Dharmafect sirna transfection reagent

For transient transfection, cells were seeded in 35 mm Petri dishes (5 × 10⁴ cells) and allowed to attach for 24 h. Transfections were carried out in 1 ml Opti-MEM using DharmaFECT siRNA transfection reagent (Thermo Fisher Scientific, Dharmacon Products, Lafayette, CO, USA), according to the manufacturers’ protocols. After 6 h incubation with the Akt1 and Akt2 siRNA or the synthetic control oligonucleotides (100 nM final concentrations), 1 ml of 10% FBS containing medium was added. After 48 h, 72 h and 120 h following transfections, cells were harvested and lysed for Western blot analysis.
For stable Akt1 and Akt2 silencing, cells were seeded at a density of 20,000 cells/well into 96-well plates and allowed to attach for 24 h. Cells were transduced with SMARTvector 2.0 Lentiviral shRNA particles targeting Akt1, Akt2 or SMARTvector 2.0 Non-Targeting control particles (Dharmacon Thermo Scientific, US). Selection of cells stably expressing Akt1-, Akt2-shRNA and the Control-shRNA started 72 h post-transfection. Growth medium was replaced with fresh selection medium containing 10 μg/mL of puromycin. Puromycin-containing medium was refreshed every 2–3 days, and selection of stable cells expressing Akt1-shRNA, Akt2-shRNA or Control-shRNA was completed after approximately 4 weeks. Multiple clones and pools of clones were expanded, harvested, and prepared for western blot analyses.

The immortalized mouse podocyte cell line (a kind gift from Dr. Peter Mundel, Harvard Medical School, USA) and HEK293FT (human embryonic kidney 293) cells were cultured as previously described^{8 (link)}. All cells tested as “mycoplasma-negative.” All DNA plasmids were transfected using X-tremeGENE^Ⓡ HP DNA transfection reagent (Roche, Mannheim, Germany) according to the manufacturers’ instructions. Experiments were conducted 48 h after transfection. Mouse siRNAs for Orai1 (M-056431-01), Orai2 (M-057985-01), Orai3 (M-054417-01), STIM1 (M-062376-01), STIM2 (M-055069-01), and VAMP2 (M-041975-01) were obtained from Dharmacon (Chicago, IL, USA). Non-targeting control oligonucleotide (sc37007) and mouse siRNA for TRPC6 (sc42673) were provided by Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Transfection of siRNA oligonucleotide was performed using DharmaFECT siRNA transfection reagent (Thermo Scientific, Lafayette, CO, USA) according to the manufacturer’s instructions.

Suppression of AKT1 function was performed by knockdown of AKT1 expression by AKT1 small interfering RNA (AKT1-siRNA) (cat# M-004364-00, Cell Signaling Technology). Transient transfections were done with cells at 60% confluence using Dharmafect siRNA transfection reagent according to the manufacturer’s (Thermo Scientific) instructions. Briefly, MLE-12 cells were seeded in six-well plates at 1.5×10⁵ cells/well and incubated at 37°C for 24 h. Each of AKT1-siRNA or non-target control siRNA (100 nmol) was transfected into the cells with 7.5 μl of Dharmafect reagent in serum-free media without antibiotics and incubated for 6 h at 37°C. After removal of siRNA transfection reagent, medium with fetal bovine serum was added and incubated further for 18 h. Cell cultures were subsequently exposed to room air or hyperoxia. The efficacy of AKT1 knockdown and its effects on Nrf2 target gene expression were assessed by real-time RT-PCR analysis.

Human survivin cDNA and Akt cDNA were obtained by RT-PCR using HCT116 and HT29 cell RNA and primers based on GenBank sequences (accession numbers U75285 [48 (link)] and NM-001014431.1, [66 (link)] respectively). PCR products were digested with EcoRI and XhoI (Takara Tokyo, Japan), and ligated into the pcDNA3.1-myc vector (Invitrogen). Transcription was specifically suppressed by the introduction of the 21-nucleotide duplex siRNA, which targeted survivin mRNA coding sequence. Briefly, HCT116 cells were plated in 6-well plates (2 × 10⁵ cells/well) and transiently transfected with 100 nM per well of survivin siRNA (Santa Cruz) mixed with the DharmaFECT siRNA transfection reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. Silencer Negative Control siRNA (Santa Cruz) was used as a negative control and introduced into the cells under the same protocol. After 5-h incubation, the medium was changed to complete culture medium, and the cells were incubated at 37°C in a CO₂ incubator for 48 h before harvesting.

The small interfering RNA (siRNA) specific for SAMHD1 (sc-76442), for Cdk1 (sc-29252), Cdk2 (sc-29259) and the non-targeting siRNA (siCtrl) (sc-37007) were from Santa Cruz Biotechnology. Three different siRNAs (1: AF520960.1s, AF520960.1as; 2: AF520960.2s, AF520960.2as; 3: AF520960.5s, AF520960.5as) targeting the UL97 gene were purchased from Sigma-Aldrich. HFFs (90% confluence) were transfected with 100–300 nM siRNA using DharmaFECT siRNA transfection reagent (Thermo Fisher Scientific), according to the manufacturer’s recommendation. One or two days after transfection, cells were infected with AD169 at the indicated MOI, and then cells and supernatants were harvested and analyzed as indicated.