Hif 1α

Corneas were carefully dissected under microscopy and rinsed in normal saline. Paraformaldehyde-fixed corneas were embedded in paraffin and sectioned at 4 μm for histological analysis. Each cornea yielded 12-16 slides for histological and immunohistological analysis. For the visualization of three layers of the cornea structure, three non-consecutive sections were stained with hematoxylin and eosin (H&E) in each cornea.
To observe the expression of angiogenic factors, sections were incubated with primary antibodies to mouse VEGF-A (1:150, Sangon Biotech, Shanghai, China) and HIF-1α (1:150, Sangon Biotech, Shanghai, China) at 4°C overnight. This was followed by three rinses in PBS. The staining was visualized through the use of a horseradish peroxidase (HRP) detection system (Dako), followed by counterstaining with hematoxylin. The integral optical density (IOD, IOD = average optic density × positive area) were quantified using Image-J software. We stained the corneas for marker of blood vessels (CD31, 1:100, Sangon Biotech, Shanghai, China). The number of vessels on microscopic images was quantified by two independent researchers, resulting in an inter-evaluator variation of less than 10%. Three non-consecutive sections were determined in each cornea and the researchers were not blinded to treatment.

Hypoxia-inducible factor 1 alpha (HIF1α) and PDK1 associated peptides used for dot blot assays were synthesized by Sangon Biotechnology. The sequences were listed as below:

HIF1: Bio-RLQFDDDMPIYPALMELDLD

PDK1-WT: Bio-FGCMQVSSSSSSHSLSA

PDK1-Δ6S: Bio-FGCMQVHSLSA

PDK1-Q387R: Bio-FGCMRVSSSSSSHSLSA

PDK1-Q387H: Bio-FGCMHVSSSSSSHSLSA

PDK1-S390L: Bio-FGCMQVSLSSSSHSLSA

Peptides were diluted into 2 mg/ml for further pulldown assays: 4 μg peptides were incubated and rocked with streptavidin beads for 1 h at 4 °C, and the beads were washed twice with NETN buffer. The peptides conjugated beads were incubated with cell lysis transfected with SPOP for another 4 h, and then the pulldown products were washed four times with NETN buffer and subjected for immunoblot analysis.

Transfection was performed using Lipofectamine 2000 (Invitrogen Life Technologies), in accordance with the manufacturer's instructions. For miRNA-20b functional analysis, the HepG2 cells were transfected with the scrambled miRNA as a negative control, miRNA-20b mimics, or miRNA-20b inhibitor (Ambion, Life Technologies, Grand Island, USA). For HIF-1α or VEGF functional analysis, the HepG2 cells were transfected with HIF-1α or VEGF-specific small interfering (si)RNA or pcDNA3.1-HIF-1α plasmid (Sangon Biotech, Shanghai, China). The transfection assay was performed as described in study [27 (link)].