Fig. 6: AIB1 interactome as a regulator of pro-EMT phenotype.

A Western blot analysis of MTA2, B-ACTIN, and EMT markers N-cadherin, E-cadherin, Vimentin, Snail and Slug. LetR cells were transfected with either siNT-1, siNT-2, siMTA2-1, or siMTA2-2 and whole cell lysate harvested. 30µg of protein was loaded per gel. Western blot gel images are cropped at the band of interest for clarity. B Light-phase microscopy images of LetR cells cellular characteristics in the presence of two distinct siRNAs against AIB1 and MTA2. Cells were steroid and serum-starved and then subsequently seeded in presence of serum. Representative images of an n = 3 biological replicates taken 48 h after seeding (×20 magnification, scale bars represent 100 μm). C, D LetR cells were depleted of AIB1 or MTA2 using a two independent siRNAs. Cellomics Cell Motility Kit was used to assess individual cell movement in collagen in a 96-well plate after 24 h. Representative images are shown as seen on the microscope after cells were fixed and stained (×20 magnification, scale bars represent 20 μm). All functional experiments were carried out under estrogen deprived conditions. Image of treatment sensitive cell line MCF7 is shown for comparison. Histogram shows mean migratory area per cell (μm.²). All results in the figure are mean ± S.E.M., n = 3 (biologically independent replicates) and two-sided t tests were used to calculate P values (*P < 0.05; **P < 0.001, ***P < 0.0001). E Representative images of E-Cadherin (E-Cad), AIB1, pAIB1Thr24, and MTA2 protein immunohistochemistry (IHC) in PDX tumor T638- PDX (left panel) and T347-PDX (right panel). All scale bars, 50 μm. F Scatter plot of IHC scores (0–300) calculated in T638-PDX and T347-PDX (n = 3 mice per PDX model; At least 500 cells were assessed in each case). G Correlation plot comparing IHC scores in T638-PDX and T347-PDX, assessing AIB1vCDH1, pAIB1vCDH1, and MTA2vCDH1. H Western blot analysis of E-cadherin (E-Cad) protein expression (from whole cell lysate) in the T347x primary culture cells transfected with either empty pcDNA3.1 plasmid vector (Control) or pcDNA3.1 plasmid containing full-length AIB1 (AIB1_OVX). Gel images display AIB1, E-Cadherin (E-Cad) and B-Actin and are representative of three biologically independent replicates.