Journal: Oncotarget

Article Title: Cooperation between ZEB2 and Sp1 promotes cancer cell survival and angiogenesis during metastasis through induction of survivin and VEGF

doi: 10.18632/oncotarget.23139

Figure Lengend Snippet: ( A ) SW480 cells were transfected with a ZEB2 expression vector for 48 h. Transfected cells were lysed and used for immunoblotting. An anti-myc antibody was used to detect myc-tagged ZEB2. GAPDH or β-actin was used as an internal control. ( B – D ) SNU-398 cells were transfected with ZEB2-specific siRNA or scrambled siRNA for 48 h. (B) Transfected cells were lysed for immunoblot analysis. Densitometry quantification was performed on the immunoblots, using GAPDH as a loading control. (C) Conditioned medium from transfected cells was collected for an additional 48 h. VEGF levels in conditioned medium were determined by an ELISA. (D) Real-time qPCR analysis of ZEB2, VEGF, cyclin D1, and survivin mRNA levels. ( E ) SNU-398 cells were co-transfected with ZEB2-specific siRNA and a VEGF promoter (−2361/+298) reporter construct. Firefly luciferase activity representing VEGF promoter activity was measured after 48 h and normalized to the fluorescence signal intensity to measure the transfection efficiency. Values represent mean standard deviation. * P < 0.05. siSCR, scrambled siRNA.

Article Snippet: Cells were incubated with an anti-ZEB2 antibody (Active Motif) followed by a Alexa546-conjugated secondary antibody and then with an anti-Sp1 antibody (Santa Cruz Biotechnology) followed by a FITC-conjugated secondary antibody.

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Construct, Luciferase, Activity Assay, Fluorescence, Standard Deviation

Journal: Oncotarget

Article Title: Cooperation between ZEB2 and Sp1 promotes cancer cell survival and angiogenesis during metastasis through induction of survivin and VEGF

doi: 10.18632/oncotarget.23139

Figure Lengend Snippet: ( A ) SW480 cells were co-transfected with a ZEB2 expression vector and VEGF promoter (−2361/+298 and −267/+50) reporter constructs for 48 h. Firefly luciferase activity representing VEGF promoter activity was measured after 48 h and normalized to Renilla luciferase activity to measure the transfection efficiency. ( B ) Mutation analysis of Sp1 sites and Egr-1 sites in the VEGF promoter (−85/+50). Reporter constructs containing Sp1 site or Egr-1 site mutations were used in the reporter assay with SW480 cells. Values represent mean standard deviation. * P < 0.05. ( C, E, F, G ) SW480 cells were co-transfected with the ZEB2 expression vector and Sp1-specific siRNA for 48 h. (C) Real-time qPCR analysis to determine the effect of Sp1-specific siRNA on VEGF mRNA induction by ZEB2 in SW480 cells. (D) Reporter assay to determine the effect of mutant ZEB2 lacking the Smad-binding domain on VEGF promoter activity. (E, F) Real-time qPCR analysis of the mRNA levels of cyclin D1 (E) and survivin (F) in SW480 cells. Values represent mean ± standard deviation. * P < 0.05 compared with empty vector + control siRNA; § P < 0.05 compared with ZEB2 + control siRNA. (G) Transfected cells were lysed for immunoblot analysis. An anti-myc antibody was used to detect myc-tagged ZEB2. Densitometry quantification was performed on the immunoblots, using GAPDH as a loading control.

Article Snippet: Cells were incubated with an anti-ZEB2 antibody (Active Motif) followed by a Alexa546-conjugated secondary antibody and then with an anti-Sp1 antibody (Santa Cruz Biotechnology) followed by a FITC-conjugated secondary antibody.

Techniques: Transfection, Expressing, Plasmid Preparation, Construct, Luciferase, Activity Assay, Mutagenesis, Reporter Assay, Standard Deviation, Binding Assay, Control, Western Blot

Journal: Oncotarget

Article Title: Cooperation between ZEB2 and Sp1 promotes cancer cell survival and angiogenesis during metastasis through induction of survivin and VEGF

doi: 10.18632/oncotarget.23139

Figure Lengend Snippet: ( A ) SNU-398 cells were transiently transfected with ZEB2-specific siRNA or scrambled siRNA for 48 h (Upper). Alternatively, ZEB2-suppressed SNU-398 stable cells were incubated for 48 h (Lower). Then, conditioned medium was collected for an additional 48 h. HUVECs were seeded into 96-well plates and incubated for 24 h before serum-starvation for 24 h. HUVECs were incubated with conditioned medium from transfected SNU-398 cells or with 10 ng/ml VEGF for 48 h (A) or 10 min ( B ). Cell proliferation was determined by the colorimetric WST assay (A) and cells were lysed for immunoblot analysis (B). siSCR, scrambled siRNA; shSCR, scrambled shRNA. ( C , D ) Serum-starved HUVECs were incubated for 48 h (C) or 10 min (D) in the presence or absence of a VEGF-blocking antibody (10 µg/ml) along with the conditioned medium from HEK293E cells transfected with the ZEB2 expression vector for 48 h. Cells treated with 10 ng/ml VEGF were included. Cell proliferation was determined by the colorimetric WST assay (C). Values represent mean standard deviation. * P < 0.05. Cells were lysed for immunoblot analysis (D). C.M., conditioned medium. Densitometry quantification was performed on the immunoblots; phospho-VEGFR2, phospho-Tie2, phospho-ERK1/2, and phospho-Akt were normalized against the corresponding total protein levels.

Article Snippet: Cells were incubated with an anti-ZEB2 antibody (Active Motif) followed by a Alexa546-conjugated secondary antibody and then with an anti-Sp1 antibody (Santa Cruz Biotechnology) followed by a FITC-conjugated secondary antibody.

Techniques: Transfection, Incubation, WST Assay, Western Blot, shRNA, Blocking Assay, Expressing, Plasmid Preparation, Standard Deviation

Journal: Oncotarget

Article Title: Cooperation between ZEB2 and Sp1 promotes cancer cell survival and angiogenesis during metastasis through induction of survivin and VEGF

doi: 10.18632/oncotarget.23139

Figure Lengend Snippet: ( A ) ZEB2-suppressed SNU-398 stable cells were seeded into 96-well plates at a density of 4 × 10 3 cells/well and incubated for up to 72 h. Cell proliferation was determined by the colorimetric WST assay. ( B ) BrdU incorporation assay. ZEB2-suppressed SNU-398 stable cells were seeded into 96-well plates at a density of 5 × 10 3 cells/well and incubated for 48 h. Then, the cells were labeled with 10 mM BrdU for 2 h and the assay was performed as described in the Materials and Methods. ( C ) Soft agar anchorage-independent growth assay. SNU-398 stable cells were seeded into 6-well plates at a density of 1 × 10 3 cells/well in triplicates in semi-solid agar and then allowed to grow for 14 days. Total numbers of colonies (>0.1 mm) and colonies with a diameter of >0.7 mm were counted. Bar, 500 µm. ( D ) To induce anoikis, SNU-398 stable cells were seeded into 96-well plates with an Ultra-Low Attachment Surface at a density of 3 × 10 4 cells/well and grown for up to 5 days. Representative images of cells at 5 days are shown. Bar, 250 µm. Cell viability was determined by the colorimetric WST assay. Values represent mean ± standard deviation. * P < 0.05. ( E ) SNU-398 stable cells were incubated for 48 h with 2% or 10% FBS under suspension culture conditions and then stained with annexin V and PI for flow cytometric analysis. ( F ) SNU-398 stable cells were incubated for 48 h and then lysed for immunoblot analysis. shSCR, scrambled shRNA. Densitometry quantification was performed on the immunoblots, using GAPDH as a loading control except that phospho-JNK was normalized against total JNK protein.

Article Snippet: Cells were incubated with an anti-ZEB2 antibody (Active Motif) followed by a Alexa546-conjugated secondary antibody and then with an anti-Sp1 antibody (Santa Cruz Biotechnology) followed by a FITC-conjugated secondary antibody.

Techniques: Incubation, WST Assay, BrdU Incorporation Assay, Labeling, Growth Assay, Standard Deviation, Suspension, Staining, Western Blot, shRNA, Control

Journal: Oncotarget

Article Title: Cooperation between ZEB2 and Sp1 promotes cancer cell survival and angiogenesis during metastasis through induction of survivin and VEGF

doi: 10.18632/oncotarget.23139

Figure Lengend Snippet: ( A ) HEK293E cells were transiently co-transfected with the ZEB2 expression vector and Sp1-specific siRNA for 48 h prior to lysis for immunoblot analysis. ( B ) HEK293E cells co-transfected with the ZEB2 expression vector and Sp1-specific siRNA for 42 h were treated with MG132 (2 µM) for 6 h before lysis for immunoblot analysis. ( C ) A cytosolic fraction and a nuclear fraction were prepared from HEK293E cells co-transfected with the ZEB2 expression vector and Sp1-specific siRNA for 48 h for immunoblot analysis. GAPDH and PARP were used as internal controls for the cytosolic and nuclear fractions, respectively. An anti-myc and an anti-ZEB2 antibodies were used to detect myc-tagged ZEB2. Densitometry quantification was performed on the immunoblots, using GAPDH or PARP as a loading control.

Article Snippet: Cells were incubated with an anti-ZEB2 antibody (Active Motif) followed by a Alexa546-conjugated secondary antibody and then with an anti-Sp1 antibody (Santa Cruz Biotechnology) followed by a FITC-conjugated secondary antibody.

Techniques: Transfection, Expressing, Plasmid Preparation, Lysis, Western Blot, Control

Journal: Oncotarget

Article Title: Cooperation between ZEB2 and Sp1 promotes cancer cell survival and angiogenesis during metastasis through induction of survivin and VEGF

doi: 10.18632/oncotarget.23139

Figure Lengend Snippet: ( A ) Scatter plots examining ZEB2 mRNA expression (x-axis) and Sp1 mRNA expression (y-axis) from colorectal adenocarcinoma data (left, TCGA, Nature 2012; right, TCGA, Provisional). Statistical analysis to assess the correlation was performed by Pearson’s test. ( B ) Kaplan–Meier analysis shows the probability of overall survival from colorectal adenocarcinoma patient data (TCGA, Provisional) in relation to ZEB2 and Sp1 mRNA expression. All tumors with an mRNA expression profile ( n = 382) were analyzed. High ZEB2 expression was defined by Z > 1.75 and high Sp1 expression was defined by Z > 1.75. ( C ) Kaplan–Meier analysis shows the probability of overall survival from gastric cancer patient data (kmPlotter; n = 631) in relation to ZEB2 and Sp1 mRNA expression. ZEB2 and Sp1 expression was stratified as high vs. low according to the auto select best cutoff, and survival plots within previously published data sets were generated using http://kmplot.com (probe: 228333_at and 1553685_s_at for ZEB2 and Sp1, respectively) . P values were calculated by the Logrank test.

Article Snippet: Cells were incubated with an anti-ZEB2 antibody (Active Motif) followed by a Alexa546-conjugated secondary antibody and then with an anti-Sp1 antibody (Santa Cruz Biotechnology) followed by a FITC-conjugated secondary antibody.

Techniques: Expressing, Generated

Journal: bioRxiv

Article Title: Epidermal stem cell-derived extracellular vesicles induce fibroblasts mesenchymal-epidermal transition to alleviate hypertrophic scar via the miR-200s/ZEBs axis

doi: 10.1101/2025.01.27.635177

Figure Lengend Snippet: miR-200s were enriched in ESC-EVs and targeting ZEBs in HSFs. (a) Heatmap of the 20 miRNAs with the largest LogFC in the microarray analysis of the miRNA expression profiles between ESC-EVs and FB-EVs. (b) Volcano annotated with miR-200s probes (ESC-EVs vs FB-EVs). Type, High expression (red), Low expression (blue), and No significance (grey). (c) Chord diagram of GO enrichment terms. (d) Chord diagram of KEGG enrichment terms. (e) RT-qPCR analysis of the expression of miR-200s in HSFs treated with ESC-EVs, FB-EVs, or PBS for 24h. Graph represented the expression of the markers relative to that of U6. (f) RT-qPCR analysis of the expression of ZEB1 and ZEB2 in HSFs relative to GAPDH. (g) Western blotting analysis of ZEB1 and ZEB2 in HSFs. (h) Quantification of the relative band density compared to GAPDH. (i) Representative images of ZEB1 and ZEB2 immunofluorescence staining in HSFs. Scale bar, 50μm. (j) Quantification of the relative fluorescence ratio. * p <0.05, ** p <0.005, *** p <0.0005, **** p <0.0001.

Article Snippet: The membrane was blocked with 5% BSA and then incubated with one of the primary antibodies: anti-alpha smooth muscle (1:1000, GB111364, Servicebio), anti-COL1A1 (1:1000, E3E1X, Cell Signaling Technology), anti-N Cadherin (1:1000, A19083, Abclonal), anti-cytokeratin 1 (1:500, PA5-113746, Invitrogen), anti-cytokeratin 15 (1:10000, ab52816, Abcam), anti-E Cadherin (1:500, ab1416, Abcam), anti-ZEB1 (1:500, ab181451, Abcam) and anti-ZEB2 (1:1000, AF5278, Affinity Biosciences) overnight at 4L.

Techniques: Microarray, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Fluorescence

Journal: bioRxiv

Article Title: Epidermal stem cell-derived extracellular vesicles induce fibroblasts mesenchymal-epidermal transition to alleviate hypertrophic scar via the miR-200s/ZEBs axis

doi: 10.1101/2025.01.27.635177

Figure Lengend Snippet: HS exhibited miR-200s deficiency and ZEBs accumulation. (a) FISH stains of miR-200s in normal skin (NS), hyperplasia stage (HS), and mature stage of hypertrophic scar (MS). Scale bar, 20μm. (b) Quantification of the relative fluorescence ratio. (c) mIHC stains of α-SMA, ZEB1, ZEB2, COLL, α-SMA, N-Cad and K14, E-Cad in normal skin, hyperplasia stage, or mature stage of hypertrophic scar. Scale bar, 200μm, 20μm. (d) Quantification of the relative fluorescence ratio. * p <0.05, ** p <0.005, *** p <0.0005, **** p <0.0001.

Article Snippet: The membrane was blocked with 5% BSA and then incubated with one of the primary antibodies: anti-alpha smooth muscle (1:1000, GB111364, Servicebio), anti-COL1A1 (1:1000, E3E1X, Cell Signaling Technology), anti-N Cadherin (1:1000, A19083, Abclonal), anti-cytokeratin 1 (1:500, PA5-113746, Invitrogen), anti-cytokeratin 15 (1:10000, ab52816, Abcam), anti-E Cadherin (1:500, ab1416, Abcam), anti-ZEB1 (1:500, ab181451, Abcam) and anti-ZEB2 (1:1000, AF5278, Affinity Biosciences) overnight at 4L.

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Epidermal stem cell-derived extracellular vesicles induce fibroblasts mesenchymal-epidermal transition to alleviate hypertrophic scar via the miR-200s/ZEBs axis

doi: 10.1101/2025.01.27.635177

Figure Lengend Snippet: ESC-EVs induced MET of HSFs via miR-200s/ZEBs axis. (a) The multiplex CRISPR/Cas9 in a single lentiviral vector. (b) Five sgRNAs are indicated by scissors targeting two genomic loci of human miR-200 family host genes. (c) RT-qPCR analysis of EVs secreted from wild-type ESC-EVs (WT), ESCs infected with scramble vectors (Scramble), or miR-200s knockdown vectors (KD). (d) Morphologic alterations in HSFs incubated with WT-EVs, Scramble-EVs, KD-EVs, or PBS (NC) for 48h. (e) RT-qPCR analysis of COLL, α-SMA, N-cad, K1, K15, and E-cad in HSFs. Graph represented the expression of the markers relative to that of GAPDH. (f) Western blotting analysis of COLL, α-SMA, N-cad, K1, K15, and E-cad in HSFs. (g) Quantification of the relative band density compared to α-tubulin. (h) Representative images of COLL,α-SMA, N-cad, K1, K15, and E-cad immunofluorescence staining in HSFs incubated with WT-EVs, Scramble-EVs, KD-EVs, or PBS for 24h. Scale bar, 50μm. (i) Quantification of the relative fluorescence ratio. (j) Representative images of ZEB1 and ZEB2 immunofluorescence staining in HSFs. Scale bar, 50μm. (k) Quantification of the relative fluorescence ratio. (l) RT-qPCR analysis of the expression of ZEB1 and ZEB2 in HSFs relative to GAPDH. (m) Western blotting analysis of ZEB1 and ZEB2 in HSFs. (n) Quantification of the relative band density compared to α-tubulin. * p <0.05, ** p <0.005, *** p <0.0005, **** p <0.0001.

Article Snippet: The membrane was blocked with 5% BSA and then incubated with one of the primary antibodies: anti-alpha smooth muscle (1:1000, GB111364, Servicebio), anti-COL1A1 (1:1000, E3E1X, Cell Signaling Technology), anti-N Cadherin (1:1000, A19083, Abclonal), anti-cytokeratin 1 (1:500, PA5-113746, Invitrogen), anti-cytokeratin 15 (1:10000, ab52816, Abcam), anti-E Cadherin (1:500, ab1416, Abcam), anti-ZEB1 (1:500, ab181451, Abcam) and anti-ZEB2 (1:1000, AF5278, Affinity Biosciences) overnight at 4L.

Techniques: Multiplex Assay, CRISPR, Plasmid Preparation, Quantitative RT-PCR, Infection, Knockdown, Incubation, Expressing, Western Blot, Immunofluorescence, Staining, Fluorescence

Journal: bioRxiv

Article Title: Epidermal stem cell-derived extracellular vesicles induce fibroblasts mesenchymal-epidermal transition to alleviate hypertrophic scar via the miR-200s/ZEBs axis

doi: 10.1101/2025.01.27.635177

Figure Lengend Snippet: ESC-EVs alleviated the RHS through the miR-200s/ZEBs axis. (a) FISH stains of miR-200s in PBS, RESC-EVs, RFB-EVs treated RHS. Scale bar, 20μm. (b) Quantification of the relative fluorescence ratio. (c) Representative images of COLL,α-SMA, N-cad, K1, K15, E-cad, ZEB1 and ZEB2 immunofluorescence staining. Scale bar, 200μm, 20μm. (d) Quantification of the relative fluorescence ratio. * p <0.05, ** p <0.005, *** p <0.0005, **** p <0.0001.

Article Snippet: The membrane was blocked with 5% BSA and then incubated with one of the primary antibodies: anti-alpha smooth muscle (1:1000, GB111364, Servicebio), anti-COL1A1 (1:1000, E3E1X, Cell Signaling Technology), anti-N Cadherin (1:1000, A19083, Abclonal), anti-cytokeratin 1 (1:500, PA5-113746, Invitrogen), anti-cytokeratin 15 (1:10000, ab52816, Abcam), anti-E Cadherin (1:500, ab1416, Abcam), anti-ZEB1 (1:500, ab181451, Abcam) and anti-ZEB2 (1:1000, AF5278, Affinity Biosciences) overnight at 4L.

Techniques: Fluorescence, Immunofluorescence, Staining

Journal: Oncology Letters

Article Title: miR-215 functions as a tumor suppressor and directly targets ZEB2 in human non-small cell lung cancer

doi: 10.3892/ol.2015.3587

Figure Lengend Snippet: Expression of miR-215 and ZEB2 in NSCLC tissues and cell lines. (A) miR-215 expression was significantly reduced in NSCLC tissues compared with that of the corresponding non-cancerous tissues. miR-215 expression levels were calculated using the 2 −∆Ct method and normalized to U6 small nuclear RNA. (B) miR-215 expression was downregulated in NSCLC cell lines A549, H460, 95D and HCC827, compared with that of NLECs. (C) Relative ZEB2 protein levels in NSCLC and corresponding non-cancerous tissues. ZEB2 protein levels were measured by western blot analysis and normalized to β-actin. (D) ZEB2 protein levels in NSCLC cells were increased compared with NLECs. (E) The inverse correlation of ZEB2 protein levels with miR-215 expression was examined by Pearson correlation analysis. Values are expressed as the mean ± standard deviation. *P<0.05; **P<0.01 vs. NLEC. miR-215, microRNA-215; NSCLC, non-small cell lung cancer; NLEC, normal lung epithelial cells, ZEB2, zinc finger E-box-binding homeobox 2.

Article Snippet: Following blocking in 5% non-fat milk in 1X Tris-buffered saline (pH 7.4) containing 0.05% Tween-20, the membranes were incubated with purified rabbit anti-ZEB2 antisera (cat. no. LS-C160768; dilution, 1:1,000; LifeSpan BioSciences, Inc., Seattle, WA, USA) at 4°C overnight.

Techniques: Expressing, Western Blot, Standard Deviation, Binding Assay

Journal: Oncology Letters

Article Title: miR-215 functions as a tumor suppressor and directly targets ZEB2 in human non-small cell lung cancer

doi: 10.3892/ol.2015.3587

Figure Lengend Snippet: ZEB2 is a direct target of miR-215. (A) miR-215-binding sites in the ZEB2 3′UTR region. ZEB2-mut indicates the ZEB2 3′UTR with a mutation in miR-215-binding sites. (B) Western blot analysis demonstrated that transfection of miR-215 reduced ZEB2 protein expression. (C) Relative luciferase assay comparing the pGL3-ZEB2 and pGL3-ZEB2-Mut vectors in A549 cells. Firefly luciferase activity was normalized to Renilla luciferase activity. Values are expressed as the mean ± standard deviation. *P<0.05 vs. control. ZEB2, zinc finger E-box-binding homeobox 2; mut, mutant; Hsa, Homo sapien .

Article Snippet: Following blocking in 5% non-fat milk in 1X Tris-buffered saline (pH 7.4) containing 0.05% Tween-20, the membranes were incubated with purified rabbit anti-ZEB2 antisera (cat. no. LS-C160768; dilution, 1:1,000; LifeSpan BioSciences, Inc., Seattle, WA, USA) at 4°C overnight.

Techniques: Binding Assay, Mutagenesis, Western Blot, Transfection, Expressing, Luciferase, Activity Assay, Standard Deviation

Journal: Frontiers in Endocrinology

Article Title: BRAF V600E Mutation-Responsive miRNA-222-3p Promotes Metastasis of Papillary Thyroid Cancer Cells via Snail-Induced EMT

doi: 10.3389/fendo.2022.843334

Figure Lengend Snippet: Downregulation of miR-222-3p suppresses cell migration and EMT of PTCs. (A) Migration images of representative microscope fields (40x, 100x). (B, C) t -test analysis of the number of B-CPAP and TPC-1 migrating cells of N.C. and IN group. *** p< 0.001,**p < 0.01. (D) q-PCR validation of expression level of miR-222-3p in B-CPAP and TPC-1 transfected with negative control (N.C.) and miR-222-3p inhibitor (IN). ***p < 0.001,****p < 0.0001. (E, F) immunoblot of E-cadherin, Zeb2, and Snail protein from extracts of B-CPAP and TPC-1 transfected N.C. and IN. *p < 0.05.

Article Snippet: Primary antibodies were listed below: anti-E-cadherin (HUABIO, EM0502, 1:1000), anti-Snail (HUABIO, ER1706-22, 1:4000), anti-Zeb2 (HUABIO, ER64964, 1:1000), anti-NF-κB-P65 (HUABIO, ET1603-12, 1:2000), anti-p-P65(S536) (CST, EP2294Y, 1:1000), anti-p-P65(S468) (ABclonal, AP0446, 1:1000), anti-β-actin (HUABIO, M1210-2, 1:5000), anti-DYKDDDDK Tag (HUABIO, M1402-2, 1:2000).

Techniques: Migration, Microscopy, Expressing, Transfection, Negative Control, Western Blot