Fig 2: CircRNF121 mediates OA progression.a The expression of circRNF121 was identified after transfection of various sicircRNAs by q-PCR. The RNF121 expression was detected by q-PCR in treated chondrocytes. b Representative proteins, MMP-13, ADAMTS-5, type II collagen, and aggrecan were analyzed in treated chondrocytes by western blot. c, d Ki-67 immunofluorescence staining and CCK-8 proliferation assay were used to identify the proliferative capability of chondrocytes after transfecting with circRNF121 or sicircRNA-2. e Apoptosis of chondrocytes was evaluated by flow cytometry. Viable and nonviable apoptotic cells are regarded as apoptotic cells. f Cleaved caspase-3 and PARP levels were measured by western blot. Data were means ± SD of three independent assays (*P < 0.05).

Fig 3: Effect of RNF121 KO on AAV genome transcription.A, Quantitative RT-PCR of AAV GFP genome expression relative to GAPDH housekeeping control in Scr and RNF121 KO Huh7 cells. B, qRT-PCR of wtAAV Rep gene expression relative to GAPDH housekeeping control in Scr and RNF121 KO Huh7 cells with and without wild type hAd5 coinfection. Chromatin immunoprecipitation of Scr and RNF121 KO HEK293 cells transduced with AAV2-Luciferase with antibodies against H3K27 acetylation (C) and serine 5 phosphorylated RNA polymerase II (D), followed by qPCR using primers against the Luciferase transgene and GAPDH. Actinomycin D mRNA stability time course profiles for Scr, 10,000 vg/cell (E) and RNF121 KO, 100,000 vg/cell (F) HEK293 cells following transduction with AAV2-GFP. A two-tailed unpaired t test was used unless otherwise indicated, with *, p<0.05; **, p<0.01; ***, p<0.005.

Fig 4: Transcriptomic and proteomic analysis of RNF121 KO cells.A, mRNAs enriched in RNF121 KO, with genes with pAdj<0.05 highlighted in blue. B, Ingenuity pathway analysis of AAV capsid binding partners in RNF121 KO cells showing pathways involved in RNA processes (green), translation (blue), viral infection (lime), DNA damage (purple), and miscellaneous (red). C, U2snRNP inhibitor PladB (20nM) treatment of Scr and RNF121 KO cells. D, DNA-PK inhibitor treatment of Scr and RNF121 KO cells (20uM). E, ATM/ATR Kinase inhibition of Scr and RNF121 KO cells (20uM). F, StringDB interactome of DNA damage and proteasomal machinery in RNF121 KO cells. G, Summary of findings highlighting VCP mediated transcriptional arrest in the absence of RNF121, an essential AAV host factor. A two-tailed unpaired t test was used unless otherwise indicated, with *, p<0.05; **, p<0.01; ***, p<0.005.

Fig 5: Effect of RNF121 knockout (KO) and overexpression (OVX) on AAV transduction.A, Western blot of whole cell lysates from Scr and RNF121 KO cell lines. B, Luciferase reporter expression of Scr and RNF121 KO Huh7 cells following transduction with AAV1 (10,000 vg/cell), AAV2 (5,000 vg/cell), AAV6 (10,000 vg/cell), and AAV9 (20,000 vg/cell). C, Luciferase reporter expression of Scr and RNF121 KO in HEK293 and U87 cell lines following transduction with AAV2. D, Luciferase reporter signal with increasing dose of AAV2 on Scr and RNF121 KO Huh7 cells. E, Luciferase reporter signal of Scr and RNF121 KO HEK293 cells following transfection with a control plasmid or plasmid containing RNF121 cDNA. A two-tailed unpaired t test was used unless otherwise indicated, with *, p<0.05; **, p<0.01; ***, p<0.005.