Fig 2: Graphical representation of the function of CORO1C-47aa in EC. CORO1C-47aa translated from circ-0000437 functions as a negative regulator in tumor angiogenesis via blocking the association between ARNT and TACC3 and then reduces the expression of VEGFA. This process leads to a decrease in both expression and secretion of VEGFA, which ultimately leads to reduced angiogenesis.

Fig 3: CORO1C-47aa competitively binds to ARNT and inhibits the binding of TACC3 to its PAS-B ligand.A, diagram of the coimmunoprecipitation experiments. B, mass spectrometric analysis of the coimmunoprecipitation experiments. C, top, CORO1C-47aa-FLAG plasmid was transfected into cells, and the CORO1C-47aa-FLAG complexes were coimmunoprecipitated by anti-FLAG antibody, and only ARNT was detected. Bottom, CORO1C-47aa-FLAG and ARNT-HA plasmids were transfected into cells, and the ARNT-HA complexes were coimmunoprecipitated by anti-HA antibody, and CORO1C-47aa-FLAG was detected. D, coimmunoprecipitation assays revealed the interaction of CORO1C-47aa with ARNT via the PAS-B region of ARNT.

Fig 4: Depletion of ROS reduces cipaltin-induced shARNT cell apoptosis.Cells were pretreated with 10-acetylcysteine (NAC) for 1 h before A375 parental cells (P), shLacZ cells (Z), and ARNT-silenced cells (shARNT #1 and #2) were treated with 5 µM cisplatin, and then were incubated for 24 h. Apoptotic cells were examined by staining with annexin V-FITC/propidium iodide (PI), and flow cytometry was used to analyze cell apoptosis. The apoptotic ratio was calculated. * late apoptosis; # early apoptosis. Similar results were obtained in three independent experiments.

Fig 5: Loss of CUL4B increases AHR protein levels and AHR transcriptional activity in MEF cells.A, WT and MEFCul4b-null cells were treated with solvent dioxane or 0.3, 1, and 10 nM TCDD for 6 h. Left panels, cell homogenates were used for Western blotting (WB) analysis with antibodies against AHR, CUL4B, and ß-actin; right bar graph shows densitometry analysis of AHR protein levels from n = 5 experiments using TCDD (1 nM) with 1 or 2 replicates per treatment group for each experiment (p = 0.0001, T versus C for both WT MEF and MEFCul4b-null; p = 0.0004, T (WT MEF) versus T MEFCul4b-null). B, WT MEF and MEFCul4b-null cells treated with TCDD (0.3 nM) for 0, 2, 4, 6, and 15 h were used to prepare homogenates for WB analysis with antibodies indicated in the figure (left panels) or to extract RNA and analyze Cyp1a1 mRNA levels (an index of AHR transcriptional activity) by RT-qPCR (right bar graph); WT versus MEFCul4b-null: p = 0.0024 (T 2 h), p = 0.0005 (T 4 h), p < 0.0001 (T 6 h), p < 0.0001 (T 15 h). C, WB using homogenates of WT MEF and MEFCul4b-null cells (TCDD, 10 nM) with antibodies against ARNT and ß-actin. Results are representative of n = 2 independent experiments. D, representative results for WB using homogenates of WT MEF and MEFCul4b-null cells treated with TCDD (1 nM, 6 h) ± MG132 (50 µM). For bar graphs in panels A and B, one-way ANOVA was used to calculate differences among the means and Tukey's honestly significant difference (HSD) test was used as a post hoc test. For this and other figures, bar graphs represent means ± SE with experimental replicates shown as black-filled circles; *p = 0.05; **p = 0.01; ***p = 0.001; ****p = 0.0001. AHR, aryl hydrocarbon receptor; ARNT, aryl hydrocarbon receptor nuclear translocator; C, control (solvent); CUL4B, cullin 4B; MEF, mouse embryonic fibroblast; MEFCul4b-null, MEF cell line in which CUL4B has been knocked out; n.s., not significant; RDU, relative densitometry units; RT-qPCR, real-time quantitative PCR; T, TCDD; T, 2,3,7,8-tetrachlorodibenzo-p-dioxin.