Optimem serum reduced medium

To overexpress ZBTB16 in HESCs, the expression vector containing the human full-length ZBTB16 gene open reading frame (pCMV6-ZBTB16) was purchased from Origene Inc. (Rockville, MD, USA). Cultured HESCs seeded in 6-well plates were transiently transfected with either the ZBTB16 or empty (pCMV6 vector as the control) vector using the Lipofectamine LTX reagent (Invitrogen, Carlsbad, CA, USA) in Opti-MEM serum reduced medium (Invitrogen) according to the manufacturer’s instructions. After transfection for 4 h, HESCs were incubated with/without EMC media for 72 h. In a parallel set, ZBTB16- or control-vector-transfected HESCs cultures ± EMC were treated with 1 U/mL of thrombin (American Diagnostic, Greenwich, CT, USA) for 6 h and stored at −80 °C for RNA isolation. The transfection efficiency for ZBTB16 overexpression was confirmed by qPCR and immunoblotting.

After thermos switching to 37 °C, immortalized human podocytes were allowed to be differentiated for 8–10 days prior to transfection. Prior to transfection, cells were incubated in Opti-Mem serum-reduced medium (Gibco, via ThermoFisher Scientific, Waltham, MA, USA) for 4 h. Podocytes were transfected with mirVana^® miRNA mimic (hsa-miR-200c-3p from ThermoFisher (Scientific, Waltham, MA, USA) at a concentration of 100 nM) using Lipofectamine 2000 (Invitrogen via ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. A random sequence was used as negative control, (miRNA MimicNegative Control #1, ThermoFisher Scientific, Waltham, MA, USA). After 4 h of transfection, media was replaced with podocyte culture medium, as above, and cells were further incubated for 48 h.
Human glomerular endothelial cells were seeded 2 days prior to transfection. The same transfection procedure as described above was also used for human glomerular endothelial cells. Cell viability and morphology were determined on an optical microscope before and after every treatment.
hsa-mir-200c-3p sequence: UAAUACUGCCGGGUAAUGAUGGA.

For all transfections, HEK293T cells were seeded at a density of 3.0 × 10⁵ cells per well in BioCoat poly-d-lysine coated 48-well plates (Corning, 356509) 16–22 h before transfection. Plasmid transfections were performed using Lipofectamine 2000 (Invitrogen, 11668-019) as previously described.^{13 (link)} Transfections with mRNA were performed using Lipofectamine MessengerMAX in accordance with manufacturer protocols, with the following specifics: 500 ng (for saturating conditions) or 62.5 ng (subsaturating conditions) of mRNA encoding for editor or control and 100 ng of synthetic gRNA were combined in 12.5 μl total volume of OptiMEM serum reduced medium (Gibco, 31985). A 12.5 μl 1:12.5 (Lipo:OptiMEM) MessengerMAX mixture was then added to the mRNA/gRNA solution, and the entire contents were left to rest at ambient temperature for 15 min. For mRNA transfections at subsaturating conditions, 437.5 ng of carrier mRNA was also added to maintain equivalent amounts of transfected material. The entire 25 μl mixture was then used to treat the preseeded HEK293T cells. The sequences of sgRNAs used in this study are specified in Supplementary Table 3. Synthetic gRNAs for mRNA transfections have 5′/3′ end-modifications as previously described.^{13 (link)}

PHH cocultures were transfected 48 h after coculture generation with 3T3-J2 murine fibroblasts. Transfections with mRNA were performed using Lipofectamine MessengerMax (Thermo Fisher Scientific, LMRNA003) in accordance with manufacturers’ protocols, with the following optimized specifics: 1 µg (for saturating conditions) of mRNA encoding for editor and 333 ng of synthetic gRNA (Synthego) were combined in 30 µl of OptiMEM serum reduced medium (Gibco, 31985). A 30 µl 1:15 (Lipofectamine:OptiMEM) mixture was added to the mRNA/gRNA solution with the resulting final mixture left to rest at ambient temperature for 15 min. The entire 60-µl solution was used to treat a well of cocultured primary human hepatocytes. Each study condition was run in triplicate and transfection amounts used were scaled up accordingly. At 9 d post-transfection, the PHH cocultures were lysed with a solution of 10 mM Tris–HCl pH8.0 (Thermo Fisher Scientific, 15568025), 0.05% SDS (Thermo Fisher Scientific, 15553027) and 500 µg proteinase K (Thermo Fisher Scientific, EO0491) at a total of 200 µl per well. Once lysed, lysate was treated at 85 °C for 15 min to inactivate proteinase K. The sequences of sgRNAs used in this study are specified in Supplementary Table 3.

The rats' retinal microvascular endothelial cells were purchased from the American Type Culture Collection (ATCC) and used to verify the exact function of lncZNRD1-AS1. The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM)/F-12 medium (12634028, Gibco, USA) supplied with 20% fetal bovine serum (FBS, 10099141C, Gibco, USA), 1% penicillin-streptomycin (TMS-AB2, Sigma, USA), 1% glutamine (G7513, Sigma, USA), and 50 μg/l vascular endothelial growth factor (VEGF, LL-0003, lifeline, USA).
Specific small-interfering RNAs (siRNAs) targeting lncZNRD1 were synthesized and purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). The cells were inoculated on a 6-well plate, and the cell density reached 60-80% confluence before the transfection of siRNA (200 nM). The mixture of transfection reagents of Lipofectamine ™ 2000 transfection reagent (11668019, Invitrogen; Thermo Fisher Scientific, Inc.), Opti-MEM™ Serum reduced medium (51985034, Gibco; Thermo Fisher Scientific, Inc.), and siRNA was slowly dripped into the 6-well plate. After being cultured at 37°C and 5% CO₂ for 4-6 h, the mixed transfection reagents were replaced with complete DMEM (without antibiotics), and the following experiment was carried out after 48 h of transfection.