Rnaase inhibitor

hPTC transduced with pRetroX-G1-Red (mCherry-G1) retrovirus were trypsinized (Euroclone) at passage 2 after transduction. The cells were then fixed with PFA 0.25%, 0.5% saponin (Merck) with the addition of 1:25 RNAase inhibitor (Promega, N2615). Then anti-DsRed (1:25, Clontech, 632496) or isotype control was incubated for 1 h at RT followed by 1 h incubation with secondary antibody Alexa Fluor 647 goat anti-rabbit (1:100, Thermo Fisher Scientific, A-21245) to detect mCherry⁺ hPTC. All the antibodies were diluted in 0,5% saponin (Merck) with the addition of 1:100 RNAase inhibitor (Applied Biosystems, N8080119). All the solutions were diluted in RNAase-free PBS prepared with DEPC water (Merck). The procedure was carried out on ice. Finally, hPTC were incubated with DAPI (1:1000, Thermo Fisher Scientific) to perform the DNA content analysis and sorted on the FACSAria III BD (Bioscience). Alexa Fluor 647 secondary antibody was excited by a 633 nm laser line, DAPI was excited by a 405 nm laser line. Data were analysed by FacsDiva software (Beckman Coulter).

After total RNA extraction of liver tissue (ReliaPrepTM RNA Tissue, Promega), cDNA was synthesised by extending a mix of random primers with the High Capacity cDNA Reverse Transcription Kit in the presence of RNAase Inhibitor (Applied Biosystems). The relative quantity of each transcript was normalized to the expression of EEF1, HPRT and GAPDH. SYBRGreen reagent was used for Real-time PCR on the ABI Prism 7000 sequence detection system (Applied Biosystems) according to the manufacturer’s instructions. Primer sequences are provided in S1 Table.

siRNA was designed according to Caplen et al. [30 (link)] with freely available software from Eurofins Genomics. The EGFP siRNA (sense: 5’-GCAAGCUGACCCUGAAGUUCA, antisense: 5’-UGAACUUCAGGGUCAGCUUGC) was directed against EGFP mRNA. As a control for the specificity of the siRNA construct, we used scramble (SCR) siRNA (sense: 5’-TTGATCGTTTGCTACGCTTTACTTC, antisense: 5’-UUGAUCGUUUGCUACGCUUUACUUC) (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA), which contains the changed nucleotide siRNA sequence for mouse endoglin and shows no significant homology to mouse transcripts according to Basic Local Alignment Search Tool analysis. siRNAs were prepared at a final concentration of 20 µM in 40 µL of siMAX universal buffer (Eurofins Genomics) containing 40 U of RNAase inhibitor (Applied Biosystems by Life Technologies, Thermo Fisher Scientific).

After total RNA extraction of liver tissue (ReliaPrepTM RNA Tissue, Promega, Madison WI, USA), cDNA was synthesized by extending a mix of random primers with the High-Capacity cDNA Reverse Transcription Kit in the presence of an RNAase inhibitor (Applied Biosystems). The relative quantity of each transcript was normalized to the expression of Eef1, Hprt, and Gapdh. SYBRGreen reagent was used for real-time quantitative polymerase chain reaction (qPCR) on the ABI Prism 7000 sequence detection system (Applied Biosystems) according to the manufacturer’s instructions. Primer sequences are provided in Supplementary Table S3.

The RF/6A cells were seeded in 6 well plates at a density of 1 million cells per well. The following day, the cells were treated with oxLDL and Etifoxine or XBD173 in media containing 0.1% DMSO at the same time for 24 h. The choice of the 24 h window to detect gene expression is based our previous studies [22 (link)] and a recent publication, which demonstrated that gene expression changes at 24 h in PK11195-treated cells were functionally related to tumorigenesis and apoptosis [68 (link)]. Total RNA was extracted using Tri Reagent (Sigma, UK) following the manufacturer’s instructions. The first-strand cDNA was synthesised applying the High Capacity cDNA Reverse Transcription Kits and with the RNAase inhibitor (Applied Biosystems, Birchwood, UK). The mRNA levels of the genes were then quantified using qRT-PCR assay employing a Platinum Syber Green QAPCR Super Mix-UDG w/ROX kit (Invitrogen, Inchinnam, UK). A 2^−ΔΔCt formula was applied to quantify the relative expression of the genes which were normalised to a housekeeping gene (β-actin). The sequences of these primers used for gene expression are shown in Table 1.

Total RNA from FFPE samples (7 sections, each 5 µm thick) was isolated with Deparaffinization Solution (Qiagen) and the RNeasy FFPE kit, which includes a DNase treatment step (Qiagen). Total RNA from cell cultures was isolated using the NucleoSpin^® RNA extraction kit (Machery-Nagel) or TRIzol reagent (Invitrogen). RNA quality and quantity were determined spectrophotometrically with the NanoDrop™ 2000c (Thermo Fisher Scientific). For cDNA synthesis, the High Capacity cDNA Reverse Transcription kit (Applied Biosystems) and the RNAase inhibitor (Applied Biosystems) were used. qPCR was performed with a CFX384 qPCR thermocycler (BioRad) using SYBR^® Green PCR Master Mix (Applied Biosystems). A standard protocol for SYBR^® Green was used with 10 min at 95°C, 39 cycles of 15 sec at 97°C and 1 min at 60°C, followed by 15 sec at 60°C and a final melting step until 95°C. The oligonucleotide primers used in this study are listed in Supplementary Table 2. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as reference gene. qPCR data are reported as –ΔCt in Figure 1C and as relative expression ratio in all other figures. The relative expression ratio was calculated according to Pfaffl’s method (equation 1) (30 (link)). As control group for the calculation of the relative expression ratio we used cells harvested at time point 0 h.