Fig 1.
Effect of 2ME2 on lens opacity.
(A) Schematic representation of the SD rat lens experiment. (B) Rat lenses were cultured in medium containing 30 mM galactose for 2–3 days (upper panel). After image acquisition, DMSO as a vehicle control or 10 μM, 20 μM, or 40 μM 2ME2 in DMSO were added to the galactose-containing medium, and culture was continued for 2–3 days. The number of days indicated in each panel represents the total days of incubation, “q” indicates samples used for RT-qPCR, and “M” indicates samples used for microarray analysis. (C) The level of lens opacity with and without 2ME2, as well as the change in opacity before and after addition of the inhibitor, were calculated [17]. Data are expressed as the mean ± SE. The two additional samples used for the quantification are shown in S2 Fig.
Table 1.
List of HIF-1 inhibitors used.
Fig 2.
Flowchart for narrowing down the genes for which expression levels were altered upon 2ME2 addition.
Probes without gene names were removed (21,282 genes remaining), genes with signal values of <5 were excluded (6,640 genes remaining), genes exhibiting more than two-fold increase between the Control and Galactose groups as well as decrease between the Galactose and 2ME2 groups were included (80 genes remaining), and genes of unknown function, ribosomal-protein genes, and genes for which primer design was difficult were excluded (43 genes remaining). Of these, 11 genes exhibited more than 10% expression decrease between the Galactose and 2ME2 groups compared with amount of increase expression between the Control and Galactose groups by RT-qPCR, which were consistent with the microarray analysis.
Fig 3.
RT-qPCR analysis of genes with altered expression levels.
RT-qPCR on 43 genes selected from the microarray analysis, Results are shown as target gene mRNA levels normalized by Gapdh mRNA levels. Data are expressed as the mean ± SE. Asterisks indicate more than a 10% decrease in expression between the galactose and 2ME2 groups compared with the amount of increase between the control and galactose groups.
Fig 4.
STRING protein interaction analysis.
STRING analysis of 11 genes, expression changes were confirmed by RT-qPCR, and their predicted functional partners. The color of each edge shows the type of relationship in the following manner: light blue = “from curated databases,” dark purple = “experimentally determined,” green = “text mining,” black = “co-expression,” and light purple = “protein homology”.
Fig 5.
Predicted mechanism of HIF-1-induced diabetic cataract.
HIF-1 is induced in high-sugar environments and forms a complex with p300. Subsequently, cataract is caused by EMT induced increase in Acta2 expression, Txnip-induced apoptosis, and inhibition of cell proliferation.