Isospin cell and tissue rna

To isolate total cellular RNA from the cultured cells, we used the High Pure RNA Isolation Kit (Roche Diagnostics K. K., Tokyo, Japan) and the ISOSPIN cell and tissue RNA (NIPPON Gene, Tokyo, Japan). We performed quantitative real-time polymerase chain reaction (PCR) analysis with iQ™ SYBR^® Green Supermix (catalog no. 170-8885; BioRad, Hercules, CA, USA) using the ViiA™7 Real-Time PCR System (Thermo Fisher Scientific). The following primers were used: Ager (catalog no. Mm_Ager_1_SG, QuantiTect Primer Assay; Qiagen, Hilden, Germany), HMGB1 (catalog no. Mm00849805_gH, TaqMan Gen Expression Assay; Applied Biosystems, Foster City, CA, USA), Tnfa (catalog no. Mm_TNF_1_SG, QuantiTect Primer Assay; Qiagen), and Actb (catalog no. 4352341E, Mouse ACTB Endogenous Control; Applied Biosystems). Data were analyzed according to the delta-delta Ct method (Iwata et al., 2017 (link)). Semiquantitative PCR for full-length RAGE, esRAGE, and β-actin were performed. The PCR products were analyzed by electrophoresis on 2% agarose in 1× TAE buffer (100 V, 20 min). Data were analyzed using the ImageJ software (https://imagej.nih.gov/ij/).

Total RNA was extracted using ISOSPIN Cell and Tissue RNA (NIPPON GENE, Tokyo, Japan) according to the manufacturer’s protocol. Total RNA purity and quantity were evaluated using a Nano Drop ND-1000 spectrophotometer (Thermo Fisher Scientific). The extracted total RNA was analyzed on 96-well plates on a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Tokyo, Japan) using 10 ng/well as the template for qRT-PCR. The reactions were performed using the One Step TB Green Prime Script PLUS PT-PCR Kit (Takara Bio, Shiga, Japan) and gene-specific primers for human ezrin, radixin, moesin, PD-L1, and β-actin (all purchased from Takara Bio) at a final concentration of 0.4 µM. The reaction program comprised a reverse transcription reaction step at 42 °C for 5 min and amplification steps at 95 °C for 10 s, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing at 60 °C for 30 s. The relative mRNA levels of the target genes normalized to those of the β-actin gene, used as an internal reference amplified from the same sample, were computed with the comparative quantification cycle (Cq) method (2^-ΔΔCq) using Bio-Rad CFX Manager software version 3.1 (Bio-Rad Laboratories). The sequences of gene-specific PCR primers are shown in Table 1.

RNA extraction was performed using ISOSPIN Cell and Tissue RNA (NIPPON GENE, Toyama, Japan) according to the manufacturer’s instruction. For tissue homogenization, FastPrep-24 (MP Biomedicals) was used. Reverse transcription from RNA to cDNA was performed by PrimeScript RT Master Mix (Takara-bio, Shiga, Japan).