Journal: Experimental Hematology & Oncology

Article Title: METTL3 modulates m6A modification of CDC25B and promotes head and neck squamous cell carcinoma malignant progression

doi: 10.1186/s40164-022-00256-3

Figure Lengend Snippet: METTL3 high expression is associated with poor prognosis of HNSCC patients. A Dot blot assay was conducted with mRNA extracted from HNSCC tissues and paired paracancerous normal tissues using an anti-m6A antibody, and MB (methylene blue) staining served as the loading control (representative images in left panel). The relative m6A contents on mRNA in HNSCC tissues and paired normal normal tissues were calculated (right panel, n = 7). B TCGA data showed that METTL3 expression was significantly upregulated in HNSCC (n = 520) than normal tissue (n = 44). C Disease-free survival (RFS) of HNSCC patients based on METTL3 expression obtained from GEPIA website ( http://gepia.cancer-pku.cn/ ). D The levels of METTL3 expression in HNSCC and paired normal tissues were measured by qRT-PCR (n = 10). E METTL3 protein levels were measured in HNSCC tissues and paired normal tissues by western blotting (n = 7). F METTL3 expression was significantly upregulated in HNSCC compared with the paired paracancerous normal tissue by IHC staining (n = 5, scale bars = 100 μm). G Kaplan–Meier OS analysis of HNSCC patients based on METTL3 expression measured by IHC of tissue microarray (n = 100). H Univariate Cox regression analysis was conducted in HNSCC patients (n = 100). All bars correspond to 95% confidence intervals. I Multivariate Cox regression analysis was conducted in HNSCC patients (n = 100). J The time-dependent receiver operating characteristic (ROC) analysis for the clinical risk score (TNM stage), the METTL3 risk score, and the combined METTL3 and clinical risk scores in HNSCC cohort

Article Snippet: We then established the stable METTL3 knockdown cells (SAS and FaDu), and METTL3-overexpressing cells (Hep2) via METTL3 shRNA or overexpression lentivirus plasmid, respectively (Fig. A–C, Additional file : Figure S2D).

Techniques: Expressing, Dot Blot, Staining, Control, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Microarray

Journal: Experimental Hematology & Oncology

Article Title: METTL3 modulates m6A modification of CDC25B and promotes head and neck squamous cell carcinoma malignant progression

doi: 10.1186/s40164-022-00256-3

Figure Lengend Snippet: METTL3 promotes HNSCC cell proliferation, migration, and invasion. A The protein levels of METTL3 in SAS cells with METTL3 knockdown were measured by western blotting (upper panel), and cell proliferation ability in METTL3 knockdown and their corresponding control SAS cells were measured by a CCK8 assay (bottom panel). B The protein levels of METTL3 in FaDu cells with METTL3 knockdown were measured by western blotting (upper panel), and cell proliferation ability in METTL3 knockdown or control FaDu cells were measured by a CCK8 assay (bottom panel). C The protein levels of METTL3 in Hep2 cells with METTL3 overexpression were measured by western blotting (upper panel), and cell proliferation ability in METTL3 knockdown or control Hep2 cells were measured by a CCK8 assay (bottom panel). D , E knockdown of METTL3 inhibits HNSCC cell proliferation by colony formation assay (left panel). Quantification of the colony formation assay results (right panel). F METTL3 overexpression promotes HNSCC cell proliferation by colony formation assay (left panel). Quantification of the colony formation assay results (right panel). G , H knockdown of METTL3 inhibits HNSCC cell migration and invasion by transwell assays. Representative images (scale bars = 100 μm, left panel) and quantification (right panel) of the cell migration and invasion assay results were shown. I METTL3 overexpression promotes HNSCC cell migration and invasion by transwell assays. Representative images (scale bars = 100 μm, left panel) and quantification (right panel) of the cell migration and invasion assay results were shown. The data are the means ± SD of three independent experiments. */# p < 0.05; **/## p < 0.01; ***/### p < 0.001; ****/#### p < 0.0001

Article Snippet: We then established the stable METTL3 knockdown cells (SAS and FaDu), and METTL3-overexpressing cells (Hep2) via METTL3 shRNA or overexpression lentivirus plasmid, respectively (Fig. A–C, Additional file : Figure S2D).

Techniques: Migration, Knockdown, Western Blot, Control, CCK-8 Assay, Over Expression, Colony Assay, Invasion Assay

Journal: Experimental Hematology & Oncology

Article Title: METTL3 modulates m6A modification of CDC25B and promotes head and neck squamous cell carcinoma malignant progression

doi: 10.1186/s40164-022-00256-3

Figure Lengend Snippet: METTL3 mediates the m6A modification on CDC25B mRNA in HNSCC. A m6A-mRNA epitranscriptomic microarray showed signal pathways in which most differentially expressed gene enriched in METTL3 knockdown cells (SAS), DE represents differentially expressed. B m6A-mRNA epitranscriptomic microarray showed signal pathways in which most differentially methylated genes enriched in METTL3 knockdown cells (SAS), DM represents differentially methylated. C m6A-mRNA epitranscriptomic microarray showed an overlap of the total differentially expressed gene, total differentially methylated gene, and differentially expressed and methylated gene enriched in the cell cycle pathway. D Genes selected from the overlap were used for qRT-PCR in METTL3 knockdown and their corresponding control cells, and CDC25B was the most significantly downregulated gene upon knockdown of METTL3. E , F CDC25B mRNA expression was confirmed by qRT-PCR in METTL3 knockdown (FaDu) and METTL3 overexpression (Hep2) cells. G CDC25B protein level was measured by western blot assay in METTL3 knockdown SAS cells. H CDC25B protein level was measured by western blot assay in METTL3 knockdown FaDu cells. I CDC25B protein level was measured by western blot assay in METTL3 overexpressed Hep2 cells. J MeRIP-qPCR was conducted to detect the m6A level of CDC25B mRNA in METTL3 knockdown (SAS) cells. K MeRIP-qPCR was conducted to detect the m6A level of CDC25B mRNA in METTL3 knockdown (FaDu) cells. L , M The levels of CDC25B expression in METTL3 knockdown and their corresponding control cells treated with actinomycin D (5 μg/mL) at the indicated time points were detected by qRT-PCR. The data are the means ± SD of three independent experiments. */# p < 0.05; **/## p < 0.01; ***/### p < 0.001; ****/#### p < 0.0001

Article Snippet: We then established the stable METTL3 knockdown cells (SAS and FaDu), and METTL3-overexpressing cells (Hep2) via METTL3 shRNA or overexpression lentivirus plasmid, respectively (Fig. A–C, Additional file : Figure S2D).

Techniques: Modification, Microarray, Knockdown, Methylation, Quantitative RT-PCR, Control, Expressing, Over Expression, Western Blot

Journal: Experimental Hematology & Oncology

Article Title: METTL3 modulates m6A modification of CDC25B and promotes head and neck squamous cell carcinoma malignant progression

doi: 10.1186/s40164-022-00256-3

Figure Lengend Snippet: CDC25B promotes SAS cell proliferation, migration, invasion, and cell cycle progression. A The qRT-PCR was conducted to confirm CDC25B knockdown efficiency at mRNA level. B Western blot assay was conducted to confirm CDC25B knockdown efficiency at protein level. C Knockdown of CDC25B inhibited cell proliferation in colony formation assay (left panel); quantification results of colony formation (right panel). D CDC25B knockdown inhibited cell migration and invasion by transwell assays. Representative images (scale bars = 100 μm, left panel) and quantification (right panel) of the cell migration and invasion assay results were shown. E CCK8 assay was conducted on SAS cell after different concentrations of CDC25B inhibitor (menadione) treatment at indicated time. F CDC25B inhibitor (menadione) concentration of 5 μM was used for constant inhibition in colony formation assay and showed that menadione can inhibit cell proliferation (upper panel); quantification results of colony formation (bottom panel). G SAS cells were treated with 5 μM menadione for 24 h and used for transwell assays, showing that menadione can inhibit cell migration and invasion. Representative images (scale bars = 100 μm, left panel) and quantification (right panel) of the cell migration and invasion assay results were shown. Cell cycle G2/M arrest was observed in CDC25B knockdown cells ( H ) and METTL3 knockdown cells ( I ). The data are the means ± SD of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: We then established the stable METTL3 knockdown cells (SAS and FaDu), and METTL3-overexpressing cells (Hep2) via METTL3 shRNA or overexpression lentivirus plasmid, respectively (Fig. A–C, Additional file : Figure S2D).

Techniques: Migration, Quantitative RT-PCR, Knockdown, Western Blot, Colony Assay, Invasion Assay, CCK-8 Assay, Concentration Assay, Inhibition

Journal: Experimental Hematology & Oncology

Article Title: METTL3 modulates m6A modification of CDC25B and promotes head and neck squamous cell carcinoma malignant progression

doi: 10.1186/s40164-022-00256-3

Figure Lengend Snippet: METTL3 accelerates HNSCC malignant progression by upregulating CDC25B. A Colony formation assay of METTL3 overexpressing Hep2 cells transfected with CDC25B siRNAs and the corresponding control (left panel), numbers of the colony formation were calculated (right panel). B Colony formation assay of METTL3 overexpressing Hep2 cells treated with 5 μM menadione or DMSO for 24 h (left panel), numbers of the colony formation were calculated (right panel). C Transwell assay of METTL3 overexpressing Hep2 cells transfected with CDC25B siRNAs and the corresponding control (left panel) to evaluate cell migration and invasion ability (scale bars = 100 μm, left panel), numbers of cells migrated and invaded were calculated (right panel). D Transwell assay of METTL3 overexpressing Hep2 cells treated with 5 μM menadione or DMOS for 24 h and the corresponding control (scale bars = 100 μm, left panel), numbers of cells migrated and invaded were calculated (right panel). E Cell cycle assay of METTL3 overexpressing Hep2 cells transfected with CDC25B siRNAs and the corresponding control (left panel), percentage of cell cycle phase was calculated (right panel). F Cell cycle assay of METTL3 overexpressing Hep2 cells treated with 5 μM menadione or DMSO for 24 h and the corresponding control (left panel), percentage of cell cycle phase was calculated (right panel). The data are the means ± SD of three independent experiments. * p < 0.05; ** p < 0.01

Article Snippet: We then established the stable METTL3 knockdown cells (SAS and FaDu), and METTL3-overexpressing cells (Hep2) via METTL3 shRNA or overexpression lentivirus plasmid, respectively (Fig. A–C, Additional file : Figure S2D).

Techniques: Colony Assay, Transfection, Control, Transwell Assay, Migration, Cell Cycle Assay

Journal: Experimental Hematology & Oncology

Article Title: METTL3 modulates m6A modification of CDC25B and promotes head and neck squamous cell carcinoma malignant progression

doi: 10.1186/s40164-022-00256-3

Figure Lengend Snippet: Overexpression of METTL3 promotes tumor growth and angiogenesis. A Knockdown of METTL3 significantly inhibits subcutaneous tumor growth in nude mice (n = 6). B The tumor volume curve was conducted by measuring every other day. C The mice were executed and tumors were extracted and weighed after 21 days. D CDC25B mRNA level in extracted tumor tissues from METTL3 knockdown and corresponding control groups was measured by qRT-PCR. E METTL3 and CDC25B protein level in extracted tumors from METTL3 knockdown and corresponding control groups was measured by western blot assay. F METTL3 and CDC25B protein level was measured by IHC on extracted tumors from METTL3 knockdown and corresponding control groups (scale bars = 100 μm). G Ki67 and CD31 protein level was measured by IHC on extracted tumors from METTL3 knockdown and corresponding control groups (scale bars = 100 μm). H Tube formation assay was conducted using HUVEC cultured in medium from METTL3 knockdown SAS cells and their corresponding control cells (upper panel), and the number of tubes was calculated (bottom panel). I Tube formation assay was conducted using HUVEC cultured in medium from METTL3 overexpressing Hep2 cells and their corresponding control cell (upper panel), and the number of tubes was calculated (bottom panel; scale bars = 100 μm). J The graphic illustration of METTL3 mediated the m6A modification of CDC25B mRNA, promoting HNSCC cell cycle progression to lead to the cell proliferation, migration, invasion, and angiogenesis. The data are the means ± SD of three independent experiments. * p < 0.05; ** p < 0.01; **** p < 0.0001

Article Snippet: We then established the stable METTL3 knockdown cells (SAS and FaDu), and METTL3-overexpressing cells (Hep2) via METTL3 shRNA or overexpression lentivirus plasmid, respectively (Fig. A–C, Additional file : Figure S2D).

Techniques: Over Expression, Knockdown, Control, Quantitative RT-PCR, Western Blot, Tube Formation Assay, Cell Culture, Modification, Migration

Journal: Aging Cell

Article Title: Liver osteopontin is required to prevent the progression of age‐related nonalcoholic fatty liver disease

doi: 10.1111/acel.13183

Figure Lengend Snippet: Liver OPN is p53 regulated. (a) Protein levels of p53 were evaluated by immunohistochemistry in liver sections of 3‐, 10‐, and 20‐month‐old (m) wild‐type (WT) mice ( n = 3–5). (b) OPN protein levels from liver homogenates were measured in WT and p53‐KO male and female mice fed a chow diet (CD) and a high‐fat diet (HFD) by immunoblotting using glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) as loading control ( n = 4–6). (c) Liver p53 and OPN protein levels were evaluated in WT mice fed a HFD injected with p53‐dominant positive adenovirus (adp53) and GFP (adGFP), using transferrin or GAPDH as a loading control ( n = 4–6). (d) Protein levels of p53 and OPN in siCtrl and sip53 HepG2 cells were measured by immunoblotting using GAPDH as loading control. Extracellular OPN was measured using an ELISA ( n = 4–5). (e) OPN protein levels from HepG2 cells silenced for p53 and Hep3B cells treated with palbociclib (Palbo) were measured by immunoblotting using GAPDH as loading control. OPN media levels from Hep3B cells treated either with vehicle (Veh) or with palbociclib (Palbo) were measured by ELISA ( n = 4–8). Values are means ± SEM . Significant differences are denoted by * p < 0.05, ** p < 0.01, and *** p < 0.001 (Student's t test)

Article Snippet: Data obtained from the Cell Line Encyclopedia (© 2019 The Broad Institute of MIT & Harvard) showed that the cell line with the least p53 expression, the Hep3B, also had the lowest OPN expression.

Techniques: Immunohistochemistry, Western Blot, Control, Injection, Enzyme-linked Immunosorbent Assay

Journal: Oncogene

Article Title: The pleiotropic effects of TNFα in breast cancer subtypes is regulated by TNFAIP3/A20

doi: 10.1038/s41388-018-0472-0

Figure Lengend Snippet: a Luminal and TNBC cell lines were treated with TNFα (50ng/ml) for 48hrs and examined for apoptotic cell death by flow cytometry analyses based on Annexin V staining. b Expression of A20 in TNFα-treated breast cancer cells was shown by qPCR assays. c TNFα-treated induced expressions of A20, BCL2 and PARP cleavage were evaluated by western blot analyses. d, e Overexpression of A20 in MCF7 and ZR75–1 cells was confirmed by qPCR and Western blotting. f Parental and A20 overexpressing MCF7 and ZR75–1 cells were treated with TNFα (50ng/ml) for 48hrs and analyzed cell death by Annexin V flow cytometry apoptotic cell staining. g, h Protein expression of A20, PARP cleavage, BAX and BCL2 was shown in parental and A20 overexpressing cells with TNFα treatment. Results are presented as mean ±SD. *P<0.05, **P<0.001, ***P<0.0001.

Article Snippet: Generation of A20 overexpression or knockdown cells Lentiviral constructs were obtained from Applied Biological Materials and stable cell lines were obtained from manufacturer’s protocol.

Techniques: Flow Cytometry, Staining, Expressing, Western Blot, Over Expression

Journal: Cancers

Article Title: ANKRD1 Promotes Breast Cancer Metastasis by Activating NF- κ B-MAGE-A6 Pathway

doi: 10.3390/cancers16193306

Figure Lengend Snippet: High ANKRD1 expression correlates with poor prognosis, aggressiveness and tumor progression of breast cancer. ( A ) Kaplan–Meier survival analysis of distant metastasis-free survival (DMFS) breast cancer patients with metastatic disease based on ANKRD1 expression using mRNA gene chip (HR; the hazard ratio) ( https://kmplot.com ). The following parameters were set: (1) lymph node status: positive; (2) probe set: only JetSet best probe; (3) grade: 3; and (4) survival: DMFS. Patients with higher ANKRD1 expression showed lower survival rates compared to those with low expression of ANKRD1. ( B , C ) Real-time PCR and Western blot results showing ANKRD1 fold change in normal breast (MCF-10A) and non-metastatic (ZR-75-30, T47D and MCF-7) and highly metastatic breast cancer cells (LM-2 and MDA-MB-231). ( D ) IHC staining (left panel) of ANKRD1 in human breast tissue array showing progressively higher ANKRD1 expression depending on the grade of breast cancer tissue. The right panel shows H&E-stained sections.

Article Snippet: For orthotopic injection, 1 × 10 6 cells per 100 μL in sterile phosphate-buffer saline (PBS) of LM-2 control vector and ANKRD1-knockdown cell lines (containing GFP) were orthotopically injected into the mammary fat pad of 6–8-week-old immunodeficient NOD/SCID mice (n = 10 per each group), which were purchased from the Jackson laboratory.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Staining

Journal: Cancers

Article Title: ANKRD1 Promotes Breast Cancer Metastasis by Activating NF- κ B-MAGE-A6 Pathway

doi: 10.3390/cancers16193306

Figure Lengend Snippet: Semi-quantitative analysis of immunohistochemical staining of ANKRD1 in different tumor grades of breast cancer tissues.

Article Snippet: For orthotopic injection, 1 × 10 6 cells per 100 μL in sterile phosphate-buffer saline (PBS) of LM-2 control vector and ANKRD1-knockdown cell lines (containing GFP) were orthotopically injected into the mammary fat pad of 6–8-week-old immunodeficient NOD/SCID mice (n = 10 per each group), which were purchased from the Jackson laboratory.

Techniques: Immunohistochemical staining, Staining

Journal: Cancers

Article Title: ANKRD1 Promotes Breast Cancer Metastasis by Activating NF- κ B-MAGE-A6 Pathway

doi: 10.3390/cancers16193306

Figure Lengend Snippet: Overexpression of ANKRD1 enhances migration of weakly metastatic MCF-7 cells. ( A , B ) Results of real-time PCR and Western blot assay showing the gene and protein expression in the ANKRD1-overexpressed cell lines (MCF-7-ANKRD1 OE, OE). ( C ) Cell proliferation of ANKRD1-overexpressing cells compared to vector and normal MCF-7 was determined by EZ-Cytox assay and ( D ) CellTrace™ CFSE Cell proliferation kit; cell numbers were counted by flow cytometry using FACS Div and calculated using FlowJo software. ( E ) Representative images depicting wound healing quantified as a percentage of the healed wound area and analyzed using Image JS. ( F – H ) Migration and invasion assays performed with MCF-7, vector and OE. OE cells showed increased wound healing, migration, and invasion compared to vector and NT cells ( I , J ) Real-time PCR and Western blot showing the gene and protein expression involved in EMT. OE cells showed reduced expression of E-cadherin and increased expression of N-cadherin and vimentin compared to vector. NT: MCF-7 parental; Vector: MCF-7-emtpy vector; OE: MCF-7-ANKRD1 overexpression. All experiments were performed in triplicate.

Article Snippet: For orthotopic injection, 1 × 10 6 cells per 100 μL in sterile phosphate-buffer saline (PBS) of LM-2 control vector and ANKRD1-knockdown cell lines (containing GFP) were orthotopically injected into the mammary fat pad of 6–8-week-old immunodeficient NOD/SCID mice (n = 10 per each group), which were purchased from the Jackson laboratory.

Techniques: Over Expression, Migration, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Plasmid Preparation, Flow Cytometry, Software

Journal: Cancers

Article Title: ANKRD1 Promotes Breast Cancer Metastasis by Activating NF- κ B-MAGE-A6 Pathway

doi: 10.3390/cancers16193306

Figure Lengend Snippet: Knockdown of ANKRD1 suppresses migration and invasion of highly metastatic LM-2 cells. ( A , B ) Real-time PCR and Western blots showing gene and protein expression of ANKRD1-knockdown cell lines (sh14 and sh49). ( C ) Cell proliferation of LM-2-ANKRD1 knockdown cells compared to vector and LM-2 was determined by EZ-Cytox assay and ( D ) by CellTrace™ CFSE Cell proliferation kit; cell numbers were counted by flow cytometry using FACS Diva and calculated using FlowJo software. Representative images depicting ( E ) wound healing ( F – H ), migration, and invasion assays performed with NT, Vector, and sh14. sh14 cells showed decrease in wound healing, migration, and invasion compared to vector and NT cells. ( I , J ) Real-time PCR and Western blots showing the expression of EMT markers. Sh14 cells showed increased expression of E-cadherin and decreased expression of N-cadherin and vimentin compared to vector and NT. NT: LM-2 parental; Vector: LM-2 with empty vector; sh14: LM-2-ANKRD1 knockdown with sh14 and sh49: LM-2-ANKRD1 knockdown with sh49. All experiments were performed in triplicate.

Article Snippet: For orthotopic injection, 1 × 10 6 cells per 100 μL in sterile phosphate-buffer saline (PBS) of LM-2 control vector and ANKRD1-knockdown cell lines (containing GFP) were orthotopically injected into the mammary fat pad of 6–8-week-old immunodeficient NOD/SCID mice (n = 10 per each group), which were purchased from the Jackson laboratory.

Techniques: Knockdown, Migration, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Plasmid Preparation, Flow Cytometry, Software

Journal: Cancers

Article Title: ANKRD1 Promotes Breast Cancer Metastasis by Activating NF- κ B-MAGE-A6 Pathway

doi: 10.3390/cancers16193306

Figure Lengend Snippet: ANKRD1 knockdown reduces metastasis efficiency of breast cancer cells in two in vivo metastasis models. ( A , B ) Representative bioluminescence (BLI) images of animals showing primary tumors (PT) at day 21, derived from orthotopic injections of LM-2 vector control (vector) and LM-2 ANKRD1 knockdown (LM-2 sh14) cells into the mammary fat pad. On day 21 after orthotopic injection into the mammary fat pad, tumor growth at the injection site and GFP was detected by IVIS and quantified based on radiant efficiency, comparing vector and LM-2 sh14 groups (day 21, n = 10 per group). ( C ) Comparison of the size of primary tumors from vector and LM-2 sh14 groups after removing the primary tumor. ( D ) Quantitation of tumor volume (cm 3 ) comparing control and LM-2 sh14 groups. The results show no significant change in tumor volume and tumorigenesis in the LM-2 sh14 cell group compared to the vector. Data presented as mean ± SD. ( E ) Radiant efficiency of the lung and liver comparing vector and LM-2 sh14 groups and ex vivo GFP signals, detected using IVIS. ( F , G ) Representative BLI images of animals on the first day of tail vein injection and quantitation of radiant efficiency in mice (day 1, n = 10 per group). ( H ) Liver metastasis with control and LM-2 sh14 cells. The results show increased metastasis of cancer cells in control compared to LM-2 sh14 group. ( I , J ) H&E staining of liver tissue from tail-vein-injected mice; red circles show the cancerous lesions in the tissues, which are increased in the control group compared to the LM-2 sh14 group. LM-2 Vector (control): LM-2 with empty vector; LM-2 sh14: LM-2-ANKRD1 knockdown with sh14. Ten NOD.SCID mice were used for each set, and all experiments were performed in triplicate.

Article Snippet: For orthotopic injection, 1 × 10 6 cells per 100 μL in sterile phosphate-buffer saline (PBS) of LM-2 control vector and ANKRD1-knockdown cell lines (containing GFP) were orthotopically injected into the mammary fat pad of 6–8-week-old immunodeficient NOD/SCID mice (n = 10 per each group), which were purchased from the Jackson laboratory.

Techniques: Knockdown, In Vivo, Derivative Assay, Plasmid Preparation, Control, Injection, Comparison, Quantitation Assay, Ex Vivo, Staining

Journal: Cancers

Article Title: ANKRD1 Promotes Breast Cancer Metastasis by Activating NF- κ B-MAGE-A6 Pathway

doi: 10.3390/cancers16193306

Figure Lengend Snippet: Impact of ANKRD1 knockdown on signaling pathway, biological process, cellular component and molecular function of LM-2 breast cancer cells. ( A ) Heatmap of differentially expressed genes obtained by RNA-SEQ comparing control (Vector) and LM-2-ANKRD1 knockdown cells (sh14). ( B ) Top 5 genes represented in volume plot between control and LM-2-ANKRD1 knockdown cells from RNA-SEQ. ( C ) Signaling pathways between control and LM-2-ANKRD1 knockdown cells from RNA-SEQ. ( D ) Biological processes (red) between control and LM-2-ANKRD1 knockdown cells from RNA-SEQ. ( E ) Cellular components (green) between control and LM-2-ANKRD1 knockdown cells from RNA-SEQ. ( F ) Molecular functions (blue) between control and LM-2-ANKRD1 knockdown cells from RNA-SEQ. Vector: LM-2 with empty vector, LM-2 sh14: LM-2-ANKRD1 knockdown with sh14 and LM-2 sh49: LM-2-ANKRD1 knockdown with sh49.

Article Snippet: For orthotopic injection, 1 × 10 6 cells per 100 μL in sterile phosphate-buffer saline (PBS) of LM-2 control vector and ANKRD1-knockdown cell lines (containing GFP) were orthotopically injected into the mammary fat pad of 6–8-week-old immunodeficient NOD/SCID mice (n = 10 per each group), which were purchased from the Jackson laboratory.

Techniques: Knockdown, RNA Sequencing Assay, Control, Plasmid Preparation

Journal: Cancers

Article Title: ANKRD1 Promotes Breast Cancer Metastasis by Activating NF- κ B-MAGE-A6 Pathway

doi: 10.3390/cancers16193306

Figure Lengend Snippet: Relationship between MAGE-A6 and ANKRD1 expression and impact of MAGE-A6 silencing on migration and invasion of breast cancer cells. ( A ) Graph showing the relationship between ANKRD1 and MAGE-A6 using the TCGA database. ( B ) Immunoblots showing high expression level of MAGE-A6 protein in MCF-7-ANKRD1-overexpressed cells and low expression level in LM-2-ANKRD1 knockdown cells. ( C ) Protein expression of MAGE-A6-knockdown in MCF-7-ANKRD1-overexpressed cells. ( D ) Wound healing of MAGE-A6-knockdown cells compared to control. ( E ) Graph showing percentage of wound enclosure in MAGE-A6-knockdown cells compared to control. ( F ) Migration assay of MAGE-A6-knockdown cells compared to control. ( G ) Graph showing percentage of cell migration in MAGE-A6-knockdown cells compared to control. ( H ) Invasion assay of MAGE-A6-knockdown cells compared to control. ( I ) Graph showing percentage of cell invasion in MAGE-A6-knockdown cells compared to control. All results showed that knockdown of MAGE-A6 reduced wound healing, cell migration, and invasion. NT: parental cells without treatment; Vector: parental cells with empty vector; ANKRD1 sh14: LM-2-ANKRD1 knockdown with sh14; Neg: MCF-7-ANKRD1-overexpressing cells treated with siRNA negative control; si1: knockdown of MCF-7-ANKRD1-overexpressing cells with MAGE-A6 si1; si2: knockdown of MCF-7-ANKRD1-overexpressing cells with MAGE-A6 si2. All experiments were performed in triplicate.

Article Snippet: For orthotopic injection, 1 × 10 6 cells per 100 μL in sterile phosphate-buffer saline (PBS) of LM-2 control vector and ANKRD1-knockdown cell lines (containing GFP) were orthotopically injected into the mammary fat pad of 6–8-week-old immunodeficient NOD/SCID mice (n = 10 per each group), which were purchased from the Jackson laboratory.

Techniques: Expressing, Migration, Western Blot, Knockdown, Control, Invasion Assay, Plasmid Preparation, Negative Control

Journal: Cancers

Article Title: ANKRD1 Promotes Breast Cancer Metastasis by Activating NF- κ B-MAGE-A6 Pathway

doi: 10.3390/cancers16193306

Figure Lengend Snippet: Recombinant MAGE-A6 increases migration and invasion of breast cancer cells. ( A ) Recombinant MAGE-A6 induced wound healing in LM-2-ANKRD1 knockdown cells compared to control. ( B ) Graph showing the percentage of wound closure in MAGE-A6-treated cells compared to control. All results showed knockdown of MAGE-A6. ( C ) Recombinant MAGE-A6 induced cell migration in LM-2-ANKRD1 knockdown cells compared to control. ( D ) Graph showing percentage of cell migration in MAGE-A6-treated cells compared to control. ( E ) Recombinant MAGE-A6 induced cell invasion in LM-2-ANKRD1 knockdown cells compared to control. ( F ) Graph showing percentage of cell invasion in MAGE-A6-treated cells compared to control. All the results showed that MAGE-A6 recombinant protein induced wound healing, cell migration, and invasion. NT: LM-2-ANKRD1 sh14; Neg: LM-2-ANKRD1 sh14 treated with negative control; MAGE-A6: LM-2-ANKRD1 sh14 treated with MAGE-A6 recombinant protein. All experiments were performed in triplicate.

Article Snippet: For orthotopic injection, 1 × 10 6 cells per 100 μL in sterile phosphate-buffer saline (PBS) of LM-2 control vector and ANKRD1-knockdown cell lines (containing GFP) were orthotopically injected into the mammary fat pad of 6–8-week-old immunodeficient NOD/SCID mice (n = 10 per each group), which were purchased from the Jackson laboratory.

Techniques: Recombinant, Migration, Knockdown, Control, Negative Control

Journal: Cancers

Article Title: ANKRD1 Promotes Breast Cancer Metastasis by Activating NF- κ B-MAGE-A6 Pathway

doi: 10.3390/cancers16193306

Figure Lengend Snippet: ANKRD1 regulates MAGE-A6 expression through the NF-κB pathway. ( A – C ) The expression level and percent band intensity of total and phosphorylated AKT/I-κK/NF-κB in MCF-7-ANKRD1-overexpressed and LM-2-ANKRD1 knockdown cells. The results show increased expression of phosphorylated IκK and NF-κB in ANKRD1 OE cells and reduced expression in ANKRD1 knockdown cells compared to NT and Vector. ( D , E ) Expression level and percentage of band intensity of total and phosphorylated NF-κB, ANKRD1, and MAGE-A6 in NF-κB-knockdown cells. ( F , G ) Expression level and percent band intensity of total and phosphorylated NF-κB, ANKRD1, and MAGE-A6 in MCF-7-ANKRD1-overexpressed cells treated with 2 µm of NF-κB inhibitor (BAY-11). The results from both siNF-κBs and NF-κB inhibitor showed no change in the expression of ANKRD1 after knockdown of NF-κB, while the expression of MAGE-A6 was reduced. NT: non-treated cells; Vector: MCF-7 or LM-2 with empty vector; ANKRD1 OE: MCF-7-ANKRD1 overexpression; sh14: LM-2-ANKRD1 knockdown with sh14; Neg: MCF-7-ANKRD1 overexpression treated with siRNA negative control; si1: MCF-7-ANKRD1 overexpression knockdown with NF-κB-siRNA1; si2: MCF-7-ANKRD1 overexpression knockdown with NF-κB-siRNA2; si3: MCF-7-ANKRD1 overexpression knockdown with NF-κB-siRNA3; si4: MCF-7-ANKRD1 overexpression knockdown with NF-κB-siRNA4; DMSO: MCF-7-ANKRD1 overexpression treated with DMSO; BAY-11: MCF-7-ANKRD1 overexpression treated with BAY-11. All experiments were performed in triplicate.

Article Snippet: For orthotopic injection, 1 × 10 6 cells per 100 μL in sterile phosphate-buffer saline (PBS) of LM-2 control vector and ANKRD1-knockdown cell lines (containing GFP) were orthotopically injected into the mammary fat pad of 6–8-week-old immunodeficient NOD/SCID mice (n = 10 per each group), which were purchased from the Jackson laboratory.

Techniques: Expressing, Knockdown, Plasmid Preparation, Over Expression, Negative Control

Journal: Cancers

Article Title: ANKRD1 Promotes Breast Cancer Metastasis by Activating NF- κ B-MAGE-A6 Pathway

doi: 10.3390/cancers16193306

Figure Lengend Snippet: A proposed pro-metastatic mechanism of ANKRD1 in breast cancer cells. ANKRD1 promotes activation of NF-κB, which upregulates MAGE-A6 expression. Increased expression of MAGE-A6 causes an increase in cell migration and invasion. Parts of the figure are adapted from Servier Medical Art, licensed by Servier under a Creative Commons Attribution 3.0 Unported License ( https://creativecommons.org/licenses/by/3.0/ ).

Article Snippet: For orthotopic injection, 1 × 10 6 cells per 100 μL in sterile phosphate-buffer saline (PBS) of LM-2 control vector and ANKRD1-knockdown cell lines (containing GFP) were orthotopically injected into the mammary fat pad of 6–8-week-old immunodeficient NOD/SCID mice (n = 10 per each group), which were purchased from the Jackson laboratory.

Techniques: Activation Assay, Expressing, Migration