Sybr green mixture

The total RNA of osteosarcoma cells was extracted using the TRIzol reagent (ELK Biotechology, China). Next, cDNA was synthesized from total RNA by using a reverse transcriptase kit (Vazyme, China). The RT-qPCR was performed by SYBR green mixture (Vazyme, China). The 2^−ΔΔCt method was employed to examine the fold change and the GAPDH serve as an internal reference gene. Besides, GAPDH and HSPB6 primers are listed in Table 1.Table 1

The primer sequences

Primer name	Sequence
GAPDH-Forward	5′-GGAGCGAGATCCCTCCAAAAT-3′
GAPDH-Reverse	5′-GCTGTTGTCATACTTCTCATGG-3′
HSPB6-Foward	5′-ACGCTCGCCCCCTACTACCT-3′
HSPB6-Reverse	5′-CGACGAATCCGTGCTCATCC-3′

Following manufacturers' instructions, RNA was isolated from Cochlea‐Orgs using RNeasy Mini Kit (Qiagen, no. 74 104), and reverse transcription was performed by using RevertAid First Strand cDNA Synthesis Kit (ThermoFisher, no. K1622). qPCR analysis was carried out in a 96‐well qPCR machine (Bio‐Rad Laboratories, CFX96) using SYBR Green Mixture (Vazyme, no. Q712‐02). Primers for qPCR were described before^[8, ^14] or checked through Primer‐BLAST and are listed in Table S1, Supporting Information.

qPCR employed an Applied Biosystems qPCR apparatus and SYBR green master mix (Vazyme, Nanjing, China), as per kit protocols. The reaction mixture was composed of 5 µl SYBR Green Mixture (Vazyme, Nanjing, China), 1 µl cDNA template, 0.2 µl primer (each), and 3.6 µl deionised water, and the conditions were set as follows: 95 °C for 5 min, 40 cycles of 95 °C for 10 s, and 60 °C for 30 s. All employed primer sequences were summarized in Table S1. GAPDH (for mRNA and circRNA) and U6 (for miRNA) served as controls for normalization purpose, and all qRT-PCR reactions were performed three times, and the average adjusted value was used for analysis. Relative gene expressions were computed using the 2^−ΔΔCt formula. All data analysis employed the unpaired 2-tailed Student’s t-tests; ^*p < 0.05, ^**p < 0.01, and data are provided as mean ± SD (standard error of mean).

Total RNAs from pig spleen tissues were extracted using Trizol (TaKaRa, Dalian, China). Then, reverse transcription of RNA was conducted using HiScript III RT SuperMix with gDNA wiper (Vazyme, Nanjing, China). The RT-qPCR reaction system contained 5 μl SYBR Green Mixture (Vazyme, Nanjing, China), 1 μl of the cDNA template, 0.2 μl of each primer, and 3.6 μl deionised water. The thermal conditions were as follows: 95°C for 5 min, 40 cycles of 95°C for 10 s, and 60°C for 30 s. The GAPDH genes were set as the internal controls. All the forward and reverse primers for the RT-qPCR assays are listed in Table 1. The expression level of each validated gene for each timepoint was calculated by the 2^−ΔΔCt method.

Total RNA was isolated from mouse liver tissue and cDNA was synthesized using HiScript II Reverse Transcriptase (Vazyme, Nanjing, China). Real-time quantitative polymerase chain reaction (RT-qPCR) analysis was performed using SYBR Green mixture (Vazyme Biotech, Q711). We set actin as the reference gene for each sample. All primers were purchased from Sangon Biotech (Shanghai, China). The primers are given in Supplementary Table 1. For western blot (WB) analysis, total proteins were extracted from the liver samples with lysis buffer (ThermoFisher, 78443) containing 1% PMSF. Proteins (15μg) of each sample were loaded onto a gradient SDS-PAGE. Proteins were then transferred to a PVDF membrane. After blocking with 1% bovine serum albumin (Sigma, A7030), the primary antibodies were used for detection: MAFB (TD8895, 1:2000) (Abmart), CX3CR1 (13885-1-AP, 1:1000) (Proteintech) and Actin (66009-1-Ig, 1. 10000). Tissue levels of MAFB and CX3CR1 were also analyzed by immunohistochemistry (IHC) using MAFB (TD8895, 1:500) (Abmart) and CX3CR1 (13885-1-AP, 1:200) (Proteintech) according to standard protocols.