Pcdna3

Super-Fidelity DNA Polymerase (Cat# P501-d1, Vazyme) was used to amplify the cDNAs of CTD-3252C9.4, IFI6 and IRF1, which were cloned into the expression vector pcDNA3.1 (Cat# V79020, Invitrogen). All PCR products were verified by DNA sequencing. In Additional file 3: Table S3, primers used for plasmid construction are listed. Cells were transfected with the above plasmids or purchased lentivirus pLVX-CTD-3252C9.4 (General Biosystems) for overexpressing the corresponding genes, and pcDNA3.1 (pcDNA-Ctrl) or pLVX-Ctrl were used as control. The plasmid construct map can be found in Additional file 4: Fig. S1. For knocking down, cells were transfected with Smart Silencers (RiboBio) that contains 3 siRNAs, coincident with si-Ctrl as negative control.

The expression plasmids of pcDNA3.1/FBXO28‐His, pEGFP‐N1/ERK5‐His, pEGFP‐N1/ERK5‐Flag, pIRES2‐EGFP/MZF1‐Flag and pIRES2‐EGFP/RGC‐32‐His were constructed by inserting the coding sequences (CDS) of rat FBXO28, ERK5, MZF1 and RGC‐32 gene into pcDNA3.1, pEGFP‐N1 and pIRES2‐EGFP respectively. Briefly, the FBXO28 gene was synthesized directly, and ERK5, MZF1 and RGC‐32 genes were amplified using polymerase chain reaction (PCR) from the cDNA of rat GMCs. The synthesized DNA or PCR products and plasmids were digested with restriction enzymes (FBXO28: HindⅢ and EcoRV; ERK5: Xho I and BamHI; MZF1 and RGC‐32: Bgl II and Sal I) and then ligated with T4 DNA ligase. pcDNA3.1/FBXO28∆1‐His (F‐box domain deletion) and pcDNA3.1/FBXO28∆2‐His (predicted coiled coil domain deletion) were constructed by General Biosystems (Chuzhou, China). The expression plasmids of pcDNA3.1/HA‐TRAF6^WT and pcDNA3.1/HA‐TRAF6^C70A (C70 → A70 mutant) were constructed in our previous study.11 The PCR primer sequences are listed in Table S1.

For transfection experiments, the empty vector (pcDNA3.1), overexpressing plasmids (hsa_circ_0000073), short hairpin RNAs (shRNAs) (sh-NC, sh-circ_0000073, sh-MDM2, and sh-CCNE2), miRNA mimics, and sponge (mimics-NC, miR-1252-5p mimics, sponge-NC, and miR-1252-5p sponge) were all designed and synthesized by General Biosystems (Anhui, China). Lipofectamine 3000 (Invitrogen, United States) was chosen for cell transfection. The shRNAs used are shown in Supplementary Table 1.