Quantity one 1 d analysis

Inactive ERK2 proteins (1 μg) were used as the substrate for an in vitro kinase assay with 1.5 μg of active TOPK. Firstly, active TOPK was incubated with FeF (200 and 400 μg/mL) in 1× kinase buffer (25 mM Tris (pH 7.5), 5 mM b-glycerophosphate, 2 mM dithiothreitol (DTT), 0.1 mM Na₃VO₄, 10 mM MgCl₂, and 5 mM MnCl₂) at 32°C for 15 min. Then inactive ERK2 and 100 mM ATP were added to reaction and incubated at 32°C for 1.5 h. Reactions were stopped by adding 5× SDS sample buffer and then were analyzed by Western blotting. Band density was determined by GS-800 Calibrated Densitometer and quantified using “Quantity One 1-D analysis” software (“Bio Rad”, USA). The percentage of inhibition of kinase activity by FeF was calculated compared with control (sample with TOPK active and ERK2 inactive).

FeF (1 mg/mL) was incubated with TOPK-Ni-NTA-agarose beads (or Ni-NTA-agarose beads alone as a control; 200 μL, 50% slurry) in the reaction buffer (50 mM Tris (pH 7.5), 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% Nonidet P-40, 2 mg/mL bovine serum albumin, 0.02 mM phenylmethylsulfonyl fluoride, and 1 mg/mL protease inhibitor mixture). After incubation with gentle rocking overnight at 4°C, the beads were washed 5 times with washing buffer (50 mM Tris (pH 7.5), 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% Nonidet P-40, 2 mg/mL bovine serum albumin, 0.02 mM phenylmethylsulfonyl fluoride).
The binding between the TOPK-Ni-NTA-agarose beads and FeF was examined by color reaction with toluidine blue stain. Firstly, FeF (control) and TOPK-Ni-NTA-agarose-FeF complex were applied to the agarose gel electrophoresis. The electrophoresis was carried out in Tris-borate buffer pH 8.3 (0.09 M Tris, 0.4 M H₃BO₃, 0.01 M EDTA) at 25 mA for 2h. The gel was stained with 0.025 % solution of toluidine blue in acetic acid. Band density was determined by GS-800 Calibrated Densitometer and quantified using “Quantity One 1-D analysis” software “Bio-Rad” (Hercules, California, USA).

Cell pellets were lysed in Winman’s Buffer (1% NP-40, 0.1 M Tris-HCl pH 8.0, 0.15 M NaCl and 5 mM EDTA) with EDTA-free protease inhibitor cocktail (Roche). Total protein content of cell lysates was quantified with “DC Protein assay kit” (Bio-Rad) and 20 μg of protein were subjected to SDS-PAGE (12% Bis-Tris gel). Following electrophoretic transfer of the proteins into nitrocellulose membranes (GE Healthcare, UK), membranes were then incubated with the following primary antibodies: goat anti-Actin (1:2000; Santa Cruz Biotechnology), mouse anti-P-gp (P7965) (1:2000; Sigma), G6PD (1:200; Santa Cruz Biotechnology), 6PGD (1:200; Santa Cruz Biotechnology) and IDH1 (1:200; Santa Cruz Biotechnology). The following secondary antibodies were then used: anti-mouse IgG-HRP; anti-rabbit IgG-HRP or anti-goat IgG-HRP (all diluted 1:2000; Santa Cruz Biotechnology). Signal was detected using the ECL Western blot Detection Reagents (GE Healthcare, UK), the Amersham Hyperfilm ECL (GE Healthcare, UK), and the Kodak GBX developer and fixer (Sigma, EUA)57 (link). The intensity of the bands obtained in each film was further analyzed using the software Quantity One – 1D Analysis (Bio-Rad, USA).

Cells were lyzed in a radioimmunoprecipitation assay (RIPA) buffer (ThermoFisher) added with Complete^TM protease inhibitor cocktail (Roche, Basel, Switzerland) and phenylmethylsulfonylfluoride (PMSF, 1 mM, Sigma). Soluble proteins were denatured in sodium dodecylsulfate (SDS, 2%) loading buffer with dl-dithiothreitol (200 mM, Sigma-Aldrich), resolved by 4–20% SDS-polyacrylamide gel electrophoresis (SDS-PAGE, BioRad) and transferred to nitrocellulose membrane (BioRad). After blocking with 5% nonfat milk, the membrane was incubated with primary antibodies against Prdx6, Na⁺K⁺ATPase, ZO-1 and housekeeping β-actin (Supplementary Table S2), followed by horseradish peroxidase-conjugated Ig antibodies (Roche). Staining signals were revealed by enhanced chemiluminescence (ThermoFisher) using a ChemiDoc XRS gel imaging system (BioRad). Band densitometry was conducted using QuantityOne 1D analysis (BioRad). Specific band density was normalized to that of β-actin. Experiments were conducted in triplicate and results were represented as mean ± SD.

PK mouse corneas were harvested 6 weeks after surgery, and three or four corneas were randomly collected within each group. Total RNA was isolated from the transplanted corneas by Trizol (Invitrogen, Carlsbad, CA, USA) and RNeasy Mini (Qiagen, Hilden, Germany). cDNA was then reverse transcribed from total RNA by using SuperScript III™ Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). Real-time PCR was performed with Taqman PCR Mastermix and FAM dye-labeled predesigned primers (VEGF-A: Mm00437306_m1, VEGF-C: Mm00437310_m1, VEGF-R2: Mm01222421_m1, VEGF-R3: Mm01292604_m1, glyceraldehyde 3-phosphate dehydrogenase (GAPDH): Mm99999915_g1, ThermoFisher, Rockford, IL, USA). GAPDH was used as an internal control for each reaction. Gene expression levels were analyzed via the comparative threshold cycle method using the analysis software (Quantity One 1-D analysis, Bio-Rad, Hercules, CA, USA), and relative expression levels for each sample were expressed as fold change compared with the untreated naive mice.

Total RNA was extracted from the hDPCs using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) followed by cDNA synthesis with QuantiTect Rev. Transcription kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The cDNA was used for real-time Polymerase Chain Reaction (RT-PCR) using SYBR Green (Takara, Shiga, Japan). The primers sequences and PCR conditions are listed in Supplementary Table S1. The quantification of the PCR product was measured by using analysis software (Quantity one 1-D analysis, Bio-Rad, Hercules, CA, USA).