Triton x 100

The chemicals used in the experiments were purchased from the following suppliers: Fetal calf serum (FCS), trypsin–EDTA, RPMI 1640, penicillin-streptomycin, L-glutamine from Biological Industries (Kibbutz Beit-Haemek, Israel); low melting point agarose (LMA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye, methylene blue dye, and normal melting point agarose (NMA) from Boehringer Mannheim (Mannheim, Germany); acetic acid, acetone, n-butanol, n-butanon, EA, formic acid, n-hekzan, chloroform (CHCl₃), methanol (MEOH), sodium chloride (NaCl), sodium hydroxide (NaOH), sulphuric acid, toluen from Merck Chemicals (Darmstadt, Germany); dimethyl sulfoxide (DMSO), ethanol, ethidium bromide (EtBr), methanol, phosphate buffered saline (PBS) from Sigma (St. Louis, MO, USA); ethylenediamine tetra acetic acid disodium salt dihydrate (Na₂-EDTA), N-lauroyl sarcosinate, tris, and Triton X-100 from ICN Biomedicals Inc. (Aurora, Ohio, USA). Water (H₂O) was supplied from in a Milli-Q (Millipore, Bedford, MA, USA) water purification system.

A 1 M stock emulsion of n-Pelargonic acid, 97% (Pelargonic acid, Acros Organics, New Jersey, USA) in water was prepared using TritonX-100, (ICN Biomedicals, Inc., Aurora, OH), Tween 80 (Fisher Chemical, Fair Lawn, NJ), Sodium Dodecyl Sulfide (SDS, Fisher Chemical), Sodium lauroyl sarcosinate (Sarkosyl, Fisher Chemical) and saponin from quillaja bark (Sigma-Aldrich, St. Louis, MO) at concentrations of 1%, 0.1% and 0.01% (w/v). Stock emulsions (1 M) were prepared by adding 1.58 g of PA to 10 ml of water with 1%, 0.1%, and 0.01% (w/v) of surfactant. The mixture was agitated on a vortexer (Vortex-2 Genie, Scientific Industries, Bohemia, NY, USA) at maximum speed setting of 10 (3200 rpm) for 1 min to form the emulsion.

Bovine serum albumin (BSA) was purchased from Sigma-Aldrich (St. Louis, MO, USA), heat inactivated fetal bovine serum (FBS) from Invitrogen (Carlsbad, CA, USA), and RPMI-1640, Dulbecco’s phosphate buffered saline (DPBS), l-glutamine, trypsin-versene and Hank’s buffered salt solution (HBSS) from Lonza, (Braine-l’Alleud, Belgium). Human recombinant IL-1α ELISA kit was purchased from R&D systems, Minneapolis, MN, USA, CytoTox 96®. The non-radioactive cytotoxicity assay, CellTiter-Glo luminescent cell viability, Cell proliferation ELISA and BrDU chemiluninescent were supplied by Roche (Mannheim, Germany), and Caspase-3/7 assay by Promega (Madison, WI, USA). BDH Chemical Ltd. (Poole, England) supplied Triton-x100 and ICN Biomedicals Inc (Santa Ana, CA, USA) supplied Tween®-20.

Transfected or infected cells (as specified in figure legends) seeded in 96 wells-plates were fixed with PBS containing 4% paraformaldehyde for 5 min at room temperature. Cells were then washed with PBS and permeabilized with PBS-0.1% Triton X-100 (ICN Biomedicals Inc.) for 5 min at room temperature. Cells were then blocked with TNB blocking reagent (Perkin Elmer) for 1 h at room temperature. Primary antibodies, diluted in TNB reagent, were incubated for 1 h at room temperature at the following dilutions: anti-PKR (rabbit, 18244-1-AP, Proteintech) 1:400, anti-eif3η (mouse, sc-137214 Santa Cruz) 1:800. Cells were then washed three times for 5 min in PBS-0.1% Tween 20 and incubated with species-matched secondary antibodies (Alexa Fluor 488- and 697-conjugated antibodies, Molecular Probes) 1:800 for 1 h at room temperature. Cells were washed three times for 5 min in PBS-0.2% Tween 20 and maintained in PBS-azide 0.02% until analysis. Fluorescence analysis was performed with a spinning disk confocal microscope (Zeiss, Germany). Image acquisition and processing (intensity, contrast and pseudocolours) were done with the Zen image acquisition sofware (Zeiss, Germany) using the same parameters across micrographs.

The actin ring formation assay was performed as described previously [29 (link)]. Briefly, BMMs cultured with M-CSF, RANKL and various concentrations of NaPPS for 7 days were washed with PBS and fixed with 4% paraformaldehyde (Wako pure chemical, Tokyo, Japan) in PBS on ice for 20 min. Osteoclasts were detergent-permeabilized with 0.2% Triton X-100 (ICN Biomedicals, Germany) in PBS for 10 min, washed and blocked in 10% normal goat serum (Sigma-Aldrich, St Louis, Missouri, USA) in PBS for 1 h. The cells were incubated with primary rabbit anti-F actin polyclonal antibody (Bioss Inc., Massachusetts, USA) (1:100 dilution) for 1 h in PBS with 1% normal goat serum, washing three times with PBS, incubating for 1 h with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit antibody (Sigma) (1:100 dilution) in PBS with 1% normal goat serum, washing three times with PBS, and finally mounting with aqueous mounting medium. The images were observed by counting the number of actin rings under a laser scanning confocal microscope (Zeiss, Illinois, USA).

Fetal bovine serum (FBS), Trypsin, cytokeratin, hyaloronidase, normal goat serum, and collagenase were purchased from Sigma- Aldrich (St Louis, MO, USA). PLZF (promyelocytic leukemia zinc finger), mouse CDH1 (cadherin-1), mouse Oct-4, and secondary FITC goat anti-mouse and goat anti-rabbit antibodies were supplied from R&D Systems, Calbiochem (San Diego, CA, USA), Chemicon (UK), and Raybiotech (Tehran, Iran), respectively. Triton X-100 was purchased from ICN Biomedicals and Dulbecco’s Modified Eagle Medium (DMEM) was the product of Gibco-BRL, (Grand Island,NY, USA).