Ruboxistaurin

The alpha mouse liver 12 (AML12) cell line, which was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), was cultured in DMEM: F12 (1:1) containing 10% (v/v) fetal bovine serum as well as insulin (10 μg/ml), transferrin (5.5 μg/ml), selenium (5 ng/ml), and dexamethasone (40 ng/ml). The cells were maintained in a humidified incubator at 5% CO₂ and 37 °C.
Small interfering RNAs (siRNAs) targeting p66Shc or circ-CBFB, pcDNA-p66Shc plasmid, Ago-185, Ant-185 or negative controls (si-control, pcDNA 3.1, Ago-NC and Ant-NC) were transfected into AML12 cells for 48 h using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). MG132 (Selleck, USA) (10 μm) was added to AML12 cells for 24 h. Mito-TEMPO (Sigma, USA) (100 μM) was added to the AML12 cells for 3 h. Ruboxistaurin (Selleck, USA) (200 nM) was added to the AML12 cells for 48 h. The siRNA sequences targeting p66Shc were sense 5′-GCUGCAUCCCAACGACAAATT-3′, antisense 3′-UUUGUCGUUGGGAUGCAGCTT-5′. The ago-185 sequences were sense 5′-UGGAGAGAAAGGCAGUUCCUGA-3′, antisense 3′-AGGAACUGCCUUUCUCUCCAUU-5′. The ant-185 sequence was sense 5′-UCAGGAACUGCCUUUCUCUCCA-3′. The siRNA sequences targeting circ-CBFB were sense 5′-GGCAGUAACUGGCUUUUGUTT-3′, antisense 3′-ACAAAAGCCAGUUACUGCCTT-5′ and sense 5′-AACUGGCUUUUGUGGCUACTT-3′, antisense 3′-GUAGCCACAAAAGCCAGUUTT-5′. AML12 cells were then treated with APAP (5 mM) for 24 h.

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.

Neutrophils were resuspended in RPMI 1640 (without phenol red) and 10 mM HEPES (Thermo Fisher, Waltham, MA, USA), and seeded (5×10⁴) in quadruplicate in a Nunc™ MicroWell™ 96-well flat bottom plate (Thermo Fisher). Cells were pre-incubated with either 200 nM ruboxistaurin (Selleckchem, Houston, TX, USA), 10 µM diphenyleneiodonium (DPI) (Cayman Chemical, Ann Arbor, MI, USA) or 10 µM dexamethasone (Sigma-Aldrich, St Louis, MO, USA) for 1 h (37°C, 5% CO₂) before stimulation with either LPS (5 µg·mL⁻¹) (Escherichia coli O111:B4) (Sigma-Aldrich), phorbol myristate acetate (PMA) (100 nM) (Sigma-Aldrich) or DMSO control. Neutrophils were incubated for a further 3 h before adding SYTOX™ Green nucleic acid stain (555 nM) (Thermo Fisher) to all wells, to measure extracellular DNA as a surrogate of NET formation. Extracellular DNA was quantified using a fluorescent plate reader (excitation/emission 490/537 nM) and median fluorescence values were reported.