Journal: eLife

Article Title: The CIC-ERF co-deletion underlies fusion-independent activation of ETS family member, ETV1, to drive prostate cancer progression

doi: 10.7554/eLife.77072

Figure Lengend Snippet: ( A ) CIC transcriptionally represses ETV1/4/5. ( B ) The 19q13.2 genomic locus demonstrating the physical location of ERF and CIC . ( C ) 15 PCa studies (cBioPortal) demonstrating the co-occurrence of ERF and CIC homozygous and heterozygous deletions. The co-occurrence of ERF and CIC alterations is highly significant (p<0.001 co-occurrence, Fisher exact test). ( D ) ERF-CIC co-deleted PCa stratified by Gleason score and tumor stage. ( E ) Frequency of ERF and CIC alterations in primary PCa (top) and metastatic castrate resistant prostate cancer (mCRPC; bottom), demonstrating enrichment in mCRPC. ( F ) Onco-print of known genetic drivers (ERG, ETV1, ETV4, SPOP, and FOXA1) of PCa aligned with CIC and ERF (cBioPortal). CIC-ERF co-deleted prostate tumors (red box) do not frequently co-occur with other known oncogenic events. ( G ) Survival analysis performed using 15 PCa datasets from cBioPortal. Disease-free survival (DFS) and progression-free survival (PFS) in patients harboring the ERF-CIC co-deletion (red) vs. no ERF-CIC co-deletion (blue). p=value, log-rank. Figure 1—source data 1. Prostate cancer studies identified in cBioPortal demonstrating the total number of patients, number of patients with shallow or deep deletions in Capicua (CIC)-ETS2 repressor factor (ERF), and the frequency of CIC-ERF alterations in each cohort. Studies that analyzed predominantly primary prostate cancers (green) and metastatic castrate resistant prostate cancer (mCRPC; yellow) are highlighted.

Article Snippet: Human CIC (Cat#. Hs00943425_g1), ERF (Cat#. Hs01100070_g1), ETV1 (Cat#. Hs00951951_m1), ETV4 (Cat#. Hs00383361_g1), ETV5 (Cat#. Hs00927557_m1), and endogenous controls GAPDH (Cat#. Hs02758991_g1) were amplified with Taqman gene expression assay (Applied Biosystems).

Techniques:

Journal: eLife

Article Title: The CIC-ERF co-deletion underlies fusion-independent activation of ETS family member, ETV1, to drive prostate cancer progression

doi: 10.7554/eLife.77072

Figure Lengend Snippet: ETS2 repressor factor ( ERF ) - Capicua ( CIC ) co-deletion frequency across 15 prostate cancer studies.

Article Snippet: Human CIC (Cat#. Hs00943425_g1), ERF (Cat#. Hs01100070_g1), ETV1 (Cat#. Hs00951951_m1), ETV4 (Cat#. Hs00383361_g1), ETV5 (Cat#. Hs00927557_m1), and endogenous controls GAPDH (Cat#. Hs02758991_g1) were amplified with Taqman gene expression assay (Applied Biosystems).

Techniques:

Journal: eLife

Article Title: The CIC-ERF co-deletion underlies fusion-independent activation of ETS family member, ETV1, to drive prostate cancer progression

doi: 10.7554/eLife.77072

Figure Lengend Snippet: ( A ) Clonogenic assay comparing prostate epithelial cells (PNT2) with ERF KD, CIC KO, or ERF KD+ CIC KO compared to control. ( B ) Number of colonies for each condition in ( A ) (n=3). ( C ) Spheroid growth assay using PNT2 cells expressing ERF KD, CIC KO, ERF KD+ CIC KO vs. control. ( D ) Size of the sphere for each condition in ( C ) (n=6). Error bars represent SD. p Values were calculated using Student’s t test. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. ( E ) Cell-titer glo viability assay (n=6), ( F ) transwell assay (n=3), and ( G ) wound healing assay comparing PNT2 ERF KD, CIC KO, and ERF KD+ CIC KO to control (n=4). Error bars represent SD. p Values were calculated using Student’s t test. ( H ) Bar graph comparing the incidence of PNT2 parental (N=3/10), PNT2 ERF KD (5/10), PNT2 CIC KO (N=5/10), or PNT2 ERF KD+CIC KO (N=6/10) tumor formation in immunodeficient mice. **p<0.01, ***p<0.001, and ****p<0.0001.

Article Snippet: Human CIC (Cat#. Hs00943425_g1), ERF (Cat#. Hs01100070_g1), ETV1 (Cat#. Hs00951951_m1), ETV4 (Cat#. Hs00383361_g1), ETV5 (Cat#. Hs00927557_m1), and endogenous controls GAPDH (Cat#. Hs02758991_g1) were amplified with Taqman gene expression assay (Applied Biosystems).

Techniques: Clonogenic Assay, Control, Growth Assay, Expressing, Viability Assay, Transwell Assay, Wound Healing Assay

Journal: eLife

Article Title: The CIC-ERF co-deletion underlies fusion-independent activation of ETS family member, ETV1, to drive prostate cancer progression

doi: 10.7554/eLife.77072

Figure Lengend Snippet: ( A ) Immunoblot of CIC, ERF, and HSP90 in PNT2 and its different variants. Representative figure; performed in duplicate. Arrows indicate CIC and ERF bands. ( B ) Tumor explants from mice in . Figure 2—figure supplement 1—source data 1. Full length western blot images of Capicua (CIC), ETS2 repressor factor (ERF) and HSP90 in prostate epithelial cells (PNT2) and its variants with associated raw images.

Article Snippet: Human CIC (Cat#. Hs00943425_g1), ERF (Cat#. Hs01100070_g1), ETV1 (Cat#. Hs00951951_m1), ETV4 (Cat#. Hs00383361_g1), ETV5 (Cat#. Hs00927557_m1), and endogenous controls GAPDH (Cat#. Hs02758991_g1) were amplified with Taqman gene expression assay (Applied Biosystems).

Techniques: Western Blot

Journal: eLife

Article Title: The CIC-ERF co-deletion underlies fusion-independent activation of ETS family member, ETV1, to drive prostate cancer progression

doi: 10.7554/eLife.77072

Figure Lengend Snippet: ( A ) Immunoblot of CIC, ERF, and beta-actin in DU-145 cells compared to prostate epithelial cells (PNT2). ( B ) Immunoblot of CIC, ERF, and beta-actin in PC-3 cells compared to PNT2 cells. ( C ) Immunoblot of CIC, ERF, and HSP90 in parental DU-145 cells and engineered DU-145 different variants. Representative figure; performed in duplicate. ( D ) Relative ERF mRNA expression in DU-145 parental, ERF rescue, CIC knockout (KO), and ERF rescue +CIC KO (n=3), Student’s t-test, ***p<0.001, and ****p<0.0001. ( E ) Wound healing assay in DU-145 cells with ERF rescue, CIC KO, or ERF rescue + CIC KO compared to parental (n=2). ( F ) CIC mRNA expression in PC-3 parental, PC-3 CIC overexpression (OE), and PC-3 ( ERF +CIC ) OE (n=3). p Value = ****p<0.0001 ( G ) Relative ERF mRNA expression in PC-3 parental, PC-3 ERF OE, PC-3 ( ERF +CIC ) OE, and PC-3 ERF knockdown (KD) (n=3). p Value = *p<0.05, **p<0.01, and ***p<0.001. ( H ) Wound healing assay in PC-3 cells expressing ERF OE, CIC OE, ( ERF +CIC ) OE, or ERF KD compared to control (n=6). ( I ) Immunoblot of ERF and HSP90 in PC-3 cells with ERF KD. Figure 3—figure supplement 1—source data 1. Full-length western blot images of basal levels of Capicua (CIC), ETS2 repressor factor (ERF), and β-actin in prostate epithelial cells (PNT2), DU145, and PC3 cells and associated raw images. Figure 3—figure supplement 1—source data 2. Full-length western blot images of CIC, ETS2 repressor factor (ERF), and HSP90 in DU145 cells with its variants and associated raw images. Figure 3—figure supplement 1—source data 3. Full-length western blot images of ETS2 repressor factor (ERF) and HSP90 in PC3 cells with its variants and associate raw images.

Article Snippet: Human CIC (Cat#. Hs00943425_g1), ERF (Cat#. Hs01100070_g1), ETV1 (Cat#. Hs00951951_m1), ETV4 (Cat#. Hs00383361_g1), ETV5 (Cat#. Hs00927557_m1), and endogenous controls GAPDH (Cat#. Hs02758991_g1) were amplified with Taqman gene expression assay (Applied Biosystems).

Techniques: Western Blot, Expressing, Knock-Out, Wound Healing Assay, Over Expression, Knockdown, Control

Journal: eLife

Article Title: The CIC-ERF co-deletion underlies fusion-independent activation of ETS family member, ETV1, to drive prostate cancer progression

doi: 10.7554/eLife.77072

Figure Lengend Snippet: ( A ) Clonogenic assay of DU-145 cells with ERF rescue, CIC knockout (KO), or ERF rescue + CIC KO compared to parental control. ( B ) Number of colonies for each condition in ( A ) (n=3). ( C ) Cell-titer glo viability assay (n=6) and ( D ) transwell assay comparing DU-145 parental cells to DU-145 with ERF rescue, CIC KO, or ERF rescue +CIC KO (n=3). p Values were calculated using Student’s t test. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Error bars represent SD. ( E ) Relative tumor volume in mice bearing DU-145 parental, DU-145 ERF , DU-145 with CIC KO, or DU-145 ERF +CIC KO xenografts (N=10). p Values were calculated using Student’s t test. *p<0.05. Error bars represent SEM. ( F ) Clonogenic assay in PC-3 cells expressing ERF knockdown (KD), ERF overexpression (OE), CIC OE, or ERF +CIC OE compared to control. ( G ) Number of colonies for each condition in ( F ) (n=3). ( H ) Cell-titer glo viability assay (n=6) and ( I ) transwell assay comparing different groups in PC-3 cells (WT, ERF KD, ERF OE, CIC OE, or ERF +CIC OE) (n=3). p Values were calculated using Student’s t test. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Error bars represent SD. ( J ) Relative tumor volume in mice bearing PC-3 parental cells, PC-3 ERF OE, or PC-3 CIC OE (N=10) over 33 days. p Values were calculated using Student’s t test. *p<0.05. Error bars indicate SEM.

Article Snippet: Human CIC (Cat#. Hs00943425_g1), ERF (Cat#. Hs01100070_g1), ETV1 (Cat#. Hs00951951_m1), ETV4 (Cat#. Hs00383361_g1), ETV5 (Cat#. Hs00927557_m1), and endogenous controls GAPDH (Cat#. Hs02758991_g1) were amplified with Taqman gene expression assay (Applied Biosystems).

Techniques: Clonogenic Assay, Knock-Out, Control, Viability Assay, Transwell Assay, Expressing, Knockdown, Over Expression

Journal: eLife

Article Title: The CIC-ERF co-deletion underlies fusion-independent activation of ETS family member, ETV1, to drive prostate cancer progression

doi: 10.7554/eLife.77072

Figure Lengend Snippet: ( A ) Percentage of CIC and ERF peaks located in defined genomic regions. ( B ) Schematic algorithm to identify shared CIC and ERF target genes in prostate cells (top). Functional Clustering Analysis of the 91 shared CIC and ERF target genes using DAVID (bottom table). ( C ) ETV1 mRNA expression in prostate epithelial cells (PNT2) ( CIC-ERF -replete) cells with ERF knockdown (KD), CIC knockout (KO), or ERF KD + CIC KO (n=3). ( D ) Schematic of CIC and ERF DNA-binding motifs in the ETV1 promoter. ( E ) Chromatin immunoprecipitation (ChIP)-PCR from PNT2 cells showing CIC occupancy on the ETV1 promoter. ( F–G ) ChIP-PCR with ERF occupancy on the ETV1 promoter. ( H ) ETV1 mRNA expression in DU-145 (ERF-deficient) cells with ERF rescue, CIC KO, or ERF rescue + CIC KO (n=3). ETV1 mRNA expression in PC-3 cells with ( I ) ERF KD (n=3) and ( J ) ERF overexpression (OE) (n=3). p Values were calculated using Student’s t test. *p<0.05, **p<0.01, and ****p<0.0001. Error bars represent SD. Performed in triplicate. ( K ) ETV1 luciferase promoter assay in 293T cells comparing Empty vector (EV) with ERF OE (n=6). Student’s t test, *p<0.05. Error bars represent SD. ( L ) Single sample gene set enrichment analysis (ssGSEA) alignments comparing gene expression patterns in PNT2 cells with ERF KD and CIC KO. IC = information coefficient. Figure 4—source data 1. Full-length PCR gel images of ETV1 after Capicua (CIC) pull down in prostate epithelial cells (PNT2). Cropped images and description shown in . Figure 4—source data 2. Full-length PCR gel images of ETV1 after ETS2 repressor factor (ERF) pull down in prostate epithelial cells (PNT2). Cropped images and description shown in . Figure 4—source data 3. Full-length PCR gel images of ETV1 after ETS2 repressor factor (ERF) pull down in DU-145 cells. Cropped images and description shown in .

Article Snippet: Human CIC (Cat#. Hs00943425_g1), ERF (Cat#. Hs01100070_g1), ETV1 (Cat#. Hs00951951_m1), ETV4 (Cat#. Hs00383361_g1), ETV5 (Cat#. Hs00927557_m1), and endogenous controls GAPDH (Cat#. Hs02758991_g1) were amplified with Taqman gene expression assay (Applied Biosystems).

Techniques: Functional Assay, Expressing, Knockdown, Knock-Out, Binding Assay, Chromatin Immunoprecipitation, Over Expression, Luciferase, Promoter Assay, Plasmid Preparation, Gene Expression

Journal: eLife

Article Title: The CIC-ERF co-deletion underlies fusion-independent activation of ETS family member, ETV1, to drive prostate cancer progression

doi: 10.7554/eLife.77072

Figure Lengend Snippet: ( A–C ) Relative ETV1 , ETV4 , and ETV5 mRNA expression in prostate epithelial cells (PNT2) parental and PNT2 CIC knockout (KO) cells (n=3). ( D–F ) Relative ETV1 , ETV4 , and ETV5 mRNA expression in PNT2 parental and PNT2 ERF knockdown (KD) + CIC KO cells (n=3). ( G ) Relative ERF , ( H ) ETV1 , ( I ) ETV4 , and ( J ) ETV5 mRNA expression in parental PNT2 and PNT2 ERF KD cells (n=3). ( K ) Co-immunoprecipitation using GFP-tagged ERF (pulldown) and immunoblotting for ERF and CIC. ( L ) Co-immunoprecipitation using Myc-tagged CIC (pulldown) and immunoblotting for ERF and CIC. ( M ) ETV1 chromatin immunoprecipitation (ChIP)-PCR fold enrichment of CIC compared to IgG control in PNT2 cells (n=3). ( N ) ETV1 ChIP-PCR fold enrichment of ERF compared to IgG control in PNT2 cells (n=3). ( O ) Gene Desert quantitative PCR (Active Motif Negative control) comparing CIC and ERF to IgG control. ( P ) Gene Desert quantitative PCR (Active Motif Negative control) comparing CIC, ERF, and IgG to input control. Relative ( Q ) ERF , ( R ) ETV1 , ( S ) ETV4 , and ( T ) ETV5 mRNA expression in parental DU-145 and DU-145 ERF cells (n=3). p Values for all figures = *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Error bars represent SD. Figure 4—figure supplement 1—source data 1. Co-immunoprecipitation using GFP-tagged ETS2 repressor factor (ERF) and immunoblotting for Capicua (CIC; bottom panel) and ETS2 repressor factor (ERF; top panel) with associated raw images. Figure 4—figure supplement 1—source data 2. Co-immunoprecipitation using myc-tagged Capicua (CIC) and immunoblotting for ETS2 repressor factor (ERF; top panel) and CIC (bottom panel) with associated raw images.

Article Snippet: Human CIC (Cat#. Hs00943425_g1), ERF (Cat#. Hs01100070_g1), ETV1 (Cat#. Hs00951951_m1), ETV4 (Cat#. Hs00383361_g1), ETV5 (Cat#. Hs00927557_m1), and endogenous controls GAPDH (Cat#. Hs02758991_g1) were amplified with Taqman gene expression assay (Applied Biosystems).

Techniques: Expressing, Knock-Out, Knockdown, Immunoprecipitation, Western Blot, Chromatin Immunoprecipitation, Control, Real-time Polymerase Chain Reaction, Negative Control

Journal: eLife

Article Title: The CIC-ERF co-deletion underlies fusion-independent activation of ETS family member, ETV1, to drive prostate cancer progression

doi: 10.7554/eLife.77072

Figure Lengend Snippet: ( A ) BRD32048 treatment of PNT2 cells expressing si CIC and si ERF compared to siSCRM control. ( B ) Relative density of crystal violet assays performed in ( A ) (n=3). ( C ) Transwell assay comparing BRD32048 treated PNT2 cells (n=3). BRD32048 (0 μM) columns were set to ‘1’ in the siSCRM and si ERF +siCIC groups for relative comparison in ( B–C ). ( D ) Relative density of crystal violet assays performed in ( A ) (n=3). ( E ) Transwell assay comparing BRD32048 treated PNT2 cells (n=3). siSCRM column (0 µM) was set to ‘1’ for relative comparison to other groups in ( D–E ). ( F ) BRD32048 treatment of PC-3 cells expressing si ERF compared to siSCRM control. ( G ) Relative density of crystal violet assays performed in ( F ) (n=3). ( H ) Transwell assay comparing BRD32048 treated PC-3 cells (n=3). BRD32048 (0 μM) columns were set to ‘1’ in the siSCRM and si ERF groups for relative comparison in ( G–H ). ( I ) Relative density of crystal violet assays performed in ( F ) (n=3). ( J ) Transwell assay comparing BRD32048 treated PC-3 cells (n=3). siSCRM column (0 µM) was set to ‘1’ for relative comparison to other groups in ( I–J ). Students t-test, p values *p<0.05, **p<0.01 for all figures.

Article Snippet: Human CIC (Cat#. Hs00943425_g1), ERF (Cat#. Hs01100070_g1), ETV1 (Cat#. Hs00951951_m1), ETV4 (Cat#. Hs00383361_g1), ETV5 (Cat#. Hs00927557_m1), and endogenous controls GAPDH (Cat#. Hs02758991_g1) were amplified with Taqman gene expression assay (Applied Biosystems).

Techniques: Expressing, Control, Transwell Assay, Comparison

Journal: eLife

Article Title: The CIC-ERF co-deletion underlies fusion-independent activation of ETS family member, ETV1, to drive prostate cancer progression

doi: 10.7554/eLife.77072

Figure Lengend Snippet:

Article Snippet: Human CIC (Cat#. Hs00943425_g1), ERF (Cat#. Hs01100070_g1), ETV1 (Cat#. Hs00951951_m1), ETV4 (Cat#. Hs00383361_g1), ETV5 (Cat#. Hs00927557_m1), and endogenous controls GAPDH (Cat#. Hs02758991_g1) were amplified with Taqman gene expression assay (Applied Biosystems).

Techniques: Transfection, Construct, shRNA, Plasmid Preparation, Sequencing

Journal: Oncogenesis

Article Title: Membrane-bound β-catenin degradation is enhanced by ETS2-mediated Siah1 induction in Helicobacter pylori -infected gastric cancer cells

doi: 10.1038/oncsis.2017.26

Figure Lengend Snippet: ETS2 binds to EBS in the 5′ UTR and induces siah1 transcription and protein expression in the H. pylori -infected GCCs. ( a ) Promoter and 5′ UTR analysis of human siah1 gene shows that an EBS located between +92 and +95 (represented by a box). We assume that the most upstream exon 1 of the Siah1 cDNA is at position +1 22 . ( b ) Western blot results showing the status of ETS2 binding with the siah1 5′ UTR ( n =3) in the presence or absence of H. pylori . ETS2 binds to the WT EBS only but not with the EBS-Mut oligo. Western blot of nuclear lysates shows the levels of ETS2 protein expression in the input lanes. HDAC1 is the loading control for nuclear lysates. ( c ) ChIP assay of ETS2 immunocomplex for siah1 EBS. IgG= immunoglobulin G; M= MW marker; NS= non-specific primer, S= specific primer. ( d ) Figure shows dual luciferase assay involving WT and ETS2-Mut siah1 5′ UTR-transfected and infected or uninfected MKN45 cells. Data are analyzed by two-way ANOVA with Tukey’s post hoc test ( n =3). Error Bars, s.e.m. *** P < 0.001, ** P < 0.01, * P < 0.05. ( e ) Bar graph of dual luciferase assay result showing transcriptional activation of WT siah1 5’ UTR with ectopic ETS2 expression and H. pylori infection. Data are analyzed by two-way ANOVA with Tukey’spost hoc test. Error bars, s.e.m. *** P <0.003; **** P< 0.0001. ( f ) Transient transfection of ETS2 siRNA followed by western blotting shows Siah1 suppression in the ETS2-suppressed MKN45 cells.

Article Snippet: ETS2 construct (#28128) was procured from Addgene, MA, USA.

Techniques: Expressing, Infection, Western Blot, Binding Assay, Control, Marker, Luciferase, Transfection, Activation Assay

Journal: Oncogenesis

Article Title: Membrane-bound β-catenin degradation is enhanced by ETS2-mediated Siah1 induction in Helicobacter pylori -infected gastric cancer cells

doi: 10.1038/oncsis.2017.26

Figure Lengend Snippet: Parallel ETS2 and Siah1 induction occurs in H. pylori- infected GCCs, as well as in human gastric adenocarcinoma biopsy samples. ( a ) A representative western blot ( n =3) showing optimal induction of ETS2 and Siah1 by 200 MOI of H. pylori at 3 h and 6 h p.i. ( b ) Cells infected for various time periods are analyzed for ETS2 and Siah1 protein expression ( n =3). ( c ) Western blot ( n =4 showing ETS2 and Siah1 proteins in uninfected and H. pylori -infected Kato III, MKN45 and AGS cells. ( d ) Western blot results ( n =3) depicting equal effectiveness of 8-1 and 26695 in inducing ETS2 and Siah1 proteins. Graphical representations of panels A–D are shown in .

Article Snippet: ETS2 construct (#28128) was procured from Addgene, MA, USA.

Techniques: Infection, Western Blot, Expressing

Journal: Oncogenesis

Article Title: Membrane-bound β-catenin degradation is enhanced by ETS2-mediated Siah1 induction in Helicobacter pylori -infected gastric cancer cells

doi: 10.1038/oncsis.2017.26

Figure Lengend Snippet: Induced ETS2 and Siah1 expression in Helicobacter -infected human and mouse gastric epithelia. ( a ) H&E staining of human non-cancer (a) and adenocarcinoma (e) biopsy samples ( n =10 for each group) and fluorescence microscopy of the staining for Siah1 (b and f), ETS2 (c and g) and DAPI (d and h). Original magnification × 100, inset × 400. Scales shown 50 μm. Inset scale 20 μm. ( b ) H&E staining of uninfected ( n =16) and infected ( n =16) antral gastric tissues from C57BL/6 mice (a and e, respectively) and their corresponding fluorescence microscopy images showing Siah1 (b and f), ETS2 (c and g) and DAPI (d and h) staining. Infected mice show inflammation (thin arrow), mucus gland metaplasia (open arrow) in the mucosa. ( c ) Data representing similar observations in another set of uninfected and infected mice gastric tissues. Original magnification × 100, inset × 400. Scales shown in b and c : 50 μm.

Article Snippet: ETS2 construct (#28128) was procured from Addgene, MA, USA.

Techniques: Expressing, Infection, Staining, Fluorescence, Microscopy

Journal: Oncogenesis

Article Title: Membrane-bound β-catenin degradation is enhanced by ETS2-mediated Siah1 induction in Helicobacter pylori -infected gastric cancer cells

doi: 10.1038/oncsis.2017.26

Figure Lengend Snippet: Siah1 promotes membrane-bound β-catenin degradation in H. pylori -infected GCCs. ( a ) Western blotting result ( n =3) of whole cell lysates prepared from Siah1 or empty vector (pcDNA3.1 + )-transfected and H. pylori -infected or uninfected MKN45 cells. Blots are incubated with Siah1 and β-catenin primary antibodies. α-tubulin is the loading control. ( b ) Decreased membrane-bound β-catenin expression is detected by western blotting ( n =3) in Siah1-overexpressed and H. pylori -infected cells. ( c ) Western blotting result ( n =3) of whole cell lysates prepared from siSiah1 or siControl duplex-transfected and H. pylori -infected or uninfected MKN45 cells. Blots are incubated with Siah1 and β-catenin primary antibodies. α-tubulin is the loading control. ( d ) Decreased membrane-bound β-catenin expression is seen in Siah1-supressed and H. pylori -infected cells. ( e ) Western blotting result ( n =3) of whole cell lysates prepared from siETS2 or siControl duplex-transfected and H. pylori -infected MKN45 cells. Blots are incubated with Siah1 and β-catenin primary antibodies. α-tubulin is the loading control. ( f ) Decreased membrane-bound β-catenin expression is detected in western blotting ( n =3) in ETS2-supressed and H. pylori -infected cells. ( g ) Siah1 is immunoprecipitated with anti-Siah1 antibody from the membrane fraction and immunoblotted to detect β-catenin and Siah1 interaction. Non-specific band=immunoglobulin heavy chain. Graphical representations are shown in .

Article Snippet: ETS2 construct (#28128) was procured from Addgene, MA, USA.

Techniques: Membrane, Infection, Western Blot, Plasmid Preparation, Transfection, Incubation, Control, Expressing, Immunoprecipitation

Journal: Oncogenesis

Article Title: Membrane-bound β-catenin degradation is enhanced by ETS2-mediated Siah1 induction in Helicobacter pylori -infected gastric cancer cells

doi: 10.1038/oncsis.2017.26

Figure Lengend Snippet: Siah1 increases the rate of cell migration in H. pylori -infected GCCs. ( a ) Graphical representations ( n =3) of wound healing assay showing enhanced migration potential of Siah1-overexpressed and H. pylori -infected cells. 24 h post transfection, wound is marked and the scratched area is monitored from 6 h-24 h. Data have been analyzed by 2-way ANOVA with Tukey’s post hoc test. Error bars, s.e.m. **** P <0.0001. ( b ) Cell migration assay performed in transwell chambers with AGS cells show decreased migration of infected cells expressing siSiah1 as compared to the siControl group. Arrowheads indicate migrated cells; scales shown: 50 μm. Bar graphs denote the average number of migrated cells (mean±s.e.m., n =3). Data are analyzed by 2-way ANOVA with Tukey’s post hoc test. Error bars, s.e.m. *** P <0.003; **** P <0.0001. Protein level of siSiah1 cells are shown in the accompanying western blot images. ( c ) Matrigel invasion assay with Siah1-suppressed AGS cells showing reduced invasiveness in H. pylori -infected GCCs. Bar graphs denote the average number of cells invaded through the Transwell matrigel ( n =3). Arrowheads indicate invaded cells. Data are analyzed by two-way ANOVA with Tukey’s post hoc test. Error bars, s.e.m. ** P <0.01; *** P <0.003; **** P <0.0001. Scales shown: 50 μm. ( d ) Soft agar colony formation assay is performed on MKN45 cells. Siah1 stably-transfected cells show a substantial increase in colony forming ability post H. pylori infection as compared to cells expressing the empty vector. Siah1 level in Siah1-stable cells are shown in the accompanying western blot result. ( e ) Soft agar assay performed with uninfected or infected MKN45 cells that stably-express either ETS2 or empty vector show a substantial increase in colony forming ability of ETS2-expressing cells. Protein level of ETS2 stable cells are shown in the accompanying western blot images. Scales shown in b and c : 100 μm.

Article Snippet: ETS2 construct (#28128) was procured from Addgene, MA, USA.

Techniques: Migration, Infection, Wound Healing Assay, Transfection, Cell Migration Assay, Expressing, Western Blot, Invasion Assay, Soft Agar Assay, Stable Transfection, Plasmid Preparation