Ultraviolet spectrophotometer

The CCK-8 kit was used to assess the ability of cells to multiply (Beyotime, Shanghai, China). Cells (3000 cells/well) were seeded in 96-well plates and transfected with miRNA-296-3p mimic or NC mimic for 24, 48, and 72 hours, after which they were incubated in the dark for 2 hours with CCK-8 solution (10 μL). An ultraviolet spectrophotometer was used to measure optical density (OD) values at 450 nm (Thermo Fisher Scientific, USA).

HYP contents were measured according to the manufacture's instruction of the kit (Jiancheng, Nanjing, China). 80 mg lung tissues were hydrolyzed with 1 ml of alkaline hydrolysate and boiled at 95°C for 20 min and then centrifuged at 3000 rpm for 10 min at 4°C. The supernatant was obtained and hydroxyproline content was measured on an ultraviolet spectrophotometer (Thermo Fisher, MA, US). Results were expressed in microgram per gram tissue (μg/g tissue).

The proliferation of FTY720- or DMSO-treated sacrum chordoma cell lines (MUG-Chor1 and U-CH1) was measuredby using the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay Kit (Promega, Madison, Wisconsin, U.S.A.) as per the established protocol. The cells were placed in 96-well plates (1 × 10³ cells/well) for 24, 48, 72, 96 and 120 h. Then, 20 μl of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was placed in every well and placed at 37°C for 3 h. Absorption was measured at 495 nm by utilizing an ultraviolet spectrophotometer (Thermo Fisher Scientific, Waltham, U.S.A.).

We extracted genomic DNA using hexadecyltrimethyl ammonium bromide (CTAB). Initially, 200 mg of colonies were placed in a 1.5-mL sterile microtube and treated with 850 µL of Tris-CTAB (100 mL of Tris, 280 mL of 5 mol/L NaCl, 40 mL of 0.5 mol/L Ethylene Diamine Tetraacetic Acid (EDTA), and 20 g CTAB, pH 8.0) in a thermostatic water bath at 65 °C for 1 h. The same volume (850 µL) of chloroform was added to the microtube with shaking up and down after cooling to room temperature. The microtube was centrifuged (3500 rpm, 5 min) after leaving the suspension undisturbed for 10 min. Then, 400 µL of the supernatant was carefully transferred to a new 1.5 mL sterile microtube. The supernatant with an equal volume of isopropyl alcohol was left undisturbed in a refrigerator at − 30 °C for 3 h. Following centrifugation at 3500 rpm for 20 min, 750 µL of 75% ethyl alcohol was added to the suspension and incubated at 3500 rpm for 15 min. The pellet was dried by placing the microtube in the fume hood with the lids open for a while. Finally, the pellets were resuspended in 100 µL of sterile ddH₂O, following which the concentration and quality of DNA samples were checked using an ultraviolet spectrophotometer (Thermo Fisher Scientific, USA) and 1% agarose gel electrophoresis.

Colonies were suspended in Mueller-Hinton Broth (MHB) for 6 h and quantified using an ultraviolet Spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, United States). The cultures were then diluted with sterilized MHB broth (about 10 times to achieve the absorbance value between 0.08 and0.10). Broth dilution technique was carried out to determine resistance to the antimicrobials (Table 2).
Antibiotic resistance testing was performed according to the broth microdilution method (Clinical and Laboratory Standards Institute [CLSI], 2015 ). Strains with an MIC less than or equal to the break points were considered susceptible; those higher than the break points were considered resistant. Susceptibility test results were interpreted using Clinical and Laboratory Standards Institute [CLSI] (2006) breakpoint criteria (Table 2).

Total mRNA was purified from peripheral blood and induced sputum cells using Trizol (Invitrogen) and quantified by an ultraviolet spectrophotometer (Thermo Fisher Scientific, USA) [36 (link)]. 1 µg RNA was reversely transcribed into cDNA using Reverse Transcriptase Kit (Qiagen, Netherlands) in accordance to the manufacturer’s instructions [37 (link)]. Then, quantitative RT-PCR was performed using SYBR® Premix Ex Taq™ II system (TaKaRa, Japan) with the CFX96 Touch™ Real-Time PCR Detection System (Bio‐Rad, USA). The PCR conditions were as follows: 95 ℃ for 30 s, 40 cycles of 95 ℃ for 15 s, and 60 ℃ for 30 s. Resulting mRNA levels were normalized to β-actin and expressed as a fold change relative to control samples. The sequences of the primers used are as follows: FOXO3 (forward, CGG ACA AAC GGC TCA CTC T; reverse, GGA CCC GCA TGA ATC GAC TAT), TP53 (forward, AAG TCT GTG ACT TGC ACG TAC TCC; reverse, GTC ATG TGC TGT GAC TGC TTG TAG) and β-actin (forward, TTC CAG CCT TCC TTC CTG GG; reverse, TTG CGC TCA GGA GGA GCA AT).