T300 thermocycler

For the intramolecular oxidation
of hLF peptides, peptides were dissolved to 5 mM in 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES; Cat No. H4034, Sigma-Aldrich, St. Louis, MO, USA) buffer,
at pH 8.0. For intermolecular disulfide bond formation, peptides were
dissolved in 50 mM HEPES at pH 9.0 at a final peptide concentration
of 50 mM. After dissolution, the pH was adjusted with 0.1 M NaOH.
In either case, 30 μL of peptide solution was added to polypropylene
tubes (Greiner Bio-One, Kremsmünster, Austria, Cat No. 673210)
and incubated in a T300 thermocycler (Biometra, Göttingen,
Germany) for 2 h at 37 °C for the formation of intramolecular
disulfide bonds (mono-hLF), and for 24 h at 37 °C for the formation
of intermolecular disulfide bonds (poly hLF). Samples were diluted
in 20 mM citrate buffer at pH 5.0 or MQ water to stabilize the formed
disulfide bonds.

Total RNA was extracted with the Universal RNA Purification Kit (Roboklon, Berlin, Germany) and reverse transcribed. In total, 20 μl reactions with 50 ng of RNA, 1× RT Buffer, 1 U RNase inhibitor, 10 mM MgCl₂, random hexamers, 250 μM pooled dNTPs and 2.5 U MuLV reverse transcriptase (all products from Applied Biosystems, Foster City, CA) were prepared. The RT reaction was carried out in a T300 thermocycler (Biometra, Göttingen, Germany) for 15 min at 42 °C, with 5 min at 95 °C for deactivation of the enzyme and final cooling to 4 °C. qRT-PCR was performed in duplicate with the GoTaq qPCR Master Mix (Promega) in a final volume of 10 μl in a LightCycler 480 (Roche, Basel, Switzerland). PCR protocol included a pre-incubation step at 95 °C for 2 min, followed by 45 cycles of incubation at 95 °C for 7 s, annealing at 60 °C for 10 s and elongation at 72 °C for 5 s. In the melting curve, the temperature was increased from 65 °C to 95 °C (0.1 °C/s). Data were analysed with the LightCycler 480 Software release 1.5.0 SP3 (Roche). MACC1 or S100P expression was normalised to the expression of GAPDH. The following gene-specific primer were used: MACC1_fow 5’-ttcttttgattcctccggtga-3’; MACC1_rev 5’-actctgatgggcatgtgctg-3’; S100P_fow 5’-aatctagcaccatgacgg-3’; S100P_rev 5’-tctgcaggaagcctggta-3’; GAPDH_fow 5’-gaagatggtgatgggatttc-3’; GAPDH_rev 5’-gaaggtgaaggtcggagt-3’.

Total cellular RNA was extracted using Trizol reagent according to the procedure provided by the manufacturer. 1 µg of isolated RNA was subsequently used for cDNA synthesis with oligo(dT) primers in a 20 µl reaction mixture containing M-MLV reverse transcriptase. The cDNA templates were used to quantify gene expression level using Maxima SYBR Green Master Mix in the following conditions: 15 min at 95°C followed by 40 cycles at 95°C for 15 s, 60°C for 30 s and 72°C for 30 s. PCR reactions were performed in an AbiPrism 7000 sequence detection system (Applied Biosciences). For each PCR amplicon, a melting curve was run. The relative fold change after normalization to Gapdh expression was calculated using a comparative 2^−ΔΔCt method [27] (link).
Conventional PCR used to estimate the efficiency of mitoSypHer transfection was carried out using Paq5000 polymerase in the following conditions: 5 min at 95°C followed by 30 cycles at 95°C for 1 min, 50°C for 1 min, 72°C for 2 min with a final extension at 72°C for 10 min in T300 thermocycler (Biometra) using cDNA obtained as described above. PCR products after staining with ethidium bromide were analyzed under UV light in GelDocEQ system (Bio-Rad). The primers used in PCR reactions are listed in Table 2.