Dlx6-os1 Overexpression and Knockdown in Podocytes

The lncRNA Dlx6-os1 (full length)-overexpressing, specific knockdown shRNA, and control adenovirus (Supplementary Methods and Materials) used in this study were designed and synthesized by Hanbio Co., Ltd. (Shanghai, China). Podocytes were seeded in six-well plates at 60–70% confluence and infected with adenovirus following the manufacturer’s instructions (4 × 10⁶ podocytes; MOI = 100). The media was replaced with fresh ones after 6–8 h of viral infection. Mouse miR-346 mimics and mutants were provided by Bo Rui Biotech. Co., Ltd. (Guangzhou, China), and transfected into cells in the presence of Lipofectamine 3000 (Thermo Fisher Scientific, CA, USA). The lentivirus carrying a mouse Dlx6-os 1 fragment (exon 3 of lncRNA dlx6-os1 containing binding sites for miR-346) and the lentivirus with specific shRNA to induce knockdown were also constructed by Hanbio Co., Ltd. (Shanghai, China). The information on the vectors is listed in the Supplementary Methods and Materials section. The lentivirus was used to treat db/m and db/db mice via tail vein injections (one single injection: virus titer 10⁸ TU/ml; 80–100 µl per mouse). The sequences of the adenovirus and lentivirus packaging fragments are listed in the Supplementary Methods and Materials. Both the overexpression and shRNA lentiviruses carried the promoter for podocin (nphs-2), a podocyte marker protein. The control lentivirus was HA-tagged and also carried the promoter for podocin (nphs-2). The transfection efficiency of lentivirus with a podocyte-specific promoter in the podocytes of glomeruli was analyzed by immunofluorescence staining showing the expression of HA-tag and podocyte marker protein podocin in the OCT-embedded frozen kidney tissues.