Sigenome non targeting sirna control pool

Pre-designed siRNA against rat dnm1l and human DNM1L (gene encoding Drp1) were purchased from Dharmacon Research, Inc. SMARTpool: siGENOME Rat Dnm1l siRNA was used for the N27 cells and SMARTpool: siGENOME Human Dnm1l siRNA was for HeLa cells. Each of this product is a mixture of four individual siRNA duplexes that target four separate sequences of the gene to maximize efficiency of gene silencing. To enhance transfection efficiency, an “in-tube” transfection procedure [15 (link)] was used with the following modifications: Cell suspension (80,000–100,000cells/ml) were mixed with jetPRIME™ DNA and siRNA Transfection Reagent (Polyplus-transfection®SA). For every 500 μL of cell suspension (RPMI + 10% FBS), 50 μL of JetPRIME buffer and 2 μL JetPRIME reagent were used. Cells were plated and left in transfection medium overnight, then media was changed the following day. Gene silencing efficiency was confirmed using western blot, with 10 nM of siRNA achieving 75–90% knockdown compared to scrambled control (siGENOME Non-Targeting siRNA Control Pools, Cat# D-001206, Dharmacon Inc) after 48 h.

2.5 × 10⁵Gsdmd-deficient iBMDMs were seeded per well of a six-well plate and incubated overnight. For the siRNA transfection, the medium was changed to OptiMEM, and siRNA transfection was carried out according to the manufacturer’s protocol, transfecting 25 pmol siRNA (non-targeting: siGENOME non-targeting siRNA control pools [D-001206-14; Dharmacon], caspase-3: Casp3 SMART POOL [M-043042-01; Dharmacon], and caspase-7: Casp7 SMART POOL [M-057362-01; Dharmacon]) with 7.5 μl Lipofectamine RNAiMax (Invitrogen) per well. Medium was exchanged for DMEM (10% FCS, 10% MCSF, 1% NeAA, and 1% Hepes) after 6 h. 48 h post-transfection, the cells were collected and reseeded in a 96-well plate at 3 × 10⁴ cells/well. The cells were primed and treated as in cell death assays.

Drp1 knockdown in HeLa and N27 cells were performed as previously described [13 (link)]. Briefly, pre-designed siRNA against human DNM1L (gene encoding Drp1) and rat Dnm1l were purchased from Dharmacon Research, Inc (now available through Horizon Discovery). SMARTpool: siGENOME Human DNM1L siRNA (Cat# 10,059) was used for HeLa cells, while SMARTpool: siGENOME Rat Dnm1l siRNA (Cat# 114,114) was used for N27 cells. Each of this product is a mixture of four siRNA targeting a single gene to enhance efficiency and specificity of Drp1-knockdown. Similarly, the scramble control, siGENOME Non-Targeting siRNA Control Pools (Cat# D-001206, Dharmacon Inc.), was designed to have a minimum of four mismatches to all human, mouse and rat genes, and confirmed by the manufacturer to have minimal targeting by genome-wide microarray analysis. To monitor LC3 levels in N27 cells, mCherry-hLC3B-pcDNA3.1 plasmid was used. All the transfection was performed using Lipofectamine™ 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions.