Protease inhibitor tablet

Single colonies were used to inoculate 50 mL of LB medium supplemented with the corresponding antibiotic. Those overnight cultures were used as starting material to inoculate large volume media (500 mL). All CYPs were grown on TB media supplemented with 15 μM FeCl₃ at 37 °C until the optical density at 600 nm (OD₆₀₀) reached 0.8–1 at which point 500 μM δ-aminolevulinic and 50 mM arabinose were added to support protein expression. After induction, cells were grown for 20–24 hours at 25 °C (CYP101A1 and CYP102A1) or 20 °C (CYP153A6) and collected by centrifugation at 4 °C. Cell pellets were resuspended (200 mg·mL⁻¹) in 50 mM potassium phosphate buffer pH 7, 200 mM NaCl and 10% glycerol. Cells were lysed using a constant cell disruptor system (Constant Systems Ltd, UK). Protease inhibitor tablets (Sigma) were added to the cell lysates to avoid enzymatic degradation of over-expressed proteins. Cell lysates were immediately used for biotransformation or kept frozen at −80 °C. Heme iron thiolate coordination was established by formation of the Fe(II)CO complex at 450 nm after dithionite-reduction and bubbling with carbon monoxide gas. CYPs concentrations were calculated using the extinction coefficient ε₄₅₀ = 96,000, as described previously^{39 (link)}.

Protein was extracted from untreated PDCLs using cell lysis buffer (10 mM Tris–Cl pH 7.4, 100 mM NaCl, 1 mM EDTA pH 8.0, 1 mM NaF, 20 mM Na₄P₂O₇, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 10% Glycerol, Milli-Q water) and complete, mini, EDTA-free protease inhibitor tablets (Sigma Aldrich). Western blots were probed with antibodies against Mre11 (Mouse monoclonal [12D7], 1:500) and Rad50 (Mouse monoclonal [13B3/2C6], 1:500). To control for protein loading, membranes were probed with EIF4E (Rabbit monoclonal [C46H6], 1:1000).

For infection 3 × 10⁶L. major promastigotes were injected intradermally in 10 μl of PBS into the ear of 8- to 12-week-old female C57BL/6 mice. The course of infection was determined by measuring the ear swelling using photo analysis with Fiji software (ImageJ) three times a week. When ear swelling started to develop, the animals were treated three times a week with the compound Eh-1 at concentrations of 5, 10, and 25 μg in 10 μl of DPBS topically applied to the ear.
After reduction of the ear swelling, the mice were sacrificed, and ear tissue, as well as lymph node tissue, was used for histological staining and gDNA isolation. Blood serum was taken every 2 weeks p.i. The cytokine profile of the serum, as well as that of the tissue lysates, was analyzed using a LEGENDplex assay kit (BioLegend). Lysates of the infected ears were obtained by mincing with zirconia beads (2 mm; Carl Roth GmbH) and incubated on ice with 200-μl protease inhibitor tablets (Sigma-Aldrich). Samples were then mixed in a TissueLyser for 10 min at 50 ms and then centrifuged at 4°C at maximum speed. The supernatant was used for cytokine analysis.

Cells were scraped from all samples, including
TCPS, and lysed with ice-cold lysis buffer (RIPA buffer 1× containing
1 mM and 1× protease inhibitor (Protease Inhibitor Tablets, SIGMA)
for 30 min on ice. The lysates were then used for Western blot analysis
according to a literature protocol.^{22 (link)} Primary
antibodies anti-vinculin (diluted 1:250), anti-β₁-integrin (diluted 1:1000), anti-FAK (diluted 1:1000), anti-phosphorylated
FAK (pY397) (diluted 1:1000), anti-β-actin (diluted 1:500),
and appropriate secondary HRP-conjugated antibodies were used. Detection
was performed with Western Chemiluminescent HRP substrate, (LI-COR)
and revealed using an ImageQuant LAS4000 imaging system (GE Healthcare).
Band densitometry analysis was carried out with ImageJ software.

Cells were scraped from all the samples, including T, and lysed with ice-cold lysis buffer (1X RIPA buffer containing 1 mM and 1X protease inhibitor (Protease Inhibitor Tablets, SIGMA) for 30 min on ice. The lysates were then used for western blot analysis according literature protocol (Cristofaro et al., 2018 (link)). Primary antibodies anti-phosphorylated AKT and anti-AKT (diluted 1:500), anti-ERK and anti-phosphorylated ERK (diluted 1:1000), anti-BAX and anti-BCL-XL (diluted 1:500), anti-β-actin (diluted 1:500) and appropriate secondary antibodies HRP-conjugated were used. Detection was performed as described in the dot-blot section. Bands densitometry analysis was carried out with Image J software.