Phalloidin ifluor 488 ab176753

Cells were fixed for 10 min in 4% paraformaldehyde in phosphate-buffered saline (PBS) and were rinsed three times for 5 min each in PBS. The cell membranes were permeabilized for 10 min in a 0.5% Triton X-100 in PBS, followed by three rinsing steps. Before immunostaining, the cells were blocked in 3% bovine serum albumin in PBS solution for 1 h. Vimentin was stained overnight with an Alexa Fluor 647 tagged human anti-vimentin V9 antibody (sc-6260; Santa Cruz) at 0.2 μg/mL in 3% bovine serum albumin in PBS. Actin was stained with fluorescently labeled phalloidin (phalloidin-iFluor 488; ab176753; Abcam) at a dilution of 1:1000, accordingly to the manufacturer protocol. After staining, the cells were rinsed three times in PBS and once in MilliQ water, and then they were mounted on glass slides using Fluoromount-G (Thermo Fisher Scientific), which contained DAPI to counterstain the cell nuclei.

The fetal bovine serum (FBS) and α-MEM were provided by Gibco (California, USA). M-CSF and RANKLwere purchased from R&D (MN, USA). The tartrate-resistant acid phosphatase (TRAP) staining kit was purchased from Sigma-Aldrich (St Louis, MO, USA). HiPerFect Transfection Reagent was purchased from Qiagen (GmbH, Hilden, Germany). SC-79 was obtained from MedChemExpress (New Jersey, USA). Trap5b and CTX-I Elisa kit were purchased from Elabscience (Wuhan, China). Phalloidin-iFluor 488 (ab176753) was purchased from Abcam (Cambridge, UK). Antibodies for NFATc1 (#64,602) was from Biolegend (San Diego, CA). S6 (#2217), phospho-S6 (#4858), p70S6K (#9202), phospho-p70S6K (#9204), Akt (#4685), phospho-Akt (#31957), mTOR (#2983) and Rock2 (#47012) antibodies were from Cell Signaling Technology (Danvers, MA, USA). RhoA (ab187027) and GAPDH (ab8245) antibodies were from Abcam (Cambridge, UK). Flag (AF0036) antibody was from Beyotime Biotechnology (Shanghai, China).