Pcdna3.1 mettl3

Mouse spermatogenic GC-1 spg cell line was purchased from the American Tissue Culture Collection (Shanghai, China) and cultured in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen, Waltham, MA, USA) containing 10% fatal bovine serum (Invitrogen) at 37°C with 5% CO₂. To establish a DITD model in vitro, GC-1 spg cells were stimulated by 30 mmol/L of glucose (Invitrogen) for 24 hours. Small interfering (si) RNAs targeting METTL3 (si-METTL3), serine and arginine rich splicing factor 1 (SRSF1; si-SRSF1), pcDNA3.1-METTL3, pcDNA3.1-TUG1, and corresponding scrambled controls were purchased from GenePharma. Target plasmid vectors or negative controls were transfected to GC-1 spg cells using Lipofectamine 3000 (Invitrogen).

Small hairpin RNA targeting lnc KCNQ1OT1 (sh-lnc KCNQ1OT1), pcDNA3.1, pcDNA3.1-METTL3, pcDNA3.1-MDR1, miR-103a-3p inhibitor/mimics, small interfering RNA against METTL3 (si-METTL3), and negative control (si-NC, sh-NC, NC inhibitor, NC mimics, empty pcDNA3.1 vector) were purchased from by GenePharma (Shanghai, China). Using Lipofectamine 2000 (Invitrogen), the above sequences, and plasmids were transfected into BC cells and DOX‐resistant BC cells according to the manufacturer’s protocol.

pcDNA3.1-METTL3 and empty pcDNA3.1 vector were purchased from GenePharma (Shanghai, China). By referring to the previously described [20 (link)], the recombinant METTL3 overexpressing adenovirus vector were established. In brief, the full length of METTL3 was cut off from pcDNA3.1-METTL3 vector between Hind III and Xhol restriction endonuclease sites, and then connected to the pENTR1A-expressing vector (Life Technologies, Carlsbad, CA, USA) to construct pENTR1A-METTL3. After the LR reaction a recombination reaction between attL and attR sites using LR Clonase II enzyme mix, the pENTR1A-METTL3 and pAd/CMV/V5-DEST (ThermoFisher Scientific, Carlsbad, CA, USA) was recombined to form pAd/CMV/V5-DEST-METTL3 (pAd-METTL3). The recombined pAd-METTL3 were transfected into 293T cells to obtain purified virus particles.