Staurosporine

iBMDMs (3 × 10⁵ cells/well) were seeded one day prior to experimentation in 12-well plates. Prior to the treatments, media was removed and replaced with fresh DMEM. iBMDMs were treated with DMSO vehicle control, Truli (N-(3-Benzylthiazol-2(3H)-ylidene)−1H-pyrrolo[2,3-b] pyridine-3- carboxamide; CSN pharm CSN26140) or Raptinal (Adipogene, AG-CR1-2902) and incubated in CO₂ humidified 37°C incubators for 3 hours. For staurosporine experiments, iBMDMs were treated with DMSO vehicle control or staurosporine (Cayman chemical, 81590) for 4 hours.

MDCK (NBL-2) cells (CVCL_0422; Cell Lines Services, Eppelheim, Germany) were cultured in Dulbecco’s modified Eagle medium F12 (DMEM/F-12; Gibco, Life Technologies, Darmstadt, Germany) with additional 5% fetal bovine serum (Gibco), 100 U/ml penicillin, 100 μg/ml streptomycin (Gibco), and 2 mM glutamine (Gibco). NRK-52E cells (ECACC 87012902; European Collection of Authenticated Cell Cultures, Salisbury, UK) were cultured in Dulbecco’s modified Eagle medium (DMEM) plus 5% newborn calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco). For experiments, cells were plated at a density of 1.5×10⁵ cells per well (MDCK) or 2×10⁵ cells per well (NRK-52E) in 6-well plates at 5% CO₂ and 37°C. After 24 h, cells were treated with PKC activator phorbol-12-myristate-13-acetate (PMA) (0.01–600 nM as indicated; Peprotech, Hamburg, Germany) and/or PKC inhibitors staurosporine (0.01–1 nM as indicated; Cayman Chemicals, Ann Arbor, MI, USA), 300 nM sotrastaurine (United States Biological, Salem, MA, USA), or 40 nM Gö6976 (Biomol, Hamburg, Germany) for further 24 h. Control cells were incubated with the respective amount of solvent dimethyl sulfoxide (DMSO; AppliChem, Darmstadt, Germany) for all experiments.