Fig 1: Identification of RINGPep1 from a SICLOPPS library. (a) Schematic of IDOL RTHS. The expression of the 434-IDOL fusion protein is induced by IPTG. IDOL dimerization leads to the formation of a functional repressor that prevents expression of the 3 reporter genes downstream: His3 (imidazole glycerol phosphate dehydratase), KanR (aminoglycoside 3′-phosphotransferase, kanamycin resistance), and LacZ (β-galactosidase). A cyclic peptide inhibitor of IDOL homodimerization will disrupt the repressor, leading to expression of the reporter genes and survival of the host colony on selective media. (b) Drop spotting analysis of RINGPep1 activity in the IDOL RTHS; serial dilutions (2.5 μL of ∼10n cells per mL) of the IDOL RTHS and the IDOL RTHS containing a SICLOPPS plasmid encoding RINGPep1. In the absence of IPTG and arabinose the strains have full growth capability, however upon addition of 25 μM IPTG (inducer of 434-IDOL) and 6.5 μM arabinose (inducer of SICLOPPS) the IDOL RTHS displays inhibition of growth, which is restored in the strain containing the plasmid encoding RINGPep1. (c) The residues on IDOL perturbed by RINGPep1 binding identified by 1H 15N HSQC NMR are shown in blue (for spectrum see ESI Fig. 5,† for additional views of the structure, see ESI Fig. 6†). Each monomer of the IDOL homodimer is shown in a different shade of pink. (d) Assessing the binding of RINGPep1 to IDOL by MST reveals a Kd of 38.5 ± 3.0 μM. Data represented as mean ± SEM, n = 3.
Fig 2: Addition of LXRα agonist (A) T0901317 or (B) 22R-HC normalized IDOL and (C and D) reduced LDLR expression, respectively, in atorvastatin-treated THP-1 cells. Each bar represents the mean and SD of triplicate experiments. *P < 0.05 compared to non-treated cells, # P < 0.05 compared to atorvastatin-treated cells. The statistical significance of differences was determined using ANOVA followed by Bonferroni’s comparison.
Fig 3: miR-19b directly targets MYLIP gene and down-regulates its expression in breast cancer cells(A) The relative expression levels of miR-19b in MCF7 cell line when transfected with miR-19b mimic, miR-19b inhibitor and their negative controls. The asterisks (**, ***) indicate a significant difference (p < 0.01, p < 0.001) respectively. (B) The relative expression levels of miR-19b in MDA-MB-231 cell line when transfected with miR-19b mimic, miR-19b inhibitor and their negative controls. The asterisks (**) indicate a significant difference (p < 0.01). (C) The protein expression levels of MYLIP and their integrated optical densities (IOD) in MCF7 cell line when transfected with miR-19b mimic, miR-19b inhibitor and their negative controls. The asterisk(s) (*, ***) indicate a significant difference (p < 0.05, p < 0.001) respectively. (D) The protein expression levels of MYLIP and their integrated optical densities (IOD) in MDA-MB-231 cell line when transfected with miR-19b mimic, miR-19b inhibitor and their negative controls. The asterisk(s) (*, ****) indicate a significant difference (p < 0.05, p < 0.0001) respectively. (E) The binding site of hsa-miR-19b with MYLIP 3′-UTR region (wild type, WT) and its mutant (MUT) sequences. (F) The relative luciferase activities of MYLIP 3′-UTR WT vector and MYLIP 3′-UTR MUT vector when co-transfected with miR-19b mimic and mimic control in HEK293 cell line. The asterisks (***) indicate a significant difference (p < 0.001).
Fig 4: The LXRα-IDOL signaling pathway is essential for DEHP-mediated inhibition of the lipid-lowering effect of simvastatin in hepatocytes. (A,B) Huh7 cells were treated with DEHP (1 μg/mL) for the indicated times (0, 1, 3, 6, 9, and 18 h). (A) Western blot analysis of IDOL, LXRα, and α-tubulin. (B) LXRα activity. (C) Cells were pre-treated with control siRNA (100 nM) or IDOL siRNA (100 nM) for 24 h, and then with DEHP (1 μg/mL) for 18 h. The levels of LDLR and α-tubulin were examined by Western blot analysis. (D) Cells were pre-treated with control siRNA (100 nM) or IDOL siRNA (100 nM) for 24 h, and then with DEHP (1 μg/mL) for 1 h and simvastatin (5 μM) for 18 h. The levels of Dil-LDL binding with LDLR were assessed. (E) Huh7 cells were pre-treated with LXRα inhibitor GSK2033 (5 μM) for 1 h, and then with DEHP (1 μg/mL) for 18 h. The levels of LDLR and α-tubulin were examined by Western blot analysis. (F) Huh7 cells were pre-treated with LXRα inhibitor GSK2033 (5 μM) for 1 h, and then with DEHP (1 μg/mL) for 1 h and simvastatin (5 μM) for 18 h. The levels of Dil-LDL binding with LDLR were assessed. Data are shown as the mean ± SEM from five independent experiments. * p < 0.05 vs. vehicle group; # p < 0.05 vs. simvastatin alone group; & p < 0.05 vs. the simvastatin DEHP group.
Fig 5: Over-expression of miR-19b and inhibition of MYLIP facilitate the migration and metastasis of breast cancer cells(A) The protein expression levels of MYLIP and their integrated optical densities (IOD) in MCF7 cell line when transfected with three different siRNA-MYLIP colonies and siRNA control. The asterisk(s) (*, ***) indicate a significant difference (p < 0.05, p < 0.001) respectively. (B) The wound healing assay for MCF7 cell line when transfected with miR-19b mimic and mimic control at 0h and 48h. The relative gap distances were calculated respectively. The asterisk (*) indicates a significant difference (p < 0.05). (C) The wound healing assay for MCF7 cell line when transfected with siRNA-MYLIP-850 and siRNA control at 0h and 48h. The relative gap distances were calculated respectively. The asterisks (**) indicate a significant difference (p < 0.01). (D) The transwell invasion assay for MCF7 cell line when transfected with miR-19b mimic and mimic control. The relative invasive cell numbers were calculated respectively. The asterisk (*) indicates a significant difference (p < 0.05). (E) The transwell invasion assay for MCF7 cell line when transfected with siRNA-MYLIP-850 and siRNA control. The relative invasive cell numbers were calculated respectively. The asterisks (**) indicate a significant difference (p < 0.01).
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