Journal: Cell Death and Differentiation

Article Title: MEST induces Twist-1-mediated EMT through STAT3 activation in breast cancers

doi: 10.1038/s41418-019-0322-9

Figure Lengend Snippet: MEST is required for transformation and migration of triple-negative breast cancer cells. a Western blot analysis of MEST expression in Hs578T, SUM159PT, and 4T1 control-shRNA or MEST-shRNA cells. β-actin was used as a loading control. b Formation of spheroid colonies of 4T1 control-shRNA or MEST-shRNA cells. Spheroid colonies in suspension were then photographed at ×200 magnification. c The soft agar colony-forming assay of the 4T1 control-shRNA or MEST-shRNA cells (n = 3). P < 0.0001, t-test. d The migration assay of in Hs578T, SUM159PT, and 4T1 control-shRNA or MEST-shRNA cells (n = 3). P < 0.0001, t-test. e Wound healing assay of Hs578T, SUM159PT, and 4T1 control-shRNA or MEST-shRNA cells. Wound closures were photographed at 0 and 24 h after wounding (magnification: ×200)

Article Snippet: Mouse (67NR, 4T07, and 4T1) and human breast cancer cells (MCF10A, MCF7, T47D, BT474, ZR75B, MDA-MB-468, SK-BR-3, BT549, Hs578T, and MDA-MB-231) and 293T cells obtained from American Type Culture Collection (Manassas, VA, USA); SUM149PT and SUM159PT breast cells were obtained from Dr. Stephen Ethier (Kramanos Institute) and were maintained as described previously [ 34 , 35 ].

Techniques: Transformation Assay, Migration, Western Blot, Expressing, Control, shRNA, Suspension, Wound Healing Assay

Journal: Cell Death and Differentiation

Article Title: MEST induces Twist-1-mediated EMT through STAT3 activation in breast cancers

doi: 10.1038/s41418-019-0322-9

Figure Lengend Snippet: High MEST expression induces EMT program. a Western blot analysis of the expression of E-cadherin, N-cadherin, fibronectin, and vimentin proteins in Hs578T, SUM159PT, and 4T1 control-shRNA or MEST-shRNA cells. β-actin was used as a loading control. b Western blot (left) and RT-PCR (Right) analysis of the expression of CAR, caludin3, occludin, E-cadherin, α-catenin, N-cadherin, fibronectin, and β-catenin proteins in MCF10A-control or MCF10A-MEST cells. GAPDH and β-actin were used as a loading control. c Immunofluorescence images of E-cadherin and N-cadherin in Hs578T, SUM159PT, and 4T1 control-shRNA or MEST-shRNA cells. The red signal represents staining of the corresponding protein, while the blue signal represents DAPI staining. d Immunofluorescence images of V5, E-cadherin, β-catenin, fibronectin, α-catenin, and N-cadherin in MCF10A-control or MCF10A-MEST cells. The red signal represents the staining of the corresponding protein, while the blue signal represents DAPI staining

Article Snippet: Mouse (67NR, 4T07, and 4T1) and human breast cancer cells (MCF10A, MCF7, T47D, BT474, ZR75B, MDA-MB-468, SK-BR-3, BT549, Hs578T, and MDA-MB-231) and 293T cells obtained from American Type Culture Collection (Manassas, VA, USA); SUM149PT and SUM159PT breast cells were obtained from Dr. Stephen Ethier (Kramanos Institute) and were maintained as described previously [ 34 , 35 ].

Techniques: Expressing, Western Blot, Control, shRNA, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Staining

Journal: Cell Death and Differentiation

Article Title: MEST induces Twist-1-mediated EMT through STAT3 activation in breast cancers

doi: 10.1038/s41418-019-0322-9

Figure Lengend Snippet: MEST knockdown is associated with loss of EMT transcription factors. a The relative expression levels of mRNA encoding Goosecoid, Foxc-1, Foxc-2, Slug, Twist-1, and Twist-2 in Hs578T, SUM159PT, and 4T1 control-shRNA or MEST-shRNA cells, as determined by quantitative RT-PCR. 18S was used as a loading control. P < 0.05, t-test. b Immunofluorescence images of Twist-1 in Hs578T, SUM159PT, and 4T1 control-shRNA or MEST-shRNA cells. The green signal represents the staining of the corresponding protein, while the blue signal represents DAPI staining. c The relative expression levels of mRNA encoding Twist-1 in Hs578T, SUM159PT, and 4T1 control-shRNA or MEST-shRNA cells, as determined by quantitative RT-PCR. 18S was used as a loading control. P < 0.05, t-test. d Promoter activity of Twist-1 gene in Hs578T, SUM159PT, and 4T1 control-shRNA or MEST-shRNA cells were measured by the Luciferase reporter assay. Each bar represents the mean ± SEM of three experiments. P < 0.0001, t-test

Article Snippet: Mouse (67NR, 4T07, and 4T1) and human breast cancer cells (MCF10A, MCF7, T47D, BT474, ZR75B, MDA-MB-468, SK-BR-3, BT549, Hs578T, and MDA-MB-231) and 293T cells obtained from American Type Culture Collection (Manassas, VA, USA); SUM149PT and SUM159PT breast cells were obtained from Dr. Stephen Ethier (Kramanos Institute) and were maintained as described previously [ 34 , 35 ].

Techniques: Knockdown, Expressing, Control, shRNA, Quantitative RT-PCR, Immunofluorescence, Staining, Activity Assay, Luciferase, Reporter Assay

Journal: Cell Death and Differentiation

Article Title: MEST induces Twist-1-mediated EMT through STAT3 activation in breast cancers

doi: 10.1038/s41418-019-0322-9

Figure Lengend Snippet: MEST led to Twist-1 upregulation through activation of STAT3. a Western blot analysis of the expression of phospho-JAK2, JAK2, phospho-STAT3, STAT3, and Twist-1 proteins in Hs578T, SUM159PT, and 4T1 control-shRNA or MEST-shRNA cells. β-actin was used as a loading control. b Western blot analysis of the expression of phospho-JAK2, JAK2, phospho-STAT3, STAT3, and Twist-1 proteins in Hs578T, SUM159PT, and 4T1 control-shRNA or MEST-shRNA cells treated with or without 20 ng/ml IL-6. β-actin was used as a loading control. c Western blot analysis of the expression of phospho-STAT3 and STAT3 proteins in Hs578T, SUM159PT, and 4T1 control-shRNA or MEST-shRNA cells after nuclear fractionation. Lamin B1 was used as a nuclear loading control. d Immunofluorescence images of phospho-STAT3 and STAT3 in Hs578T, SUM159PT, and 4T1 control-shRNA or MEST-shRNA cells. The green signal represents the staining of the corresponding protein, while the blue signal represents DAPI staining

Article Snippet: Mouse (67NR, 4T07, and 4T1) and human breast cancer cells (MCF10A, MCF7, T47D, BT474, ZR75B, MDA-MB-468, SK-BR-3, BT549, Hs578T, and MDA-MB-231) and 293T cells obtained from American Type Culture Collection (Manassas, VA, USA); SUM149PT and SUM159PT breast cells were obtained from Dr. Stephen Ethier (Kramanos Institute) and were maintained as described previously [ 34 , 35 ].

Techniques: Activation Assay, Western Blot, Expressing, Control, shRNA, Fractionation, Immunofluorescence, Staining

Journal: Cell Death and Differentiation

Article Title: MEST induces Twist-1-mediated EMT through STAT3 activation in breast cancers

doi: 10.1038/s41418-019-0322-9

Figure Lengend Snippet: MEST induces STAT3 activation through induction of JAK2–STAT3 complex formation. a Identification of the complex formation of endogenous MEST/STAT3 in Hs578T, SUM159PT and 4T1 cells. b Western blot analysis of the whole-cell lysates and immunoprecipitates derived from 293T cells transfected with the V5-MEST and Flag-STAT3 constructs, as indicated. c Identification of the STAT3 region that interacted with MEST. Western blot analysis of whole-cell lysates and immunoprecipitates derived from 293T cells transfected with V5-MEST, Flag-STAT3 wild, and the STAT3 deletion construct (STAT3 mt) as indicated. d Western blot analysis of the whole-cell lysates and immunoprecipitates derived from 293T cells transfected with the V5-MEST, V5-MESTΔC, and Flag-STAT3 constructs, as indicated. e Western blot analysis of the expression of phospho-STAT3 and STAT3 proteins in Hs578T, SUM159PT, and 4T1 MEST-shRNA cells after transient transfection of MEST or MESTΔC constructs. β-actin was used as a loading control. f Western blot analysis of whole-cell lysates and immunoprecipitates derived from 293T cells after transfection of JAK2, STAT3, or MEST constructs. β-actin was used as a loading control. g Identification of the complex formation of endogenous JAK2/STAT3 in Hs578T and SUM159PT control-shRNA or MEST-shRNA cells. h Western blot analysis of whole-cell lysates and immunoprecipitates derived from 293T cells after transfection of JAK2, STAT3, MEST, or MESTΔC constructs. β-actin was used as a loading control

Article Snippet: Mouse (67NR, 4T07, and 4T1) and human breast cancer cells (MCF10A, MCF7, T47D, BT474, ZR75B, MDA-MB-468, SK-BR-3, BT549, Hs578T, and MDA-MB-231) and 293T cells obtained from American Type Culture Collection (Manassas, VA, USA); SUM149PT and SUM159PT breast cells were obtained from Dr. Stephen Ethier (Kramanos Institute) and were maintained as described previously [ 34 , 35 ].

Techniques: Activation Assay, Western Blot, Derivative Assay, Transfection, Construct, Expressing, shRNA, Control

Journal: Cell Death and Differentiation

Article Title: MEST induces Twist-1-mediated EMT through STAT3 activation in breast cancers

doi: 10.1038/s41418-019-0322-9

Figure Lengend Snippet: Suppression of MEST inhibited the ability of 4T1 cells to metastasize to the lung. a Lung metastasis by 4T1 control-shRNA or 4T1 MEST-shRNA cells. The total numbers of lung metastatic nodules from each mouse harboring 4T1 tumors expressing control-shRNA or MEST-shRNA were counted using a dissection scope (n = 6 mice per group). P < 0.05, ANOVA. b Representative images of the lungs from the mice harboring 4T1 control-shRNA or 4T1 MEST-shRNA cells for 30 days after implantation of mouse mammary fat pads of mice. c Representative H&E staining in the sections of the lungs from Fig. 7b. N normal lung tissue, M metastatic nodule. d Western blot analysis of MEST, phospho-STAT3, STAT3, and Twist-1 in tumor cells recovered from the lungs of individual mice expressing either 4T1 control-shRNA or 4T1 MEST-shRNA. β-actin was used as a loading control. e Quantitative RT-PCR of Twist-1 in tumor cells recovered from the lungs of individual mice expressing either 4T1 control-shRNA or 4T1 MEST-shRNA. 18S mRNA was used as a loading control. P < 0.001, t-test

Article Snippet: Mouse (67NR, 4T07, and 4T1) and human breast cancer cells (MCF10A, MCF7, T47D, BT474, ZR75B, MDA-MB-468, SK-BR-3, BT549, Hs578T, and MDA-MB-231) and 293T cells obtained from American Type Culture Collection (Manassas, VA, USA); SUM149PT and SUM159PT breast cells were obtained from Dr. Stephen Ethier (Kramanos Institute) and were maintained as described previously [ 34 , 35 ].

Techniques: Control, shRNA, Expressing, Dissection, Staining, Western Blot, Quantitative RT-PCR

Journal: Oncoimmunology

Article Title: Germinal center hypoxia in tumor-draining lymph nodes negatively regulates tumor-induced humoral immune responses in mouse models of breast cancer

doi: 10.1080/2162402X.2021.1959978

Figure Lengend Snippet: pVHL deletion in class-switched B cells alters the phenotype of T cells and macrophages infiltrating 4T07 mammary tumors

Article Snippet: To determine whether hypoxia could develop among B cells responding to tumors, we injected BALB/c mice bearing orthotopic 4T07 mammary tumors with pimonidazole (Hypoxyprobe), a 2-nitroimidazole that is reduced and bound in cells under low oxygen conditions.

Techniques: