Anti n cadherin

The following primary antibodies and dilutions were used: mouse anti-Elavl4 (anti-HuD; Santa Cruz Biotechnology (SCB), 1:1,000, sc-28299), rabbit anti-Pax6 (Biolegend previously Covance, 1:250, 901301 previously PRB-278P), rabbit anti-Tbr2 (Abcam, 1:250, ab23345), rabbit anti-Cdp (SCB, 1:250, sc13024), rat anti-Bcl11b (SCB, 1:250, sc-98514), mouse anti-Celf1 (SCB, 1:100, sc20003), rat anti-Ctip2 (Abcam, 1:250, ab18465), mouse anti-Tle4 (SCB, 1:250, sc-365406), chicken anti-GFP (Aves, 1:1000, GFP-1020), mouse anti-Gapdh (Millipore Sigma, 1:2000, MAB374), mouse anti-Satb2 (Abcam, 1:250, ab51502), rabbit anti-Nestin (Sigma Aldrich, 1:100, N5413), chicken anti-N-Cadherin (Takara, 1:250, M110 Clone NCD-2), rabbit anti-NeuN (Millipore Sigma, 1:500, ABN78), rabbit anti-pH3 (Millipore Sigma, 1:1000, 06-570), rat anti-Brdu/Cldu (Novus, NB500-169), mouse anti-Brdu/IdU (BD Biosciences, 1:100, B44), and rabbit anti-Renilla (Thermo Fisher, 1:1000, PA1-180). Appropriate species-specific Donkey secondary antibodies were used at a 1:250 dilution and were obtained from Jackson ImmunoResearch.

BM-MSCs were seeded on cover slips coated with type 1P collagen (1 × 10⁴ cells/cm²) and cultured in the culture medium for 24 h. Then, the cells were starved with serum-free DMEM supplemented with 1% l-glutamine and 1% P/S for another 18 h. The cells were then treated with PC3 CM or CON CM for 24 h, and subsequently fixed with 4% PFA. After fixation, immunocytochemistry was performed according to standard protocols [32 (link)]. The following primary antibodies were used: Anti-N-cadherin (1:30; Invitrogen), Anti-N-cadherin (1:100; Takara, Shiga, Japan), anti-β-catenin (1:100; Bethyl Laboratories, Montgomery, TX, USA), and anti-α-catenin (1:100; BD Biosciences, San Jose, CA, USA). Actin was stained with Alexa Fluor 633-tagged phalloidin (1:1000; Invitrogen). Nuclei were stained using DAPI for 10 min. Images were taken using a Zeiss LSM 700 confocal microscope (Carl Zeiss) at a 400× magnification. The number and the length of N-cadherin-positive structures at the borders between BM-MSCs and the width of N-cadherin-positive borders between BM-MSCs were measured (Figure S1) using Adobe Photoshop CS6 (Adobe Systems Incorporated, San Jose, CA, USA).

Cells grown on glass coverslips were fixed with 4% paraformaldehyde for 10 minutes, and treated with 0.05% Triton-X for 5 min. For immunostaining, fixed cells were incubated with blocking buffer (3% BSA in 1× PBS) for 1 h and then stained with primary antibodies for 1 h at 37 °C. Antibodies used included: anti-GRHL2 (HPA004820, Sigma-Aldrich); anti-E-cadherin (610182, BD); anti-N-cadherin (M142, Takara); anti-pan-Cytokeratin (M3515) and anti-Vimentin (M7020) from Dako; anti-β-Catenin (8480), anti-phospho-Myosin Light Chain 2 (Ser19) (3671), anti-LC3A (4599), anti-EEA1 (3288), anti-LAMP1 (9091), anti-RCAS1 (12290) from Cell Signaling Technology. Alexa Fluor 488-conjugated anti-mouse/rabbit (A11029/A11034) and Alexa Fluor 594-conjugated anti-mouse/rabbit (A11032/A11037) from Invitrogen were used as secondary antibodies. Rhodamine phalloidin probe (R415, Life Technologies) was used for F-actin staining. Cover slips were mounted onto glass slides using Vectashield mounting medium with/without DAPI (H-1200, H-1000) from Vector Laboratories. Images were taken using a Nikon A1R confocal system or a Zeiss AxioImager M2 epifluorescence imaging system.