Hs rrn18s 1 sg quantitect primer assay

THP‐1 cells were differentiated, loaded, and treated as described earlier. Total RNA was extracted from differentiated THP‐1 macrophages using the peqGOLD Total RNA Kit (PeqLab), according to the manufacturer's instructions.56 The quantitation of RNA was performed with NanoDrop 2000c (peqlab; Thermo Scientific). A ratio of the absorbance at 260 and 280 nm (A_260/280) close to 2.0 was considered to indicate sufficient RNA quality. cDNA was synthesized with 1 μg total RNA based on the protocol from the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) with RNase Inhibitor (Applied Biosystems). Quantitative reverse transcription polymerase chain reaction was conducted using the LightCycler 480 SYBR Green I Master Kit (Roche) with 40 ng cDNA for each sample. A LightCycler 480 system of Roche was used for detection of the amplification cycles. Primers used for quantitative reverse transcription polymerase chain reaction were specific for ABCA1 (HS_ABCA1_1_SG QuantiTect primer assay, catalog no. QT00064869; Qiagen), ATP‐binding cassette transporter G1 (ABCG1; Hs_ABCG1_1_SG QuantiTect Primer Assay, catalog no. QT00021035; Qiagen), and 18S (Hs_RRN18S_1_SG QuantiTect Primer assay, catalog no. QT00199367; Qiagen). Relative quantification of ABCA1 and ABCG1 gene expression was performed with the ΔC_T method, using 18S as endogenous control.

THP-1 cells were seeded in 6-well plates and differentiated into macrophages with a 200 nM PMA treatment for 72 h. The macrophages were treated with the indicated compounds at the indicated time points, which are described in detail in the figure legends. Total cellular RNA was extracted from the treated THP-1 macrophages using peqGOLD Total RNA kits (PeqLab, Linz, Austria) according to the manufacturer's instructions. One microgram of total RNA was used for cDNA synthesis according to the protocol of the High Capacity cDNA Reverse Transcription Kit together with RNase Inhibitor (Applied Biosystems). LightCycler® 480 SYBR Green I Master kit (Roche) was used for quantitative real-time PCR (qRT-PCR) with 40 ng cDNA from each sample for triplicate measurements. Amplification cycles were detected using the LightCycler 480 system (Roche). ABCA1 (HS_ABCA1_1_SG QuantiTect primer assay, Cat.no.: #QT00064869, QIAGEN) and 18S (Hs_RRN18S_1_SG QuantiTect Primer assay, Cat.no.: #QT00199367, QIAGEN) primers were used, and the quantification was performed with the ΔC_T method.

Total RNA from tumor tissue and corresponding healthy mucosa was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer´s protocol. Reverse transcription was performed using an ImProm-II™ Reverse Transcription System (Promega, Mannheim, Germany). qPCR quantification with LightCycler^® 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannheim, Germany) was assessed using five nanograms of cDNA and a Roche Light Cycler^® 480 System (Roche Diagnostics GmbH, Mannheim, Germany). Ready to use primers for granulin (Hs_GRN_1_SG QuantiTect Primer Assay, QT00236635) and 18s (Hs_RRN18s_1_SG Quanti tect Primer Assay), both from Qiagen, Hilden, Germany, were acquired. Negative controls were included according to the manufacturer’s instructions using each primer, nuclease-free water and master mix.