Fig 2: The regulation of the stabilities of MCL-1, Cyclin A in HeLa cells. a Representative confocal images showing the expressions and distributions of the MCL-1 (green in merged images) in unperturbed HeLa cells at different cell cycle stages as indicated. Cyclin A in red (merged images, Santa Cruz, sc-271682, mouse monoclonal anti-cyclin A (B-8) 1:500 dilutions). These cells were fixed with 4% formamide-PBS solution and stained with a primary anti-Mcl-1 (S-19, Santa Cruz, sc-819) antibody (1:500 dilutions) or anti-Cyclin A (B-8) antibody (Santa Cruz, sc-271682) (1:500 dilutions). DNA was stained with DAPI (blue in merged images). The distributions of the fluorescent signals of Cyclin A throughout the cell cycle used for timing the cell cycle stages for comparison purpose. b Representative confocal images showing the expressions of MCL-1 (green) in nocodazole arrested (top panel) or nocodazole and MG132 arrested (bottom panel) HeLa cells. DNA was stained with DAPI (blue in merged images) and the mitotic status highlighted by staining with the phosphor-histone H3 S-10 antibody (red, Cell signalling, 9706, 1:200 dilutions). HeLa cells were treated with nocodazole or nocodazole + MG132 for 8 h

Fig 3: 1,6-Hexanediol suppresses endothelial function through cyclin A1. A HUVECs were infected with lentiviral particles encoding CCNA1 open reading frame. After 36 h, the infected cells were treated with indicated 1,6-HD for 24 h. Tube formation assay was carried out following. B Quantification analysis of A. C Cell proliferation was examined by CCK8 assay in HUVECs overexpressed by CCNA1. D, E The overexpression of Cyclin A1 was analyzed. F The expression of endogenous cyclin A1 and exogenous Flag-cyclin A1 was detected. Mean viability was shown and standard error of the mean presented the standard deviations of triplicate samples. **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 200 µm

Fig 4: 1,6-Hexanediol suppresses the expression of cyclin A1. A Western blot analysis of the G1/S transit-related genes in HUVECs treated with or without 1,6-HD. GAPDH was used as the loading control. B The mRNA expression of CCNA1 at different concentrations of 1,6-HD treatment was determined by qRT-PCR. C HUVECs were treated with 1,6-HD at indicated time points. Cells were collected and the expression of cyclin A1 was analyzed by western blot. D In the parallel experiment, the mRNA expression of CCNA1 was determined by qRT-PCR. E The expression of cyclin A1 was examined in Matrigel plugs containing 1,6-HD by Western blot. F The mRNA expression of CCNA1 in cornea was determined by qRT-PCR. G, H HUVECs were treated with bortezomib or MG132 for 1 h and then treated by 1,6-HD at indicated times. The expression of cyclin A1 was analyzed by Western blot. GAPDH was used as the loading control. Mean viability was shown and standard error of the mean presented the standard deviations of triplicate samples. *p < 0.05, **p < 0.01, ***p < 0.001