Highgene transfection reagent

TC-1 mice lung epithelial cells were provided by the Stem Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The TC-1 cells within the 10th passage were used for the subsequent experiments and were cultivated in Dulbecco’s modified Eagle medium (Gibco, Brooklyn, NY, USA) containing 10% fetal bovine serum (Gibco, Brooklyn, NY, USA) at 37 °C with 5% CO₂ in an incubator and were exposed to varying concentrations (0, 1, 5, and 10 ng/mL) of TGF-β1 for 48 h to construct cell models. TC-1 cells were then treated with 10 mM 3-methyladenine (Sigma Aldrich, Burlington, VT, USA) or 20 nM rapamycin (Sigma Aldrich, Burlington, VT, USA) to inhibit and activate autophagy, respectively. The miRNA mimics/inhibitors and small interfering RNA (siRNAs) were constructed by GenePharma Co., Ltd. (Shanghai, China), and the sequences are listed in Supplementary Table S1. When TC-1 cells reached 60% confluence in 6-well plates, they were transiently transfected with 30 nM miR-29a-3p mimics or inhibitor or 50 nM siR-Akt3 using HighGene transfection reagent (ABclonal, Wuhan, China).

HEK293T cells were cultured in DMEM (Gibco, C11995500BT) supplemented with 10% fetal bovine serum (ExCell Biotech, FSP500). SH-SY5Y cells were cultured in DMEM-F12 (Gibco, 11,320,033) supplemented with 10% fetal bovine serum (Thermo, 10,099,141). For transfection, 1 μg of plasmids was introduced into HEK293T cells using Highgene transfection reagent (Abclonal, RM09014). For the SH-SY5Y cells, 1ug of plasmids was transfected using the Lipofectamine™ 3000 transfection reagent (Thermo, L3000001). The cells were cultured for 48 h before immunofluorescence staining, and for 72 h before Western blotting analysis.

To locate the critical sequence for LDLRAD4 mRNA disruption in lncRNA LDLRAD4-AS1, we established five truncated mutants of lncRNA LDLRAD4-AS1. Based on the full-length lncRNA LDLRAD4-AS1 sequence (2196 bp), we established truncated mutants as follows (5′–3′): mutant TM-549-5′ (TM indicates truncated mutant) is the 1–549 bp region, mutant TM-1098–5′ is the 1–1098 bp region, mutant TM-1098-M is the 549–1647 bp region, mutant TM-1098–3′ is the 1098–2196 bp region, and mutant TM-549-3′ is the 1647–2196 bp region. All five truncated mutants and full-length lncRNA LDLRAD4-AS1 are illustrated in Fig. 5l. The sequences were cloned into the pCDH-CMV-MCS-EF1-Puro empty vector and sequenced. Then, the recombinant plasmids were transiently transfected into 293T cells using HighGene Transfection reagent (ABclonal, Shanghai, China). Forty-eight hours after transfection, RNA was harvested and reverse transcribed into cDNA. qRT-PCR was applied to detect the levels of LDLRAD4 mRNA.

Breast cancer MDA-MB-231 cells (FuHeng Biology, Shanghai, China) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) basic culture medium (Gibco, Carlsbad, USA) supplemented with 10% fetal bovine serum (FBS; AusGeneX, Brisbane, Australia) in 5% CO
₂. The cell lines used in our experiments were free of mycoplasma infections. HighGene transfection reagent (RM09014; Abclonal, Wuhan, China) was used for the transfection of MDA-MB-231 cells. The cells were inoculated into 6-well plates and considered ready for transfection when the cell density reached 70% to 90%. A total of 3 μg plasmid (General Biol) was added to 200 μL of serum-free opti-MEM medium (Gibco) and mixed well. Afterward, 6 μL of HighGene transfection reagent was added and mixed well. The mixture was evenly dripped into 6-well plates and mixed. After 4–6 h of transfection, the medium was replaced by complete medium with 10% FBS, and continued to cultivate for 24–48 h before the subsequent experiments.

T cells isolated from blood samples of control mice were cultured in RPMI 1640 Medium supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin at 37 °C with 5% CO₂. Anti-CD3 and -CD28 were added to activate T cells. sh-ANRIL/sh-NC and miR-181b-5p mimic (Forward: 5′-AACAUUCAUUGCUGUCGGUGGGU-3′, reverse 5′-CCACCGACAGCAAUGAAUGUUUU-3′)/mimic NC (Forward: 5′-UUCUCCGAACGUGUCACGUTT-3′, reverse 5′-ACGUGACACGUUCGGAGAATT-3′) (RiboBio) were transfected into T cells using Highgene transfection reagent (ABclonal, Wuhan, China).