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: 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