Lens epithelium-derived growth factor

Cell lysates boiled in LDS sample buffer with reducing agent (Fisher Scientific, Pittsburgh, PA, USA, Cat# NP0004) were loaded into individual lanes (20 μg/lane) of 4–12% bis-tris SDS-polyacrylamide gels and electrophoresed in 1X MOPS running buffer (Fisher Scientific, Pittsburgh, PA, USA, Cat# NP0001) containing antioxidant (Fisher Scientific, Pittsburgh, PA, USA, Cat# NP0005) at constant 175 volts for 70 min. Proteins were then transferred to Immobilon-FL polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Burlington, MA, USA, Cat# IPFL00010) in transfer buffer (Fisher Scientific, Pittsburgh, PA, USA, Cat# NP00061) for 90 min at constant 25 volts. Protein transfer was assessed by Ponceau S staining. Membranes were blocked for 1 h in tris-buffered saline containing 0.05% tween-20 (TBS-T) and 5% dry milk followed by incubation with primary antibodies for 12 h at 4 °C. Primary antibodies included mouse monoclonal anti-GR (BD Biosciences, Franklin Lakes, NJ, USA, Cat# 611226,) and anti-total β-catenin (BD Biosciences, Franklin Lakes, NJ, USA, Cat# 610153); rabbit monoclonal anti-non phosphorylated (active) β-catenin (Cell Signaling Technology, Danvers, MA, USA, Cat# 8814S, clone D13A1), anti-AR (Cell Signaling Technology, Danvers, MA, USA, Cat# 5153S, clone D6F11) and anti-AR-V7 (Cell Signaling Technology, Danvers, MA, USA, Cat# 19672S, clone E3O8L); rabbit polyclonal anti-histone H3 (GeneTex, Irvine, CA, USA, Cat# GTX122148), anti-MDR1/ABCB1 (Cell Signaling Technology, Danvers, MA, USA, Cat# 13342S, clone E1Y7B), and anti-Lens Epithelium Derived Growth Factor p75 (LEDGF/p75, Bethyl Laboratories/Fortis Life Sciences, Montgomery, TX, USA, Cat# A300-848A); rabbit monoclonal anti-GAPDH (Cell Signaling Technology, Danvers, MA, USA, Cat# 2118S, clone 14C10) and anti-alpha/beta-tubulin (Cell Signaling Technology, Danvers, MA, USA, Cat# 2148S), or horseradish peroxidase (HRP)-conjugated rabbit monoclonal anti-β-actin (Cell Signaling Technology, Danvers, MA, USA, Cat# 5125S clone 13E5). After incubation with primary antibodies, membranes were washed with TBS-T, followed by incubation for 1 h with appropriately diluted HRP-linked secondary antibodies, anti-rabbit IgG (Cell Signaling Technology, Danvers, MA, USA, Cat# 7074S), or anti-mouse IgG (Cell Signaling Technology, Danvers, MA, USA, Cat# 7076S). Membranes were washed with TBS-T, and immunoreactive bands detected with SuperSignal West Pico PLUS Chemiluminescent reagents in autoradiography film (Midwest Scientific, Fenton, MO, USA, Cat# XC6A2). Signals were quantified using ImageJ (https://imagej.nih.gov/ij/ (accessed on 23 March 2023)).

This research was carried out at King Abdullah University Hospital (KAUH) between December 2020 and June 2022. KAUH is a university hospital affiliated with Jordan University of Science and Technology (JUST), and it is a tertiary referral center for surgical vitreoretinal cases in the North of Jordan. The study was approved by the IRB committee at KAUH (Approval No. 58/137/2021). Patients who had been scheduled to undergo pars plana vitrectomy (PPV) for any reason were asked to obtain a vitreous sample during the vitrectomy. The vitreous samples were tested for vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). All patients gave written informed consent, and the study was conducted according to the guidelines of the Declaration of Helsinki and its subsequent amendments. The patients’ age, sex, past ophthalmic history, previous medical history, and co-morbidities were recorded. Additionally, the indication for PPV, the type of tamponade and the associated surgical procedures were documented. Lastly, the visual outcomes were measured.
The included participants were those patients who had been scheduled to undergo PPV. The exclusion criteria included patients who had previously undergone vitrectomy, patients with occlusion of the retinal veins, those who had less than two months duration of vitreous hemorrhage (VH), cases where obtaining a vitreous sample can result in a complication, patients who had received prior chemotherapy, and those who had previously undergone complicated cataract surgery where the fluid could dilute the vitreous.
Similar to our previous study, the participants were divided into two groups; cases vs. control [17 ]. The case group included patients with persistent VH and/or fibrovascular membranes (FVM) with tractional retinal detachment (TRD) (advanced proliferative diabetic retinopathy (PDR)). The modified Airlie house classifications was used for the grading [18 , 19 (link)].
The control group comprised patients with vitreomacular interface diseases (i.e., vitreomacular traction (VMT), macular hole (MH), and non-diabetic epiretinal membranes (ERM)), rhegmatogenous retinal detachment (RRD), endophthalmitis, and drop of crystalline lens materials and/or drop of intraocular lens implants after complicated cataract surgery.
Intravitreal anti-VEGF injections were used in most patients in the first group preoperatively. They were analyzed and correlated with the level of the biomarkers according to the time and the number of injections given in the case group. Either aflibercept or ranibizumab were utilized in this study. In addition, the effect of panretinal photocoagulation laser sessions was studied. The possible effect of the associated cataract surgery in some patients during PPV was also studied. Moreover, the type of retinal tamponade after the PPV was collected, which included silicone oil, gas (SF6), and air.
A special analysis was performed for RRD cases alone. Certain characteristic of RRD was investigated and related to PEDF level including macula status (either the macula was attached or not), the location of the tear (superiorly or inferiorly), and the duration of symptoms of RRD. Using Snellen decimal projectors, the best correct visual acuity (BCVA) was assessed. Visual acuity was converted to LogMAR visual acuity.

HIV-1 infection of HeLa P4 cells was performed as described before [7 (link)]. A total of 3 × 10⁴ cells per well were infected with HIV(NL4.3) in a6 well plate for 6 h. After 6 h, the virus was washed away with PBS (Invitrogen, Waltham, MA, USA) and replaced with 5 mL of medium. Replication of HIV-1 was monitored by quantifying p24 antigen in the supernatant via ELISA (Alliance HIV-1 p24 ELISA kit; PerkinElmer, Mechelen, Belgium).

All LEDGF constructs were transformed in E. coli BL21(DE3)pLysS (Oneshot, Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA) competent cells by heat shock and grown in Lysogeny broth (LB, Sigma, St. Louis, MO, USA) supplemented with 100 μg/mL ampicillin (Sigma, St. Louis, MO, USA), and 2 g/L glucose (Sgima, St. Louis, MO, USA) for MBP-tagged proteins. Bacterial cultures were grown at 37 °C (flag-tag) or 30 °C in case of His₆-tagged proteins. After an OD₆₀₀ of 0.6 was reached, protein expression was induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG, Sigma, St. Louis, MO, USA). Proteins were induced for four hours of induction at 30 °C for flag-tagged proteins, four hours of induction at 37 °C for GST-tagged proteins, and overnight induction at 18 °C for His₆-tagged proteins. After incubation, cultures were harvested by centrifugation for 10 min at 5000 rpm using the Fiberlite™ F12-6 × 500 LEX Fixed Angle Rotor (Thermo Fisher Scientific, Waltham, MA, USA). Pellets were washed in cold STE buffer containing 10 mM Tris-HCl (Sigma, St. Louis, MO, USA), pH 7.3, 100 mM NaCl (Sigma, St. Louis, MO, USA), and 0.1 mM EDTA (Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA), and centrifuged as described before. Pellets were stored at −20 °C.
Cells were resuspended in lysis buffer (50 mM Tris-HCl(Sigma, St. Louis, MO, USA), pH 7.4, 100 mM NaCl(Sigma, St. Louis, MO, USA), 1 mM dithiothreitol (DTT; Sigma, St. Louis, MO, USA), 0.1 μg/mL DNase (Thermo Fisher Scientific, Waltham, MA, USA) and protease inhibitor (cOmplete, EDTA-free, Roche, Basel, Switzerland) and lysed by sonication using the MSE 150 Watt Ultrasonic Disintegrator. The lysate was cleared by 30 min of centrifugation at 15,000 rpm using the Fiberlite™ F21 × 50y Fixed Angle Rotor (Thermo Fisher Scientific, Waltham, MA, USA) and 4 °C.
For Flag-tagged proteins, the cleared lysate was first filtered through a Millex-GS Syringe Filter Unit of 0.22 µm (Millipore, Burlington, MA, USA) and subsequently loaded on an Äkta Purifier (GE Healthcare, Chicage, IL, USA), equilibrated with running buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl and 1 mM DTT; all reagents from Sigma, St. Louis, MO, USA) and connected with a 5 mL HiTrap heparin column (GE Healthcare, Chicage, IL, USA). The protein was eluted by a NaCl gradient in 50 mM Tris, pH 7.5 (reagents from Sigma, St. Louis, MO, USA). Elution fractions were run on an in-house-made SDS-PAGE gel to select the correct LEDGF fraction, which was pooled and concentrated by use of an Amicon^® Ultra Centrifugal Filter, until volumes of maximum 1 mL. The sample was further purified on a Superdex 75 10/300 GL column (GE Healthcare, Chicago, IL, USA). At last, fractions were analyzed for protein content with an in-house-made SDS-PAGE gel and stained with Coomassie blue.
GST-tagged proteins were purified as described before [24 (link)].
For His₆-tagged proteins, 20 mM imidazole (Acros Organics, Geel, Belgium) was added to the lysis buffer. Purification by affinity chromatography was carried out on a Ni-NTA-resin (Thermo Fisher Scientific, Waltham, MA, USA). The resin was washed in 50 mM Tris-HCl (Sigma, St. Louis, MO, USA), pH 7.5, 150 mM NaCl (Sigma, St. Louis, MO, USA), and 20 mM imidazole (Acros Organics, Geel, Belgium), and His₆-tagged proteins were eluted in wash buffer with increased imidazole concentration (230 mM). The protein was additionally purified over a HiTrap Heparin HP resin (GE Healthcare, Chicage, IL, USA) equilibrated with 50 mM Tris-HCl, pH 7.5, and 150 mM NaCl (all reagents from Sigma, St. Louis, MO, USA). The protein was eluted by a NaCl gradient in 50 mM Tris, pH 7.5, using the FPLC Äkta Purifier system (GE Healthcare, Chicage, IL, USA).
MBP-tagged proteins were purified by applying the supernatant onto 2 mL of previously washed (50 mM Tris-HCl (Sigma, St. Louis, MO, USA), pH 7.2, 500 mM NaCl (Sigma, St. Louis, MO, USA), 1 mM EDTA (Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA) amylose resin (NEB, Ipswich, MA, USA). Here, 10 mM maltose was supplemented to the wash buffer for elution. Peak fractions were pooled and maltose was removed by dialysis against 50 mM Tris-HCl (Sigma, St. Louis, MO, USA), pH 7.2, 500 mM NaCl (Sigma, St. Louis, MO, USA), and 10% (v/v) glycerol (Acros Organics, Geel, Belgium).
HIV-1 integrase was expressed from pKB-IN6H and grown in similar conditions as LEDGF constructs, except for the addition of 25 mg/L chloramphenicol (Sigma, St. Louis, MO, USA) in LB medium (Sigma, St. Louis, MO, USA). Protein expression was induced after addition of 0.5 mM IPTG (Sigma, St. Louis, MO, USA) at an OD₆₀₀ of 0.8. The bacteria were grown for 3 h at 29 °C and harvested by spinning down for 30 min at 5000 rpm using the Fiberlite™ F12-6 × 500 LEX Fixed Angle Rotor (Thermo Fisher Scientific Waltham, MA, USA). Pellets were washed in STE buffer, and stored at −20 °C. The cells were lysed in buffer containing 25 mM HEPES (Sigma, St. Louis, MO, USA), pH 7.4, 1 M NaCl (Sigma, St. Louis, MO, USA), 1 mM MgCl₂ (Sigma, St. Louis, MO, USA), 7.5 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS; VWR, Radnor, PA, USA), and 3 mM dithiothreitol (DTT; Sigma, St. Louis, MO, USA)), supplemented with 0.1 μg/mL DNase (Thermo Fisher Scientific, Waltham, MA, USA), protease inhibitor (cOmplete, EDTA-free, Roche, Basel, Switzerland), and 20 mM imidazole (Acros Organics, Geel, Belgium), and lysed by sonication (MSE 150 Watt Ultrasonic Disintegrator). The lysate was cleared by 30 min of centrifugation at 15 000 rpm, using the Fiberlite™ F21 × 50y Fixed Angle Rotor (Thermo Fisher Scientific Waltham, MA, USA), and 4 °C, after which the supernatant was applied onto 2 mL of previously washed (25 mM HEPES (Sigma, St. Louis, MO, USA), pH 7.4, 1 M NaCl (Sigma, St. Louis, MO, USA), 1 mM MgCl₂ (Sigma, St. Louis, MO, USA), 7.5 mM CHAPS (VWR, Radnor, PA, USA), and 3 mM DTT (Sigma, St. Louis, MO, USA) Ni-NTA-agarose (Qiagen, Hilden, Germany). The protein was eluted with wash buffer supplemented with 180 mM imidazole. Pooled peak fractions were cleared from imidazole by dialysis against wash buffer containing 10% glycerol (Acros Organics, Geel, Belgium).
MLL1_1–160-GST was expressed from the pET-20b MLL1_1–160-GST plasmid and grown similarly as LEDGF constructs. After 5 h of induction, cells were harvested by centrifugating for 10 min at 5000 rpm using the Fiberlite™ F12-6 × 500 LEX Fixed Angle Rotor (Thermo Fisher Scientific, Waltham, MA, USA). After washing the pellet in STE buffer, the pellet was stored at −20 °C. Glutathione Sepharose-4 Fast Flow (GE Healthcare, Chicago, IL, USA) was used to selectively purify the GST-tagged protein after the cells were sonicated in buffer containing 50 mM Tris-HCl (Sigma, St. Louis, MO, USA) pH 7.3, 150 mM NaCl (Sigma, St. Louis, MO, USA), 1 mM DTT (Sigma, St. Louis, MO, USA), 0.1 μg/mL DNase (Thermo Fisher Scientific, Waltham, MA, USA) and protease inhibitor (cOmplete, EDTA-free, Roche, Basel, Switzerland). The resin was equilibrated with wash buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl; all reagents from Sigma, St. Louis, MO, USA). Proteins were eluted in wash buffer with 25 mM glutathione (Acros Organics, Geel, Belgium). The protein was further purified over a HiTrap Heparin HP resin (GE Healthcare, Chicago, IL, USA) equilibrated with 50 mM Tris-HCl pH 7.5, 150 mM NaCl, and 1 mM DTT (all reagents from Sigma, St. Louis, MO, USA). The protein was eluted by a NaCl gradient in 50 mM Tris pH 7.5 using the FPLC Äkta Purifier system (GE Healthcare, Chicago, IL, USA).
Elution fractions were analyzed for protein content on SDS-PAGE, followed by a Coomassie (Brilliant blue G 250, Merck, Darmstadt, Germany) staining. All selected protein fractions were pooled, and if not dialyzed, supplemented with 10% (v/v) glycerol (Acros Organics, Geel, Belgium) before aliquoting and snap-freezing. Protein samples were stored at −80 °C. Coomassie stains of the used proteins are available in supporting Figure 1. The amount of DNA was measured using the Qubit 4 (Thermo Fisher Scientific, Waltham, MA, USA) and the 1× Qubit dsDNA HS assay kit (Thermo Fisher Scientific, Waltham, MA, USA).