Table 1

Total RNA was extracted from ipsilateral cerebral cortex tissues and primary microglia cells by TRIzol reagent (Takara, Kyoto, Japan). Samples were then reverse-transcribed to cDNA using a cDNA Synthesis kit (Takara). Then, cDNA was amplified using a SYBR Premix Ex Taq kit (Takara) on a 7500 Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA). All procedures were performed according to the manufacturers’ instructions. Data were normalized to β-actin and expressed as a fold-change compared to the control. The primers used for the amplification are shown in Table 1 (Sangon Biotech Co., Ltd., Shanghai, China).

We cloned the DNA fragments for the expression of control and specific shRNAs (Table 1, Sangon Biotech, Shanghai, China) or the cDNA for C3orf21 expression into pLKO-ZSG-Puro, together with pCMV△R8.92 and pVSVG-I, to generate different types of lentiviruses in 293T cells. Subsequently, we transduced A549 cells with control or lentivirus for the expression of C3orf21-specific shRNA (Addgene) at multiplicity of infection of 4 in the presence of 8 mg/ml hexadimethrine bromide (Sigma). Similarly, we transduced PC-9 cells with control or lentivirus for the over-expression of C3orf21. Three days later, we treated the cells with puromycin (500 ng/ml) or blasticidin (10 mg/ml) to generate shCon, sh-C3orf21 stably silencing A549 and control, C3orf21 over-expressing PC-9 cells, respectively. We tested the efficacy of C3orf21 silencing or over-expression by fluorescent microscopy and Western blot assays.

The full open reading frame (ORF) of CD69 was amplified by PCR using specific primers as listed in Table 1 (Sangong Biotech, Shanghai, China). Subsequently, the CD69 ORF was cloned into the retroviral vector pMX containing internal ribosomal entry site-green fluorescent protein (IRES-GFP) to generate the plasmid of pMX-CD69. The pMX-CD69 and control pMX were transfected into Plat-E cells to generate retrovirus with pMX-CD69 or pMX, respectively. Retroviral transfection was performed as described previously^{27 (link),28 (link)}. Briefly, EL4 cells were transfected with retrovirus at MOI (multiplicity of infection) of 50 in the presence of 5 μg/ml polybrene (Millipore) for 48 h, then the cells were collected for further experiments.

Based on the Watson–Crick base‐pairing principles, tFNAs nanomaterial was self‐assembled from four single‐stranded sequence‐specific DNA (ssDNA) fragments in equimolar quantities (1 μM), as outlined in Table 1 (Sangon Co., Ltd.), using TM buffer (composed of 50 mM MgCl₂ and 10 mM TrisHCl (pH 8.0) solutions) and heating at 95°C for 10 min followed by rapid cooling to 4°C for 20 min. The obtained tFNAs were further stored in a fridge at 4°C until required for use.

Total RNA was extracted from lung tissues and cells using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). RNA was reverse-transcribed into cDNA using a reverse transcription kit (Thermo Fisher Scientific). Quantitative real-time PCR (Q-PCR) was performed. The Q-PCR conditions were: 95 °C for 2 min, followed by 40 cycles of 95 °C for 3 s and 60 °C for 30 s, with a melting curve of 60–95 °C. The primer sequences used in this experiment are listed in Table 1 (Sangon Biotech).Table 1

The primer sequences.

Gene	Forward [5'-3']	Reverse [5'-3']
Mouse β-Actin	GTGCTATGTTGCTCTAGACTTCG	ATGCCACAGGATTCCATACC
Mouse α-SMA	TGGCTATTCAGGCTGTGCTGTC	CAATCTCACGCTCGGCAGTAGT
Mouse collagen I	GAGCGGAGAGTACTGGATCG	GCTTCTTTTCCTTGGGGTTC
Mouse PPARɣ	AGCCCTTTACCACAGTTGATTTCTCC	GCAGGTTCTACTTTGATCGCACTTTG
Rat β-Actin	TGTCACCAACTGGGACGATA	GGGGTGTTGAAGGTCTCAAA
Rat α-SMA	GCGTGGCTATTCCTTCGTGACTAC	CATCAGGCAGTTCGTAGCTCTTCTC
Rat collagen I	TGTTGGTCCTGCTGGCAAGAATG	GTCACCTTGTTCGCCTGTCTCAC
Rat PPARɣ	CGCCAAGGTGCTCCAGAAGATG	AGGGTGAAGGCTCATATCTGTCTCC
Human β-Actin	CCTGGCACCCAGCACAAT	GGGCCGGACTCGTCATAC
Human α-SMA	TCCGGAGCGAAATACTCTG	CCCGGCTTCATCGTATTCCT
Human collagen I	CCACCAATCACCTGCGTACA	CACGTCATCGCACAACACCT
Human PPARɣ	TGAATCCAGAGTCCGCTGACCTC	ATCGCCCTCGCCTTTGCTTTG

Extraction of total RNA from choroidal tissues using Tissue RNA Purification Kit (Yishan Biotechnology, China). The gene transcription was quantified by quantitative RT-PCR with PrimeScript™ RT Master Mix Kit (Takara, Shiga, Japan). The sequence of the primers are shown in Table 1 (Sangon Biotechnology, China).