Cell culture
MIN6 Cells were grown in Dulbecco's modified eagle medium (DMEM; Invitrogen, Carlsbad, CA, USA) at a condition of 37°C, 5% CO2, and 88% humidity. The MIN6 cells were next separated into five subgroups: the control, high glucose, asprosin at different doses (50, 100, and 150 nmol/L); alternatively, the cells were separated into four groups: the control, high glucose, vehicle, and asprosin transfected subgroups. Min6 cells in high glucose subgroup were incubated using 35 mmol/L glucose. The cells in asprosin subgroup were incubated using recombinant mouse asprosin (50nmol/L, 100nmol/L ,150nmol/L, solved in DMEM, Biolegend) or transfected using Fbn1-del pCDH-GFP-Puro vector (Yixiang Biotechnology Corp., Tianjin, China). The pCDH-GFP-puro vector was used to transfect the cells in vehicle subgroup. STM2457 (100µmol/L, solved in DMSO, MedChemExpress) was served as METTL3 inhibitor.
Cell viability assay
Cell viability assay of MIN6 cells was measured using an Cell Counting Kit-8 (UElandy Inc, SuZhou, China). A density of 5x103 cells was plated in 96-well plates at 100 ul per well for 24 hours, followed by the addition of high glucose or asprosin for 48 hours. 10 ul of CCK-8 mixture was incorporated into every well with serum-free medium for 3 hours at 37°C. The absorbance at 450 nm was used to determine the vitality of the cells.
Glucose-Stimulated Insulin Secretion (GSIS)
In 24-well plates, Min6 cells were planted. Following that, the cells were treated for one hour at 37°C in Krebs-Ringer bicarbonate HEPES (KRBH, Yuanye Bio-Technology Corp., Shanghai, China) buffer. Cells in different subgroups were subsequently administered with 1 ml KRBH containing 5.0 or 20.0 mM glucose for 1 hour at 37°C after the KRBH buffer was removed. The insulin concentrations were measured by an ELISA Kit (MLBI Biotechnology Corp., Shanghai, China) in compliance with the guidelines provided by the manufacturer.
Measurements of IL-1β and IL-18 concentrations
IL-1β and IL-18 concentrations were assessed using the ELISA kit (MLBIO Biotechnology Corp., Shanghai, China) in compliance with the guidelines provided by the manufacturer.
Immunofluorescence
µ-Slide eight wells (Ibidi, Germany) were used to plant MIN6 cells. High glucose and asprosin were administered as previously mentioned. After three PBS washes, the cells were fixed for 15 minutes with 10% (w/v) formaldehyde. Following three PBS washes, the sample was permeabilized for 15 minutes using 0.1% (v/v) Triton X-100 in PBS. After blocking the cells for 60 minutes in 5% (w/v) bovine serum albumin buffer, the cells were treated at 4°C for an entire night with a 1:100 dilution of the caspase-1 antibody (Proteintech, Ca 22915-1-AP) in PBS. Subsequent to incubation, the cells underwent three PBS washes, one hour at 37°C of incubation with 1:200 dilution of fluorophore-conjugated secondary antibodies (goat anti-rabbit, Abcam, ab150081 Alexa Fluor 488) in PBS, and two PBS washes. Lastly, 4,6-diamidino-2-phenylindole (DAPI, UElandy Inc) was used to counterstain nuclei for 15 minutes at room temperature. Images were captured using a Leica confocal microscope (Leica, Germany). Using ImageJ 1.8s, the relative fluorescence intensity in the indicated groups was assessed.
Quantitative real time polymerase chain reaction (RT-PCR)
MIN6 cells were subjected to total RNA isolation using the Trizol kit (Invitrogen Life Technologies) in compliance with the guidelines provided by the manufacturer. The cDNA was synthesized using the cDNA Synthesis kit (Kemix, Beijing, China). SYBR Green Mastermix (Vazyme Suzhou, China) was used for RT-PCR amplification. The sequences of primers used in the RT-PCR were as follows: NLRP3, 5’-GTGGAGATCCTAGGTTTCTCTG-3’(forward) and 5’-CAGGATCTCATTCTCTTGGATC-3’(reverse); ASC, 5’-CTTGTCAGGGGATGAACTCAAAA-3’(forward) and 5’-GCCATACGACTCCAGATAGTAGC-3’(reverse); caspase-1, 5’-ACACGTCTTGCCCTCATTATCT-3’(forward) and 5’-ATAACCTTGGGCTTGTCTTTCA-3’(reverse); GSDMD, 5’-CCATCGGCCTTTGAGAAAGTG-3’(forward) and 5’-ACACATGAATAACGGGGTTTCC-3’(reverse); METTL3, 5’-CAAGCTGCACTTCAGACGAA-3’(forward) and 5’-GCTTGGCGTGTGGTCTTT-3’(reverse); METTL14, 5’-AGAAACTTGCAGGGCTTCCT-3’(forward) and 5’-TCTTCTTCATATGGCAAATTTTCTT-3’(reverse); WTAP, 5’-GGCGAAGTGTCGAATGCT-3’(forward) and 5’-CCAACTGCTGGCGTGTCT-3’(reverse); FTO, 5’-ACCTCCAGCATTAGATTC-3’(forward) and 5’-GAAACTACCGCATTTACC-3’ (reverse); ALKBH5, 5’-CCCGAGGGCTTCGTCAACA-3’(forward) and 5’-CGACACCCGAATAGGCTTGA-3’(reverse); GAPDH, 5’-AAATCCCATCACCATCTTCCAG-3’(forward) and 5’-AGGGGCCATCCACAGTCTTCT-3’(reverse). Every reaction was carried out in three duplicates. GAPDH was used as an internal reference to standardize the relative expression of the target gene.
Western blot
The cells' proteins were abstracted with a buffer of RIPA lysis. A DC protein kit was used to measure the protein content. Following 10% sulfate polyacrylamide gel electrophoresis (SDS-PAGE), protein samples were transferred to PVDF membranes. After blocking the membranes in 5% milk for 1 hour at room temperature, the primary antibodies were added, and the membranes were then incorporated at 4°C for an entire night: asprosin (1:1000, LSBio), NLRP3 (1:1000, Abcam), ASC (1:1000, Abcam), Cleaved caspase-1 (1:1000, Cell Signaling Technology), GSDMD (1:1000, Abcam), Cleaved GSDMD (Asp276, 1:1000, Cell Signaling Technology), METTL3 (1:1000, Abcam), WTAP (1:1000, Cell Signaling Technology), FTO (1:1000, Abcam), ALKBH5 (1:1000, Abcam), and GAPDH (1:5000, Abcam). The membranes were treated with HRP-linked secondary antibodies for two hours at room temperature the next day. Chemiluminescence was used to detect the bands, and ImageJ 1.8s was used to quantify the proteins.