Sirna oligo

15-HETE was purchased from Cayman Chemicals (Ann Arbor, MI), Anti-phospho-p38, anti-p38, cyclin E, FGF-2, α-SMA, TGF-β1 and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). FGF-2 and Egr-1 siRNAs, FGF-2 and SP600125 were obtained from Sigma, USA. siRNA oligo was synthesized by GenePharma Co., Ltd (Shanghai, China).

HeLa cells were seeded on tissue-culture-treated six-well plates at a density of 300,000 cells per well and incubated at 37 °C (5% CO₂) for 24 h to ensure adhesion onto the tissue-culture plate surface. Briefly, 100 pmol small interfering RNA (siRNA) oligo (GenePharma Biotechnology, China) for each well were transfected into cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Two siRNA sequences (siRNA-2423 and siRNA-2061, respectively) were synthesized as follows: for siRNA-2423, sense strand was 5′-GAACUUGAAACUGCGUAAATT-3′ and antisense strand was 5′-UUUACGCAGUUUCAAGUUCTT-3′; for siRNA-2061, sense strand was 5′-GCUGGUCAGUUCGUGAUUATT-3′ and antisense strand was 5′-UAAUCACGAACUGACCAGCTT-3′. The silencing effects were confirmed by western blot experiments (Supplementary Fig. 1). Cells were transfected with siRNAs for 48 h in all experiments before exposure to CdSe nanoparticles. After silencing, cells were washed by DMEM medium and then replaced by nanoparticle dispersions (i.e., diluted by DMEM with 10% FBS to concentration of 40 µg mL⁻¹) and incubated for 3 h before harvesting. The samples were digested by dry baths/block heaters (Thermo Scientific, China), and the cellular content of Cd was quantified using an inductively coupled plasma–mass spectrometer (ICP-MS, Agilent 8800, Agilent Technologies, Inc., China).

Cells were transfected with siRNA or plasmid using Lipofectamine 2000 (Invitrogen, USA). C2C12 cells were seeded in 6-well plates and cultured until the cell density reached ~60–70%; cells were transfected after 12 h with 4 μg of expression vector or approximately 1.44 µM siRNA oligo (GenePharma, China) in each well. After incubating for ~6 h, the cell media were replaced with culture media containing serum. Transfection efficiency was measured using the GFP vector after 48 h under the same transfection conditions.

Three siRNAs targeting TUG1 were conducted and are presented in Table S1. In brief, siRNA oligo (GenePharma, Suzhou, China) were added into six-well plates with PMA-induced macrophages (2×105/well) and then treated by using GP-Transfect-Mate (GenePharma, Suzhou, China) according to the operation manual. After 24 or 48 h, macrophages were collected for further experiments. Then, qPCR analysis was applied to detect lncRNA TUG1 expression and to validate the transfected efficiencies. According to the interfering efficiencies, lncRNA TUG1 shRNA-1 was selected for the next experiment. In brief, lncRNA TUG1 shRNA-1 with or without miR-29c inhibitor (GenePharm, Suzhou, China) were added into macrophages for 6h and co-cultured with naive CD4⁺ T cells described above for another 24 h. After that, the supernatant in the lower chamber and CD4⁺ T cells in the upper chamber was collected.

The siRNA oligo was purchased from Genepharma Co., Ltd. (Shanghai, China) (for sequences, see Table S3). When cells at 30% confluence, targeting siRNA or non-targeting control siRNA was diluted in Opti-MEM Reduced Serum Medium (11058021, Gibco, Grand Island, NY, USA) and mixed with Lipofectamine RNA-iMAX Transfection Reagent (13778150, Invitrogen), then added to the culture medium for 6 h.