Journal: Journal of Molecular Signaling

Article Title: Direct RNA sequencing mediated identification of mRNA localized in protrusions of human MDA-MB-231 metastatic breast cancer cells

doi: 10.1186/1750-2187-8-9

Figure Lengend Snippet: MDA - MB - 231 protrusion localized mRNA

Article Snippet: The following primary antibodies were used for protein detection: Anti Histone H3 (1:10000, Rabbit, Abcam ab1791, 19 kDa); Anti α-Tubulin (1:5000, Rabbit, Rockland 600-401-880, 51 kDa), Anti β-Actin (1:2500, Rabbit, Sigma A2103, 42 kDa); Anti Zeb1 (1:500, Rabbit, Sigma HPA027524, 200 kD); Anti ANP32B (1:100, Rabbit, Sigma SAB4500125, 30 kDa).

Techniques: Multiple Displacement Amplification

Journal: Journal of Molecular Signaling

Article Title: Direct RNA sequencing mediated identification of mRNA localized in protrusions of human MDA-MB-231 metastatic breast cancer cells

doi: 10.1186/1750-2187-8-9

Figure Lengend Snippet: DRS transcriptome analysis of purified RNA from MDA- MB -231 protrusions. (A) Comparison of DRS and RT-qPCR analyses. RNA localization ratios were normalized to ARPC3 mRNA given the value 1. (B) Annotation analysis of the 100 most MDA-MB-231 protrusion localized mRNA compared to the total group of mRNAs expressed with ≥ 5 tpm. Annotations were made using the IPA Ingenuity platform. (C) RT-qPCR confirmation analysis of selected MDA-MB-231 protrusion localized RNA identified by DRS. RNA localization ratios are normalized to ARPC3 mRNA. (D) Western blot analysis. Western blotting was performed on pooled material from three independent Boyden chamber experiments representing cell body fraction (CF) and protrusion fraction (PF). α-Tubulin Western blot analysis was used to equalize the loaded protein amounts. Subsequent western blot analyses were performed with antibodies for Histone H3, ZEB1, ANP32B and ACTB.

Article Snippet: The following primary antibodies were used for protein detection: Anti Histone H3 (1:10000, Rabbit, Abcam ab1791, 19 kDa); Anti α-Tubulin (1:5000, Rabbit, Rockland 600-401-880, 51 kDa), Anti β-Actin (1:2500, Rabbit, Sigma A2103, 42 kDa); Anti Zeb1 (1:500, Rabbit, Sigma HPA027524, 200 kD); Anti ANP32B (1:100, Rabbit, Sigma SAB4500125, 30 kDa).

Techniques: Purification, Quantitative RT-PCR, Western Blot

Journal: Cancer research

Article Title: A NOTCH3-mediated squamous cell differentiation program limits expansion of EMT competent cells that express the ZEB transcription factors

doi: 10.1158/0008-5472.CAN-11-0846

Figure Lengend Snippet: Representative images for H&E and corresponding IHC for ZEB1 in two primary ESCC cases featuring poorly differentiated invasive tumor nests (A) and tumor cells surrounding a well-differentiated lesion with keratin pearl formation (B). The selected areas were enlarged in the respective lower panels. Note that ZEB1 positive tumor cells tend to show spindle-cell differentiation (arrows). Stromal inflammatory cells and fibroblasts (arrow heads) are also positive for ZEB1. Scale bar, 100 μm.

Article Snippet: In brief, cells were fixed and permeabilized in cold acetone at −20°C for 10 min, and washed twice, followed by incubation on ice for 30 min with primary rabbit monoclonal anti-ZEB1 antibody (1:200)(Cell Signaling, #3396) or rabbit IgG as a control, and then on ice for 30 min with Alexa Fluor 633 dye-conjugated secondary anti-rabbit IgG (1:200)(Invitrogen).

Techniques: Cell Differentiation

Journal: Cancer research

Article Title: A NOTCH3-mediated squamous cell differentiation program limits expansion of EMT competent cells that express the ZEB transcription factors

doi: 10.1158/0008-5472.CAN-11-0846

Figure Lengend Snippet: EPC2-T cells expressing either DNMAML1 or GFP (control) were stimulated with TGF-β1 for 2 weeks in (A)-(E). In (C)-(E), cells expressing tetracycline-inducible (Tet-On) shRNA directed against either ZEB1, ZEB2 or a non-silence control sequence (Scramble) were subjected to TGF-β1 stimulation along with or without 1 μg/ml of doxycycline (DOX) for 2 weeks. Representative data are shown with comparable results using two independent shRNA sequences.

Article Snippet: In brief, cells were fixed and permeabilized in cold acetone at −20°C for 10 min, and washed twice, followed by incubation on ice for 30 min with primary rabbit monoclonal anti-ZEB1 antibody (1:200)(Cell Signaling, #3396) or rabbit IgG as a control, and then on ice for 30 min with Alexa Fluor 633 dye-conjugated secondary anti-rabbit IgG (1:200)(Invitrogen).

Techniques: Expressing, shRNA, Sequencing

Journal: Cancer research

Article Title: A NOTCH3-mediated squamous cell differentiation program limits expansion of EMT competent cells that express the ZEB transcription factors

doi: 10.1158/0008-5472.CAN-11-0846

Figure Lengend Snippet: Notch signaling regulates squamous differentiation in the normal esophageal epithelium. Notch signaling also contributes to keratin pearl formation in well-differentiated tumor nests. ZEB1 is expressed at the invasive fronts where tumor cells do not undergo squamous differentiation. In early lesions such as carcinoma in situ, ZEBs may be induced in response to oncogene activation (e.g. EGFR) to negate oncogene-induced senescence as a cellular fail safe mechanism (23). In advanced ESCC, NOTCH3 may be downregulated, resulting in dedifferentiated status where microenvironmental cues (e.g. TGF-β and hypoxia) may promote EMT in concert with ZEBs, leading to invasive growth and tumor cell dissemination. In this study, either DNMAML1 or shRNA directed against NOTCH3 (N3) was used to suppress canonical Notch-mediated squamous differentiation to facilitate ZEB-mediated EMT.

Article Snippet: In brief, cells were fixed and permeabilized in cold acetone at −20°C for 10 min, and washed twice, followed by incubation on ice for 30 min with primary rabbit monoclonal anti-ZEB1 antibody (1:200)(Cell Signaling, #3396) or rabbit IgG as a control, and then on ice for 30 min with Alexa Fluor 633 dye-conjugated secondary anti-rabbit IgG (1:200)(Invitrogen).

Techniques: In Situ, Activation Assay, shRNA

Journal: American Journal of Cancer Research

Article Title: YEATS4 is associated with poor prognosis and promotes epithelial-to-mesenchymal transition and metastasis by regulating ZEB1 expression in breast cancer

doi:

Figure Lengend Snippet: YEATS4 regulates ZEB1 expression transcriptionally. (A) Fold changes in the mRNA expression levels of epithelial-to-mesenchymal transition (EMT) transcriptional factors in four breast cancer cell lines after YEATS4 depletion. Fold changes calculated by normalizing mRNA levels of YEATS4 deletion cells to that of the control cells. (B) Immunofluorescence images of YEATS4 and ZEB1 staining in BT-474 cells. YEATS4 and ZEB1 simultaneously expressed in cancer cells. Scale bar, 20 µm. (C) Expression of YEATS4 and ZEB1 in stable YEATS4-deleting MDA-MB-231 cells, determined by Western blot analysis. (D) YEATS4 and ZEB1 expression in YEATS4-overexpressing T-47D cells, detected by Western blot analysis. (E) Expression changes in ZEB1 and E-cadherin after restoring YEATS4 in YEATS4-deleting MDA-MB-231 cells. (F) Comparison of EMT markers and migration capabilities between YEATS4-deleting or ZEB1-deleting MDA-MB-231 cells and the control group. (G-I) ZEB1 mRNA (G), protein expression levels (H), and promoter activities (I) were investigated after 48 h transient transfection of YEATS4 overexpression or empty vector plasmids in ZR-75-1 cells. (J) Enhancement of the ZEB1 promoter activity by YEATS4 overexpression in a dose-dependent manner. (K) The abundance of H3 lysine acetylation was assessed by Western blot analysis; total H3 was used as a loading control. (L) Schematic of three regions relative to the ZEB1 transcription start site used as primers to test histone-occupied abundance. (M, N) ChIP was conducted to assess H3K27ac occupancy in T-47D-YEATS4 (M) and MDA-MB-231-shYEATS4 (N) cells. IgG was used as a negative control. Percentage of input indicates the ratio of the DNA fragment of each promoter region bound by H3K27ac to the total of the input DNA fragment without the H3K27ac antibody pull-down. Data are presented as mean ± s.d. for 3 independent assays. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Slides were then immunoreacted for 1 h with the anti-YEATS4 primary antibody (diluted at 1:100; Sigma-Aldrich) or the anti-ZEB1 primary antibody (diluted at 1:100; Cell Signaling Technology) and then with a secondary antibody for 30 min.

Techniques: Expressing, Immunofluorescence, Staining, Western Blot, Migration, Transfection, Over Expression, Plasmid Preparation, Activity Assay, Negative Control

Journal: American Journal of Cancer Research

Article Title: YEATS4 is associated with poor prognosis and promotes epithelial-to-mesenchymal transition and metastasis by regulating ZEB1 expression in breast cancer

doi:

Figure Lengend Snippet: ZEB1 mediates YEATS4-induced EMT and metastasis. (A) Recovery of epithelial marker expression and reduction of mesenchymal marker expression in T-47D-YEATS4 cells by ZEB1 deletion. (B, C) Reduction of YEATS4-induced migration and invasion abilities in T-47D-YEATS4 cells by ZEB1 depletion, as determined by wound healing (B) and transwell invasion (C) assays. Scale bar, 200 µm for (B); 50 µm for (C). (D) Representative images of H&E-stained lung tissues from the T-47D-YEATS4 cells with or without ZEB1 deletion. The scale bars represent 1 mm in upper panel and 100 µm in lower panel. (E) Total number of mice with lung metastasis in different groups. (F) Relative number of lung metastasis nodules in each group. (G) Overall survival of mice in each group. (H) Positive association of YEATS4 expression with ZEB1 expression in breast cancer tissues. Left, representative staining images of YEATS4 and ZEB1. Scale bar, 50 µm for 200×; 20 µm for 400×. (I) Representative images of ZEB1 staining of ANTs, DCIS, ICW, and ICLNM specimens. Scale bar, 50 µm for 200×; 20 µm for 400×. (J) Proportions of ANTs, DCIS, ICW, and ICLNM specimens with ZEB1-positive staining. (K) DMFS curves of patients with breast cancer, plotted based on the expression profiles of YEATS4 and ZEB1 as indicated. Co-expression of YEATS4 and ZEB1 (Group 4) indicating the shortest distant metastasis-free period vs. other groups. (L) Schematic demonstrating the role of YEATS4 in mediating ZEB1 expression, EMT, and metastasis in breast cancer. Data are presented as mean ± s.d. for 3 independent assays. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Slides were then immunoreacted for 1 h with the anti-YEATS4 primary antibody (diluted at 1:100; Sigma-Aldrich) or the anti-ZEB1 primary antibody (diluted at 1:100; Cell Signaling Technology) and then with a secondary antibody for 30 min.

Techniques: Marker, Expressing, Migration, Staining