zeb2 anti sip1  (Bioss)

Journal: iScience

Article Title: STEAP3 promotes triple-negative breast cancer growth through the FGFR1-mediated activation of PI3K/AKT/mTOR signaling

doi: 10.1016/j.isci.2025.112526

Figure Lengend Snippet: Modulating STEAP3 expression alters breast cancer cell proliferative, migratory, invasive, and EMT activity in vitro (A–M) Knockdown Effects: (A-G) The effects of STEAP3 KD on MDA-MB-231 and MDA-MB-468 cell proliferative activity were assessed through analyses of Ki67 and PCNA expression, CCK-8 assays, and colony formation assays. (H) The effects of STEAP3 KD on migration in a wound healing assay. Scale bars: 50 μm. (I–J) The effects of STEAP3 KD on invasion and migration in Transwell assays. Scale bars: 25 μm. (K–M) Effects of STEAP3 KD on EMT marker expression ( E-cadherin , N-cadherin , ZEB1 , ZEB2 ) as confirmed through RT-qPCR, Western immunoblotting, and TCGA database analyses. (N–T) Overexpression Effects: (N–P) The effects of STEAP3 OE on BT-549 cell proliferative activity were assessed through analyses of Ki67 and PCNA expression, CCK-8 assays, and colony formation assays. (Q) The effects of STEAP3 OE on migration in a wound healing assay. Scale bars: 50 μm. (R) The effects of STEAP3 OE on invasion and migration in Transwell assays. Scale bars: 25 μm. (S and T) Effects of STEAP3 OE on EMT marker levels were assessed via RT-qPCR and Western immunoblotting. Data are presented as mean ± SD (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001).

Article Snippet: ZEB2/Anti-SIP1 , Bioss USA , Cat# bs-20485R; RRID: AB_11097973.

Techniques: Expressing, Activity Assay, In Vitro, Knockdown, CCK-8 Assay, Migration, Wound Healing Assay, Marker, Quantitative RT-PCR, Western Blot, Over Expression

Journal: Cell

Article Title: Structural Remodeling of the Human Colonic Mesenchyme in Inflammatory Bowel Disease

doi: 10.1016/j.cell.2018.08.067

Figure Lengend Snippet: Human Colonic Mesenchymal Heterogeneity in Health (A) Flow cytometry analysis of the indicated surface markers on colonic single-cell suspensions following removal of epithelial and hematopoietic cells by MACS. Column flow-through is shown in red, and column-retained fraction is in blue. (B) t-SNE plot of the healthy human colonic mesenchyme dataset. Single cells colored by cluster annotation. (C) Violin plots for pan-fibroblast marker genes vimentin ( VIM ) and collagen types 1 and 3 ( COL1A2 , COL3A1 ) across clusters. (D) Violin plots for high-ranked transcriptional regulators and marker genes sharing GO annotation for significantly enriched terms for (i) S1 subset, (ii) S2 subset, (iii) S3 subset, (iv) S4 subset, and (v) myofibroblasts. Crossbars indicate median expression. (E) Single-molecule ISH staining of healthy human colonic tissue showing distribution of S1 markers ( ADAMDEC1 , DCN , SLIT2 , and CXCL12 ) (left) and S2 markers ( F3 (CD142) , WNT5A , HSD17B2 , WNT5B , POSTN , BMP2 , FRZB , BMP5 ) (right). (F) Identification of SOX6 − ZEB2 + /ZEB1 − ZEB2 + S1 and SOX6 + ZEB2 − /ZEB1 + ZEB2 − S2 subsets in healthy human colon. (G) Single (left) and co-staining with CD45 (right) and F3/CD142 (S2), ZEB2 (S1), and SMAD7 (S3) by IHC in colonic sections. The lower far-right panel is a quadruple stain of all 4 markers. (H) Differential expression analysis between S2a and S2b reveals 302 differentially expressed genes. (I) t-SNE plots showing examples of genes differentially expressed between S2a and S2b. (J) GO enrichment terms for S2a and S2b. See also – and – .

Article Snippet: Mouse monoclonal anti-human ZEB2/SIP1 , Bio-Techne , Cat#MAB73782; RRID: AB_2737260.

Techniques: Flow Cytometry, Marker, Expressing, Staining

Journal: Cell

Article Title: Structural Remodeling of the Human Colonic Mesenchyme in Inflammatory Bowel Disease

doi: 10.1016/j.cell.2018.08.067

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-human ZEB2/SIP1 , Bio-Techne , Cat#MAB73782; RRID: AB_2737260.

Techniques: Recombinant, Coagulation, Antibody Labeling, Membrane, Plasmid Preparation, Polymer, Blocking Assay, Staining, Imaging, Software, Hybridization

Journal: iScience

Article Title: ZEB2 and MEIS1 independently contribute to hematopoiesis via early hematopoietic enhancer activation

doi: 10.1016/j.isci.2023.107893

Figure Lengend Snippet: Requirement of Notch signaling and hematopoietic cytokines for hematopoietic enhancer activation (A) Heatmap and boxplots showing the effects of Notch signaling inhibition by DAPT and of the absence of hematopoietic cytokines on the H3K27ac ChIP-seq signal at the hematopoietic enhancers defined in Figure 1 C. DAPT was added from day 4 at a concentration of 10 μM, and hematopoietic cytokines (SCF, TPO, FLT3L and FP6) were removed the same day. ns, p > 0.05; ∗∗∗∗ P ≦ 0.0001 (repeated-measures one-way ANOVA followed by Dunnett’s multiple comparisons test). (B) Flow cytometry of control and DAPT-treated live cells on day 10 for identifying HPCs by the expression of CD34 and CD45. DAPT was added from days 4–10 at a concentration of 10 μM. Representative (left) and summary data (right) are shown. ∗ P ≦ 0.05 (paired t-test). (C) Percentage of hematopoietic enhancers occupied by Notch1 on day 7. Hematopoietic enhancers are divided into 3 clusters, as shown in Figure 1 C. (D) Fold-change of the indicated gene expressions in day 7 CD34 + cells by DAPT treatment. Genes were selected from the transcription factors whose binding motifs were enriched at cluster 2 enhancers (see Figure 2 A). ns, FDR >0.05; ∗∗FDR ≦ 0.01; ∗∗∗∗FDR ≦ 0.0001 (calculated by DESeq2). (E) Binding of the indicated transcription factors to hematopoietic enhancers on day 7. The ChIP-seq signal distribution at enhancer regions (5′ to 3′ ends) +/− 1 kB (top) and the proportions of hematopoietic enhancers bound by the transcription factors (bottom) are shown. Hematopoietic enhancers were grouped according to the timing of their activation, as shown in Figure 1 C. (F) Expression of the indicated genes during hematopoietic cell differentiation. Genes were selected from candidate regulators of cluster 1 and 2 hematopoietic enhancers, as shown in Figure 2 A. (G) Expression patterns of ZEB2 and MEIS1 during in vitro hematopoietic cell differentiation. The averages and standard deviations are shown. (H) Occupancy of the genomic loci of the genes shown in Figure 3 E by the five indicated regulators. The average of two independent experiments (A, C, D, G), representative and summary of four independent experiments (B), individual data of two or three independent experiments (F), and representative data from one or two experiments (E, H) are shown.

Article Snippet: Anti-SIP1 (ZEB2) , Bethyl Laboratories , A302474A; RRID: AB_1944271.

Techniques: Activation Assay, Inhibition, ChIP-sequencing, Concentration Assay, Flow Cytometry, Control, Expressing, Binding Assay, Cell Differentiation, In Vitro

Journal: iScience

Article Title: ZEB2 and MEIS1 independently contribute to hematopoiesis via early hematopoietic enhancer activation

doi: 10.1016/j.isci.2023.107893

Figure Lengend Snippet: Complete impairment of HPC differentiation in the absence of ZEB2 (A) Flow cytometry of WT and two lines of ZEB2-deficient live cells on day 10 for identifying HPCs and mature hematopoietic cells by the expression of CD34 and CD45. (B) Percentages (left) and numbers (right) of WT and ZEB2-deficient CD34 + CD45 + and CD34 – CD45 + cells on day 10. Individual results, mean and standard deviation are shown. ∗ P ≦ 0.05; ∗∗ P ≦ 0.01; ∗∗∗ P ≦ 0.001; ∗∗∗∗ P ≦ 0.0001 (two-way ANOVA followed by the Holm–Sidak multiple comparisons test). (C) PCA of the global gene expression during WT and ZEB2-deficient HPC differentiation. (D) Gene ontology analysis of genes down-regulated in ZEB2-deficient day 10 CD34 + CD45 – cells compared to WT counterparts. The top 10 gene sets are shown. (E) The expression of RUNX1 splice variants in WT and ZEB2-deficient day 7 and 10 endothelial cells, shown as transcripts per million (TPM) of RNA-seq data. ns, p > 0.05; ∗ P ≦ 0.05; ∗∗ P ≦ 0.01 (one-way ANOVA followed by Tukey’s multiple comparisons test). (F) Heatmap showing the H3K27ac ChIP-seq signal at the hematopoietic enhancers along the hematopoietic cell differentiation of WT and ZEB2-deficient ESCs. Hematopoietic enhancers are grouped by the timing of activation in WT, as shown in Figure 1 C ns, p > 0.05; ∗∗ P ≦ 0.01; ∗∗∗∗ P ≦ 0.0001 (repeated-measures one-way ANOVA followed by Dunnett’s multiple comparisons test). (G) Gene ontology analysis of genes nearby cluster 2 enhancers whose activity was reduced by ZEB2 deletion, as shown in Figure 4 F. The top 10 gene sets are shown. Representative (A) and summary (B) data from five independent experiments, and individual (C, E) and average (F) data from two or three independent experiments are shown.

Article Snippet: Anti-SIP1 (ZEB2) , Bethyl Laboratories , A302474A; RRID: AB_1944271.

Techniques: Flow Cytometry, Expressing, Standard Deviation, Gene Expression, RNA Sequencing, ChIP-sequencing, Cell Differentiation, Activation Assay, Activity Assay

Journal: iScience

Article Title: ZEB2 and MEIS1 independently contribute to hematopoiesis via early hematopoietic enhancer activation

doi: 10.1016/j.isci.2023.107893

Figure Lengend Snippet: Severe reduction of HPC differentiation in the absence of MEIS1 (A) The enrichment of transcription factor motifs at cluster 2 hematopoietic enhancers, whose activity was affected or unaffected by ZEB2 deletion (left). The top 15 differentially enriched transcription factors and ZEB2 are shown. The expression of the indicated transcription factors in ZEB2-deficient cells compared to WT counterparts on day 7 (middle) and occupation of their gene loci by ZEB2 in WT (right) are shown by the color code. (B) H3K27ac modification, binding of ZEB2, and mRNA transcription at the MEIS1 gene locus in WT and ZEB2-deficient CD34 + CD45 – cells on day 7 and 10. Location of cluster 2 enhancer regions are also indicated. (C) Expression pattern of MEIS1 during in vitro hematopoietic cell differentiation in WT and ZEB2-deficient lines. The averages and standard deviations are shown. ∗∗∗∗FDR ≦ 0.0001 (calculated by DESeq2). (D) Flow cytometry of WT and two lines of MEIS1-deficient live cells on day 10 for identifying HPCs and mature hematopoietic cells by the expression of CD34 and CD45. (E) Percentages (left) and numbers (right) of WT and MEIS1-deficient CD34 + CD45 + and CD34 – CD45 + cells on day 10. Individual results, mean and standard deviation are shown. Ns, p > 0.05; ∗∗∗∗ P ≦ 0.0001 (two-way ANOVA followed by Holm–Sidak multiple comparisons test). (F) PCA of the global gene expression during WT and MEIS1-deficient hematopoietic progenitor cell differentiation. (G) The expression of RUNX1 splice variants in WT and MEIS1-deficient CD34 + CD45 – cells on day 7 and 10 endothelial cells, shown as transcripts per million (TPM) in the RNA-seq data. ns, p > 0.05; ∗ P ≦ 0.05; ∗∗ P ≦ 0.01 (one-way ANOVA followed by Tukey’s multiple comparisons test). Representative (B) and individual (F, G) data from two or three independent experiments, average of two independent experiments (C), and representative (D) and summary (E) data from four to five independent experiments are shown.

Article Snippet: Anti-SIP1 (ZEB2) , Bethyl Laboratories , A302474A; RRID: AB_1944271.

Techniques: Activity Assay, Expressing, Modification, Binding Assay, In Vitro, Cell Differentiation, Flow Cytometry, Standard Deviation, Gene Expression, RNA Sequencing

Journal: iScience

Article Title: ZEB2 and MEIS1 independently contribute to hematopoiesis via early hematopoietic enhancer activation

doi: 10.1016/j.isci.2023.107893

Figure Lengend Snippet: Transcriptional dysregulation by ZEB2 deletion in arterial HE (A) Flow cytometry of WT and two lines of ZEB2-deficient and MEIS1-deficient CD34 + CD73 – CD43 – cells on day 7 for identifying arterial HE by the expression of DLL4 and CXCR4. (B) Number of WT, ZEB2-deficient, and MEIS1-deficient CD34 + CD73 – CD43 – DLL4 + CXCR4 + arterial HE cells. The individual results, means, and standard deviations are shown. ns, p > 0.05 (one-way ANOVA followed by Holm–Sidak multiple comparisons test). (C) PCA of global gene expression in WT, ZEB2-deficient, MEIS1-deficient, and ZEB2/MEIS1 double-deficient CD34 + CD73 – CD43 – DLL4 + CXCR4 + arterial HE, and CD34 + KDR + CD73 + CD43 – DLL4 + CXCR4 + arterial non-HE on day 7. (D) Heatmap and boxplots showing the expression patterns of genes associated with Notch signaling (GO:0007219) in WT, ZEB2-deficient, MEIS1-deficient, and ZEB2/MEIS1 double-deficient CD34 + CD73 – CD43 – DLL4 + CXCR4 + arterial He. ns, p > 0.05; ∗∗∗ P ≦ 0.001; ∗∗∗∗ P ≦ 0.0001 (repeated-measures one-way ANOVA followed by Dunnett’s multiple comparisons test). (E) Average Notch1 ChIP-seq signal at the Notch1 binding sites in WT and ZEB2-deficient CD34 + cells on day 7. Notch1 binding sites were defined in WT CD34 + cells on day 7. (F) Number of differentially expressed genes by ZEB2 deletion, MEIS1 deletion, or ZEB2/MEIS1 double deletion compared to WT on day 7. Representative (A) and summary (B) of four independent experiments and individual (C) or average (D, E, F) data from two independent experiments are shown.

Article Snippet: Anti-SIP1 (ZEB2) , Bethyl Laboratories , A302474A; RRID: AB_1944271.

Techniques: Flow Cytometry, Expressing, Gene Expression, ChIP-sequencing, Binding Assay

Journal: iScience

Article Title: ZEB2 and MEIS1 independently contribute to hematopoiesis via early hematopoietic enhancer activation

doi: 10.1016/j.isci.2023.107893

Figure Lengend Snippet: Cooperative activation of hematopoietic enhancers by ZEB2 and MEIS1 (A) Heatmap and boxplots showing the z-score-normalized H3K27ac ChIP-seq signal at hematopoietic enhancers in WT, ZEB2-deficient, and MEIS1-deficient day 7 CD34 + KDR + , day 10 CD34 + CD45 – , and day 10 CD34 + CD45 + (HPC) cells. Hematopoietic enhancers were grouped by the timing of activation in WT, as shown in Figure 1 C, and clusters 2 and 3 were further clustered by the effects of ZEB2 and MEIS1 deletion. ns, p > 0.05; ∗∗ P ≦ 0.01; ∗∗∗∗ P ≦ 0.0001 (repeated-measures one-way ANOVA followed by Dunnett’s multiple comparisons test). (B) H3K27ac modification and binding of ZEB2 and MEIS1 at RUNX1 and SPI1 gene loci in WT, ZEB2-deficient, and MEIS1-deficient CD34 + CD45 – cells on day 7 and 10. (C) The overlap of ZEB2 and MEIS1 genomic occupancy outside and inside hematopoietic enhancers. (D) Heatmap showing the MEIS1 ChIP-seq signal at ZEB2 binding sites (5′ to 3′ ends) +/− 1 kB within hematopoietic enhancers in WT and ZEB2-deficient CD34 + cells on day 7. (E) Expression patterns of genes down-regulated and up-regulated by ZEB2 deficiency in WT, ZEB2-deficient, MEIS1-deficient, MEIS1-induced ZEB2-deficient, and ZEB2-overexpressed ZEB2-deficient CD34 + KDR + cells on day 7. Black circles indicate medians. ns, p > 0.05; ∗ P ≦ 0.05; ∗∗∗∗ P ≦ 0.0001 (repeated-measures one-way ANOVA followed by Dunnett’s multiple comparisons test). (F) Histograms showing the H3K27ac ChIP-seq signal at the hematopoietic enhancers defined in Figure 4 F in WT, ZEB2-deficient, MEIS1-induced ZEB2-deficient, and ZEB2-overexpressed ZEB2-deficient CD34 + KDR + cells on day 7. (G) Heatmap and violin plots showing the z-score-normalized H3K27ac ChIP-seq signal at the subfraction of cluster 2 hematopoietic enhancers, whose activity is reduced by ZEB2 deletion, in cells shown in E. Black circles indicate medians. ∗∗ P ≦ 0.01; ∗∗∗∗ P ≦ 0.0001 (repeated-measures one-way ANOVA followed by Dunnett’s multiple comparisons test). (H) Flow cytometry of WT, ZEB2-deficient, and MEIS1-induced ZEB2-deficient, and ZEB2-overexpressed ZEB2-deficient live cells on day 10 for identifying HPCs and mature hematopoietic cells by the expression of CD34 and CD45. Both MEIS1 and ZEB2 were induced by the addition of doxycycline (dox) from day 4. Representative (top) and summary data (bottom) are shown. ns, p > 0.05; ∗∗∗∗ P ≦ 0.0001 (two-way ANOVA followed by Sidak’s multiple comparisons test). Average (A, C, E, F, G) and representative (B, D) from two independent experiments, and representative and summary results of four independent experiments (H) are shown.

Article Snippet: Anti-SIP1 (ZEB2) , Bethyl Laboratories , A302474A; RRID: AB_1944271.

Techniques: Activation Assay, ChIP-sequencing, Modification, Binding Assay, Expressing, Activity Assay, Flow Cytometry

Journal: iScience

Article Title: ZEB2 and MEIS1 independently contribute to hematopoiesis via early hematopoietic enhancer activation

doi: 10.1016/j.isci.2023.107893

Figure Lengend Snippet:

Article Snippet: Anti-SIP1 (ZEB2) , Bethyl Laboratories , A302474A; RRID: AB_1944271.

Techniques: Recombinant, Sequencing, CRISPR, Software

Journal: International Journal of Oncology

Article Title: SIP1 serves a role in HBx-induced liver cancer growth and metastasis

doi: 10.3892/ijo.2019.4884

Figure Lengend Snippet: Primer and siRNA sequences.

Article Snippet: The following primary antibodies were used in the present study: Rabbit anti-E-cadherin monoclonal antibody (Cell Signaling Technology, Inc., Beverly, MA, USA; cat. no. 3195), rabbit anti-N-cadherin monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 4061P), rabbit anti-Slug monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 9585P), mouse anti-vimentin monoclonal antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-15393), mouse anti-SIP1 monoclonal antibody (E-11; Santa Cruz Biotechnology, Inc.; cat. no. sc-271984), rabbit anti-SIP1 monoclonal antibody (Abcam, Cambridge, UK; cat. no. ab-138222), mouse anti-HBx polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-57760), mouse anti-HDAC1 polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-81598), rabbit anti-HDAC1 monoclonal antibody (GeneTex, Inc., Irvine, CA, USA; cat. no. GTX100513222) and mouse anti-β-actin monoclonal antibody (Boster Biological Technology, Ltd., Wuhan, China; cat. no. BM0627).

Techniques: Sequencing, Control, Chromatin Immunoprecipitation

Journal: International Journal of Oncology

Article Title: SIP1 serves a role in HBx-induced liver cancer growth and metastasis

doi: 10.3892/ijo.2019.4884

Figure Lengend Snippet: SIP1 is crucial in the HBx-induced epigenetic silencing of E-cadherin. (A) Epithelial-mesenchymal transition-related protein levels in HepG2 cells transfected with pcDNA3.1 and pHBx were examined by western blotting. (B) mRNA levels of SIP1 in HBx-expressing HepG2 cells were verified by reverse transcription-quantitative PCR analysis ( * P<0.05, ** P<0.01). (C) Expression levels of SIP1 and E-cadherin in HepG2-X and HepG2 cells were examined by western blotting. (D) Western blot analysis of E-cadherin and SIP1 in HepG2-X cells transfected with siHBx or siCont. (E) Western blotting results. shRNA reduced the expression of SIP1 in HBx-expressing HepG2 cells, with nonspecific shRNA serving as a negative control. (F) Transwell assay of HepG2 cells transfected with pcDNA3.1 or pHBx and treated with shSIP1 or scramble control. (G) Immunofluorescence analysis. The knockdown of SIP1 restored the epigenetic repression of E-cadherin induced by ectopic HBx. Vimentin concomitantly exhibited an inverse change in expression. DAPI was used to visualize nuclei. Scale bar, 10 µ m. Magnification, ×200. SIP1, Smad-interacting protein 1; HBx, hepatitis B virus X; pHBx, pcDNA3.1-HBx; si, small interfering RNA; sh, short hairpin RNA.

Article Snippet: The following primary antibodies were used in the present study: Rabbit anti-E-cadherin monoclonal antibody (Cell Signaling Technology, Inc., Beverly, MA, USA; cat. no. 3195), rabbit anti-N-cadherin monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 4061P), rabbit anti-Slug monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 9585P), mouse anti-vimentin monoclonal antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-15393), mouse anti-SIP1 monoclonal antibody (E-11; Santa Cruz Biotechnology, Inc.; cat. no. sc-271984), rabbit anti-SIP1 monoclonal antibody (Abcam, Cambridge, UK; cat. no. ab-138222), mouse anti-HBx polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-57760), mouse anti-HDAC1 polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-81598), rabbit anti-HDAC1 monoclonal antibody (GeneTex, Inc., Irvine, CA, USA; cat. no. GTX100513222) and mouse anti-β-actin monoclonal antibody (Boster Biological Technology, Ltd., Wuhan, China; cat. no. BM0627).

Techniques: Transfection, Western Blot, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, shRNA, Negative Control, Transwell Assay, Control, Immunofluorescence, Knockdown, Virus, Small Interfering RNA

Journal: International Journal of Oncology

Article Title: SIP1 serves a role in HBx-induced liver cancer growth and metastasis

doi: 10.3892/ijo.2019.4884

Figure Lengend Snippet: HBx recruits endogenous SIP1 to the E-cadherin promoter. (A) Dual-luciferase assay of human E-cadherin promoters in Mock treated, pcDNA3.1 or pHBX transfected HepG2 cells and HepG2-X cells. Data were normalized to the luciferase activity of cells treated with pcDNA3.1 and pGL3-Basic. Data are representative of at least three independent experiments. * P<0.05 HepG2 + pcDNA3.1 vs. HepG2 + pHBx; # P<0.05 HepG2 + pcDNA3.1 vs. HepG2-X. (B) ChIP primers were designed on E-cadherin gene regulatory regions. Distal primers correspond to downstream regulatory regions of -1895 to -1707 nt of the E-cadherin gene. The ChIP primers were designed adjacent to the TSS locations across the E-box region of the human E-cadherin promoter. pHBx-transfected HepG2 cell lysates were subjected to ChIP using an anti-SIP1 antibody. PCR was conducted using the indicated primer pairs. An empty vector and IgG served as external and internal negative controls. (C) Sequence homology of the consensus E-box in the E-cadherin promoter of mammalian. E-boxes 1, 3 and 4, CCAAT box and GC box are conserved regulatory elements, as shown in the diagram. The arrow indicates the putative TSS. (D) Mutations generated in the E-boxes carried the E-box 1 mutation CAGGTG → AAGGTA and E-box 3 mutation CACCTG → AACCTA. The wild-type E-cadherin promoter and promoter comprising two mutated E-boxes were cloned into a luciferase vector to construct the proE-cad-Luc and proE-cad-Luc-mEbox plasmids. (E) Dual-luciferase assay of E-cadherin promoter constructs with proE-cad-Luc or proE-cad-Luc-mEbox in pHBx- or pcDNA3.1-transfected HepG2 cells. ** P<0.01. (F) Relative E-cadherin promoter activities inshSIP1/shCont and pcDNA3.1/pHBX treated HepG2 cells. * P<0.05; (shSIP1 + WT, vs. shCont + WT). (G) Co-immunoprecipitation in protein extracts of pcDNA3.1-transfected HepG2 cells and HBx-expressing HepG2 cells with anti-SIP1 or anti-HBx antibodies and western blot detection of HBx and SIP1, respectively. (H) Immunofluorescent staining of HepG2-X cells with anti-HBx and anti-SIP1 to show the subcel-lular co-localization of HBx and SIP. DAPI was used to visualize nuclei. Scale bar=10 µ m. SIP1, Smad interacting protein 1; HBx, hepatitis B virus X; pHBx, pcDNA3.1-HBx; sh, short hairpin RNA. TSS, transcription start site; WT, proE-cad-Luc; Mut, proE-cad-Luc-mEbox; ChIp, chromatin immunoprecipitation .

Article Snippet: The following primary antibodies were used in the present study: Rabbit anti-E-cadherin monoclonal antibody (Cell Signaling Technology, Inc., Beverly, MA, USA; cat. no. 3195), rabbit anti-N-cadherin monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 4061P), rabbit anti-Slug monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 9585P), mouse anti-vimentin monoclonal antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-15393), mouse anti-SIP1 monoclonal antibody (E-11; Santa Cruz Biotechnology, Inc.; cat. no. sc-271984), rabbit anti-SIP1 monoclonal antibody (Abcam, Cambridge, UK; cat. no. ab-138222), mouse anti-HBx polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-57760), mouse anti-HDAC1 polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-81598), rabbit anti-HDAC1 monoclonal antibody (GeneTex, Inc., Irvine, CA, USA; cat. no. GTX100513222) and mouse anti-β-actin monoclonal antibody (Boster Biological Technology, Ltd., Wuhan, China; cat. no. BM0627).

Techniques: Luciferase, Transfection, Activity Assay, Plasmid Preparation, Sequencing, Generated, Mutagenesis, Clone Assay, Construct, Immunoprecipitation, Expressing, Western Blot, Staining, Virus, shRNA, Chromatin Immunoprecipitation

Journal: International Journal of Oncology

Article Title: SIP1 serves a role in HBx-induced liver cancer growth and metastasis

doi: 10.3892/ijo.2019.4884

Figure Lengend Snippet: HBx recruits SIP1 and HDAC1 to the E-cadherin promoter to repress its expression. (A) Western blot analysis of E-cadherin and HDAC1 in pcDNA3.1 or pHBX transfected HepG2 cells +/- TSA. E-cadherin promoter activities in TSA-treated cells following transfection with (B) shSIP1 or (C) SIP1 expression plasmids. Results are reported as the relative luciferase activity, vs. activity of pGL3-Basic. * P<0.05, ** P<0.01 (mean ± SD). (D) ChIP of lysates from HepG2 cells transfected with pHBx using anti-HDAC1 antibody. An empty vector pcDNA3.1 and protein G beads served as external and internal controls, respectively. (E) ChIP performed using HDAC1 antibody on the lysates of HepG2 cells treated with shSIP1/shCont and pcDNA3.1/pHBX. (F) Co-immunoprecipitation of HBx-expressing HepG2 cell-protein extracts with anti-SIP1 or anti-HBx antibodies and western blot detection of HDAC1, HBx and SIP1, respectively. (G) Immunofluorescent staining of HepG2-X cells with anti-HDAC1, anti-SIP1 and DAPI. The merged image showed HDAC1 and SIP1 co-localization in the nucleus. Scale bar=10 µ m. SIP1, Smad-interacting protein 1; HBx, hepatitis B virus X; pHBx, pcDNA3.1-HBx; sh, short hairpin RNA; Cont, control; HDAC1, histone deacetylase 1; ChIp, chromatin immunoprecipitation; TSA, trichostatin A.

Article Snippet: The following primary antibodies were used in the present study: Rabbit anti-E-cadherin monoclonal antibody (Cell Signaling Technology, Inc., Beverly, MA, USA; cat. no. 3195), rabbit anti-N-cadherin monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 4061P), rabbit anti-Slug monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 9585P), mouse anti-vimentin monoclonal antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-15393), mouse anti-SIP1 monoclonal antibody (E-11; Santa Cruz Biotechnology, Inc.; cat. no. sc-271984), rabbit anti-SIP1 monoclonal antibody (Abcam, Cambridge, UK; cat. no. ab-138222), mouse anti-HBx polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-57760), mouse anti-HDAC1 polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-81598), rabbit anti-HDAC1 monoclonal antibody (GeneTex, Inc., Irvine, CA, USA; cat. no. GTX100513222) and mouse anti-β-actin monoclonal antibody (Boster Biological Technology, Ltd., Wuhan, China; cat. no. BM0627).

Techniques: Expressing, Western Blot, Transfection, Luciferase, Activity Assay, Plasmid Preparation, Immunoprecipitation, Staining, Virus, shRNA, Control, Histone Deacetylase Assay, Chromatin Immunoprecipitation

Journal: International Journal of Oncology

Article Title: SIP1 serves a role in HBx-induced liver cancer growth and metastasis

doi: 10.3892/ijo.2019.4884

Figure Lengend Snippet: SIP1 mediates cell proliferation and apoptosis affected by ectopic expression of HBx in HepG2 cells. (A) HepG2 and HepG2-X cells were trans-fected with shSIP1 or shCont. Cell counting kit-8 assays were performed to determine cell proliferation ability. (B) Colony formation assay in HepG2 and HepG2-X cells transfected with shSIP1 or shCont. The colonies were stained with crystal violet and counted. Images are representative of three replicate experiments. (C) Apoptosis of HepG2 and HepG2-X cells were analyzed by flow cytometry with Annexin V-FITC/propidium iodide. * P<0.05, ** P<0.01. SIP1, Smad-interacting protein 1; HBx, hepatitis B virus X; sh, short hairpin RNA; Cont, control.

Article Snippet: The following primary antibodies were used in the present study: Rabbit anti-E-cadherin monoclonal antibody (Cell Signaling Technology, Inc., Beverly, MA, USA; cat. no. 3195), rabbit anti-N-cadherin monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 4061P), rabbit anti-Slug monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 9585P), mouse anti-vimentin monoclonal antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-15393), mouse anti-SIP1 monoclonal antibody (E-11; Santa Cruz Biotechnology, Inc.; cat. no. sc-271984), rabbit anti-SIP1 monoclonal antibody (Abcam, Cambridge, UK; cat. no. ab-138222), mouse anti-HBx polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-57760), mouse anti-HDAC1 polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-81598), rabbit anti-HDAC1 monoclonal antibody (GeneTex, Inc., Irvine, CA, USA; cat. no. GTX100513222) and mouse anti-β-actin monoclonal antibody (Boster Biological Technology, Ltd., Wuhan, China; cat. no. BM0627).

Techniques: Expressing, Cell Counting, Colony Assay, Transfection, Staining, Flow Cytometry, Virus, shRNA, Control

Journal: International Journal of Oncology

Article Title: SIP1 serves a role in HBx-induced liver cancer growth and metastasis

doi: 10.3892/ijo.2019.4884

Figure Lengend Snippet: HBx accelerates tumor growth through SIP1 in vivo . (A) Representative images of nude mice implanted with HepG2 and HepG2-X cells treated with shSIP1 or shCont. (B) Growth curves of tumors from nude mice implanted with the indicated cells (xenograft mice). (C) Images of tumors from xenograft mice. (D) Average tumor weights from xenograft mice. (E) Expression of SIP1 and E-cadherin in tumor tissues of mice was detected using western blotting. (F) Representative images of liver tissues and H&E staining of intrahepatic metastasis tumors from each group of orthotopic transplantation mice were shown. The arrows indicate visible intrahepatic metastatic tumors. (G) Immunohistochemical analysis of the transplanted tumors from each group of orthotopic transplantation mice. (H) H&E staining of the diaphragm metastases was detected only in the shCont-treated HepG2-X cells group of mice. Scale bar, 150 µ m. SIP1, Smad-interacting protein 1; HBx, hepatitis B virus X; si, small interfering RNA; sh, short hairpin RNA; Cont, control; H&E hematoxylin and eosin.

Article Snippet: The following primary antibodies were used in the present study: Rabbit anti-E-cadherin monoclonal antibody (Cell Signaling Technology, Inc., Beverly, MA, USA; cat. no. 3195), rabbit anti-N-cadherin monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 4061P), rabbit anti-Slug monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 9585P), mouse anti-vimentin monoclonal antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-15393), mouse anti-SIP1 monoclonal antibody (E-11; Santa Cruz Biotechnology, Inc.; cat. no. sc-271984), rabbit anti-SIP1 monoclonal antibody (Abcam, Cambridge, UK; cat. no. ab-138222), mouse anti-HBx polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-57760), mouse anti-HDAC1 polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-81598), rabbit anti-HDAC1 monoclonal antibody (GeneTex, Inc., Irvine, CA, USA; cat. no. GTX100513222) and mouse anti-β-actin monoclonal antibody (Boster Biological Technology, Ltd., Wuhan, China; cat. no. BM0627).

Techniques: In Vivo, Expressing, Western Blot, Staining, Transplantation Assay, Immunohistochemical staining, Virus, Small Interfering RNA, shRNA, Control

Journal: International Journal of Oncology

Article Title: SIP1 serves a role in HBx-induced liver cancer growth and metastasis

doi: 10.3892/ijo.2019.4884

Figure Lengend Snippet: Schematic diagram showing how HBx regulates E-cadherin via SIP1 and histone deacetylation. (A) Abundant enrichment of CAC and TFs are recruited to E-cadherin promoter region in the absence of HBx protein, leading to the transcription of E-cadherin. (B) HBx protein increases the levels of SIP1 and HDAC1. The three factors form a repressive triple complex locates at the E-cadherin promoter, and then induces the epigenetic silencing of E-cadherin. SIP1, Smad-interacting protein 1; HBx, hepatitis B virus X; TF, transcription factor; Pol II, RNA polymerase II.

Article Snippet: The following primary antibodies were used in the present study: Rabbit anti-E-cadherin monoclonal antibody (Cell Signaling Technology, Inc., Beverly, MA, USA; cat. no. 3195), rabbit anti-N-cadherin monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 4061P), rabbit anti-Slug monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 9585P), mouse anti-vimentin monoclonal antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-15393), mouse anti-SIP1 monoclonal antibody (E-11; Santa Cruz Biotechnology, Inc.; cat. no. sc-271984), rabbit anti-SIP1 monoclonal antibody (Abcam, Cambridge, UK; cat. no. ab-138222), mouse anti-HBx polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-57760), mouse anti-HDAC1 polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-81598), rabbit anti-HDAC1 monoclonal antibody (GeneTex, Inc., Irvine, CA, USA; cat. no. GTX100513222) and mouse anti-β-actin monoclonal antibody (Boster Biological Technology, Ltd., Wuhan, China; cat. no. BM0627).

Techniques: Virus