Fig 2: Hsa_circ_0024093 affects migration of PDGF-BB-treated VSMCs by miR-889-3p/miR-4677-3p/USP9X signaling(A and B) Transwell assay (scale bar, 50 μm) and wound-healing assay (scale bar, 100 μm) examined the migration of PDGF-BB-treated VSMCs with different transfections. ∗p < 0.05, ∗∗p < 0.01.

Fig 3: ZNF598 is destabilized in USP9X KO cells. (A) HCT116 or HCT116 USP9X−/0 lysates were analyzed by immunoblot with the indicated antibodies (representative of three independent experiments). (B) Quantitative RT-PCR reactions for ZNF598, USP9X, and USP9Y (normalized to Actin) were performed with cDNA derived from the indicated cell lines. The mean of three independent biological replicates is shown, and error bars indicate the standard deviation. (C) HCT116 or HCT116 USP9X−/0 cells were transfected with 0, 0.2, 0.4, 0.8, or 1.6 µg HA-ZNF598 and 0.2 µg GFP as a transfection control, and lysates analyzed by immunoblot with the indicated antibodies. Graph shows HA-ZNF598 relative to cotransfected GFP. Panel is representative of three experiments. (D) HCT116 or HCT116 USP9X−/0 cells were transfected with 0.2 µg HA-ZNF598 and treated for the indicated times with 100 µg/ml cycloheximide (Chx). Lysates (8 µg for HCT116 and 20 µg HCT116-USP9X−/0) were probed with the indicated antibodies. Graph represents the results from four independent experiments. Error bars represent the standard deviation. TPS, total protein stain.

Fig 4: Hsa_circ_0024093 stabilizes YAP1 protein(A) YAP1 protein level was detected under CHX treatment in VSMCs with or without hsa_circ_0024093 overexpression. (B) The protein level of YAP1 was examined under CHX treatment in PDGF-BB-treated VSMCs under hsa_circ_0024093 inhibition. (C and D) YAP1 protein level was analyzed in VSMCs under diverse conditions. (E and F) IP assay assessed the ubiquitination level of YAP1 in indicated VSMCs. (G and H) The mRNA and protein levels of USP9X in VSMCs under different transfections were investigated. ∗∗p < 0.01.

Fig 5: Hsa_circ_0024093 sponges miR-889-3p and miR-4677-3p to upregulate USP9X(A and B) Subcellular fractionation and FISH assays (scale bar, 20 μm) monitored the cellular location of hsa_circ_0024093. (C) Enrichment of hsa_circ_0024093 and USP9X in anti-Ago2 in VSMCs and PDGF-BB-treated VSMCs was calculated. (D) The binding sites between hsa_circ_0024093 and miR-4677-3p/miR-889-3p were predicted on ENCORI. (E) Luciferase activity of hsa_circ_0024093-WT/MUT in indicated VSMCs with the transfection of miR-4677-3p mimics or miR-889-3p mimics was detected. (F) The binding sites of miR-889-3p/miR-4677-3p in the USP9X 3′ UTR were also predicted on ENCORI. (G) Luciferase activity of USP9X-WT/MUT in indicated VSMCs with overexpression of miR-889-3p or miR-4677-3p was detected. (H) Luciferase activity of USP9X-WT under different treatment and transfections was investigated. ∗∗p < 0.01; n.s., no significance.