Fig 2: CCND1 overexpression counteracts the effects of miR-466 transfection in OS cells. (A) The mRNA and protein levels of CCND1 were measured by RT-qPCR and western blot in 143B and U2OS cells. CCK8 assays were used to determine the proliferation of (B) 143B and (C) U2OS cells. Cell cycle distribution in (D) 143B and (E) U2OS cells were analyzed by FACS. (F) Cellular apoptosis was measured by FACS in 143B and U2OS cells stained with Annexin V/PI. *P<0.05 and **P<0.01 vs. control group. OS, osteosarcoma.

Fig 3: miR-144 is downregulated in PTC tissues and inversely correlates with E2F8 expression. a TCGA dataset showed that miR-144 was significantly downregulated in PTC tissues compared with adjacent normal tissues. b qRT-PCR analysis showed that miR-144 was significantly decreased along with more advanced T stage in PTC tissues. U6 was used as an internal control to normalize expression data. c Pearson correlation scatter plot of fold change in miR-144 levels and E2F8 mRNA levels in PTC tissues (n = 64). d A hypothetical model of the miR-144/E2F8/CCND1 axis controlling PTC cell proliferation. ***p < 0.001, # No significance

Fig 4: In vitro characterization, pharmacokinetics and toxicity profile of VNLG-152R and its two enantiomers. (A) HPLC chromatograms racemic VNLG-152R (left panel) and the resolved enantiomers VNLG-152E1 and VNLG-152E2 (right panel). The conditions of HPLC procedure are presented in Supplementary Materials. (B) Table summarizing GI50 values (µM) each cell line treated with VNLG-152R and its enantiomers. TNBC cell subtypes were treated with listed compound (0.1 nmol/L–100 µmol/L) for 7 days and the GI50 values for the anti-proliferative effects of the compounds were determined from dose response curves (by a nonlinear regression analysis using Graph Pad Prism). Data represents the results from six independent experiments for each cell line. (C) Plasma concentration versus time profile after intravenous (IV, 2 mg/kg each, top panel) or oral (PO, 20 mg/kg each, bottom panel) of VNLG-152R (red), VNLG-152E1 (purple) and VNLG-152E2 (blue) to female CD1 mice. Values represent the mean plasma concentrations from three mice per time point. (D) Table of the pharmacokinetic parameters of various compounds after IV and PO dosing. AUC = area under the curve, T1/2 = elimination half-life, Cmax = maximum plasma concentration, F (%) = oral bioavailability. (E) Effects of VNLG-152R, VNLG-152E1 and VNLG-152E2 on body weight and clinical observations at chronic dose termination of study. Three different doses, i.e., 10, 25, and 50 mg/kg/day (formulated in 40% β-cyclodextrin in saline) of each compound were administered daily by oral gavage for 14 days. Each group consisted of three mice. Loss in body weight, a clinical sign of toxicity was observed during the 14 days of dosing and the maximum tolerated dose (MTD) was estimated following established procedures. Changes in body weight were scored on a scale of 0–4 as follows as described under methods [55].

Fig 5: ABCC6 knockdown suppressed PPARα expression in HCC cells. (A) ATP level of ABCC6 knockdown and control 97H and Huh7 cells. (B) qPCR analysis showing the expression of PPARA, ACOX1 and CCND1 in ABCC6 knockdown 97H cells. (C) Western blot showing the protein levels of ABCC6, Cyclin D1, PPARα and ACOX1 in ABCC6 knockdown 97H and Huh7 cells. (D) Western blot showing the protein levels of ABCC6, Cyclin D1, PPARα and ACOX1 in ABCC6 overexpression 7721 and LM3 cells. (E) MDA assay showing the relative lipid peroxidation level of ABCC6 knockdown and control 97H and Huh7 cells. (F) Seahorse assay displaying the baseline and stressed energy phenotype of ABCC6 knockdown and control 97H cells. (G) Flow cytometry analysis showing the intracellular ROS level of vector and ABCC6 overexpression 97H cells. (H) CCK-8 assay showing the effect of PPARα agonist fenofibrate in the proliferation of ABCC6 knockdown and control 97H cells. (I) Colony formation assay showing the effect of PPARα agonist fenofibrate in the proliferation of ABCC6 knockdown and control 97H cells. Error bars indicate means ± SEM. P-values were determined by two tailed t-test (A, E) and one-way ANOVA (B, H, I). *P < 0.05, **P < 0.01, ***P < 0.001.