Sybr green based system

Total RNA was isolated from cells by using TRIzol® (Life Technologies) according to the manufacturer’s protocol. After reverse-transcription with qScript™ cDNA SuperMix (Quanta Biosciences), quantitative PCR assays were performed to analyze mRNA levels by using a SYBR-Green-based system (Applied Biosystems). mRNA levels were normalized to levels of Rpl32. Primer sequences are listed in Additional file 2: Table S2. MicroRNA levels were quantified by using TaqMan™ miRNA Assays (Applied Biosystems) according to the manufacturer’s protocol and normalized to levels of snoRNA-135.

Total RNA was isolated with TriFast (PeqLab). The following primers were used: TPO, forward, 5′-ACC AAC TCC AGT GTC TCA G-3′ and reverse, 5′-TCC TTG TGT CCC GTT CAG-3′ GAPDH, forward,5′-AAG GTC ATC CCA GAG CTG AA-3′ and reverse, 5′-CTG CTT CAC CAC CTT CTT GA-3′. Triplicate PCR reactions were performed using a SYBR green-based system (Applied Biosystems, Waltham, MA, USA), and the expression levels were normalized to those of GAPDH.

Total RNA was extracted from tissues and cell lines using TRIzol Reagent (Thermo), according to the manufacturer’s instructions. Subsequently, RNA samples were sent to Majorbio Technology Co., Ltd. (Shanghai, China) for high-throughput sequencing analysis. For quantitative real-time PCR, cDNA from isolated RNA was reverse-transcribed using the TaKaRa PrimeScript RT Reagent kit (TaKaRa, Japan) and then used for qPCR using SYBR Green-based system (Applied Biosystems) with gene-specific primers, and mRNA levels were normalized to GAPDH as a control. Primer sequences used are listed in Additional file 1: Table S1.

The differentiated osteoblasts and osteoclasts were lysed in a radioimmune assay precipitation buffer (Thermo Fisher Scientific), and western blotting was performed as described previously (Cho et al., 2021 (link)). Rabbit anti-Prdx1 (Invitrogen), rabbit anti-Prdx2 (Ab Frontier), rabbit anti-Prdx3 (Ab Frontier), rabbit anti-Prdx4 (Abcam), mouse anti-Prdx5 (Invitrogen), rabbit anti-Prdx6 (Invitrogen), mouse anti-AR (Santa Cruz), rabbit anti-hnRNPK (CST), rabbit anti-Lamin β (Ab Frontier), and mouse anti-β-Actin (Sigma-Aldrich) antibodies were used to detect proteins.
Nuclear proteins were isolated from osteoblasts at day 7 under BMP2 stimulation using NE-PER Nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific) according to the manufacturer’s instruction.
Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific), and cDNA was synthesized as previously described (Cho et al., 2021 (link)). Quantitative PCR was performed using a SYBR Green-based system (Thermo Fisher Scientific), and data were calculated using the 2^-ΔΔCT method. Three separate experiments were performed. The primers used are listed in Table 4.

Individual total RNA from 5 × 10⁵ tumor cells, 5 × 10⁵ CD8⁺ T cells, or 5 × 10⁶ non-CD8⁺ PBMCs with indicated treatments was isolated. The procedure is described previously [33 (link)]. In brief, cells were collected and lysed in 200 μL of TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) mixed with 80 μL of 1-bromo-3-chloropropane. After 13,000 rpm centrifugation for 15 min at 4 °C, RNA remained in 200 μL of the aqueous phase and was transferred to a fresh tube and precipitated by adding 200 μL of isopropanol. The pellet was collected after 13,000 rpm centrifugation for 10 min at 4 °C and washed with 200 μL of 70% ethanol. Furthermore, the pellet was dissolved in RNase-free water after air-drying and the concentration was subsequently detected using a SpectraMax iD3 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA). Consequently, the complementary DNA was synthesized from 1 μg of RNA using a ToolScript MMLV RT Kit (Biotools, New Taipei, ROC) according to the manufacturer’s protocol. qPCR was performed using an SYBR Green-based system (Thermo Fisher Scientific, Waltham, MA, USA). The expression of specific genes was quantified based on a 3-times repeat with normalization to GAPDH. The primer sequences for the reaction are listed in Table S1.