cst 39444  (Cell Signaling Technology Inc)

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: Fig. 2 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: ABI1 was immunoprecipitated with MBL Mab 1B9: For Western blotting we used Abi1 CST 39444 (bottom panel), STAT3 CST 30835 (middle panel); FYN immunoreactivity was detected with BioRad VPA00019. (B) Recombinant ABI1 interacts with STAT3 (middle panels), and co-immunoprecipitated active phospho-SRC activity as indicated by pan phospho-SRC family kinase antibody to pY416 (CST 2101), lower panels.

Techniques: Expressing, Western Blot, Control, 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: Fig. 3 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: ABI1 was immunoprecipitated with MBL Mab 1B9: For Western blotting we used Abi1 CST 39444 (bottom panel), STAT3 CST 30835 (middle panel); FYN immunoreactivity was detected with BioRad VPA00019. (B) Recombinant ABI1 interacts with STAT3 (middle panels), and co-immunoprecipitated active phospho-SRC activity as indicated by pan phospho-SRC family kinase antibody to pY416 (CST 2101), lower panels.

Techniques: Membrane, 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: Fig. 4 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: ABI1 was immunoprecipitated with MBL Mab 1B9: For Western blotting we used Abi1 CST 39444 (bottom panel), STAT3 CST 30835 (middle panel); FYN immunoreactivity was detected with BioRad VPA00019. (B) Recombinant ABI1 interacts with STAT3 (middle panels), and co-immunoprecipitated active phospho-SRC activity as indicated by pan phospho-SRC family kinase antibody to pY416 (CST 2101), lower panels.

Techniques: Migration, Generated, Software, Overlay Assay, Control, 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: Fig. 6 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: ABI1 was immunoprecipitated with MBL Mab 1B9: For Western blotting we used Abi1 CST 39444 (bottom panel), STAT3 CST 30835 (middle panel); FYN immunoreactivity was detected with BioRad VPA00019. (B) Recombinant ABI1 interacts with STAT3 (middle panels), and co-immunoprecipitated active phospho-SRC activity as indicated by pan phospho-SRC family kinase antibody to pY416 (CST 2101), lower panels.

Techniques: Gene Expression, RNA Sequencing, Western Blot, Phospho-proteomics, Activation Assay, Immunoprecipitation, Control, Expressing, Clone Assay, Immunofluorescence, Disruption, Migration, Activity Assay