Block it u6 rnai entry vector kit

Adenoviral vectors expressing shRNA against β-Galactosidase (βGal) or RetSat were cloned using the BLOCK-iT U6 RNAi Entry Vector Kit and Adenoviral RNAi Expression System according to the manufacturer’s instructions (ThermoFisher). Adenoviruses expressing GFP or a GFP- murine ChREBP fusion protein are described elsewhere^{25 (link)}. Viruses were amplified in HEK293 cells (Clontech) and purified by standard CsCl gradient centrifugation and dialyzed against 0.9% saline. Titers were determined by the Adeno-X Rapid Titer Kit (Clontech). For liver-selective RetSat depletion, ~10E10 infectious units of shβGal-or shRetSat-expressing adenoviruses were injected via the tail vein into lean or obese, insulin-resistant mice.

Three pairs of siRNA primers (Z1, Z2, Z3) targeting human LOCCS were synthesized and purified by Shanghai Haike Corporation. Annealing was performed in a 10 μL reaction mixture including 4.5 μL forward primer (50 μM), 4.5 μL reverse primer (50 μM) and 1 μL annealing buffer at 95 °C for 5 min and decreased to 30 °C gradually (0.1 °C/s). BLOCK-iT U6 RNAi Entry Vector kit (Invitrogen) was used for ligation in a 10 μL reaction volume containing 1 μL annealed primers, 1 μL pENTR/U6 plasmid, 1 μL T4 ligase buffer, 1 μL T4 ligase and 6 μL deionized H₂O, and the reactions were placed at 16 °C for 2 h. Then, 5 μL of the ligated product was added to 100 μL DH5X cell solution, and the mixture was placed at 4 °C for 10 min, 42 °C for 90 s, and 4 °C for 5 min. After 300 μL Luria-Bertani medium was added, the mixture was shaked at 220 rpm for 1 h. Finally, transformants were transferred to kanamycin-containing plates at 37 °C overnight. Kanamycin-resistant clones were chosen, and the plasmids were isolated using the lyticase method. The inserted sequences in the plasmid were verified by DNA sequencing. Spheroid cells were transfected with 500 ng of each of the three pENTR/U6-siLOCCS plasmids (Z1, Z2, Z3) with Fugene 6 Transfection kit (Roche). The transfected cells were harvested 48 h later, and expression level of miR-93 was mensurated using quantitative PCR.

pBABE-puro, pBABE-puro-PDK2 (pBABE-PDK2; Addgene, USA) and lentivirus-encoded short hairpin RNA (shRNA)-PDK2, shRNA-negative control (shNC) (Open Biosystems, Inc., USA) were constructed with BLOCK-iT U6 RNAi entry vector kit (Invitrogen, USA). MSCs were transfected with lentivirus in serum-free DMEM at 37°C for 4 h. Then, 10% DMEM was added to culture MSCs for the further studies.

Conditionally immortalised mouse podocytes were cultured as described previously^{25 (link)}. To generate Rac1 KD podocytes, we used a Gateway System (BLOCK-iT U6 RNAi Entry Vector Kit and BLOCK-iT Adenoviral RNAi Expression System; Invitrogen, CA, USA) following the manufacturer’s instructions. Briefly, adenoviral particles expressing Rac1 shRNA were produced in 293 A cells. LacZ non-silencing adenoviral shRNA was used as control. Differentiated cultured podocytes were transduced with the adenoviral particles and transduction was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and WB analysis. For the ADR-administration experiments, control and Rac1 KD podocytes were cultured in the presence of ADR at a concentration of 3 ng/ml for 0, 1, 3, 6, and 12 h. Podocytes were lysed on ice in lysis buffer, and then protein samples were subjected to SDS-PAGE and WB analysis. β-Actin was measured in cell lysates that were used as controls.