ankrd1-knockdown cell lines (containing gfp)  (Jackson Laboratory)

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