Gene pulser 2 with rf module

HDAC-specific and ScI control siRNAs (Supplementary Table S1) were transfected into Ba/F3 and Ba/F3–1*6 cells by electroporation using either the Bio-Rad Gene Pulser II with RF Module (Supplementary Figure S4 A–D) as previously described (41 (link)), or the Bio-Rad Gene Pulser Xcell with CE Module (Supplementary Figure S4 E–G) as follows. Ba/F3–1*6 cells were subjected to two rounds of electroporation (at t = 0 and at 24 h) and harvested at 48–72 h for gene expression and western blot analyses. Per transfection, PBS-washed 1 × 10⁶ cells were resuspended in 100 μl Gene Pulser Electroporation Buffer (Bio-Rad) containing 0.5 to 1 μM siRNA duplexes, transferred to a 0.1 cm gap cuvette (VWR) and electroporated through delivery of two square wave pulses of 95V, 5 ms duration and 0.1 s interval. Transfected cells were returned to RPMI 1640 supplemented with 10% FCS and 1% penicillin/streptomycin. Multiple siRNA transfections were performed using equivalent amounts of each siRNA up to the final siRNA concentration indicated.

Plasmid DNA of the HotG plasmid (52 (link)) was extracted from liquid bacterial culture using the EndoFree Plasmid Maxi Kit (QIAGEN). Twenty to 25 animals per treatment were transferred into an electroporation cuvette (Gene Pulser/Micro Pulser Electroporation Cuvette 0.4-cm gap or Bio-Rad electroporation cuvettes 4-mm gap, VWR), washed with Milli-Q water twice and then incubated at 4° for 30 min followed by RT for 30 min. Residual water was removed, and 200 μl of a plasmid solution (10 μg of plasmid and 10 mM Hepes to 200 μl) was added to each cuvette. Animals were incubated with this solution for 5 min. After relaxation of the animals, two pulses (150-V range) were applied for 75 ms (Gene Pulser II with RF module, Bio-Rad). After electroporation, 500 μl of ice-cold HM was added to the animals. Animals were carefully transferred into a new petri dish filled with prechilled HM. Electroporations were performed in this manner on 3 days (0, 2, and 4). Animals were used for experiments when first GFP⁺ cells appeared (~ 1 week after the last electroporation).