Fig 1: 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/).
Fig 2: 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.
Fig 3: 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.
Fig 4: 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.
Supplier Page from Abcam for Recombinant Human MAGEA6 protein (His tag C-Terminus)