Calphostin c

Unless indicated, all reagents and solutions were as previously described40 (link)43 (link)77 (link)78 (link). Fluo-4 and DAF-FM were from Invitrogen (Grand Island, NY). Aflatoxins B₁ (AFB₁) and B₂ (AFB₂) were from Cayman (Ann Arbor, MI). Gö6983, BIIE-0246, [Leu³¹,Pro³⁴]-NPY, NPY-(16–36), antagonist G, and calphostin C were from Tocris (Minneapolis, MN USA). All other reagents were from Sigma-Aldrich (St. Louis, MO USA). Stock solutions of aflatoxin were 10 mM in DMSO. Working solutions (10, 1, and 0.1 μM) contained 0.1%, 0.01%, and 0.001% DMSO, respectively, and were made up immediately before use; activities of aqueous solutions were observed to be markedly reduced after ~1–2 hr at room temp. Anti-aflatoxin antibodies (recognizing AFB₁, AFB₂, and AFG) were from Thermo Scientific (MA-7386) and Sigma Aldrich (A9555).

Cell culture and experiments with UMR106 rat osteoblast-like cells (purchased from ATCC, Manassas, VA, USA) were conducted as described before [39 (link)]. Briefly, cells were cultured in DMEM high-glucose medium containing 10% FBS and penicillin-streptomycin at 37°C and 5% CO₂.
IDG-SW3 bone cells (purchased from Kerafast, Boston, MA, USA) were cultured as described earlier [40 (link)]. Briefly, non-differentiated cells were kept at 33°C in AlphaMEM medium (with L-glutamine and deoxyribonucleosides) containing 10% FBS, penicillin-streptomycin and interferon-gamma (INF-γ; 50 U/ml). For differentiation, cells were plated on collagen-coated dishes at 37°C in medium with 50 μg/ml ascorbic acid and 4 mM β-glycerophosphate but without INF-γ. All reagents were from ThermoFisher unless indicated.
IDG-SW3 osteocytes were used after 35 days of differentiation, and UMR106 cells were pretreated with 100 nM 1,25(OH)₂D₃ (Tocris, Wiesbaden-Nordenstadt, Germany) for 24h before the experiment. Cells were then incubated with activator phorbol ester 12-O-tetradecanoylphorbol-13-acetate (PMA; Sigma, Schnelldorf, Germany; 0.1 μM; 6 h) with or without 1 μM PKC inhibitors calphostin C (Tocris), Gö6976 (Tocris), sotrastaurin (Selleckchem, München, Germany), ruboxistaurin (Selleckchem), or NFκB inhibitor withaferin A (Tocris; 0.5 μM), or with vehicle only for another 24 h.

PDGF-BB was provided by Peprotech (Hamburg, Germany). Imatinib mesylate, amlodipine, fasudile, calphostin C, SC51322, PF04418948, L798106 and L161982 were purchased from Tocris Bioscience (Ellisville, Missouri, USA). Ponatinib was acquired from SelleckChem (Munich, Germany). SQ29548, RO-1138452, SU6668, PD98059 and U0126 were acquired from Cayman Europe (via Biomol, Hamburg, Germany). ET-1 was purchased from Biotrends (Wangen, Switzerland). Cytochalasin D or standard laboratory chemicals were provided by Sigma (Steinheim, Germany).

Chinese hamster ovary-derived recombinant rat IFN-β protein (U-CyTech, Utrecht, Netherlands) was dissolved in sterile double-distilled water to a concentration of 10⁵ IU and stored at –20°C. The final concentration was 1,000 IU IFN-β ml^-1, as this was previously shown to effectively increase suprathreshold responses [5 (link)] and is assumed to occur during viral infections [17 (link),18 (link)]. PKC activators 4β-phorbol 12-myristate 13-acetate (4β-PMA) or Bryostatin1 and PKC inhibitors GF109203X (also known as BisI or Gö6850) or calphostin C (all Tocris Bioscience, Bristol, UK) were dissolved in 99.8% Dimethyl sufoxide (Sigma-Aldrich, Steinheim, Germany) to stock concentrations of 10 mM (4β-PMA, Bryostatin1, GF109203X) or 1 mM (calphostin C) and stored at –20°C. The final concentrations in artificial cerebrospinal fluid (ACSF, for content see patch clamp recordings) were 1 μM (4β-PMA, Bryostatin1, GF109203X, for all: 10 μl to 100 ml ACSF) and 100 nM (calphostin C, 10 μl to 100 ml ACSF). In all cases the final Dimethyl sulfoxide percentage accounted for 0.01%.

Fluoxetine HCl (FLX, 100 µM, LKT Laboratories, St. Paul, MN, USA), escitalopram oxalate (ESC, 100 µM, BioTrend, Miramar Beach, FL, USA), ω-agatoxin TK (AgaTx, 0.4 µM, Tocris, Bristol, UK), BAPTA-AM (10 µM, Tocris, Bristol, UK), phorbol 12,13-dibutyrate (PMA, 200 nM, Tocris, Bristol, UK), and calphostin C (1 µM, Tocris, Bristol, UK) were used. In all experiments, cells were treated with FLX or ESC for 90 min, and other drugs were added 20 min before FLX application.