Steponeplus real time system

Total RNA was extracted from HCa-1 cells using TRIzol reagent (Life technologies, Carlsbad, CA). One μg of total RNA was reverse transcribed to cDNA using Omniscript RT Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Real-time PCR was performed on the Step One Plus-Real Time System (Applied Biosystems, Tokyo, Japan) using the Power SYBR Green Master Mix (Applied Biosystems). The primers were as follows: mouse PD-L1;5’-AAA TCG TGG TCC CCA AGC-3’ and 5’-TCC TCA TGT TTT GGG AAC TAT CT-3’, mouse INF-γ; 5’-CCT AGC TCT GAG ACA ATG AAC GCT-3’ and 5’-TGC CAG TTC CTC CAG ATA TCC AAG-3’, mouse TNF-α;5’-AAA ATT CGA GTG ACA AGC CTG TAG-3’ and 5’-CCC TTG AAG AGA ACC TGG GAG TAG-3’, mouse STAT1; 5’- TGA GAT GTC CCG GAT AGT GG-3’ and 5’-CGC CAG AGA GAA ATT CGT GT-3’, mouse STAT3; 5’-GTC TGC AGA GTT CAA GCA CCT-3’ and 5’-TCC TCA GTC ACG ATC AAG GAG-3’, mouse IRF-1; 5’-GGA CTC AGC AGC TCT ACC CTA CCT-3’ and 5’-GCT GGA GTT ATG TCC CTT TCC ATA TC-3’, and mouse GAPDH; 5’-CGA CTT CAA CAG CAA CTC CCA CTC TTC C-3’ and 5’-TGG GTG GTC CAG GGT TTC TTA CTC CTT-3’. The relative levels of mRNA were determined by normalization of the data to the expression levels of GAPDH mRNA.

Candidates of the upregulated genes associated with the glutathione synthesis pathway, phospholipid synthesis pathway, cell-wall synthesis pathway, pentose phosphate pathway, and protein refolding were selected to validate the transcriptome data by using quantitative real-time PCR (RT-qPCR). The list of the genes and primers is displayed in Table 2. Amplification and detection were performed by using a StepOnePlus™ Real-Time System (Applied Biosystems, Foster City, CA, USA).
The reaction mixtures consisted of 5 μL of SYBR Premix Ex Taq II (Takara Bio., Kyoto, Japan), 1 μL of each primer (50 μM), 1 μL of prepared cDNA, and 2 µL of nuclease-free water, which made up a total volume of 10 μL.
Forty cycles were run for the PCR program after primary denaturation at 95 °C for 10 s. The conditions were as follows: 95 °C for 10 s, 55 °C for 10 s, and 72 °C for 10s. The β-actin gene, ACT1, was used as an internal gene-expression control. The whole experiment was repeated three times. The gene-expression level of the non-treated group was defined as 1.0, and the expression level in the HPCD-treatment group (4.0 MPa, 4 h) was evaluated.

The total RNAs were isolated from MEF-TFEB-EGFP cells treated with indicated chemicals for indicated time using a QIAzol lysis reagent (QIAGEN, Hilden, Germany). The cDNAs were synthesized with a high-capacity cDNA RT kit (Applied Biosystems, Waltham, CA, USA) for quantitative polymerase chain reaction (qPCR). The qPCRs were carried out with a TOPreal™ qPCR 2X PreMIX (SYBR Green with High ROX) (RT501M, Enzynomics, Daejeon, Korea) using a StepOnePlus Real Time System (Applied Biosystems, Waltham, CA, USA). The specificity of each primer pair was confirmed using the melting curve analysis. The copy number relative to β-actin mRNA was calculated as previously described [92 (link)]. Primer sequences are presented in Supplemental Table S1.

Quantitative RT-PCR (qRT-PCR) was performed as previously described (Watanabe et al., 2022 (link)). Total RNA was isolated from the cells using ISOGENⅡ (Nippon Gene, Tokyo, Japan), and first-strand cDNA was synthesized using PrimeScript RT Master Mix (Takara Bio, Otsu, Japan). qRT-PCR was performed using the Luna Universal qPCR Master Mix (NEW ENGLAND BioLabs Inc.) on a StepOnePlus Real-Time System (Applied Biosystems, Carlsbad, CA, United States) under the following conditions: 95°C for 1 min, 40 cycles of 95°C for 15 s, and 60°C for 30 s. Primer sequences used in this study are listed in Supplementary Table S1.

qRT-PCR was performed as previously described^{16 (link)}. Total RNA was isolated from cells using ISOGEN II (Nippon Gene, Tokyo, Japan), and first-strand cDNA was synthesized using PrimeScript RT Master Mix (Takara Bio, Otsu, Japan). qRT-PCR was performed using the KAPA SYBR® FAST qPCR Kit Master Mix (Kapa Biosystems, Woburn, MA, USA) on a StepOnePlus™ Real-Time System (Applied Biosystems, Carlsbad, CA, USA) under the following conditions: 95 °C for 3 min, 40 cycles of 95 °C for 3 s, and 60 °C for 30 s. To correct varying copies of first-strand cDNA templates, a passive reference dye (Rox) was added to the PCR master mix. The results were recorded and analyzed using StepOne™ software V2.2.2 (Applied Biosystems, Life Technologies Corporation) utilizing the auto-calculated threshold cycle. The ΔΔC_T method was used to calculate the relative expression levels of the individual genes, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Primer sequences used in this study are listed in Supplementary Table 2.

The gingiva of the right (lig-) and left (lig+) maxillary tissues were harvested and promptly immersed in ISOGEN II for total RNA extraction. cDNA was synthesized using the PrimeScript RT Master Mix (Takara Bio, Otsu, Japan). Quantitative reverse transcriptional polymerase chain reaction (qRT-PCR) was performed using Luna Universal qPCR Master Mix (New England BioLabs, Ipswich, MA, USA) on a StepOne Plus™ Real-Time System (Applied Biosystems, Carlsbad, CA, USA) under the following conditions: 95 °C for 3 min, 40 cycles of 95 °C for 3 s, and 60 °C for 30 s. A passive reference dye (Rox) was added to the PCR master mix to correct for varying copies of first-strand cDNA templates. The results were recorded and analyzed using StepOne™ software V2.2.2 (Applied Biosystems) utilizing an auto-calculated threshold cycle. The ΔΔCT method was used to calculate the relative expression levels of the individual genes, and 18s rRNA was used as an internal control. Primer sequences used in this study are listed in Supplementary Table S2.