Ab20680

EBs were obtained by culturing 1,500 iPSCs in 20 μl hanging drops in ES medium without LIF for 2 days, before seeding on gelatin-coated coverslips in 24-well plates.
After 5 days of culture, an immunofluorescence was performed on part of the cells using antibodies against Alphafetoprotein (1:50, AFP, R&D Systems, MAB1368) and Brachyury (1:100, Abcam, ab20680). The remaining cells were treated with retinoic acid (PeproTech) to induce ectoderm differentiation. After 19 days of culture, immunofluorescence was performed using an antibody against Nestin (1:100, Abcam, ab11306). To further validate mesoderm differentiation, EBs were seeded at day 5 of differentiation and checked for cardiomyocyte beating activity starting from day 7.

At 72 h following transfection, U-CH2 cells were lysed in ice-cold radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). The protein concentration was estimated using a bicinchoninic acid protein assay (Beyotime Institute of Biotechnology). A total of 50 µg protein was separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat dry milk in 0.1% Tween-20 at 4°C for 1 h, then incubated with primary antibodies against brachyury (1:1,000; ab20680; Abcam, Cambridge, MA, USA) or β-actin (1:2,000; sc-130656; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) overnight at 4°C. Membranes were washed with TBS and incubated with alkaline phosphatase-conjugated goat anti-rabbit secondary antibody (1:3,000; BA1011; Wuhan Boster Biological Technology, Ltd., Wuhan, China). Respective bands from these blots were observed using the BCIP/NBT Color Development Substrate (Promega Corporation). The experiments were repeated three times. The mean densities of the bands were analyzed using Quantity One 4.6.2 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and normalized to that of β-actin.

Culture plates were washed with phosphate-buffered saline (PBS) solution and placed on ice. Total breast cancer cell proteins were extracted using mammalian protein extraction reagent (Thermo Fisher Scientific, Waltham, MA), containing a protease inhibitor cocktail (Thermo Fisher Scientific). Equal amounts of protein were electrophoresed using 10% sodium dodecyl sulfate-gel electrophoresis and transferred on to polyvinylidene fluoride membranes (Millipore, Billerica, MA). The membranes were blocked using Tris-buffered saline and 0.1% Tween 20, containing 5% non-fat dry milk for 1 h, and then incubated overnight with primary antibodies. The membranes were washed three times and incubated with the secondary antibodies (Multi-sciences Biotechnology, Zhejiang, China). The membranes were then visualized using a chemiluminescence (Multi-sciences Biotechnology) detection system. The primary antibodies were used as follows: anti-Bry (Abcam, ab20680, 1:2000), anti-SOX5 (Abcam, ab94396, 1:1000), anti-E-cadherin (Abcam, ab1416, 1:50), anti-N-cadherin (Abcam, ab18203, 1:1000), snail family transcriptional repressor 1 (anti-Snai1; Abcam, ab53519, 1:1000), anti-Vimentin (Abcam, ab8978, 1:500), epithelial cell adhesion molecule (anti-EpCAM; Abcam, ab213500, 1:1000), anti-Fibronectin (Abcam, ab32419, 1:1000) and anti-β-actin (Multi-sciences Biotechnology, 70-ab008-100, 1:2000).