Blebbistatin b0560

(−) blebbistatin (B0560), Y-27632 dihydrochloride (Y0503), Staurosporine (S3939, IC₅₀ 20 nM)43 (link)44 (link), Chelerythrine (C2932, IC₅₀ 1.77 μM)23 (link), G_Ö6976 (G1171)24 (link)45 (link), DMSO vehicle, protease inhibitors cocktail (P8340) and phosphatase inhibitor I and II cocktails (P2850 and P5726), neuraminidase (N2876), 4′, 6-diamidino-2 phenylindole (DAPI, D8417) Kodak-developer (P5670) and fixer (P6557), were purchased from Sigma-Aldrich. Octadecyl Rhodamine B Chloride (R18, O-246) and (+) blebbistatin (203392) were purchased from Life Technologies (Carlsbad, CA, USA) and EMD4 Biosciences, respectively.

Samples were incubated with drugs at day 3 for MDCK cells and day 6 for J3B1A cells (immediately after the cell monolayer formation) for observations over time. To maximize inhibition of cell proliferation and minimize drug toxicity, cells were incubated for 1 h with 15 µM mitomycin-C (M4287; Sigma-Aldrich), then washed out with warm medium, and 6 h later, incubated for a further 1 h with 15 µm mitomycin-C. To inhibit myosin activity, 20 µM blebbistatin (B0560; Sigma-Aldrich) in DMSO was added and kept in the growth media until day 7 (MDCK cells) or day 9 (J3B1A cells). Cells were incubated in DMSO for the same amount of time as control.

Rabbit monoclonal anti-Rab5 antibody (3547) from Cell Signaling Technology (Danvers, MA, USA) was used at 1∶200 dilution for immunocytochemistry. Goat polyclonal anti-Syntenin antibody (ab53552) and rabbit polyclonal anti-calnexin antibody (ab137336) from abcam were used at 1∶100 dilution for immunocytochemistry. Rabbit polyclonal anti-GFP antibody (ab6556) from abcam was used at 1∶1,000 dilution for immunocytochemistry. Transferrin, Alexa Fluor 647 conjugate (T-23366) was purchased from Invitrogen. (−)-Blebbistatin (B0560) was purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA) and used at 100 µM. ML141 (71203-35-5) and NSC23766 (sc-204823) were purchased from R&D Systems (Minneapolis, MN, USA) and Santa Cruz Biotechnology (Dallas, TX, USA), respectively. AllStars negative control siRNA (1027281) was purchased from Qiagen (Hilden, Germany), and Silencer Cy3 Labeled GAPDH siRNA (AM4649) was purchased from invitrogen. Dynasore (D7693), 5-(N-ethyl-N-isopropyl) amiloride (A3085), Y27632 (Y0503), bafilomycin A1 (B1793) and W56 (W4392) were purchased from Sigma. siRNAs against ARF1, ARF6, ANKFY1 and CtBP were purchased from Santa Cruz Biotechnology and used at 12.5 nM final concentration with lipofectamine RNAiMAX (invitrogen).