Hs02786624 g1

KCs (2 × 10⁴ cells/well) and HDF cells (2 × 10⁴ cells/well) were seeded into 12-well plates and treated with 0.05% H.ECM™ liposome or positive control (1 μM retinoic acid (Sigma-Aldrich) or 10 ng/mL recombinant epidermal growth factor (EGF; R&D systems, Minneapolis, MN, USA) when the cells reached around 80% confluence. Then, the UVB-irradiated human skin specimens were homogenized using TissueLyser Ⅱ (Qiagen, Hilden, Germany). Total RNA was extracted using the RNAiso Plus (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. Total RNA was quantified using a NanoDrop 2000 spectrophotometer (ThermoFisher) and reverse transcribed using an RNA to cDNA EcoDry Premix Kit (Takara Sake, Berkley, CA, USA). The synthesized cDNA, Taqman Gene Expression Master Mix (Applied Biosystems), and Taqman primer of each target (HAS3; Hs00193436_m1, AQP3; Hs00185020_m1, COL1A1: Hs00164004_m1, MMP1: Hs00899658_m1; Filaggrin; Hs00856927_g1, Loricrin; Hs01894962_s1, Applied Biosystems) were used for the qRT-PCR experiments. The expression level of each gene was normalized to that of the housekeeping gene GAPDH (Hs02786624_g1, Applied Biosystems) and calculated using the 2^−ΔΔCt method.

Total RNA was extracted from TPC1, FTC133 and BCPAP thyroid cancer cells lines, using TRIzol reagent (Invitrogen, Waltham, MA, USA) and purified using an RNeasy Mini Kit (Qiagen, Venlo, The Netherlands). For gene expression, 500–1000 ng of total RNA was reverse transcribed using a High Capacity Reverse Transcription cDNA kit (cat no. 4374967; Applied Biosystems, Waltham, MA, USA), and the resulting cDNA was diluted and amplified with specific primers, according to the manufacturer’s instructions, using a real-time polymerase chain reaction (RT-PCR) system (Quant Studio 5; Applied Biosystems, Waltham, MA, USA). Gene expression levels were normalized using the GAPDH gene expression as an endogenous control. Results obtained were analyzed using the ΔΔCt method with SDS software (Applied Biosystems, Waltham, MA, USA).
The taqman probes for detecting gene expression of human NOP53 (Hs00414236_m1, Catalog # 4448892) and internal control GAPDH (Hs02786624_g1, Catalog #4331182) were purchased from Thermo Fisher (Waltham, MA, USA). The amplicon length for NOP53 was 118 bp and it spanned the exon boundary 1–2. The sequence of the probe is proprietary and would not be released.

Cryopreserved differentiated HepaRG™ cells (HPRGC10, ThermoFisher Scientific, Waltham, MA, USA) were seeded according to the manufacturer’s protocol and cells were treated by studied compounds after 72 h of stabilization (control (DMSO 0.1%), CITCO 10 µM, diazepam 30 and 50 µM, nordazepam, temazepam, and oxazepam 50 µM, respectively) for 48 h. Total RNA was isolated using TRIZOL^® and reverse transcription was performed with the RevertAid RT Reverse Transcription Kit. qPCR was performed using TaqMan™ Fast Advanced Master Mix with TaqMan probes for CYP2B6 (Hs04183483_g1), GAPDH (Hs02786624_g1), and B2M (Hs00187842_m1) genes (all reagents for qRT-PCR were obtained from Thermo Fischer Scientific Catalog number: 4331182, Waltham, MA, USA). The delta-delta method was used for gene expression quantification normalized to GAPDH and B2M gene expression average. Three technical replicates were used for each reaction.

Total RNA was isolated from cell lines using RNeasy Mini kit (QIAGEN, Hilden, Germany). 1ug of RNA was reverse transcribed using High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). Quantitative PCR was performed using a CFX96 Real-Time system (Bio-Rad, Hercules, CA) and Taqman gene expression assays for Cd274, Pdcd1lg2, Gata2 and Gata3 (Hs00204257_m1, Hs00228839_m1, Hs00231069_m1, Hs00231119_m1, Hs00231122_m1, respectively Thermo Fisher Scientific). GAPDH was the endogenous control (Hs02786624_g1, Thermo Fisher Scientific).

Cellular senescence was analyzed by quantitative RT-PCR (qRT-PCR) to measure and compare the gene expression of the cell senescence markers CDKN1A (Hs00355782_m, Taqman, ThermoFisher), CDKN2A (Hs00923894_m1, Taqman, ThermoFisher), and CDKN2B (Hs00793225_m1, Taqman, ThermoFisher) of TCs incubated with conditioned media from either tumor-free or tumor-bearing tissues. Transcriptional levels were normalized with internal controls GAPDH (Hs02786624_g1, Taqman, ThermoFisher) and 18S (Hs99999901_s1, Taqman, ThermoFisher). A minimum of four biological replicates per assay were analyzed. n = 4 with triplicates per condition.

RT-qPCR was performed using Taqman probes as described [46 (link)]. Analyses were performed using the delta-delta-Ct method with relative gene expression first normalized to GAPDH mRNA and then expressed relative to the experimental controls. Primers for human PDIA3 (Hs04194196_g1) and GAPDH (Hs02786624_g1) or mouse PDIA3 (Mm00433130_m1) and GAPDH (Mm99999915_g1) were from ThermoFisher and designed to cross exon borders to specifically target cDNA.

cDNAs were synthesized with 1 μg of total RNA using FastGene Scriptase Basic cDNA Synthesis (NE‐LS62, NIPPON Genetics, Tokyo, Japan). qRT‐PCR was performed with TaqMan^® Fast Advanced Master Mix (444556, Thermo Fisher Scientific) on a QuantStudio™ 6 Pro (A43180, Thermo Fisher Scientific). Signals were normalized against Gusb (β‐glucuronidase). Predesigned TaqMan Gene Expression Assays, including primer sets and TaqMan probes (Gusb; Mm01197698_m1, Nqo1; Mm01253561_m1, Ugdh; Mm00447643_m1 and Gstm1; Mm00833915_g1, GAPDH; Hs02786624_g1, NQO1; Hs00168547_m1, UGDH; Hs01097550_m1, GCLC; Hs00155249_m1, SOD1; Hs00533490_m1, SQSTM1; Hs00177654_m1) were purchased from Thermo Fisher Scientific.