Neon transfection system 100 μl kit

Constitutively active FOXO1 and RNF144B were overexpressed in PTEN CR cells. One day before transfection, cells were plated at a density of about 1300 cells/cm² to ensure optimal growth. For transfection, we used the Neon Transfection System 100 μl Kit (Invitrogen; Thermo Fisher Scientific, Inc). Cells were harvested via trypsinization and washed with DPBS. A control vector (c-Flag pcDNA3 was a gift from Stephen Smale [Addgene plasmid #20011; http://n2t.net/addgene:20011;RRID:Addgene_20011 (47 (link))]), a vector for overexpression of constitutively active FOXO1 with mutated phosphorylation site (pcDNA3 Flag FKHR AAA mutant was a gift from Kunliang Guan [Addgene plasmid #13508; http://n2t.net/addgene:13508;RRID:Addgene_13508] (32 (link))) or a vector for RNF144B overexpression (RNF144B pcDNA3.1+ C-(K)-DYK #OHu07981D, GenScript) was added to the cell pellets (final concentration of 1 μg/ml for FOXO1 and 2 μg/ml for RNF144B in the culture medium after transfection). Pellets were resuspended in 100-μl R-buffer for transfection and electroporated in Neon 100 μl tips at 1300 V, 2 pulses, 20 ms. After electroporation, cells were transferred to the prewarmed culture medium and distributed to multiwell tissue culture plates for functional assays.

To electroporate LPS or MDP, the Neon Transfection System (MPK5000, Invitrogen) and the Neon Transfection System 100 μL Kit (MPK10025, Invitrogen) were used. Per each tip, 5 ×10^5 cells were transfected with the indicated amount of ultrapure lipopolysaccharide (LPS) from E. coli 111:B4 (tlrl-3pelps, Invivogen) or MDP (tlrlmdp, Tocris). Electroporation parameters were set at 1500 V, 30 ms and pulse number 1 for IMR90 cells and 1100 V, 20 ms and pulse number 2 for HEK293T cells. Once electroporated, the tip content was unloaded into a clean Eppendorf tube and tubes were centrifuged on a bench-top centrifuge at 3000 rpm 3 min. The supernatant was removed to avoid any traces of MDP or LPS in the extracellular media prior to plating. For CAPAN-1, PSN-1, A549, and HCT116, 1 to 10 μg/mL MDP and LPS were transfected in DMEM media with 10% FugeneHD (Promega).

The Gibco™ Minimum Essential Medium (MEM), Gibco™ MEM Non-Essential Amino Acids, GlutaMAX, penicillin, streptomycin, Neurobasal medium, glutamine, TranscriptAid T7 High Yield transcription Kit, Pierce™ RNA 3′ End Biotynylation Kit, LightShift® Chemiluminescent RNA EMSA Kit, and HuR siRNA (Elavl1 Silencer® Select siRNA, Ambion) were purchased from Thermo Fisher Scientific, USA. Poly-d-lysine, glutamic acid, β-mercaptoethanol, cytosine-β-d-arabinofuranoside, actinomycin D, TRI Reagent, and pentylenetetrazole (PTZ) were obtained from Sigma-Aldrich, USA. Papain latex was from Worthington, USA. The electroporation Buffer R was procured from the Neon Transfection System 100 μL Kit, Invitrogen, USA.

One day before transfection, cells were plated at a density of about 1300 cells/cm² to ensure optimal growth. For transfection, we used the Neon Transfection System 100 μl Kit (Invitrogen; Thermo Fisher Scientific, Inc). Cells were harvested via trypsinization and washed with DPBS. Either control siRNA (Silencer Negative Control No. 1 siRNA, Ambion, Thermo Fisher Scientific, Inc) or a combination of PTEN siRNA (s325 and s326, both Ambion, Thermo Fisher Scientific, Inc) was added to the cell pellets (final concentration of 10 nM in the culture medium after transfection). Pellets were resuspended in 100 μl R-buffer for transfection and electroporated in Neon 100 μl tips at 1300 V, 2 pulses, 20 ms. After electroporation, cells were transferred to a prewarmed culture medium and distributed to multiwell tissue culture plates for functional assays.

Anp32a-knockout HEK293T and A549 cells were generated by using the CRISPR/Cas9 system. HEK293T cells grown in a 10-cm dish were transfected with 10 μg of pSpCas9(BB)-2A-GFP (pX458) containing a target sequence (5′-CTTTGGTAAGTTTGCGATTG-3′) complementary to exon 2 of human Anp32a by using TransIT-LT1 (Mirus). For A549 cells, 1 × 10⁶ cells were electroporated using the Neon transfection system 100-μl kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. At 48 h posttransfection, cells were trypsinized and sorted into 96-well plates at 1 cell/well using fluorescence-activated cell sorting (FACS) with a FACS-Aria II cell sorter (BD BioSciences). GFP-expressing cells were expanded to obtain individual clones. The knockout of HuANP32A expression was confirmed by Western blotting using an anti-ANP32A antibody (15491; Cell Signaling Technology).
The luciferase assay in Anp32a-knockout HEK293T cells was performed as described above.
To study the effect of Anp32a knockout on the growth of parental or mutant PG/S1421(H7N9) viruses, Anp32a-KO A549 cells were infected with the indicated virus at an MOI of 0.1. Supernatants were collected at 24 h p.i., and virus titers were determined in embryonated chicken eggs.