz Biotechnology); E-Cadherin (Cat. No. 4065) and phospho-Akt S473 (Cat. No. #9271) (Cell Signaling Technology, Inc., Danvers, MA, USA); B-catenin (Cat. No. 610154, BD Biosciences, San Jose, CA, USA); Ki-67 (Cat. No. VP-K451, Vector Laboratories); WAVE2 (H-110, #sc-33548; #07-410, Santa Cruz Biotechnology; rabbit polyclonal (#1735) and NAP1 were kind gifts from Theresia Stradal).

Techniques: Expressing, In Vitro, Inhibition, Transfection, Mutagenesis, Xenograft Assay, Plasmid Preparation, Comparison

Journal: Oncogenesis

Article Title: Disruption of Abi1/Hssh3bp1 expression induces prostatic intraepithelial neoplasia in the conditional Abi1/Hssh3bp1 KO mice

doi: 10.1038/oncsis.2012.28

Figure Lengend Snippet: The onset of histopathological changes in Abi1 KO mice at 8 months. ( a ) Confirmation of Abi1 gene disruption in mouse prostate. DNA was isolated from Abi1 (fl/fl) mouse prostates (anterior and dorsal lobes) positive for the Cre recombinase-expressing allele (+) or with no Cre presence (−), as shown in the indicated genotypes. PCR using nested primers encompassing the recombined DNA region was carried out as described in Materials and methods. Positive bands were detected only in samples with Cre expression. As controls-immortalized Abl floxed MEF cells were used, parental, that is, of the genotype Abi1 (fl/fl) or upon Cre-mediated disruption of ABI1 gene (clone names above the panel). The fidelity of the Cre-mediated recombination was confirmed by DNA sequencing of PCR fragments. ( b ) The earliest time of observation for mPIN in mice-lacking Abi1, that is, of the genotype Abi1(fl/fl);PBCre(+), is 8 months. Comparison of changes noted in the anterior and dorsal lobes of 8-month-old Abi1(fl/fl);PBCre(+) mice vs age-matched Cre (−) and background control strain mice. Abi1(fl/fl);PBCre(+) mice feature hyperplasia/mPIN changes, with multiple glands lined by crowded epithelial cells, as well as disorderly piling up of cells in the more severely affected areas. Atypical nuclear changes (best visualized in the inserts), including karyomegaly and hyperchromasia, are also present in many glands. Glands from both the Cre (−) and control strain mice are unremarkable. Number of mice evaluated per genotype at 8 months, n >5.

Article Snippet: For evaluating protein levels, the following antibodies were used: Abi1 (clone 1B9, MBL International, Woburn, MA, USA); Abi2 (p-20, #sc-20327, Santa Cruz Biotechnology); E-Cadherin (Cat. No. 4065) and phospho-Akt S473 (Cat. No. #9271) (Cell Signaling Technology, Inc., Danvers, MA, USA); B-catenin (Cat. No. 610154, BD Biosciences, San Jose, CA, USA); Ki-67 (Cat. No. VP-K451, Vector Laboratories); WAVE2 (H-110, #sc-33548; #07-410, Santa Cruz Biotechnology; rabbit polyclonal (#1735) and NAP1 were kind gifts from Theresia Stradal).

Techniques: Disruption, Isolation, Expressing, DNA Sequencing, Comparison, Control

Journal: Oncogenesis

Article Title: Disruption of Abi1/Hssh3bp1 expression induces prostatic intraepithelial neoplasia in the conditional Abi1/Hssh3bp1 KO mice

doi: 10.1038/oncsis.2012.28

Figure Lengend Snippet: Disruption of ABI1 does not lead to invasive adenocarcinoma in up to 12-month-old mice. ( A ) Summary of observations at 4–12 months of mice lacking both Abi1 alleles, Abi1(fl/fl);PBCre(+). (a–d) At 4 months of age, all prostatic lobes from the Abi1 KO mice appeared normal. (e–h) At 8 months of age, the anterior (e) and dorsal (f) lobes of Abi1 KO mice featured multiple gland profiles lined by crowded cells, many of which had enlarged hyperchromatic nuclei. In several affected areas, cells were piled up in a disorderly fashion (consistent with PIN). Lateral (g) and ventral (h) lobes were unremarkable. (i–l) At 10 months of age, a few glands within the lateral (k) lobes are also affected. Ventral (l) lobes are virtually unaffected. (m–p) At 12 months of age, all lobes of Abi1 KO mice demonstrated some degree of hyperplasia/mPIN changes. These changes were most prominent in the anterior lobe (m), in which the epithelium in the region shown here demonstrated a high degree of cellular atypia. All panels represent hematoxylin and eosin staining (h and e). At least four mice were evaluated per each time point; all mice were positive for histopathological changes at 10 and 12 months. ( B ) Nuclear atypia in Abi1 KO prostate. Enlarged selected panels from ( A ) to indicate nuclear atypia (arrows).

Article Snippet: For evaluating protein levels, the following antibodies were used: Abi1 (clone 1B9, MBL International, Woburn, MA, USA); Abi2 (p-20, #sc-20327, Santa Cruz Biotechnology); E-Cadherin (Cat. No. 4065) and phospho-Akt S473 (Cat. No. #9271) (Cell Signaling Technology, Inc., Danvers, MA, USA); B-catenin (Cat. No. 610154, BD Biosciences, San Jose, CA, USA); Ki-67 (Cat. No. VP-K451, Vector Laboratories); WAVE2 (H-110, #sc-33548; #07-410, Santa Cruz Biotechnology; rabbit polyclonal (#1735) and NAP1 were kind gifts from Theresia Stradal).

Techniques: Disruption, Staining

Journal: Oncogenesis

Article Title: Disruption of Abi1/Hssh3bp1 expression induces prostatic intraepithelial neoplasia in the conditional Abi1/Hssh3bp1 KO mice

doi: 10.1038/oncsis.2012.28

Figure Lengend Snippet: Prostate tissue lacking Abi1 exhibit enhanced proliferation and loss of cellular adhesion markers downstream of WAVE2 complex downregulation. ( a ) Cells and prostate tissues lacking Abi1 exhibit downregulation of WAVE2 complex components but upregulation of Abi2 as indicated by western blotting analysis. Examination of WAVE2 complex integral proteins as indicated in Abi1 KO MEF cells ( a ); and in prostate tissue ( b ). Please note co-incidental downregulation of E-cadherin in prostate tissue and upregulation of Abi2 evident at 12 months in Abi1 KO MEF cells. Anterior prostates were analyzed at 10 and 12 months, ‘−' indicates absence of probasin Cre recombinase transgene and ‘+' indicates its presence in Abi1 floxed mice. ( b ) Enhanced cellular proliferation in prostate tissue lacking Abi1 correlates with enhanced Abi2 levels in prostate tissue. Top: immunostaining of Abi1 KO prostate tissues with Abi2 antibody. Staining with Abi2 antibody (P20, Santa Cruz Biotechnology) reveals striking upregulation of Abi2 levels in Abi1 KO lesions. Middle: enhanced proliferation as indicated by positive staining with Ki67. Bottom: immunostaining with phospho-Akt S473 antibody indicating staining in the PIN lesion (circle). Right panels, prostate tissue from Abi1 lacking (Abi1(fl/fl);PBCre(+)); left panels, prostate tissue from with Abi1 expression (Abi1(fl/fl);PBCre(+)). Staining of tissue was performed as described in Materials and methods.

Article Snippet: For evaluating protein levels, the following antibodies were used: Abi1 (clone 1B9, MBL International, Woburn, MA, USA); Abi2 (p-20, #sc-20327, Santa Cruz Biotechnology); E-Cadherin (Cat. No. 4065) and phospho-Akt S473 (Cat. No. #9271) (Cell Signaling Technology, Inc., Danvers, MA, USA); B-catenin (Cat. No. 610154, BD Biosciences, San Jose, CA, USA); Ki-67 (Cat. No. VP-K451, Vector Laboratories); WAVE2 (H-110, #sc-33548; #07-410, Santa Cruz Biotechnology; rabbit polyclonal (#1735) and NAP1 were kind gifts from Theresia Stradal).

Techniques: Western Blot, Immunostaining, Staining, Expressing

Journal: Oncogenesis

Article Title: Disruption of Abi1/Hssh3bp1 expression induces prostatic intraepithelial neoplasia in the conditional Abi1/Hssh3bp1 KO mice

doi: 10.1038/oncsis.2012.28

Figure Lengend Snippet: Enhanced activation of Akt and colony formation of cells lacking Abi1. ( a , b ) MEF cells lacking Abi1 exhibit enhanced sensitivity to activation of phospho-Akt downstream from EGFR receptor. Cells were starved overnight and subsequently incubated with epidermal growth factor (10 ng/ml) for indicated amounts of time. ( a ) Cell lysates were prepared as described in Materials and methods, and proteins were analyzed by western blotting with indicated antibodies. ( b ) Quantification of phospho-Akt S473 levels indicate significant increase of p-Akt S473 signal of the Abi1 KO clone #3–11 at 1 min and 5 min ( P <0.01, two-way analysis of variance); clone #3–6 demonstrated enhanced response vs control #3 cell line at 1 min ( P <0.05, t -test). ( c , d ) Disruption of Abi1 expression promotes colony formation in soft agar. Syngeneic MEF cells containing or lacking Abi1 were incubated in soft agar as indicated in Materials and methods, and total numbers of colonies were scored. Cell lines lacking the Abi1 gene exhibit enhanced colony formation ( P <0.001, #3–6; and P <0.01, #3–11) vs control cell line #3 ( c ). Representative images of soft agar colonies from two independent experiments ( d ). #3 represents Abi1 floxed cell line; #3–6 and #3–11 represent Abi1 KO cell lines.

Article Snippet: For evaluating protein levels, the following antibodies were used: Abi1 (clone 1B9, MBL International, Woburn, MA, USA); Abi2 (p-20, #sc-20327, Santa Cruz Biotechnology); E-Cadherin (Cat. No. 4065) and phospho-Akt S473 (Cat. No. #9271) (Cell Signaling Technology, Inc., Danvers, MA, USA); B-catenin (Cat. No. 610154, BD Biosciences, San Jose, CA, USA); Ki-67 (Cat. No. VP-K451, Vector Laboratories); WAVE2 (H-110, #sc-33548; #07-410, Santa Cruz Biotechnology; rabbit polyclonal (#1735) and NAP1 were kind gifts from Theresia Stradal).

Techniques: Activation Assay, Incubation, Western Blot, Control, Disruption, Expressing