Amaxa nucleofector

The synthetic miR-1246 mimic (forward, 5′- AAUGGAUUUUUGGAGCAGG - 3′; reverse, 5′- UGCUCCAAAAAUCCAUU TT- 3′), miR-1246 inhibitor (5′- CCUGCUCCAAAAAUCCAUU- 3′), mimic control (forward, 5′-UUCUCCGAACGUGUCACGUTT-3′; reverse, 5′-ACGUGACACGUUCGGAGAATT-3′) and inhibitor control (5′-UUGUACUACACAAAAGUACUG-3′) were purchased from GenePharma (GenePharma Inc., Shanghai, China). The siRNAs for CADM1 was synthesized (Invitrogen Inc.) and the sequence was list in Additional file 1: Table S1. The nucleotide sequences were delivered into human HCC cell lines by Amaxa Nucleofector® following the manufacturer’s instructions. Briefly, cell pellets were collected by 90 × g centrifugation at room temperature for 10 min and resuspended in 100 μl of Nucleofector Solution to a final concentration of 1 × 10⁶ cells/100 μl. Each 100 μl sample was transferred into an Amaxa-certified cuvette and underwent nucleofection using the appropriate Nucleofector program. The program for transfecting HepG2 and SMMC7721 was T028. After nucleofection, each sample was immediately transferred from the cuvette to a culture plate in 2 ml of medium [14 (link)].

Retroviral expression vector (pBabe-puro) encoding Syndecan-4 fused to an extracellular HA-tag was kindly provided by Dr. Mark Morgan (University of Liverpool, UK) [36 ]. Syndecan-4-HA fragment was subcloned into EcoRI/XhoI sites of pcDNA3.1(+) plasmid. For the live cell experiments we used mCherry-vinculin (modified pEGFP-C1-mCherry plasmid harboring a deletion of the GFP gene sequence), kindly provided by Dr. Vicente Torres (Universidad de Chile, Chile) [37 ]. pEGFP-C1-Tiam1-PHnCCEx containing a mutation in the RhoK phosphorylation site encoding for a dominant negative mutant of Tiam1 was kindly provided by Dr. María Paz Marzolo (P. Universidad Católica de Chile, Chile) [38 ].
To downregulate PAR-3 expression we used siRNA ON-TARGET plus SMART pool (Thermo Scientific) against rat PAR-3 or FlexiTube siRNA Mm_Pard3_4 (Qiagen) targeting mouse PAR-3. A mix of two Tiam1 siRNA targeting the human Tiam1 with 88% homology with the rat Tiam1 sequence (Dharmacon) was used to lower Tiam1 expression. Silencer® Negative Control No. 1 (Ambion™) or AllStars (Qiagen) were used as siRNA negative control. Plasmids and siRNAs transfections of DI TNC1 cells were performed with Amaxa Nucleofector following manufacturer’s recommendations for astrocytes (Amaxa Biosystems, Lonza). Lipofectamine RNAiMAX Reagent and Lipofectamine 3000 (Thermo Fisher Scientific) were used to transfect MEFs.

Electroporation in primary microglia was performed as described by us previously (8 (link)). Briefly, knockdown of CSF1R with Csf1r-specific siRNA (GenePharma) was achieved by electroporation using an Amaxa Nucleofector and a glial specific Nucleofector kit (LONZA) according to the manufacturer’s instructions. Each electroporation reaction contained 10⁶ cells and 200 nM siRNA. Transfected cells were plated and used for qRT-PCR, Western blotting, MTS assay.

For silencing STAT6 (NM_009284.2), siRNA knockdown was performed in RAW264.7 cells using siRNA duplexes purchased from RiboBio (Guangzhou, China). The negative control siRNA (scrambled siRNA) was provided by RiboBio. The two independent oligonucleotides designed for STAT6 were as follows: (1) 5′-CCAAGACAACAACGCCAAA dTdT-3′ and (2) 5′-GCUGAUCAUUGGCUUUAUU dTdT-3′. The siRNA fragments were transfected into RAW264.7 cells by electroporation using the Cell Line Nucleofector Kit V (Lonza) and program D-32 of an AmaxaNucleofector (Amaxa, Cologne, Germany) according to the manufacturer's protocol. Cells were then recovered for 48 h, and the silencing effect was detected by Q-PCR.