nm23-h1/h2 (#3345)  (Cell Signaling Technology Inc)

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: Figure 1. 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: Figure 2. 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: Figure 7. 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: Figure 9. 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: Figure 10. 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

Journal: Oncogene

Article Title: Novel roles of NM23 proteins in skin homeostasis, repair and disease.

doi: 10.1038/sj.onc.1209822

Figure Lengend Snippet: Figure 1 KGF-regulated expression of NM23-H1 and NM23-H2 in HaCaT keratinocytes. Cells were rendered quiescent by serum starvation and then treated with KGF as described in Materials and methods. (a) RPAs were performed with total cellular RNA (10 mg) from quiescent and KGF-treated cells to analyse the NM23- H1 (upper panel) and NM23-H2 (lower panel) mRNA levels. As a loading control, the RNA was hybridized with a probe for GAPDH (lower panel). tRNA (50 mg) was used as a negative control. The hybridization probes (1000 c.p.m.) were loaded in the lanes labeled ‘probe’ and used as size markers. (b) Western blot analysis of whole cell lysates (50 mg) from quiescent and KGF- stimulated HaCaT cells. The lysates were analysed for the presence of NM23-H1 and -H2 and of b-actin (loading control).

Article Snippet: The following primary antibodies were used: a mouse monoclonal antibody directed against keratin 10 (DAKO, Glostrup, Denmark; dilution 1:1000), and rabbit polyclonal antibodies directed against K14 (BabCO, Richmond, CA, USA; dilution 1:5000), loricrin (kindly provided by Professor D Hohl, Lausanne, Switzerland; dilution 1:250), cleaved caspase 3 (Cell Signaling Technology, Beverly, MA, USA; dilution 1:100), and NM23-H1/H2 (Santa Cruz Biotechnology Inc., La Jolla, CA, USA; dilution 1:200).

Techniques: Expressing, Control, Negative Control, Hybridization, Labeling, Western Blot

Journal: Oncogene

Article Title: Novel roles of NM23 proteins in skin homeostasis, repair and disease.

doi: 10.1038/sj.onc.1209822

Figure Lengend Snippet: Figure 2 NM23-M1 and NM23-M2 mRNA levels during cutaneous wound repair in BALB/c mice. Mice were wounded and killed at different time points after injury as described in Materials and methods. Total cellular RNA (20 mg) from normal and wounded skin were analysed by RPA for the expression of NM23-M1 and NM23-M2. Lanes labeled ‘probe’ and ‘tRNA’: see Figure 1. Ethidium bromide-stained 1% agarose gel loaded with each RNA sample (1 mg) served as a loading control (shown below the protection assays). dw: days after wounding.

Article Snippet: The following primary antibodies were used: a mouse monoclonal antibody directed against keratin 10 (DAKO, Glostrup, Denmark; dilution 1:1000), and rabbit polyclonal antibodies directed against K14 (BabCO, Richmond, CA, USA; dilution 1:5000), loricrin (kindly provided by Professor D Hohl, Lausanne, Switzerland; dilution 1:250), cleaved caspase 3 (Cell Signaling Technology, Beverly, MA, USA; dilution 1:100), and NM23-H1/H2 (Santa Cruz Biotechnology Inc., La Jolla, CA, USA; dilution 1:200).

Techniques: Expressing, Labeling, Staining, Agarose Gel Electrophoresis, Control

Journal: Oncogene

Article Title: Novel roles of NM23 proteins in skin homeostasis, repair and disease.

doi: 10.1038/sj.onc.1209822

Figure Lengend Snippet: Figure 3 Localization of NM23-M1 (a–d) and NM23-M2 mRNAs (e–h) and protein (i–k) in 5-day wounds. (a–h) in situ hybridization: paraformaldehyde-fixed frozen sections from the middle of 5-day full-thickness excisional wounds from BALB/c mice were hybridized with 35S-labeled sense (c, g) or antisense (a, b, d, e, f, h) riboprobes. (a, e) Show an overview over one wound margin, the rectangles mark the area of the wounds, which are shown in (d, h), respectively. Signals appear as black dots in the bright field survey (a, d, e, h) and as white dots in the dark field survey (b, c, f, g). Sections were counterstained with H/E. (i–j) immunofluorescence: sections from the middle of acetic acid/ethanol fixed paraffin-embedded 5-day wounds were incubated with an antibody directed against NM23-M1 and -M2 (i). As a control staining without the primary antibody is shown is (j). D: dermis, E: epidermis, ES: eschar, G: granulation tissue; HE: hyperproliferative epithelium.

Article Snippet: The following primary antibodies were used: a mouse monoclonal antibody directed against keratin 10 (DAKO, Glostrup, Denmark; dilution 1:1000), and rabbit polyclonal antibodies directed against K14 (BabCO, Richmond, CA, USA; dilution 1:5000), loricrin (kindly provided by Professor D Hohl, Lausanne, Switzerland; dilution 1:250), cleaved caspase 3 (Cell Signaling Technology, Beverly, MA, USA; dilution 1:100), and NM23-H1/H2 (Santa Cruz Biotechnology Inc., La Jolla, CA, USA; dilution 1:200).

Techniques: In Situ Hybridization, Labeling, Incubation, Control, Staining

Journal: Oncogene

Article Title: Novel roles of NM23 proteins in skin homeostasis, repair and disease.

doi: 10.1038/sj.onc.1209822

Figure Lengend Snippet: Figure 4 Increased expression of NM23 in psoriatic skin. (a) Total RNA (10 mg) isolated from biopsies of normal (ctrl) and psoriatic skin (patient) was analysed by RPA for the presence of NM23-H1 and -H2 transcripts. As a loading control RNA was also hybridized to a GAPDH probe. Lanes labeled ‘probe’ and ‘tRNA’: see Figure 1. (b) The extent of overexpression of NM23-H1 and -H2 compared to control skin (normalized to GAPDH) was quantified and is shown schematically.

Article Snippet: The following primary antibodies were used: a mouse monoclonal antibody directed against keratin 10 (DAKO, Glostrup, Denmark; dilution 1:1000), and rabbit polyclonal antibodies directed against K14 (BabCO, Richmond, CA, USA; dilution 1:5000), loricrin (kindly provided by Professor D Hohl, Lausanne, Switzerland; dilution 1:250), cleaved caspase 3 (Cell Signaling Technology, Beverly, MA, USA; dilution 1:100), and NM23-H1/H2 (Santa Cruz Biotechnology Inc., La Jolla, CA, USA; dilution 1:200).

Techniques: Expressing, Isolation, Control, Labeling, Over Expression

Journal: Oncogene

Article Title: Novel roles of NM23 proteins in skin homeostasis, repair and disease.

doi: 10.1038/sj.onc.1209822

Figure Lengend Snippet: Figure 5 Overexpression of NM23-H1, NM23-H2 or NM23-H1/ H2 in HaCaT keratinocytes. (a) Samples of 10 mg RNA from NM23-H1, NM23-H2 or NM23-H1/H2 overexpressing cells and vector-transfected control cells were analysed by RPA for the presence of NM23-H1 (upper panel) and NM23-H2 mRNAs (lower panel). Lanes labeled ‘probe’ and ‘tRNA’: see Figure 1. (b) Samples of 60 mg total cellular protein of exponentially growing cells were analysed by western blotting for the expression of NM23- H1 and -H2 and of b-actin (loading control).

Article Snippet: The following primary antibodies were used: a mouse monoclonal antibody directed against keratin 10 (DAKO, Glostrup, Denmark; dilution 1:1000), and rabbit polyclonal antibodies directed against K14 (BabCO, Richmond, CA, USA; dilution 1:5000), loricrin (kindly provided by Professor D Hohl, Lausanne, Switzerland; dilution 1:250), cleaved caspase 3 (Cell Signaling Technology, Beverly, MA, USA; dilution 1:100), and NM23-H1/H2 (Santa Cruz Biotechnology Inc., La Jolla, CA, USA; dilution 1:200).

Techniques: Over Expression, Plasmid Preparation, Transfection, Control, Labeling, Western Blot, Expressing

Journal: Oncogene

Article Title: Novel roles of NM23 proteins in skin homeostasis, repair and disease.

doi: 10.1038/sj.onc.1209822

Figure Lengend Snippet: Figure 6 Overexpression of NM23-H1 and/or H2 does not affect HaCaT cell proliferation in a monoculture. (a) NM23-transfected and vector-transfected cells (100 000) were seeded into 3.5 cm cell culture dishes. At the time points indicated the cells were trypsinized and counted in triplicate. The growth medium was changed every second day. (b) NM23-transfected and control cells (100 000) were seeded into 3.5 cm cell culture dishes. At 2 days after plating, BrdU was added to the medium. 2 h later cells were fixed, and proliferating cells were stained with an antibody against BrdU. The ratio of BrdU positive cells versus total number of cells was determined. All results were reproduced in three independent experiments and with two different clones each.

Article Snippet: The following primary antibodies were used: a mouse monoclonal antibody directed against keratin 10 (DAKO, Glostrup, Denmark; dilution 1:1000), and rabbit polyclonal antibodies directed against K14 (BabCO, Richmond, CA, USA; dilution 1:5000), loricrin (kindly provided by Professor D Hohl, Lausanne, Switzerland; dilution 1:250), cleaved caspase 3 (Cell Signaling Technology, Beverly, MA, USA; dilution 1:100), and NM23-H1/H2 (Santa Cruz Biotechnology Inc., La Jolla, CA, USA; dilution 1:200).

Techniques: Over Expression, Transfection, Plasmid Preparation, Cell Culture, Control, Staining, Clone Assay

Journal: Oncogene

Article Title: Novel roles of NM23 proteins in skin homeostasis, repair and disease.

doi: 10.1038/sj.onc.1209822

Figure Lengend Snippet: Figure 7 NM23 affects keratinocyte differentiation in a mono- culture. (a) Phase contrast pictures of vector-transfected control cells (upper left panel), NM23-H1-transfected (upper right panel), NM23-H2-transfected (lower left panel) and NM23-H1/H2-trans- fected cell lines (lower right panel). (b) NM23-transfected and control cells were seeded into 3.5 cm cell culture dishes; when they reached 70% confluence they were fixed and stained with an antibody against K10. The ratio of K10 positive cells versus total number of cells was determined. All results were reproduced in three independent experiments and with two different clones each. (c) Samples of 10 mg total cellular RNA were analysed by RPA at different time points after induction of differentiation. Analysis of K10 expression (third panel) was used as a marker for the onset of differentiation. As a loading control, the RNA was hybridized with a probe for b-actin (bottom panel). Lanes labeled ‘probe’ and ‘tRNA’: see Figure 1.

Article Snippet: The following primary antibodies were used: a mouse monoclonal antibody directed against keratin 10 (DAKO, Glostrup, Denmark; dilution 1:1000), and rabbit polyclonal antibodies directed against K14 (BabCO, Richmond, CA, USA; dilution 1:5000), loricrin (kindly provided by Professor D Hohl, Lausanne, Switzerland; dilution 1:250), cleaved caspase 3 (Cell Signaling Technology, Beverly, MA, USA; dilution 1:100), and NM23-H1/H2 (Santa Cruz Biotechnology Inc., La Jolla, CA, USA; dilution 1:200).

Techniques: Plasmid Preparation, Transfection, Control, Cell Culture, Staining, Clone Assay, Expressing, Marker, Labeling

Journal: Oncogene

Article Title: Novel roles of NM23 proteins in skin homeostasis, repair and disease.

doi: 10.1038/sj.onc.1209822

Figure Lengend Snippet: Figure 8 Overexpression of NM23-H1/-H2 affects proliferation, differentiation and apoptosis of HaCaT keratinocytes in organotypic cultures. The stably transfected cell lines were cultured in three-dimensional organotypic cultures for 14 days, embedded in paraffin and sectioned. (a) Sections were stained with H/E. Vector-transfected control cells: upper left panel, NM23-H1-transfected cells: upper right panel, NM23-H2-transfected cells: lower left panel, and NM23-H1/H2-transfected cells: lower right panel. (b) The epithelial area was measured with the OpenLab program (n ¼ 10 with n: number of pictures measured per culture; N ¼ 3 with N: number of independent cultures per clone). (c) Cultures were incubated in BrdU-containing medium for 2 h before harvesting and stained with a peroxidase-coupled anti-BrdU antibody, followed by peroxidase staining and counterstaining with hematoxylin. BrdU positive basal keratinocytes were counted and their number per mm basement membrane was determined. (d) Sections were stained with an antibody directed against cleaved caspase 3. Cleaved caspase 3 positive cells were counted and the number of positive cells per 100 mm2 epithelium was determined. For the quantitative analyses shown in (b), (c) and (d) 10 pictures from each of three cultures per clone were counted. (e, f) Sections were immunostained with antibodies to keratin 10 (green) and keratin 14 (red) (e) or loricrin (red) (f). (f) Nuclei were stained with Hoechst (blue).

Article Snippet: The following primary antibodies were used: a mouse monoclonal antibody directed against keratin 10 (DAKO, Glostrup, Denmark; dilution 1:1000), and rabbit polyclonal antibodies directed against K14 (BabCO, Richmond, CA, USA; dilution 1:5000), loricrin (kindly provided by Professor D Hohl, Lausanne, Switzerland; dilution 1:250), cleaved caspase 3 (Cell Signaling Technology, Beverly, MA, USA; dilution 1:100), and NM23-H1/H2 (Santa Cruz Biotechnology Inc., La Jolla, CA, USA; dilution 1:200).

Techniques: Over Expression, Stable Transfection, Transfection, Cell Culture, Staining, Plasmid Preparation, Control, Incubation, Membrane