Journal: Scientific Reports

Article Title: CTCF and EGR1 suppress breast cancer cell migration through transcriptional control of Nm23-H1

doi: 10.1038/s41598-020-79869-9

Figure Lengend Snippet: Analysis of Nm23-H1 protein, mRNA, and promoter activity in breast cancer cell lines. Confluent cell cultures were lysed and analyzed for Nm23-H1 expression by Western blot with corresponding antibodies ( a ) or RT-qPCR ( b ). ( c ) MCF-7 and MDA-MB-231 cells were transiently transfected with Nm23-H1 promoter constructs and pRL-TK control vector and lysed 48 h after transfection. Luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega). RLU values of reference constructs: MCF-7/pGL3-Empty, Firefly luciferase (10,431 ± 311), Renilla luciferase (92,978 ± 1766); MDA-MB-231/pGL3-Empty, Firefly luciferase (1967 ± 75), Renilla luciferase (10,371 ± 183). Luciferase activities were calculated using firefly luciferase values normalized to renilla luciferase values. Data represent the mean and standard deviation of three independent trials. *p < 0.05.

Article Snippet: Antibodies for EGR1 (#4153), IgG-Mouse (#5415), IgG-Rabbit (#3900), and Nm23-H1/H2 (#3345), were purchased from Cell Signaling Technology (Danvers, USA).

Techniques: Activity Assay, Expressing, Western Blot, Quantitative RT-PCR, Transfection, Construct, Control, Plasmid Preparation, Luciferase, Reporter Assay, Standard Deviation

Journal: Scientific Reports

Article Title: CTCF and EGR1 suppress breast cancer cell migration through transcriptional control of Nm23-H1

doi: 10.1038/s41598-020-79869-9

Figure Lengend Snippet: In silico screening of transcription factor binding sites. ( a ) The proximal (− 244 to − 109 bp) and minimal promoter (− 109 to − 1 bp) region of the Nm23-H1 promoter were analyzed in MatInspector (matrix similarity threshold > 0.8) and TRANSFAC (matrix score threshold > 0.8) for potential transcription factor binding events. Consistently predicted transcription factors and corresponding binding sites 1–11 were selected for mutagenesis. Mutants of the proximal promoter ( b ) and minimal promoter ( c ) were transiently transfected in MCF-7 or MDA-MB-231 cells and lysed 48 h after transfection. Luciferase assays were performed using the Dual-Luciferase Reporter Assay System (Promega). RLU values of reference constructs: MCF-7/pGL3-244, Firefly luciferase (2,749,183 ± 106,184), Renilla luciferase (208,951 ± 7441); MCF-7/pGL3-109, Firefly luciferase (689,809 ± 49,773), Renilla luciferase (131,826 ± 3867); MDA-MB-231/pGL3-244, Firefly luciferase (138,241 ± 401), Renilla luciferase (24,492 ± 1526); MDA-MB-231/pGL3-109, Firefly luciferase (689,809 ± 49,773), Renilla luciferase (131,826 ± 3867). Data represent the mean and standard deviation of three independent trials. *Significance with wild-type promoter, p < 0.05.

Article Snippet: Antibodies for EGR1 (#4153), IgG-Mouse (#5415), IgG-Rabbit (#3900), and Nm23-H1/H2 (#3345), were purchased from Cell Signaling Technology (Danvers, USA).

Techniques: In Silico, Binding Assay, Mutagenesis, Transfection, Luciferase, Reporter Assay, Construct, Standard Deviation

Journal: Scientific Reports

Article Title: CTCF and EGR1 suppress breast cancer cell migration through transcriptional control of Nm23-H1

doi: 10.1038/s41598-020-79869-9

Figure Lengend Snippet: Transcription factors regulating Nm23-H1 in MDA-MB-231 cells. ( a ) MDA-MB-231 cells were transiently transfected with HA-tagged transcription factors and pGL3-1291 and lysed 48 h after transfection. Luciferase assays were performed using the Dual-Luciferase Reporter Assay System (Promega). RLU values of pGL3-1291/pcDNA3.1: Firefly luciferase (310,113 ± 8524), Renilla luciferase (60,130 ± 5091). *Significant stimulation as compared to pcDNA3.1, p < 0.05. ( b ) RNA extraction was performed with TRIzol in transiently transfected MDA-MB-231 cells, followed by cDNA synthesis and qPCR. *Significance with pcDNA3.1, p < 0.05. Data represent the mean and standard deviation of three independent trials. ( c ) Proteins were separated on 15% acrylamide gels and immunoblots were probed with corresponding antibodies. Corresponding protein bands are marked by red boxes. Quantification of Nm23-H1/H2 protein bands was performed with ImageJ. Data are shown as a representative experiment from three independent trials. ( d ) Protein-DNA interactions were crosslinked and the chromatin was sheared into 200–500 bp fragments by sonication. Complexes were pulled down by the HA-antibody and protein G agarose beads. ‘Input’ is the chromatin collected before antibody incubation. ‘Beads only’ represents the chromatin containing protein G agarose beads without antibodies and serves as a negative control. Data are shown as a representative experiment from three independent trials.

Article Snippet: Antibodies for EGR1 (#4153), IgG-Mouse (#5415), IgG-Rabbit (#3900), and Nm23-H1/H2 (#3345), were purchased from Cell Signaling Technology (Danvers, USA).

Techniques: Transfection, Luciferase, Reporter Assay, RNA Extraction, cDNA Synthesis, Standard Deviation, Western Blot, Sonication, Incubation, Negative Control

Journal: Scientific Reports

Article Title: CTCF and EGR1 suppress breast cancer cell migration through transcriptional control of Nm23-H1

doi: 10.1038/s41598-020-79869-9

Figure Lengend Snippet: siRNA knockdown of CTCF and EGR1 in MCF-7 cells. ( a , d ) Untransfected MCF-7 cells or MCF-7 cells transiently transfected with siCTCF, siEGR1, or siScramble, were lysed 48 h after transfection. Proteins were separated on 12% polyacrylamide gels and immunoblots were probed with corresponding antibodies. Quantification of Nm23-H1 bands was performed with ImageJ. Data are shown as a representative experiment from three independent trials. ( b ) Luciferase assays were performed in transiently transfected cells using the Dual-Luciferase Reporter System (Promega) *Significant inhibition as compared to siScramble, p < 0.05. ( c ) RNA extraction was performed in transiently transfected MCF-7 cell, followed by cDNA synthesis and qPCR. *Significance with siScramble, p < 0.05. Data represent the mean and standard deviation of three independent trials.

Article Snippet: Antibodies for EGR1 (#4153), IgG-Mouse (#5415), IgG-Rabbit (#3900), and Nm23-H1/H2 (#3345), were purchased from Cell Signaling Technology (Danvers, USA).

Techniques: Knockdown, Transfection, Western Blot, Luciferase, Inhibition, RNA Extraction, cDNA Synthesis, Standard Deviation

Journal: Scientific Reports

Article Title: CTCF and EGR1 suppress breast cancer cell migration through transcriptional control of Nm23-H1

doi: 10.1038/s41598-020-79869-9

Figure Lengend Snippet: Cell migration of MCF-7 cells upon Nm23-H1 knockdown and CTCF or EGR1 overexpression. ( a , c ) Transiently transfected cells were grown to full confluency and scratched with a yellow pipette tip. Photos were taken at different time points with a microscope at ×10 magnification. Cell migration was quantified as percentage of wound closure using ImageJ and MRI Wound Healing Tool plugin. ( b , d ) Transiently transfected cells were seeded into the upper chamber of Transwell inserts and allowed for migration for 48 h. Photos were taken with a microscope at ×10 magnification and the number of migrated cells was quantified using ImageJ. Data represent the mean and standard deviation of three independent trials. *Significance with siScramble + pcDNA3.1, p < 0.05.

Article Snippet: Antibodies for EGR1 (#4153), IgG-Mouse (#5415), IgG-Rabbit (#3900), and Nm23-H1/H2 (#3345), were purchased from Cell Signaling Technology (Danvers, USA).

Techniques: Migration, Knockdown, Over Expression, Transfection, Transferring, Microscopy, Standard Deviation

Journal: Scientific Reports

Article Title: CTCF and EGR1 suppress breast cancer cell migration through transcriptional control of Nm23-H1

doi: 10.1038/s41598-020-79869-9

Figure Lengend Snippet: Proposed model of pathways regulating Nm23-H1 expression. CTCF and EGR1 are recruited to the promoter of NME1 to activate transcription, potentially contributing to metastasis suppression. The expression of Nm23-H1 can be further regulated by the action of AKT, which activates EGR1 and inhibits FOXO3, a negative regulator of NME1 transcription. In addition, CRE regions in the promoters of EGR1 and NME1 may be targeted by CREB to drive the expression of Nm23-H1. The low expression of CTCF and EGR1 in aggressive breast cancer cells may contribute to decreased Nm23-H1 protein levels and metastasis.

Article Snippet: Antibodies for EGR1 (#4153), IgG-Mouse (#5415), IgG-Rabbit (#3900), and Nm23-H1/H2 (#3345), were purchased from Cell Signaling Technology (Danvers, USA).

Techniques: Expressing