Tgf β2

EMCMs or neonatal mouse cardiomyocytes were treated with 1 ng/ml TGF-β2 (R&D Systems) and then were exposed to stretch conditions for 24 hours, or, to study long-term effects, were cultured in static conditions for 3 days, with daily changes of medium supplemented with 1 ng/ml TGF-β2 before analysis. Untreated cardiomyocytes were used as controls.

Primary human umbilical vein endothelial cells (HUVECs) were purchased from Lonza (batch p1032 and p1028) and were cultured in endothelial cell medium (ScienceCell, Carlsbad, CA, USA, 1001), supplemented with endothelial cell growth supplement (ECGS) (ScienceCell, Carlsbad, CA, USA, 1052), penicillin/streptomycin (ScienceCell, Carlsbad, CA, USA, 0503), and 5% fetal bovine serum (ScienceCell, Carlsbad, CA, USA, 0025). Primary HUVECs were cultured between passage 1 and 5 for experiments. Induction of EndMT in HUVECs was performed with IL-1β (10 ng/mL, R&D Systems, Minneapolis, MN, USA, 201-LB) and TGF-β2 (10 ng/mL, R&D Systems, 302-B2-002) for 72 h. EndMT conditions are defined as the HUVECs treated with IL-1β and TGF-β2. Hek293T cells were acquired from ATCC and cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher, Waltham, MA, USA, 31966021), supplemented with 10% FCS, 1% Pyruvate, 1% D-glucose, 1% penicillin/streptomycin and 1% minimum essential media with non-essential amino acid mix (Sigma-Aldrich, St. Louis, MI, USA, M7145). Hek293T and primary HUVECs were cultured at 37 °C with 5% CO₂. Cells were counted with the Countess II cell counter (Thermo Fisher). All cell types tested negative for mycoplasma.

As described previously, ACAID APCs were generated in vitro [7 (link),19 (link)]. ACAID APCs from C57BL6 and DBA/1 mice were generated as described before [20 (link)]. Briefly, at concentrations of TGF-β2 that are similar to those present in the aqueous humor of the eye, APCs (2 × 10⁶ cells/ml) were cultured overnight in complete RPMI 1640 with 10 mg/ml CII and 2–5 ng/ml TGF-β2 (R&D Systems).

All procedures involving animals were performed following the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research, and were approved by the Animal Use and Care Committee of Zhongshan Ophthalmic Center (Permit number: 2020-039; Approval date: 3 September 2020). Whole lenses of 21-day-old mice maintained in the C57BL/6J background were collected and cultured in serum-free M199 medium supplemented with 0.1% BSA, 0.1 μg/mL L-glutamine, 100 IU/mL penicillin, and 100 IU/mL streptomycin [33 (link),34 (link)]. Whole lenses from human donors were also freshly collected and cultured with the same media. After isolation, lenses were kept in the media for at least 24 h before any treatment. Those with cloudiness caused during surgical collection were excluded. After transfection or infection for 24 h, TGF-β2 (R&D Systems) was added to the medium (10 ng/mL). The medium was changed every other day. Mouse whole lenses were cultured for up to 6 days and photographed under a stereoscope before being harvested. Human whole lens explants were cultured for up to 10 days. RNA levels of H19 in lens epitheliums were further detected to ensure the efficiency of silencing or overexpressing agents.

CD4⁺CD25^– T cells were isolated from splenocytes using the CD4⁺CD25⁺ regulatory T Cell Isolation Kit (Miltenyi Biotec) as a negative fraction by MACS separator. The purity of the isolated cell population (>95%) was confirmed by flow cytometry.
CD4⁺CD25^– T cells (5 × 10⁵ cells/ml/well) were incubated with vehicle or HDAC6 inhibitor (1 to 10 μM) in the presence of antiCD3/CD28 beads (T cell Activation/Expansion kit, Miltenyi Biotec), and recombinant mouse transforming growth factor (TGF)-β2 (40 pg/ml; R&D systems Inc., Minneapolis, MN, USA) in a 48-well plate (Becton Dickinson) for 6 days at 37 °C in a humidified 5% CO₂ incubator.
For surface staining, cells were incubated with PE-Cy7-labeled antimouse CD4 (eBioscience), and APC-labeled antimouse CD25 (eBioscience) for 20 min at room temperature (RT). For intracellular staining, the cells were fixed and permeabilized with fix/permeabilization buffer (eBioscience) for 20 min at 4 °C, washed twice with wash buffer, and then incubated with AlexaFluor488-labeled antimouse forkhead box P3 (Foxp3) and PE-labeled antimouse CTLA-4 (eBioscience) for 20 min at 4 °C in the dark. CTLA-4 expression of induced Foxp3⁺ Treg cells was analyzed by FACS CantoII flow cytometry (BD bioscience, San Jose, CA, USA) and the results were analyzed with FlowJo software (TreeStar Inc. Ashland, OR, USA).

Recombinant human TGF-β1 and TGF-β2 was purchased from R and D Systems. miR-335 antagomirs were obtained from the following companies: antagomir one from Ambion; antagomir two from Exiqon (miRCURY LNA microRNA Power Inhibitor; 4100464–002), and antagomir three from Thermo Scientific Dharmacon (miRIDIAN hairpin inhibitor; IH-300708–07). miR-335 Mimic oligonucleotide was obtained from Exiqon (473600–001). The following chemical reagents were used for cell treatment: Erlotinib Hydrochloride 99% from LGM Pharmaceutical Inc, pyridone 6 (P6) from Calbiochem, Trichlostatin (TSA), and 5-aza-2-deoxycytidine from Sigma-Aldrich.