Protein Kinase C

The study was approved by the local ethics committee (Bayerische Landesärztekammer, Munich) and subjects gave written, informed consent. Monocytes were enriched from whole blood by negative selection using the Rosette Sep® monocyte enrichment cocktail (STEMCELL Technologies) according to the manufacturer's instructions. Briefly, blood from healthy donors was collected in Falcon tubes containing EDTA at 2 mM final concentration and incubated with enrichment antibody cocktail (50 µl per ml of whole blood) at room temperature for 20 minutes. Cells were then separated by density gradient using Ficoll-Paque™ PLUS (GE Healthcare). Platelets present in the enriched monocyte fraction were discarded by 3 washing steps in PBS, 2% FBS. Finally, monocytes were seeded in either XVivo 10 (Cambrex) or RPMI 10% FBS, 4 mM L-Glutamine with Pen/Strep at a concentration of 5×10⁵ cells/ml in 12-well tissue culture treated plates for 6 days in the presence of either 100 ng/ml rHuGM-CSF (M1) or 100 ng/ml rHuM-CSF (M2). For M2a and M2c polarization, monocytes were incubated in XVivo 10 with rHuM-CSF and 10 ng/ml rHuIL-4 or 10 ng/ml rHuIL-10, respectively. For M1 activation, monocytes were first incubated with rHuM-CSF or rHuGM-CSF for 3 days followed by stimulation with 10 ng/ml LPS and 50 ng/ml rHuIFN-γ for 3 additional days. As indicated by the manufacturer, XVivo 10 media contains human albumin, recombinant human insulin, and human transferrin. It does not contain any exogenous growth factors, artificial stimulators of cellular proliferation, undefined supplements, or protein kinase C stimulators.
Changes in cell morphology were assessed by phase contrast microscopy (Axiovert 135, Zeiss). Phenotypical and functional characterization of MDM was performed after 6 days.

Antifungal tolerance and resistance were determined in flat bottom, 96-well microtiter plates (Sarstedt) using a modified broth microdilution protocol as described [25] (link), [27] (link). Dimethyl sulfoxide (DMSO, Sigma Aldrich Co.) was the solvent for fenpropimorph (FN, Sigma Aldrich Co) and terbinafine (TB, Sigma Aldrich Co.); fluconazole (FL, Sequoia Research Products) and micafungin (MF, generously provided by Julia R. Köhler) were dissolved in sterile ddH₂O. Geldanamycin (GdA, A.G. Scientific, Inc.) was used to inhibit Hsp90 at the indicated concentrations. Cyclosporin A (CsA, Calbiochem) was used to inhibit calcineurin at the indicated concentrations. Cercosporamide and staurosporine (STS, A.G. Scientific, Inc.) were used to inhibit protein kinase C at the indicated concentrations. DMSO was the solvent for GdA, CsA, STS, and cercosporamide.
Minimum inhibitory concentration (MIC) tests were set up in a total volume of 0.2 ml/well with 2-fold dilutions of FL, FN, TB and cercosporamide. FL gradients were from 256 µg/ml down to 0 with the following concentration steps in µg/ml: 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25. FN gradients were from 25 µg/ml down to 0 with the following concentration steps in µg/ml: 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625, 0.1953125, 0.09765625, 0.04882813, 0.02441406. TB gradients were from 250 µg/ml with the following concentration steps in µg/ml: 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.90625, 1.953125, 0.9765625, 0.48828125, 0.24414063. Cercosporamide gradients were from 100 µg/ml with the following concentration steps in µg/ml: 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625, 0.1953125, 0.09765625. Cell densities of overnight cultures were determined and dilutions were prepared such that ∼10³ cells were inoculated into each well. Plates were incubated in the dark at 30°C or 35°C for the period of time indicated in the figure legend, at which point plates were sealed with tape and re-suspended by agitation. Absorbance was determined at 600 nm using a spectrophotometer (Molecular Devices) and corrected for background from the corresponding medium. Each strain was tested in duplicate on at least 3 occasions. MIC data was quantitatively displayed with color using the program Java TreeView 1.1.1 (http://jtreeview.sourceforge.net).
Checkerboard assays were set up in a total volume of 0.2 ml/well with 2-fold dilutions of cyclosporin A across the x-axis of the plate and 2-fold dilutions of STS across the y-axis of the plate. STS gradients were from 0.5 µg/ml to 0 in the following concentrations steps in µg/ml: 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0.0078125. CsA gradients were from 48 µg/ml down to 0 in the following concentration steps in µM: 48, 24, 12, 6, 3, 1.5, 0.75, 0.375, 0.1875, 0.09375, 0.046875. Plates were inoculated and growth was measured as with MIC tests. To test for synergy, the fractional inhibitory concentration (FIC) was calculated as follows: [(MIC₈₀ of drug A in combination)/(MIC₈₀ of drug A alone)] + [(MIC₈₀ of drug B in combination)/(MIC₈₀ of drug B alone)]. Values of ≤0.5 indicate synergy, those of >0.5 but <2 indicate no interaction and those ≥2 indicate antagonism.

First, following prediction of the protein-coding gene set for H. contortus, each inferred amino acid sequence was assessed for conserved protein domains using InterProScan [114 (link),115 (link)], employing default settings. Second, amino acid sequences were subjected to BLASTp (e-value ≤10^-5) against the following protein databases: C. elegans in WormBase [116 (link)]; Swiss-Prot and TrEMBL within UniProtKB [117 (link)]; Kinase SARfari [118 (link)] and the protein kinase database for C. elegans [119 (link)], which contains all domain information for C. elegans kinases [120 (link)]; GPCR SARfari [118 (link)]; Transporter Classification Database [121 (link),122 (link)]; KEGG [123 (link),124 (link)]; LGICs [125 (link)]; ChEMBL [126 (link)]; NCBI protein nr [127 (link)]; and an in-house RNAi machinery database for nematodes. Finally, the BLASTp results were used to infer key protein groups, including peptidases, kinases, phosphatases, GTPases, GPCRs, channel and transporter proteins, TFs, major sperm proteins, vitellogenins, SCP/TAPS proteins, and RNAi machinery proteins.
Each coding gene was assessed against the known KEGG Orthology (KO) term BLAST hits. These BLAST hits were clustered to a known protein family using the KEGG-BRITE hierarchy in a custom script. ES proteins were predicted using SignalP (version 4.0) [128 (link)] and TMHMM (version 2.0c) [122 (link),129 (link),130 (link)] and by BLASTp homology searching of the validated Signal Peptide Database [131 (link)] and of an ES database containing published proteomic data for A. suum [14 (link)], B. malayi [15 (link)]. C. elegans [116 (link)], and T. spiralis [16 (link)]. In the final annotation, proteins inferred from genes were classified based on a homology match (e-value cut-off, ≤10^-5) to: (i) a curated, specialist protein database, followed by (ii) the KEGG database, followed by (iii) the Swiss-Prot database, followed by (iv) the annotated gene set for a model organism, including C. elegans, followed by (v) a recognized, conserved protein domain based on InterProScan analysis. Any inferred proteins lacking a match (e-value cut-off, ≤10^-5) in at least one of these analyses were designated hypothetical proteins. The final annotated protein-coding gene set for H. contortus is available for download at WormBase [116 (link)] in nucleotide and amino acid formats.

In vitro stimulation of purified blood neutrophils was performed based on a previously published method for bovine neutrophils with minor changes [20 (link),23 (link)]. Isolated neutrophils were suspended in HBSS at a final concentration of 1 × 10⁷ cells/mL. For the in vitro priming of neutrophils, 1 × 10⁶ cells in 100 µL medium were incubated for 30 min at 37 °C and 5% CO2 with 1 μg/mL of the TLR4-ligand LPS, 1 μg/mL of the TLR2/1-ligand Pam3CSK4, 0.2 µg/mL of the TLR7/8-ligand R848 (Resiquimod), 10 µg/mL of the TLR3-ligand Poly IC, or 10 ng/mL of the protein kinase C activator phorbol 12-myristate 13-acetate (PMA), or only HBSS medium without stimulants. The concentrations of the stimulants were chosen based on previously described concentrations for the stimulation of bovine cells [20 (link)].

The total protein content was obtained from the colon tissues of the C57BL/6 mice by applying Pro-Prep Protein Extraction Solution (Intron Biotechnology Inc., Seongnam, Republic of Korea) in accordance with the manufacturer’s protocol. After centrifugation at 13,000 rpm/min for 5 min, the protein concentrations were determined using a SMART™ Bicinchoninic Acid Protein Assay Kit (Thermo Fisher Scientific Inc.). Proteins were separated by 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 2 h and then transferred to nitrocellulose membranes at 40 V for 2 h. Membranes were then incubated at 4 °C with the following primary antibodies overnight: anti-p38 (Cell Signaling Technology Inc., Cambridge, MA, USA), anti-p-p38 (Cell Signaling Technology Inc.), anti-Protein kinase C (PKC) (Cell Signaling Technology Inc.), anti-phospho-PKC (p-PKC) (Cell Signaling Technology Inc.), anti- Akt serine/threonine kinase (AKT) (Cell Signaling Technology), anti-p-AKT (Cell Signaling Technology), anti-Extracellular signal-regulated kinase (ERK) (Cell Signaling Technology Inc.), anti-p-ERK (Cell Signaling Technology Inc.), anti-PI3K (Cell Signaling Technology), anti-p-PI3K (Cell Signaling Technology), anti- c-Jun N-terminal kinases (JNK) (Cell Signaling Technology Inc.), anti-p-JNK (Cell Signaling Technology Inc.), or anti- Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (Cell Signaling Technology Inc.). The membranes were then washed with washing buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 0.05% Tween 20) and incubated with 1:2000 diluted horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Invitrogen) at room temperature for 1 h. Blots were developed using Amersham ECL Select Western Blotting detection reagent (GE Healthcare, Little Chalfont, UK). Chemiluminescence signals from specific bands were detected using FluorChemi^®FC2 (Alpha Innotech Co., San Leandro, CA, USA).

Phorbol 12-myristate 13-acetate (PMA), a protein kinase C inducer, induced cell differentiation into macrophages.THP-1 cells were cultured in 1640 medium containing 10% FBS, 1% penicillin-streptomycin, and 0.05 mM β-mercaptoethanol (Sigma, St. Louis, MO, USA), and then passed on to the third generation. After 1 mg PMA (Sigma, St. Louis, USA) was dissolved in DMSO, it was divided and diluted to a final concentration of 100 nM. THP-1 cells were cultured for 48 h. Morphological changes in cells were observed under a microscope.