Electroporator

To study the role of TLR4 in the mediation of SAA1-induced effects, amnion fibroblasts were transfected with 50 nM siRNA (GenePharma) against TLR4 (5′-CCCACAUUGAAACUCAAAUtt-3′) or randomly scrambled siRNA using an electroporator (Nepa Gene, Chiba, Japan) at 175 V for 5 ms following a protocol as described previously^{28 (link)}. The cells were then incubated in DMEM containing 10% FBS and 1% antibiotics for three days before treatment. The efficiency of knockdown is shown in Supplementary Figure S1.

Pancreatic cancer was induced by in situ electroporation to transfect the pancreatic parenchyma with oncogenic plasmids in a locally restricted manner [9 (link)]. Engin Gürlevik generously provided transposon plasmids coding for KRAS-G12V, shRNA that silences p53, a gain-of-function mutant of p53, and a constitutively active version of Akt2 (generated by myristoylation of its N-terminus-myrAkt2). These four oncogenic plasmids were injected into the tail of the pancreas. Simultaneous co-delivery of the SB13-transposase achieved somatic integration of the transposon plasmids into the genome. The electroporation reproduces characteristic mutations of human PDAC. As a result, the clinical course of the model is very similar to the human disease. The application of the plasmids does not lead to increased induction of cancer stem cells. The emerging tumors showed local infiltration and developed distant metastases in the peritoneum, the liver, and the lungs. For the procedure, mice were anesthetized, and the pancreatic tail was mobilized after laparotomy. Plasmid DNA (50 μL of 0.5 μg/μL) was injected into the tail of the pancreas using a 27/30-gauge needle. Using an Electroporator (NepaGene), four electric pulses were administered twice (duration: 35 ms at 35 V, interval between pulses: 500 ms). The peritoneal cavity was washed and closed by suturing.

α-E catenin siRNA was transfected to RAW 264.7 macrophages using Nepagene electroporator as per instruction manual. Briefly, 250 nM of siRNA was mixed with 1 × 10⁷ cells/100 μl of RAW 264.7 cells in Opti-Mem and electroporated using below conditions. Pouring pulse conditions are voltage at 175 V, pulse length 5 ms, pulse interval 50 ms, 2 pulses, 10% decay rate and + polarity. Transfer pulse conditions are 20 V voltage, 20 ms pulse length, 50 ms pulse interval, 5 pulse, 40% decay rate. Cells were used for experiments 48–72 h after transfection.

To study the role of the ST2 in the mediation of IL-33 effects, siRNA-mediated knockdown of ST2 was performed. Briefly, after isolation, amnion fibroblasts were transfected with 50 nM of two sets of siRNA against ST2 (1#: 5’-CTCTGTTTCCAGTAATCGGAGCC-3’; 2#: 5’- GCAGCCAAGAACTGAGTGCCTT-3’; GenePharma, Shanghai, China) or randomly scrambled siRNA (5′-UUCUCCGAACGUGUCACGUTT-3′) in Opti-MEM (Life Technologies Inc.) using an electroporator (Nepa Gene, Chiba, Japan) at 165 V for 5ms. The cells were then incubated in DMEM containing 10% FBS for 72 h before IL-33 treatment. The knockdown efficiency was assessed with qRT-PCR and Western blotting.