Amaxa nucleofector 1 device

STAT3 shRNA plasmid clones (Human pTRIPZ vector) were purchased from Open Biosystems (Lafayette, CO, USA) and used to transfect JVM-3 cells. Nucleofection was performed using the Amaxa Nucleofector I device. JVM-3 cells (3×10⁶ cells) were resuspended in 100 μl of Cell line Solution Kit V (Amaxa, Seattle, WA, USA) and nucleofected with 6 μg of shRNA using the Amaxa Nucleofector I device (program X-001). Transfection efficiency by this protocol was ~80% as measured by a comparable GFP plasmid provided by the manufacturer.

For RNA interference assays, human monocytes (5 × 10⁶ cells) were transfected with RAGE (Thermo Fischer Catalog # AM16708 ID: 110857, 200 nM) or CD32 siRNA (Thermo Fischer Catalog #4392420 ID: s194407, 200 nM) using an Amaxa Nucleofector I Device (Lonza). Transfection efficiency was >40%. The efficiency of the knockdown was determined by immunofluoresence microscopy and q-PCR. Following transfection, cells were cultured for 48 h in 24 wells tissue culture plates in X-VIVO 15 culture medium, and then washed and incubated with indicated under indicated conditions.

WT and SK2KO BMMs were transfected with siRNA using the Amaxa nucleofector I device (Lonza, Basel, Switzerland) using Amaxa Cell Line Nucleofector Kit T (Lonza). Four–5 × 10⁶ cells were resuspended in 100 µl nucleofector solution. Control scrambled all-stars negative siRNA (Qiagen) or a pool of four different siRNA sequences targeting RhoA (Qiagen) were added to a final concentration of 2 µM. Cells were added to cuvettes and nucleofected using program T-20. Cells were transferred to prewarmed culture media and plated into tripartition petri dishes and cultured for 48 h. Cell solutions were trypsinized and plated into coverslip microscopy dishes for microscopy experiments or six-well plates overnight for assessment of knockdown efficiency. Typically, RhoA protein levels 72 h posttransfection were reduced ≥60% by RhoA siRNA in comparison with scrambled siRNA control.