Journal: Breast Cancer : Targets and Therapy

Article Title: Anticarcinogenic Effects of Odorant Substances Citral, Citrathal R and Cyclovertal on Breast Cancer in vitro

doi: 10.2147/BCTT.S322619

Figure Lengend Snippet: MTT-assay. ( A ) MTT-assay T-47D cells, ( B ) MTT-assay BT474 cells, ( C ) MTT-assay MDA-MB-231 cells.

Article Snippet: BT474 cells (#300131) and T-47D cells (#300353) were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany), MDA-MB-231 cells (#92020424) from Sigma-Aldrich (Taufkirchen, Germany).

Techniques: MTT Assay

Journal: Breast Cancer : Targets and Therapy

Article Title: Anticarcinogenic Effects of Odorant Substances Citral, Citrathal R and Cyclovertal on Breast Cancer in vitro

doi: 10.2147/BCTT.S322619

Figure Lengend Snippet: Ki67 in T-47D, MDA-MB-231 and BT474 cells. Relative intracellular expression of Ki67 RNA in T-47D cells ( A ), MDA-MB-231 cells ( B ) and BT474 cells ( C ) after treatment with 50µM citral (Cit), 50µM citrathal R (CR) and 50µM Cyclovertal (Cy). CTRL: DMSO-Control.

Article Snippet: BT474 cells (#300131) and T-47D cells (#300353) were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany), MDA-MB-231 cells (#92020424) from Sigma-Aldrich (Taufkirchen, Germany).

Techniques: Expressing, Control

Journal: Breast Cancer : Targets and Therapy

Article Title: Anticarcinogenic Effects of Odorant Substances Citral, Citrathal R and Cyclovertal on Breast Cancer in vitro

doi: 10.2147/BCTT.S322619

Figure Lengend Snippet: Survivin in T-47D, MDA-MB-231 and BT474 cells. Relative intracellular expression of survivin RNA in T-47D cells ( A ), MDA-MB-231 cells ( B ) and BT474 cells ( C ) after treatment with 50µM citral (Cit), 50µM citrathal R (CR) and 50µM Cyclovertal (Cy). CTRL: DMSO-Control.

Article Snippet: BT474 cells (#300131) and T-47D cells (#300353) were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany), MDA-MB-231 cells (#92020424) from Sigma-Aldrich (Taufkirchen, Germany).

Techniques: Expressing, Control

Journal: Breast Cancer : Targets and Therapy

Article Title: Anticarcinogenic Effects of Odorant Substances Citral, Citrathal R and Cyclovertal on Breast Cancer in vitro

doi: 10.2147/BCTT.S322619

Figure Lengend Snippet: Cyclin A2 in T47-D, MDA-MB-231 and BT474 cells. Relative intracellular expression of cyclin A2 RNA in T-47D cells ( A ), MDA-MB-231 cells ( B ) and BT474 cells ( C ) after treatment with 50µM citral (Cit), 50µM citrathal R (CR) and 50µM Cyclovertal (Cy). CTRL: DMSO-Control.

Article Snippet: BT474 cells (#300131) and T-47D cells (#300353) were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany), MDA-MB-231 cells (#92020424) from Sigma-Aldrich (Taufkirchen, Germany).

Techniques: Expressing, Control

Journal: Breast Cancer : Targets and Therapy

Article Title: Anticarcinogenic Effects of Odorant Substances Citral, Citrathal R and Cyclovertal on Breast Cancer in vitro

doi: 10.2147/BCTT.S322619

Figure Lengend Snippet: Western blots. Western Blot showing control, capsazepine alone, citral, citral + capsazepine, citrathal R, citrathal R + capsazepine, cyclovertal and cyclovertal + capsazepine. ß-tubulin served as loading control in ( A ) T-47D, ( B ) BT474, ( C ) MDA-MB-231.

Article Snippet: BT474 cells (#300131) and T-47D cells (#300353) were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany), MDA-MB-231 cells (#92020424) from Sigma-Aldrich (Taufkirchen, Germany).

Techniques: Western Blot, Control

Journal: Breast Cancer : Targets and Therapy

Article Title: Anticarcinogenic Effects of Odorant Substances Citral, Citrathal R and Cyclovertal on Breast Cancer in vitro

doi: 10.2147/BCTT.S322619

Figure Lengend Snippet: Immunocytochemistry. 1: T-47D PCNA. Immunocytochemical staining of T47D cells with PCNA antibody (dilution 1:7500, microscopic enlargement 400x). (a) control, (b) citral, (c) citrathal R, (d) cyclovertal. Compared to control reduced nuclear–cytoplasmic ratio and increased nuclear condensation. 2: T-47D Annexin V. Immunocytochemical staining of T47D cells with Annexin V antibody (dilution 1:250, microscopic enlargement 400x). (a) control, (b) citral, (c) citrathal R, (d) cyclovertal. Compared to control reduced nuclear–cytoplasmic ratio and increased nuclear condensation. Slightly increased staining of the outer cell membrane after citral treatment, more after citrathal R and cyclovertal, compared to the control. 3: BT474 PCNA. Immunocytochemical staining of BT474 cells with PCNA antibody (dilution 1:7500, microscopic enlargement 400x). (a) control, (b) citral, (c) citrathal R, (d) cyclovertal. Compared to control reduced nuclear–cytoplasmic ratio and increased nuclear condensation. 4: BT474 Annexin V. Immunocytochemical staining of BT474 cells with Annexin V antibody (dilution 1:250, microscopic enlargement 400x). (a) control, (b) citral, (c) citrathal R, (d) cyclovertal. Clearly increased staining of the outer cell membrane after citral, cylovertal and mostly after citrathal R treatment compared to the control. 5: MDA-MB-231 PCNA. Immunocytochemical staining of MDA-MB-231 cells with PCNA antibody (dilution 1:7500, microscopic enlargement 400x). (a) control, (b) citral, (c) citrathal R, (d) cyclovertal. Compared to control reduced condensation and increased fragmentation of the nucleus and loss of spindle-shape after treatment mostly with citral and citrathal R, but also cyclovertal. Less intensive staining of the nuclei after treatment with citral, citrathal R and cyclovertal compared to the control. 6: MDA-MB-231 Annexin V. Immunohistochemical staining of MDA-MB-231 cells with Annexin V antibody (dilution 1:250, microscopic enlargement 400x). (a) control, (b) citral, (c) citrathal R, (d) cyclovertal. Compared to control reduced condensation and increased fragmentation of the nucleus and loss of spindle-shape after treatment with citral, citrathal R and cyclovertal. Increased staining of the outer cell membrane after citral, citrathal R and mostly after cyclovertal treatment compared to the control.

Article Snippet: BT474 cells (#300131) and T-47D cells (#300353) were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany), MDA-MB-231 cells (#92020424) from Sigma-Aldrich (Taufkirchen, Germany).

Techniques: Immunocytochemistry, Staining, Control, Membrane, Immunohistochemical staining

Journal: NPJ Breast Cancer

Article Title: NDRG4 promoter hypermethylation is a mechanistic biomarker associated with metastatic progression in breast cancer patients

doi: 10.1038/s41523-019-0106-x

Figure Lengend Snippet: NDRG4 depletion in breast tumor cells increases lymph node adhesion and cell migration toward VN. a NDRG4 expression meta-analysis in human breast cancer cell lines using GOBO application . Breast cancer subtypes can be identified by different colors: red = Basal A, gray = Basal B and blue = Luminal cells. b Analysis of NDRG4 mRNA expression levels in MCF-7 cells transfected with NDRG4-shRNAs or scramble control (shSCR). Expression levels are relative to wild type cells and normalized to hydroxymethylbilane synthase (HMBS) gene. Error bars represent SEM of biological replicates ( n = 5). *** p < 0.001, ns = not significant, by one-way ANOVA. c Western blot analysis of NDRG4 expression in cytoplasmic extracts of MCF-7 shNDRG4s or shSCRs cells, using antibodies against NDRG4 and Actin. MCF-7 cells express all the three isoforms of NDRG4: 37 kDa (NDRG4-B), 39 kDa (NDRG4-B var ) and 41 kDa isoform (NDRG4-H). For simplicity, results obtained for two independent shNDRG4 clones were grouped and presented as the shNDRG4 group. ( d , left) Representative images of adherent red fluorescent MCF-7 cells on frozen rat lymph node sections. d , e Quantification of adherent MCF7 ( d ) and T47D ( e ) breast tumor cells in lymph nodes sections (right, n = 4 independent experiments for MCF-7 and 3 for T47D, ** p < 0.01 by two-tailed t test). f Stationary adhesion assays showed that NDRG4 knockdown promotes ‘adhesive switch’ between FN and VN. Error bars represent SEM of biological replicates ( n = 4). Cells do not adhere to albumin control (BSA, data not shown). * p < 0.01, ** p < 0.01, ns = not significant by two-tailed t tests. g , h Haptotactic cell migration toward VN was analyzed by transwell migration assay in MCF-7 ( g ) and T47D ( h ) cell lines. Error bars represent SEM of biological replicates ( n = 4). *** p < 0.001 by two-tailed t tests

Article Snippet: For stable transfection of NDRG4 shRNA into MCF-7 and T47D breast tumor cells, the pGFP-V-RS vector containing shRNA inserts which targeted the 3′-unttranslated region of NDRG4 (TRCN0000134583 and TRCN0000137216) was purchased (Origene).

Techniques: Migration, Expressing, Transfection, Western Blot, Clone Assay, Two Tailed Test, Transwell Migration Assay

Journal: NPJ Breast Cancer

Article Title: NDRG4 promoter hypermethylation is a mechanistic biomarker associated with metastatic progression in breast cancer patients

doi: 10.1038/s41523-019-0106-x

Figure Lengend Snippet: NDRG4 knockdown promotes clustering of β1-integrin at the leading edge of T47D cells. Representative confocal images of β1 integrin subunit (MAB1965, green) at the ventral cell surface of VN-adherent T47D shNDRG4 or shSCR cells

Article Snippet: For stable transfection of NDRG4 shRNA into MCF-7 and T47D breast tumor cells, the pGFP-V-RS vector containing shRNA inserts which targeted the 3′-unttranslated region of NDRG4 (TRCN0000134583 and TRCN0000137216) was purchased (Origene).

Techniques:

Journal: Cell Death & Disease

Article Title: NEDDylation negatively regulates ERRβ expression to promote breast cancer tumorigenesis and progression

doi: 10.1038/s41419-020-02838-7

Figure Lengend Snippet: a Relative ERRβ mRNA expression level of MCF10A, MCF7, T47D and MDA-MB-231 cells were analysed using qRT-PCR. Three technical repeats ( N = 3) were performed and the data represent the means ± SD. (two-tailed t test; Significant: ns no significant difference) (upper panel). Representative western blot analysis of the ERRβ in MCF10A, MCF7, T47D and MDA-MB-231 cells. GAPDH was used as a loading control (lower panel). b MDA-MB-231 cells were treated with 1 µM MG132 and Chloroquine independently for 12 h and the ERRβ protein expression was analysed by western blotting. Representative Western blot is shown. GAPDH was used as a loading control. ( N = 3). c Co-immunoprecipitation was performed with antibodies against Ubiquitin (Ub) and Cullin1 and analysed with ERRβ antibodies using western blotting. Anti-IgG antibody was used as a negative control. d Western blot analysis of the NEDD8 in in MCF7, MCF10A, T47D and MDA-MB-231 cells. GAPDH was used as a loading control. ( N = 3). e APP-BP1 and UBA3 expression was analysed by western blotting in MCF10A, MCF7, T47D and MDA-MB-231 cells. GAPDH was used as a loading control. ( N = 3). f , g Immunohistochemical (IHC) staining of normal (left) and carcinoma (right) breast tissue samples using the APP-BP1 and NEDD8 antibody, respectively. Graphical representation (below) was the IHC composite score of each tissue microarray sample. A composite score (<3 = low; 3–5 = moderately; ≥6 = highly categorised) was calculated for each sample using intensity score and percentage of APP-BP1 and and NEDD8 stained-positive cells. (2-sample t test; significant: **** p < 0.001, very significant). Bar = 100 μm.

Article Snippet: Breast cancer cell lines MCF7, T47D and MDA-MB-231 were purchased from National Centre for Cell Sciences (NCCS, Pune, India).

Techniques: Expressing, Quantitative RT-PCR, Two Tailed Test, Western Blot, Control, Immunoprecipitation, Ubiquitin Proteomics, Negative Control, Immunohistochemical staining, Immunohistochemistry, Microarray, Staining

Journal: Cell Death & Disease

Article Title: NEDDylation negatively regulates ERRβ expression to promote breast cancer tumorigenesis and progression

doi: 10.1038/s41419-020-02838-7

Figure Lengend Snippet: a MCF7 cells were treated with varying concentrations of MLN4924 (0, 0.1, 0.3, 0.5 and 1.0 µM) for 24 h prior to western blot analysis of NEDDylated Cullin1. The NEDD8-modified Cullin1 was determined by probing the whole western blot membrane with an anti-NEDD8 antibody. The characteristic predominant band (just below 80kD) correspond to the NEDD8-modified Cullin1, were shown. Tubulin was used as a loading control (left panel). MCF7 cells were treated with varying concentrations of MLN4924 (0, 0.1, 0.3, 0.5 and 1.0 µM) for 48 h prior to western blot analysis of hyper-NEDDylated and hypo-NEDDylated Cullin1. GAPDH was used as a loading control (right panel). b MCF7, T47D and MDA-MB-231 cells were treated with varying concentrations of MLN4924 (0, 0.1, 0.5 and 1.0 µM) for 48 h prior to western blot analysis of ERRβ. GAPDH was used as a loading control. ( N = 3). c Expression levels of ERRβ and Cullin1 in MCF-7 cells were analysed by western blotting after transfection with siRNA targeting Cullin1 or non-targeting control siRNA. Western blots are representative of three independent experiments. The ratio to GAPDH expression was calculated for the relative ERRβ and Cullin1 expression levels. The relative expression levels (right panels) are shown. Data represent means ± SD. ( n = 3; t tests). Significant * P < 0.05; ** P < 0.01.

Article Snippet: Breast cancer cell lines MCF7, T47D and MDA-MB-231 were purchased from National Centre for Cell Sciences (NCCS, Pune, India).

Techniques: Western Blot, Modification, Membrane, Control, Expressing, Transfection

Journal: Cell Death & Disease

Article Title: NEDDylation negatively regulates ERRβ expression to promote breast cancer tumorigenesis and progression

doi: 10.1038/s41419-020-02838-7

Figure Lengend Snippet: a MCF-7, T47D and MDA-MB-231 cells were treated with 25 μg/mL cycloheximide (CHX) in the presence of 0.3 µM MLN4924 or DMSO as a control. Cells were harvested in 3 h intervals (0, 3, 6, 9, 12 h) for western blot analysis of ERRβ protein expression. α-Tubulin was used as a loading control. Densitometry was used to quantify the ERRβ and α-Tubulin levels from which independent background readings were subtracted. Western blots are representative of three independent experiments. The relative expression levels (lower panels) are shown. b MCF7 and MDA-MB-231 cells were treated with 25 μg/mL cycloheximide (CHX) in the presence of 0.3 µM MLN4924 or DMSO as a control. Cells were harvested after 24 h for western blot analysis of ERRβ protein expression. GAPDH was used as a loading control. Densitometry was used to quantify the ERRβ and GAPDH levels from which independent background readings were subtracted. Western blots are representative of three independent experiments. The relative expression levels (lower panels) are shown. Data represent means ± SD. ( n = 3; t tests). Significant * P < 0.05; ** P < 0.01; *** P < 0.001. c Western blot analysis of ERRβ expression in different combinations of MLN4924, MG132 and chloroquine-treated MCF7 (left) and MDA-MB-231 (right) cells. GAPDH and α-Tubulin were used as a loading control, respectively. ( N = 3). d Co-immunoprecipitation was performed on 0.3 µM MLN4924-treated and control untreated MCF7 cells (24 h) with antibodies against Ubiquitin (Ub) and Cullin1 and analysed with ERRβ and Cullin1 antibodies using western blotting. Anti-IgG antibody was used as a negative control. ( N = 3).

Article Snippet: Breast cancer cell lines MCF7, T47D and MDA-MB-231 were purchased from National Centre for Cell Sciences (NCCS, Pune, India).

Techniques: Control, Western Blot, Expressing, Immunoprecipitation, Ubiquitin Proteomics, Negative Control

Journal: Cell Death & Disease

Article Title: NEDDylation negatively regulates ERRβ expression to promote breast cancer tumorigenesis and progression

doi: 10.1038/s41419-020-02838-7

Figure Lengend Snippet: a MCF-7 and MDA-MB-231 cells were treated with varying concentrations of MLN4924 (0, 0.1, 0.5, 1.0 μM) for 48 h and analyse for the cell proliferation rates with MTT assay. Graphical representation was the optical density (wavelength = 570 nM) of the MLN4924-treated MCF-7 and MDA-MB-231 cells relative to the untreated control counterparts. ( N = 3). b MCF-7 and MDA-MB-231 cells were treated with varying concentrations of MLN4924 (0, 0.1, 0.3 and 1.0 µM) for 10 days. The representative images showed cell clonogenic survival of the MLN4924-treated MCF7 and MDA-MB-231 cells (left panels). Data represent means ± SD. ( n = 3; t tests). Significant * P < 0.05; ** P < 0.01; *** P < 0.001. c MDA-MB-231 cells were treated with varying concentrations (0, 0.1, 0.5 and 1.0 µM) of MLN4924 and injected onto the chorioallantoic membrane of 9 days old fertilised chick egg xenograft model (left panel). The representative images showed the dissected tumour after a 5 days incubation (central panel). ( N = 3). Data are representative of three independent experiments. Data represent means ± SD. ( n = 3; t tests). Significant ** P < 0.01; *** P < 0.001. d MDA-MB-231 cells were treated with 1 µM MLN4924 for 24 h and the protein expression of p21 Waf1/Cip1 and p27 Kip1 were analysed by western blotting. α-Tubulin was used as a loading control. ( N = 3). e MDA-MB-231 cells were transfected with ERRβ shRNA and/or treated with 1.0 µM MLN4924. The representative images showed cell clonogenic survival of the MLN4924-treated, with and without ERRβ-knockdown, MDA-MB-231 cells. ( N = 3) (top right). Data represent means ± SD. ( n = 3; t tests). Significant; * P < 0.05; ns non-significant. (bottom right). f MDA-MB-231 cells were treated with 1.0 µM MLN4924 in the presence or absence of p21 Waf1/Cip1 shRNA-mediated knockdown. The protein expression of p21 Waf1/Cip1 was analysed by western blotting with or without p21 Waf1/Cip1 depletion in the presence of 1.0 µM MLN4924. GAPDH was used as a loading control. The representative images showed cell clonogenic survival of the MLN4924-treated, with and without p21 Waf1/Cip1 -knockdown, MDA-MB-231 cells. ( N = 3) (middle). Data represent means ± SD. ( n = 3; t tests). Significant; * P < 0.05 (right).

Article Snippet: Breast cancer cell lines MCF7, T47D and MDA-MB-231 were purchased from National Centre for Cell Sciences (NCCS, Pune, India).

Techniques: MTT Assay, Control, Injection, Membrane, Incubation, Expressing, Western Blot, Transfection, shRNA, Knockdown

Journal: Cell Death & Disease

Article Title: NEDDylation negatively regulates ERRβ expression to promote breast cancer tumorigenesis and progression

doi: 10.1038/s41419-020-02838-7

Figure Lengend Snippet: a MCF7 cells were treated varying concentrations of MLN4924 (0, 0.1, 0.5 and 1.0 µM) for 48 h, and relative mRNA expression level of p21 Waf1/Cip1 were analysed with qRT-PCR. ( N = 3) The data represent the means ± SD. (two-tailed t test; Significant: ** P < 0.01, significant) b MCF7 and c MDA-MB-231 cells were transfected with ERRβ shRNA and relative mRNA levels of p21 Waf1/Cip1 were analysed after 1 µM MLN4924 treatment for 48 h. ( N = 3). The data represent the means ± SD. (two-tailed t test; Significant: * P < 0.05; ** P < 0.01; *** p < 0.001; **** p < 0.0001, significant). d MCF7 cells were transfected with p21 Waf1/Cip1 gene cloned upstream of the pGL2 luciferase reporter and ERRβ shRNA, with and without 1.0 µM MLN4924 treatment. Graphical representation showed relative luciferase unit of the Renilla luciferase reporter activity. ( N = 3). The data represent the means ± SD (One-way ANOVA test; Significant: * P < 0.05; ** P < 0.01, significant). e Chromatin Immunoprecipitation was performed with ERRβ antibody on the promoter region of endogenous p21 Waf1/Cip1 and analysed with Electrophoresis (left; image colour inverted) and qRT-PCR (right). Anti-IgG antibody was used as a negative control. ( N = 3). The data represent the means ± SD. (two-tailed t test; Significant: **** p < 0.0001, significant). f Co-immunoprecipitation was performed with p300 antibody in untreated and 1 µM MLN4924-treated MCF7 cells and analysed with ERRβ and p300 using western blotting. Anti-IgG antibody was used as a negative control. ( N = 3). g Chromatin Immunoprecipitation was performed with p300 antibody on the promoter region of endogenous p21 Waf1/Cip1 and analysed with electrophoresis (left; image colour inverted) and qRT-PCR (right). Anti-IgG antibody was used as a negative control. ( N = 3). The data represent the means ± SD. (two-tailed t test; Significant: * P < 0.05; *** p < 0.001, significant).

Article Snippet: Breast cancer cell lines MCF7, T47D and MDA-MB-231 were purchased from National Centre for Cell Sciences (NCCS, Pune, India).

Techniques: Expressing, Quantitative RT-PCR, Two Tailed Test, Transfection, shRNA, Clone Assay, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Electrophoresis, Negative Control, Immunoprecipitation, Western Blot

Journal: Cell Death & Disease

Article Title: NEDDylation negatively regulates ERRβ expression to promote breast cancer tumorigenesis and progression

doi: 10.1038/s41419-020-02838-7

Figure Lengend Snippet: a MDA-MB-231 and b MCF7 cells were treated with 1 µM MLN4924 for 24 h prior to Transwell-migration analysis. Graphical representation determined the average number of migrated cells. ( N = 3). The data represent the means ± SD. (One-way ANOVA test; Significant: ** P < 0.01, significant). c MDA-MB-231 cells were treated with 1 µM MLN4924 for 24 h prior to invasion analysis. Graphical representation determined the average number of cells. ( N = 3). The data represent the means ± SD. (One-way ANOVA test; Significant: * P < 0.05, significant) d MDA-MB-231 cells were untreated, treated with 1 µM MLN4924 and/or ERRβ shRNA prior to invasion analysis. Graphical representation determined the average number of cells. ( N = 3). The data represent the means ± SD. (One-way ANOVA test; Significant: * P < 0.05, significant; ns non-significant). Size bar:100 µm. e MDA-MB-231 cells were untreated, treated with 1 µM MLN4924 and/or ERRβ shRNA prior to western blot analysis for N-Cadherin, E-Cadherin, Vimentin, ERRβ and GAPDH expression. Representative blots are shown.

Article Snippet: Breast cancer cell lines MCF7, T47D and MDA-MB-231 were purchased from National Centre for Cell Sciences (NCCS, Pune, India).

Techniques: Migration, shRNA, Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: NEDDylation negatively regulates ERRβ expression to promote breast cancer tumorigenesis and progression

doi: 10.1038/s41419-020-02838-7

Figure Lengend Snippet: a MDA-MB-231 were treated with varying concentrations (0.1, 0.3, 0.5 and 1.0 µM) of MLN4924 for 24 h prior to western blot analysis. α-Tubulin was used as a loading control. ( N = 3). b MDA-MB-231 (left panel) and MCF7 (right panel) cells were treated with 1 µM MLN4924 for 24 h prior to qRT-PCR analysis. ( N = 3). The data represent the means ± SD. (Two-tailed t test; Significant: ** P < 0.01; **** p < 0.0001, significant). c MDA-MB-231 (right) and MCF7 (left) cells were transfected with ERRβ shRNA and relative mRNA level of E-Cadherin were analysed after 1 µM MLN4924 treatment for 48 h. ( N = 3). The data represent the means ± SD. (two-tailed t test; Significant: * P < 0.05; ** P < 0.01, significant) d MCF7 cells were transfected with E-Cadherin promoter cloned upstream of the pGL3 luciferase reporter and ERRβ shRNA, with and without 1.0 µM MLN4924 treatment. Graphical representation showed relative luciferase unit of the Renilla luciferase reporter activity. ( N = 3). The data represent the means ± SD (One-way ANOVA test; Significant: ** P < 0.01, significant) . e Chromatin Immunoprecipitation was performed with the ERRβ antibody on the promoter region of E-Cadherin and analysed with electrophoresis (left; image colour invested) and qRT-PCR (right). Anti-IgG antibody was used as a negative control. ( N = 3). f Chromatin immunoprecipitation was performed with a p300 antibody on the promoter region of endogenous E-Cadherin and analysed with electrophoresis (left; image colour invested) and qRT-PCR (right). Anti-IgG antibody was used as a negative control. ( N = 3). The data represent the means ± SD. (two-tailed t test; Significant: * P < 0.05, significant).

Article Snippet: Breast cancer cell lines MCF7, T47D and MDA-MB-231 were purchased from National Centre for Cell Sciences (NCCS, Pune, India).

Techniques: Western Blot, Control, Quantitative RT-PCR, Two Tailed Test, Transfection, shRNA, Clone Assay, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Electrophoresis, Negative Control

Journal: Frontiers in Medicine

Article Title: ADAM9 Mediates Triple-Negative Breast Cancer Progression via AKT/NF-κB Pathway

doi: 10.3389/fmed.2020.00214

Figure Lengend Snippet: ADAM9 expression in breast clinical samples and breast cell lines. (A) ADAM9 was markedly upregulated in clinical samples of TNBC primary tumor ( n = 34) compared to ER+ breast cancer primary tumor ( n = 37) in NCBI GEO (GSE58135) database (PT = primary tumor); (B) ADAM9 protein expression was upregulated in MDA-MB-231 cells compared to the other non-TNBC cell lines (MCF-7, MDA-MB-361, SKBR3, and MCF-10A); (C) Western blotting showed the increased ADAM9 protein expression in MDA-MB-231 and Hs578t cell lines; (D) RT-qPCR analysis indicated upregulated ADAM9 mRNA expression in TNBC cell line; (E) TNBC cell lines showed significantly overexpressed ADAM9 mRNA compared with non-TNBC cell lines in GSE58135 dataset; (F) ADAM9 mRNA expression from CCLE showed overexpressed ADAM9 in TNBC cell lines compared with non-TNBC cell lines. (* p -value < 0.05, ** p -value < 0.01, *** p -value < 0.001).

Article Snippet: MDA-MB-231, Hs578t, T47D, MCF-7, and MCF-10A were purchased from China Center for Type Culture Collection.

Techniques: Expressing, Western Blot, Quantitative RT-PCR

Journal: Frontiers in Medicine

Article Title: ADAM9 Mediates Triple-Negative Breast Cancer Progression via AKT/NF-κB Pathway

doi: 10.3389/fmed.2020.00214

Figure Lengend Snippet: ADAM9 silencing in TNBC cells inhibits cell growth, migration, and invasion in vitro . (A) Scrambled siRNA had a satisficed transfection efficiency in MDA-MB-231 cells at 3 and 6 h by Lipofectamine 2000. (scale bars = 40 μm); (B) ADAM9 protein expression in MDA-MB-231 cells was markedly inhibited by ADAM9 siRNA; (C) RT-qPCR analysis indicated that ADAM9 mRNA was significantly suppressed by ADAM9 siRNA; (D) Inhibited cell proliferation was observed in MDA-MB-231 (top panel) and Hs578t (bottom panel) cells transfected with ADAM9 siRNA by the MTT assay; (E) Representative images indicated the inhibited colony formation in MDA-MB-231 (left panel) and Hs578t (right panel) cells transfected with ADMA9 siRNA compared to the NC and BC. Mean colonies were counted in six-well plate for each group. Bars = mean ± S.D., n = 3; (F) Suppressed cell migration in siRNA group were observed in MDA-MB-231 (top panel) and Hs578t cells (bottom panel) by wound healing assay; (G) ADAM9 silencing inhibited MDA-MB-231 (top panel) and Hs578t (bottom panel) cells invasion. (* p -value < 0.05, ** p -value < 0.01, *** p -value < 0.001).

Article Snippet: MDA-MB-231, Hs578t, T47D, MCF-7, and MCF-10A were purchased from China Center for Type Culture Collection.

Techniques: Migration, In Vitro, Transfection, Expressing, Quantitative RT-PCR, MTT Assay, Wound Healing Assay

Journal: Frontiers in Medicine

Article Title: ADAM9 Mediates Triple-Negative Breast Cancer Progression via AKT/NF-κB Pathway

doi: 10.3389/fmed.2020.00214

Figure Lengend Snippet: ADAM9 is involved in AKT/NF-κB pathway in TNBC cells. (A) No change of HB-EGF protein levels was detected in supernatant of MDA-MB-231 cells transfected with ADAM9 siRNA, NC and BC by HB-EGF ELISA assay; (B,C) Down-regulated ADAM9 expression inhibited the phosphorylation of AKT in MDA-MB-231 (B) and Hs578t cells (C) ; (D) Specific p-AKT inhibitor, MK-2206 2HCl, significantly Inhibited phosphorylation of AKT without influencing the ADAM9 expression; (E,F) AKT (GSK690693) inhibitor (E) and p-AKT (MK-2206 2HCl) inhibitor (F) suppressed the phosphorylation of NF-κBp65 and IκBα, respectively; (G) Phosphorylation of NF-κBp65 and IκBα were inhibited as down-regulated ADAM9 expression in MDA-MB-231 (left panel) and Hs578t cells (right panel); (H) ADAM9 mRNA levels were significantly correlated with phosphorylation status of NF-κBp65 in Ser536 in TNBC patients from TCGA database ( n = 68).

Article Snippet: MDA-MB-231, Hs578t, T47D, MCF-7, and MCF-10A were purchased from China Center for Type Culture Collection.

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Expressing, Phospho-proteomics