Fluorescence multi detection microplate reader

The production of reactive oxygen species (ROS) in the mitochondria of cells was measured using an ROS assay kit (Beyotime, China) according to the manufacturer’s protocol. Dichlorofluorescein (DCF) fluorescence was determined using a Fluorescence/Multi-Detection Microplate Reader (BioTek, USA) as previously described [11 (link)]. Data were normalized to the control group and are expressed as a percentage of control levels.

ATP levels were measured using an ATP assay kit (Beyotime, China) according to the manufacturer’s protocol. A Fluorescence/Multi-Detection Microplate Reader (BioTek, USA) was used to determine luminescence level.

To identify the promoter region of chCH25H, the 2077 bp nucleotide sequences before the CH25H coding region were truncated into 6 segments to construct different dual luciferase reporter vectors. The first nucleotide before the chCH25H coding region is numbered −1, and the different dual luciferase reporter vectors are named PGL3- p2077(−2077/−1), p1000 (−1000/−1), p500 (−500/−1), p250 (−250/−1), p125 (−125/−1) and p75 (−75/−1), respectively (Figure 4A). The primers used to construct PGL3 plasmids were showed in Supplementary Table S1.
The PGL3 plasmids (p2077, p1000, p500, p250, p125, p75) were co-transfected with pRL-TK in DF1 cells, respectively. The pGL3-basic was co-transfected with pRL-TK as a control. Firefly and Renilla luciferase activities were measured at 24 h post-transfection using a Dual-GLO Luciferase Assay System Kit (Promega, Madison, WI, USA). Luminescence was measured using a Fluorescence/Multi-Detection Microplate Reader (BioTek, Shoreline, WA, USA). Firefly luciferase activities were normalized to Renilla luminescence in each well.
Furthermore, the common interferon stimulated response elements (ISRE) consensus 5’ A/GGTTTCN(1–2)TTTCC/T3’ or its reverse complement, and the common gamma activated sequence (GAS) consensus 5’ TTNCNNNAA3’ were screened at upstream of chCH25H according to previous studies [23 (link)].

We transfected 800 ng microRNA expression vector, 400 ng firefly luciferase reporter plasmid pMIR-Report Luciferase (circRNA-Zfp609 wild type or mutant), and 80 ng Renilla luciferase reporter plasmid pRL-TK 293T cells in 12-well plates were transfected for 48 h. Co-transfected cells were lysed with 250 μL of cell lysis buffer. The Dual-GLO Luciferase Assay System kit (Promega, Madison, WI, USA) was employed to detect luminescent signals of firefly and Renilla Luciferase with a Fluorescence/Multi-Detection Microplate Reader (BioTek, Winooski, VT, USA), according to the manufacturer. Firefly luciferase activities were normalized to Renilla luminescence in each well.

In order to verify the binding relationship between gga-miR-12207-5P and circPLXNA2 and MDM4, we designed the plasmids for several groups of co-transfection experiments, i.e., a. gga-miR-12207-5P mimic+Pmir-GLO-circPLXNA2-WT, b. mimic NC+Pmir-GLO-circPLXNA2-WT, c. gga-miR-12207-5P mimic+Pmir-GLO-circPLXNA2-MUT, d. mimic NC+Pmir-GLO-circPLXNA2-MUT, e. gga-miR-12207-5P mimic+Pmir-GLO-MDM4-WT, f. mimic NC+Pmir-GLO-MDM4-WT, g. gga-miR-12207-5P mimic+Pmir-GLO-MDM4-MUT, and h. mimic NC+Pmir-GLO-MDM4-MUT were transfected into DF-1 cell lines with 60–80% confluence (96-well plate). At 48 h post-transfection, luciferase activity analysis was examined using a fluorescence/multi-detection microplate reader (BioTek, Winooski, VT, USA) and Dual-GLO^® Luciferase Assay System Kit (Promega). Firefly luciferase activities were normalized to Renilla luminescence in each well.

Gastrocnemius muscle was dissected and immediately frozen in liquid nitrogen, and then stored at −80 °C [36 (link)]. We used commercial assay kits (BC0515, BC3235, BC3245, BC0945; Solarbio, Beijing, China) to measure the enzyme activity of mitochondrial OXPHOS complexes of gastrocnemius muscle and DF-1 cells according to the manufacturer’s protocol. Complex I enzyme activity was determined by the change in absorbance of NADH as measured at 340 nm. Complex II enzyme activity was determined by the change in absorbance of DCIP as measured at 600 nm. The enzyme activity of complex III and complex IV was determined by the change in absorbance of reduced cytochrome c as measured at 550 nm. Absorbance was determined using a Fluorescence/Multi-Detection Microplate Reader (BioTek, Winooski, VT, USA) according to the manufacturer’s protocol. Data were normalized to the control group and expressed as a percentage of control levels.