Silencer select pre designed sirna

siRNAs were transfected into cells according to the protocol recommended for Lipofectamine^® RNAi MAX reagent (ThermoFisher Scientific Japan Ltd., Tokyo, Japan). MLEC and HLECs were transiently transfected with siRNAs against a mixture of mouse, and human Tpm1 (Silencer^® Select pre‐designed siRNA, ID:s75390, ThermoFisher Scientific Japan Ltd.) and mouse and human Tpm2 (Silencer^® Select pre‐designed siRNA, ID:s75393) or negative control (Silencer^® Select Negative control#1 siRNA: NC‐siRNA). The final concentrations of the siRNAs were 10 nM.

ON-TARGETplus SMART pool siRNA (GE Healthcare, Little Chalfont, UK) and Silencer Select Pre-designed siRNA (Thermo Fisher Scientific, Waltham, MA, USA) were used for the knockdown of MYC expression. ON-TARGETplus Non-targeting Pool (GE Healthcare) or Silencer Select Pre-designed siRNA (Thermo Fisher Scientific) was used as a control. HT-29 cells were seeded at a density of 8×10⁴ cells/well on a six-well plate for immunoblot analysis and at a density of 3×10³ cells/well on a 96-well plate for cell viability assay, and were transfected for 24 h with 20 nM of each siRNA in Opti-MEM (Thermo Fisher Scientific) with lipofectamine RNAiMAX (Thermo Fisher Scientific), in accordance with the manufacturer’s reverse transfection protocol. After 48 h, the cells were used for further experiments.

To silence RhoB, we used Silencer Select Predesigned siRNA (catalog No. 4390817, locus ID 388, Ambion, Austin, TX). As a negative control we used non-targeting control siRNA Silencer Select Predesigned siRNA (Negative control #1, catalog No. 4390844, Ambion). Cells were transfected at a confluency of 30–50% in six-well dishes, using Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer’s protocol for forward transfection. In forward transfections, cells are plated in the wells, and the transfection mix is prepared and added the next day. Twenty-four hours after transfection cells were replated for subsequent experiments, evaluation of Ad5-mediated transgene expression or Western blot. Silencing of RhoB expression was verified by Western blot on whole cell lysates 48 hours after transfection using polyclonal antibody against RhoB (Santa Cruz Biotechnology, Santa Cruz, CA). At the same time cells were infected with Ad5 vectors for measurement of Ad5-mediated transgene expression.

The podocytes were subjected to transient transfection with silencing RNAs (siRNAs) to inactivate RAB3A and RAB27A genes, using Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. Pre-designed siRNAs were utilised (Ambion™ Silencer™ Select Pre-designed siRNAs (Ambion™ Silencer™ Select Pre-designed siRNA) for RAB27A gene (ID: s11693) and a pool of two siRNAs for RAB3A (ID: s11667 and s11668) were employed. To control the correct transfection efficiency, we used Cy3™ Dye-Labeled Negative Control (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). As a negative control or scramble (scr), we used Silencer™ Select Negative Control siRNA (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), and as a positive control we used a siRNA from the constitutively expressed gene GAPDH (Silencer™ Select GAPDH siRNA, 4404024, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) (Supplementary Figure S1). After 24 h, the transfection medium was replaced by RPMI at 1% P/S and ITS. Subsequently, D-(+)-Glucose (Sigma Aldrich, St. Louis, MO, USA) was added at normal (NG; 5.5 mM) and high (HG; 30 mM) concentrations, and the cells were incubated for 24 h at 37 °C.

For the knockdown expression studies, we used four cell lines (143B, MG63, KHOS, and U2OS). XBP1 siRNA knockdown was also performed using pre-designed XBP1 siRNA (sc-38627: Santa Cruz or siRNA negative control, Sigma-Aldrich), EIF2A siRNA (s38344, s38345: Silencer™ Select Pre-Designed siRNA or AM4611: Invitrogen™ Silencer™ Negative Control No. 1 siRNA), and ATF4 siRNA (s1702, s1703: Silencer™ Select Pre-Designed siRNA or AM4611: Invitrogen™ Silencer™ Negative Control No. 1 siRNA) using Lipofectamine™ RNAiMAX reagent (Thermo Fisher Scientific). After 72 h, RNA from each cell line was isolated, and its expression was validated using quantitative real-time PCR.