Hs00174103 m1

The T84 cells were incubated with HDL 50–200 μg/mL for 18 hours and then with tumor necrosis factor (TNF) 25 ng/mL for 3 hours. Total RNA was isolated using TRIzol reagent (Invitrogen). We reverse transcribed 1–2 μg RNA with a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Rotkreuz, Switzerland) before real-time polymerase chain reaction (RT-PCR) analysis (7900HT; Applied Biosystems) using various TaqMan assays: Hs00174128_m1 (human TNF), Mm00443258_m1 (murine TNF), Hs00174103_m1 (human interleukin-8 [IL-8]), Hs00164932_m1 (human intracellular adhesion molecule [ICAM]), Mm00516023_m1 (murine ICAM), Hs00222677_m1 (human β-actin), 4352341E_mACTB (murine β-actin), Hs00797944_s1 (LC3), Hs00250530_m1 (ATG16L1), and endogenous controls for human and animals (Applied Biosystems). Constitutively expressed β-actin was measured as an internal standard for normalization. Relative mRNA levels were calculated using the comparative threshold cycle method. For each experiment, all tests were performed in triplicate. The mRNA levels obtained in control conditions were set to 1, and the results are shown relative to those.

To examine IL-6 mRNA expression, human BSMCs were incubated for 5 hours in 0.1% BSA-DMEM containing 300 μM Ni, 300 μM Co, or the control vehicle. To investigate the effect of DEX on IL-6 mRNA expression, DEX (100 nM) or the control vehicle was added to the medium 30 minutes before replacing the medium containing Ni, Co, and DEX or control vehicles. Total RNA was isolated from BSMCs in 6-well plates using the RNeasy^Ⓡ Plus Mini Kit (Qiagen, Hilden, Germany) or Direct-zol^TM RNA MiniPrep (Zymo Research, Irvine, CA) according to the manufacturer’s instructions. Up to 2.5 g of total RNA was reverse-transcribed using random priming and reverse transcriptase (SuperScript^TM VILO^TM Master Mix, Thermo Fisher Scientific, Waltham, MA). To evaluate expression levels of OGR1, IL-6, IL-8, and GAPDH mRNA, quantitative RT-PCR was performed using real-time TaqMan technology with a StepOne^TM Real-Time PCR system (Thermo Fisher Scientific). TaqMan probes specific for human OGR1 (Hs00268858_s1), IL-6 (Hs00174131_m1), IL-8 (Hs00174103_m1), and GAPDH (Hs02758991_g1) were purchased from Thermo Fisher Scientific. The expression levels of IL-6, IL-8, and OGR1 mRNA were normalized to GAPDH mRNA expression levels, as previously reported.8 (link)

RNA was isolated from GC monolayers, GC spheroids and gingiva ex vivo tissue 24‐hr after seeding onto biomaterials or plastic using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). Control samples without cells were included. RNA quality was verified photometrically by the 260/280 ratio.
Undiluted RNA was transformed into cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer's protocol.
Quantitative polymerase chain reaction (qPCR) was performed to analyze changes in COL1A1 (Hs00164004_m1; Thermo Fisher Scientific), VEGF (Hs00900055_m1; Thermo Fisher Scientific), ANG (Hs04195574_sH; Thermo Fisher Scientific), IL6 (Hs00985639_m1; Thermo Fisher Scientific) and IL8 (Hs00174103_m1; Thermo Fisher Scientific) at mRNA levels. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH; Hs02758991_g1; Thermo Fisher Scientific) was used as reference gene. Results of qPCR were evaluated by the ∆∆CT method and normalized to the respective plastic control.