A total of 40 14-week-old WT or Eaf2 KO mice (14) were divided into four groups (n=10 in each): i) WT-nonUV, ii) WT-UV, iii) Eaf2 KO-nonUV and iv) Eaf2 KO-UV. The right eyes of the WT-UV mice and Eaf2 KO-UV mice were exposed to UV radiation, while the left eyes received no radiation. WT-nonUV mice and Eaf2 KO-nonUV mice were not exposed to UV. The Eaf2 KO mice were obtained from Professor Yi Sin Liu (University of Southern California, Los Angeles, CA, USA). The mice were maintained in an environment with a constant temperature of 23 ± 2°C, a relative humidity of 50±5 % and a 12 h light-dark cycle. Chow and water were provided ad libitum. The mice were sacrificed by decapitation. All animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals. The present met the standards of, and was approved by, the ethics committee of China Medical University (Shenyang, China).

The Eaf2 coding sequence (CDS) was amplified by polymerase chain reaction (PCR) using murine cDNA, which was reverse transcribed from total RNA extracted from the rat cells. This was maintained in our laboratory as a template. The primers used were as follows: Eaf2-CDS forward, 5′-GTATGAAAGCTTAAGGCCAAAAGCGG-3′ and reverse, 5′-GCCCGAATTCATCTCACAAATGTTTTCTCTGT-3′. The primers were synthesized by Sangon Biotech (Shanghai, China). The PCR was performed using a Life Express PCR Instrument (Bioer, Hangzhou, China) and the following cycling conditions were used: 95 °C for 5 min; 95°C for 30 sec, 55°C for 30 sec, 72°C for 60 sec, 30 cycles; 72°C for 5 min and 4°C for 2 min. The products were purified using a multifunctional DNA purification kit (BioTeke, Beijing, China). The sequencing was performed by Sangon Biotech). The coding sequence was inserted into the p enhanced green fluorescence protein-N1 vector (Clontech, Mountain View, CA, USA) by the restriction enzymes FastDigest HindIII and FastDigest EcoRI (Fermentas, Burlington, CA, USA). The vector was sequenced for verification and was named as Eaf2 OE.

The rat crystalline lens epithelial cell line α-TN4 was cultured in Dulbecco's modified Eagle's medium (Gibco Invitrogen Life Technologies, Carlsbad, CA, USA) containing 10% (v/v) fetal bovine serum (Hyclone, Logan, UT, USA). The cells were incubated in an incubator at 37°C in an atmosphere containing 5% (v/v) CO₂. Cells were seeded into six-well plates and transfected at 70–80% confluence with the empty vector (Vector) or the Eaf2 overexpression plasmid (Eaf2 OE) using Lipofectamine 2000 reagent (Invitrogen Life Technologies) according to the manufacturer's instructions. 48 h after transfection, the transfection efficiency was analyzed by quantitative real-time PCR and western blotting.

UV at a wavelength of 302 nm and intensity of 200 mW/cm² was generated by a UV transilluminator (Spectroline XX-15N/F; Spectronics, Westbury, NY, USA). The UV transilluminator was covered with aluminum foil, leaving only a small hole with a diameter of 5 mm for irradiating the eyes of the mice. The mice were not anesthetized, and only the right eyes were irradiated with UV. The left eyes were not irradiated and used as a control. 5 min prior to UV irradiation, the eyes of the mice were injected with 1% (w/v) tropicamide (cat. no. T9778; Sigma-Aldrich, St. Louis, MO, USA) and 0.1% (w/v) atropine sulfate to induce mydriasis. Prior to UV irradiation, the mice were checked with a slit lamp to exclude pre-existing cataracts. One eye of each mouse was exposed to UV for 100 sec twice per week for three weeks. 48 h after the last UV irradiation, the mouse lens opacity was observed by slit lamp examination.

48 h after UV irradiation, the eyeballs of all the mice were surgically removed, together with a ~2-mm optic nerve. The eyeballs were incubated in fixing buffer, containing acetic acid, formaldehyde solution (Kemiou Chemical Reagent Co., Lt., Tianjing China), physiological saline (Cisen Pharmaceutical Co., Ltd., Shandong, China) and 75% ethanol at a ratio of 1:2:7:10. After fixing, the crystalline lenses were removed under a microscope OLYMPUS DP73 (Olympus Corporation, Tokyo, Japan) for subsequent experiments.

Apoptosis was analyzed using the In Situ Cell Death Detection kit (cat. no. 11684817910; Roche, Basel, Switzerland). Fixed eyes were paraffin-embedded and cut into tissue sections of 5 µm. Cultured cells were seeded and fixed with 4% (w/v) paraformaldehyde. Prior to the TUNEL assay, the samples were permeabilized with 0.1% (v/v) TritonX-100 (Beyotime Institute of Biotechonlogy, Shanghai, China) and blocked with 3% (v/v) H₂O₂. The TUNEL reagent was mixed with Enzyme Solution and Label Solution and then added to the sample surfaces dropwise. The samples were incubated at 37°C in dark in a humid environment for 60 min. The Converter-POD was added to the sample surfaces dropwise and incubated at 37°C for 30 min followed by diaminobenzidine substrate (Solarbio, Beijing, China). The samples were counterstained with hematoxylin (Solarbio), mounted with neutral balsam and subjected to microscopic imaging.

Proteins in tissues were extracted using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) containing 1% phenylmethanesul-fonylfluoride (Beyotime Institute of Biotechnology). Proteins in cultured cells were extracted using NP-40 lysis buffer (Beyotime Institute of Biotechnology). Proteins extracted were quantified using a Bicinchoninic Acid Protein Assay kit (cat. no. P0012; Beyotime Institute of Biotechnology). Equal amounts of each protein sample were subjected to SDS-PAGE. The separated proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). PVDF membranes were blocked with 5% (w/v) skimmed milk or 5% (w/v) bovine serum albumin (Biosharp, Hefei, China) and incubated at 4°C overnight with the corresponding primary rabbit antibodies against bax (rabbit anti-mouse polyclonal antibody; 1:500; cat. no. WL0101; Wanleibio, Shenyang, China), bcl-2 (rabbit anti-mouse polyclonal antibody; 1:500; cat. no. WL0104; Wanleibio), ERK (rabbit anti-mouse polyclonal antibody; 1:1,000; cat. no. WL0323; Wanleibio), p-ERK (rabbit anti-mouse polyclonal antibody; 1:1,000; cat. no. WLP002; Wanleibio), Eaf2 (rabbit anti-mouse polyclonal antibody; 1:1,000; cat. no. WL0333; Wanleibio), cleaved-caspase-3 (rabbit anti-mouse polyclonal antibody; 1:1,000; cat. no. ab2575; Abcam, Cambridge, MA, USA) and cleaved-caspase-9 (rabbit anti-mouse polyclonal antibody; 1:1,000; cat. no. WL0001; Wanleibio). The PVDF membranes were then incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:5,000; cat. no. A0208; Beyotime Institute of Biotechnology) at 37°C for 45 min. Using β-actin as an internal reference, the objective proteins were detected by an enhanced chemiluminescence detection system (cat. no. E002-5; 7Sea Biotech, Shanghai, China). The signal intensities of the protein bands were analyzed using Gel-Pro-Analyzer 4.5 software.

The crystalline lens tissues were mixed with nine volumes of phosphate-buffered saline, homogenized using a High speed Homogenizer (Scientz, Ningbo, China) and then frozen and thawed three times in liquid nitrogen. The samples were centrifuged at 10010 × g at 4°C for 10 min, and the supernatants were retained for the experiments. The protein concentrations were measured using the Bradford Protein Assay kit (Beyotime Institute of Biotechnology) and diluted to equal concentrations. Caspase-3 and caspase-9 activities were analyzed using Caspase-3 Activity Assay kit (cat. no. C1116) and Caspase-9 Activity Assay kit (cat. no. C1158; Beyotime Institute of Biotechnology) according to the manufacturer's instructions. The absorbance at 409 nm was measured using a microplate reader (ELX-800; BioTek, Winooski, VT, USA).

Total RNA was extracted using the RNA simple Total RNA kit (cat. no. DP419; Tiangen, Beijing, China), according to the manufacturer's instructions. The total RNA was reverse transcribed into cDNA by Moloney mouse leukemia virus reverse transcriptase (BioTeke) and oligo (dT)₁₅ (Sangon Biotech). Eaf2 mRNA levels were determined by RT-qPCR using the SYBR GREEN method with cDNA as template. SYBR GREEN reagent was purchased from Solarbio. The primers used are as follows: Eaf2 forward, 5′-CTTGCATACCTGGACCGT-3′ and reverse, 5′-GTTCACCTTTGCCAACCTCA-3′; β-actin forward, 5′-CTGTGCCCATCTACGAGGGCTAT-3′ and reverse, 5′-TTTGATGTCACGCACGATTTCC-3′. The primers were synthesized by Sangon Biotech. The RT-qPCR reaction volume (20 µl), contained cDNA (1 µl), forward primer (10 µM; 0.5 µl), reverse primer (10 µM; 0.5 µl), SYBR GREEN mastermix (10 µl) and water. An Exicycler™ 96, BIONEER Real-Time PCR system was used (Bioneer Corporation, Daejeon, Korea) and the reaction conditions were as follows: 95°C for 10 min followed by 40 cycles of 95°C for 10 sec, 60°C for 20 sec and 72°C for 30 sec, and final incubation at 4°C 5 min. The relative mRNA expression levels for each sample were calculated using the 2^−ΔΔCt method with β-actin as an internal reference (15).

Values are expressed as the mean ± standard deviation. All experiments were repeated three times. Differences between groups were analyzed using one-way analysis of variance and Bonferroni's Multiple Comparison. Statistical analysis was performed using GraphPad Prism 5 software (GraphPad Software, Inc., San Diego, CA, USA). P<0.05 was considered to indicate a statistically significant difference between values.

In order to study the role of Eaf2 in UV-induced formation of cataracts in mice, UV was used to irradiate the crystalline lenses of WT mice and Eaf2 KO mice, and a TUNEL assay was used to quantify the apoptosis in the crystalline lenses. For Eaf2 KO and WT mice, the apoptosis rates of the crystalline lenses after receiving UV irradiation were higher than those in animals that were not exposed to UV (Fig. 1). These results indicated that UV induced apoptosis in the crystalline lenses. In addition, the apoptotic rates in crystalline lenses in the Eaf2 KO-UV group were lower than those in the WT-UV group (Fig. 1). These results implied that Eaf2 knockout reduces UV-induced apoptosis in the crystalline lenses, thereby mitigating the formation of UV-induced cataracts.

To further study the role of Eaf2 in UV-induced cataract formation in mice, the activities of caspase-3 and caspase-9 were examined. In WT-UV and Eaf2 KO-UV mice, after UV irradiation, the activities of caspase-3 and caspase-9 were significantly increased. Compared with WT-UV mice, Eaf2 KO-UV mice had signifi-cantly lower caspase-3 and caspase-9 activities (Fig. 2A and B; P<0.01). These results suggested that Eaf2 knockout can reduce UV-induced apoptosis, which was consistent with the results of the TUNEL assays. Protein levels of bax, bcl-2, ERK and p-ERK were also detected by western blotting. In WT-UV mice, bax protein levels were increased, while bcl-2 and p-ERK protein expression levels were decreased as compared with those in the WT-nonUVmice. Of note, compared with WT-UV mice, Eaf2 KO-UV mice had decreased bax levels and elevated bcl-2 and p-ERK levels (Fig. 2C). These results, on the molecular level, suggested that Eaf2 knockout can reduce UV-induced apoptosis in crystalline lenses.

To investigate the role of Eaf2 in UV-induced apoptosis in crystalline lenses, a plasmid named Eaf2 OE to over-express Eaf2 in murine crystalline lens cells was constructed and the over-expression efficiency was detected by RT-qPCR and western blotting. The RT-qPCR results showed that after transfection with Eaf2 OE, the Eaf2 mRNA levels were significantly increased (Fig. 3A; P<0.01). The western blotting results also showed that after transfection with Eaf2 OE, Eaf2 protein levels were significantly increased (Fig. 3B; P<0.01). These results indicated that transfection of Eaf2 OE effectively increased the levels of Eaf2 in murine crystalline lens cells.

The TUNEL assay was used to detect cell apoptosis after transfection with Eaf2 OE. Compared with non-transfected (Control) cells and cells transfected with empty vector (Vector), cells transfected with Eaf2 OE exhibited a significantly increased level of apoptosis (Fig. 4A). Western blotting was then used to examine protein levels of cleaved caspase-3 and cleaved caspase-9. After transfection with Eaf2 OE, the levels of cleaved caspase-3 and cleaved caspase-9 were significantly increased (Fig. 4B; P<0.01), indicating an increase in the activation of caspase-3 and caspase-9. Western blotting was also used to detect the protein levels of the pro-apoptotic protein bax and the anti-apoptotic protein bcl-2. In cells over-expressing Eaf2, bax expression was significantly elevated, whereas bcl-2 expression levels were significantly reduced (Fig. 4C; P<0.01). These results illustrated that Eaf2 promoted the activation of caspase-3 and caspase-9 and affected the expression of apoptosis-associated proteins, thereby inducing apoptosis.