Lipofectamine 8000

We purchased reporter plasmids of pGL3-Basic-luc or pGL3-Basic- GSK3β-luc from Jinmai biotechnology (Chongqing, China), U87MG cells were seeded in 24-well plates at 5 × 10⁴ cells/well and transfected with 1 μl of Lipofectamine 8000 (Beyotime Biotechnology), and 0.5 μg of reporter plasmids of pGL3-Basic-luc or pGL3-Basic- GSK3β-luc in 25 μl of Opti-MEM. After 24 h of incubation, the cells were treated with quercetin or DMSO for 48 h and then harvested for luciferase assay. The luciferase reporter assay was performed with a Dual Luciferase Reporter Gene Assay Kit (RG027, Beyotime Biotechnology) according to the manufacturer’s protocol. The relative luciferase activity was calculated. Small interfering RNA (siRNA) targeting GSK3β was purchased from Sangon biotechnology (Shanghai, China), U87MG and CHG-5 cells were transfected with siRNA and cultured for 48 h before performing the assays according to the manufacturer’s instructions.

Transfection of siRNAs was performed with Lipofectamine 8000 (Beyotime, Beijing, China) according to the suppliers’ protocol. The relevant siRNA sequences were as follows: SDHD-si#1: 5′-GCTCACAATAAGGAAGAAATA-3′; SDHD-si#2: 5′-GCCGAGCTCTGTTGCT TCGAA-3′; STAT1-si#1: 5′-CTGGAAGATTTACAAGATGAA-3′; STAT1-si#2: 5′-CCCTGAAGTATCTGTATCCAA-3′.

We received human NSCLC cell lines (A549 and H1299) from the Chinese Academy of Sciences (Shanghai, China). A549 and H1299 cells were cultivated in Dulbecco's modified eagle medium (Gibco, Grand Island, USA) supplemented with 10% fetal bovine serum (Gibco), penicillin, and streptomycin. These cell lines were maintained at 37 °C and 5% CO2 and then exposed to siRNA (Nanning Gensis Biotechnology Ltd, China) using Lipofectamine 8000 (Beyotime, China) in a serum-free medium after cells were cultured for 24 hours. The siSGO2 sequence used was 5′-GAACACAUUCUUCGCCUATT-3′, while non-targeted siRNAs were used with the sequence 5′-UUCUCCGAACGUGUCACGU-3′.13 (link)

Plasmid transfections were performed using polyethylenimine (PEI, Polyscience, USA) or Lipofectamine 8000 (Beyotime, Shanghai, China). For PEI transfection, the plasmid and PEI were added into serum-free DMEM with vigorous shaking, and the mixture was incubated for 15 min before being added into the cell culture medium. For Lipofectamine 8000 transfection, the plasmid and Lipofectamine 8000 were added into serum-free DMEM, mixed gently with a pipette, and then immediately added to the cell culture medium. The culture medium was replaced every 12–16 h after transfection. Cells were harvested after 36 h.

The putative binding sites of miR-874-3p with ATF3 mRNA 3′-UTR were predicted using the TargetScan database [28 (link)]. The luciferase reporter vectors (psiCHECK2-Firefly luciferase-Renilla luciferase containing ATF3 wild-type (WT) sequence or mutant (MUT) sequence) were obtained from Guangzhou Geneseed Biotech Co. (Guangzhou, China). Human embryonic kidney (HEK) 293T cells were added to 24-well plates at a density of 1 × 10⁵ cells per well. Subsequently, 1 μg vectors and 100 μL miR-874-3p mimic or mimic NC were cotransfected to HEK-293T cells using 2 μL Lipofectamine 8000 (Beyotime, China). The Luciferase Assay Kit (Promega, Madison, WI, USA) was used to measure the luciferase activity of WT ATF3 or MUT ATF3. The reporter genes' activation degree was calculated between different samples according to the obtained ratio of the relative light unit (RLU) value detected by the Renilla luciferase is divided by the RLU value detected by the Firefly luciferase.

RIP assays were performed with the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) following the manufacturer’s instructions. Cells were washed in PBS, lysed in co-IP buffer, and then sonicated and incubated for 3 h at 4 °C. The probe-streptavidin-dynabeads were added, and the mixtures were incubated at 30 °C for 12 h. Next, lysis buffer and proteinase K were added. RNA was extracted according to the TRIzol Reagent and then subjected to qRT-PCR analysis.
MiRNA pull-down assays were performed by transfecting biotinylated miR-345-3p or control probe (Geneseed, Guangzhou, China) into HRVECs with Lipofectamine 8000 (Beyotime, China). The HRVECs lysates were precleared by centrifugation. Then, the remaining lysates and M-280 streptavidin magnetic beads (Invitrogen, USA) were incubated overnight at 4 °C. The biotin-coupled RNA complex bound to the beads was purified using TRIzol reagent. qRT-PCR was conducted to analyze circRSU1 levels in bound fractions.

The empty vector: pcDNA3.1 + Circ Mini (5607 bp), as well as over-expression vector: pcDNA3.1 + Circ Mini-circ_0040039 (6333 bp) and pcDNA3.1 + Circ Mini- circ_0004354 (5765 bp) were designed and synthesized by Hy cell biotechnology (Wuhan, China). MiR-345-3p mimic and miR-345-3p inhibitor were ordered from Guangzhou Geneseed Biotech Co. (Guangzhou, China). The pcDNA3.1 + FAF1 (7337 bp) and pcDNA3.1 + TP73 were obtained from JIAMAY BIOLAB (Beijing, China). The effects of overexpression were detected by qRT-PCR and western blot experiments. Lipofectamine 8000 (Beyotime, China) was used to transfect plasmids or miR-345-3p or NCs into NPCs following the manufacturer's guidance. After 48 h transfection, NPCs were used to perform the subsequent functional identification experiments.