The lentiviral vector of Nrf2-shRNA (shNrf2) was packaged into virus particles by the method reported previously37 (link), which was provided by Sang-Min Jeon, Professor, College of Pharmacy, Ajou University (Gyeonggido, Rep. of Korea). 293T cells were transfected with a lentiviral vector using Lipofectamine® 2000 according to the recommended protocol on the Addgene website. Lentivirus-containing conditioned medium (LCCM) was aliquoted into 1 ml stock in each cryovial. Then, HeLa stable cells that do not express Nrf2 (tet-shNrf2, + Dox) were prepared as follows. Briefly, HeLa 1 × 105 cells were incubated in each well of the 6-well plate overnight. Cell culture in 500 μl medium of each well was mixed with 1 ml LCCM and 1.2 μl polybrene (Millipore TR-1003-G). Culture medium was changed with 2 ml fresh medium containing 250 μg/ml hygromycin (Cayman 14,291). The infected shNrf2-positive control cells (tet-shNrf2, -DOX) were selected by the treatment with hygromycin every 3 days. Nrf2-knockdown (KD) cells were obtained and maintained by the treatment with 0.2 μg/ml doxycycline (Cayman 14,422) every 2 days.
Hygromycin
Manufactured by Cayman Chemical
Sourced in United States
Hygromycin is a laboratory reagent used as a selective antibiotic in cell culture and molecular biology applications. It inhibits protein synthesis in eukaryotic cells, making it useful for selecting cells that have been genetically modified to express resistance to the compound.
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5 protocols using hygromycin
1
Generation and Maintenance of Nrf2-knockdown Cells
2
Cultivation of Francisella tularensis Strains
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The Francisella tularensis subsp. holarctica live vaccine strain (LVS) was obtained from the Centers for Disease Control and Prevention (Atlanta, GA), and F. tularensis subsp. tularensis Schu S4 was obtained from BEI Resources. Wild-type (WT) F. tularensis Schu S4 was initially cultured on chocolate agar supplemented with 1% IsoVitaleX (see the supplemental material for the recipe). WT F. tularensis Schu S4 harboring a luciferase (LUX) plasmid (pJB3 [29 (link)]) and WT F. tularensis LVS pEDL20 (53 (link)) were cultured on chocolate agar supplemented with 1% IsoVitaleX and 200 μg/mL hygromycin (Cayman Chemicals). WT F. tularensis Schu S4 expressing green fluorescent protein (GFP) was cultured on chocolate agar supplemented with 1% IsoVitaleX and 10 μg/mL kanamycin (Sigma-Aldrich). All strains were grown overnight at 37°C with aeration in Chamberlain’s defined broth medium (CDM) following growth on plates (54 (link)).
3
Lentiviral Selection System for IFNAR2 KO
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To construct the
selection system, the ISG54 promoter57 (link) and
iCasp9 sequences were synthesized and inserted into the pGreenFire1-ISRE-Neo
vector (System Biosciences, TR016PA-N) and replaced the original sequences
of mCMV promoter and luciferase, respectively. Then the whole selection
cassette (4 × ISRE-ISG54-dscGFP-T2A-iCasp9) was cloned and inserted
into the lentiviral vector pCDH-CMV-MSC-EF1α-Hygro (System Biosciences,
CD515B-1) to replace the original CMV promoter. To pack the selection
system in VSVG-pseudotyped lentiviral particles, the selection plasmid
and packaging plasmids pMD2.G, psPAX2 were cotransfected with polyethylenimine
(Polysciences, linear, MW ≈ 25000) in 293FT cells (Thermo Scientific,
R70007). After 48 h, the virus-containing supernatant was collected
and spun for 10 min at 4 °C (500g) to remove
cells. Lentiviral particles were concentrated ∼10× in
volume with a Lenti-X Concentrator reagent (Clontech, PT4421-2). THP-1-Dual
KO-IFNAR2 cells were transduced by addition of concentrated viral
suspension at a multiplicity of infection (MOI) of 1 in the full medium
containing 8 μg/mL Polybrene (Sigma-Aldrich, TR-1003-G). After
a 24 h of incubation at 37 °C, the virus-containing medium was
removed and replenished with a fresh medium. After an extra 24-h recovery,
the transduced cells were selected in 1 μg/mL hygromycin (Cayman,
14291) for 7 days.
selection system, the ISG54 promoter57 (link) and
iCasp9 sequences were synthesized and inserted into the pGreenFire1-ISRE-Neo
vector (System Biosciences, TR016PA-N) and replaced the original sequences
of mCMV promoter and luciferase, respectively. Then the whole selection
cassette (4 × ISRE-ISG54-dscGFP-T2A-iCasp9) was cloned and inserted
into the lentiviral vector pCDH-CMV-MSC-EF1α-Hygro (System Biosciences,
CD515B-1) to replace the original CMV promoter. To pack the selection
system in VSVG-pseudotyped lentiviral particles, the selection plasmid
and packaging plasmids pMD2.G, psPAX2 were cotransfected with polyethylenimine
(Polysciences, linear, MW ≈ 25000) in 293FT cells (Thermo Scientific,
R70007). After 48 h, the virus-containing supernatant was collected
and spun for 10 min at 4 °C (500g) to remove
cells. Lentiviral particles were concentrated ∼10× in
volume with a Lenti-X Concentrator reagent (Clontech, PT4421-2). THP-1-Dual
KO-IFNAR2 cells were transduced by addition of concentrated viral
suspension at a multiplicity of infection (MOI) of 1 in the full medium
containing 8 μg/mL Polybrene (Sigma-Aldrich, TR-1003-G). After
a 24 h of incubation at 37 °C, the virus-containing medium was
removed and replenished with a fresh medium. After an extra 24-h recovery,
the transduced cells were selected in 1 μg/mL hygromycin (Cayman,
14291) for 7 days.
4
Generating Nrf2-Knockdown Cell Lines
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Lentiviral vector of Nrf2-shRNA (shNrf2) was packaged into virus particle by the method reported previously44 (link), which was provided by Sang-Min Jeon, Professor, College of Pharmacy, Ajou University, Gyeonggido, Republic of Korea. 293T cells were transfected with lentiviral vector using Lipofectamine® 2000 according to the recommended protocol on the Addgene website. Lentivirus-containing conditioned medium (LCCM) was aliquoted into 1 ml stock in each cryovial. Then, WiL2-NS stable cells that do not express Nrf2 (tet-shNrf2, +Dox) were prepared as follows. Briefly, WiL2-NS 1 × 105 cells were incubated in each well of six-well plate overnight. Cell culture in 500 μl medium of each well was mixed with 1 ml LCCM and 1.2 μl polybrene (Millipore TR-1003-G). Culture medium was changed with 2 ml fresh medium containing 250 μg/ml hygromycin (Cayman 14291). The infected shNrf2-positive control cells (tet-shNrf2, -Dox) were selected by the treatment with hygromycin B (250 µg/ml) every 3 days. Nrf2-KD cells were obtained and maintained by the treatment with 0.2 μg/ml doxycycline (Cayman 14422) every 2 days.
5
Plasmid Construction for Dual Resistance
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The pCEV-G2-Km plasmid was a gift from Lars Nielsen and Claudia Vickers (Addgene plasmid #46815) [70] (link). This plasmid has TEF1 and PGK1 promoter-controlled expression cassettes associated with geneticin (G418) resistance. To add hygromycin (hph) resistance, the corresponding cassette hphMX6 was amplified from the pJET1,2-attB-hph-attP plasmid [72] (link) using Fw_hph_ho_pCev and Rv_hph_ho_pCev primers (Table 2). An amplicon was used to transform S. cerevisiae BY4741 by homologous recombination together with EcoRV digested pCEV-G2-Km [10] (link), and pCevhph was obtained. The same strategy was used to generate pCe-vWaSTL1, but pCEV-G2-Km was digested with EcoRV and SalI instead. WaSTL1 was amplified with specific primers (primers Fw_WaSTL1_ho_pCev and Rv_WaSTL1_ho_pCev in Table 2). Transformants were selected for resistance to 500 µg mL -1 hygromycin (Cayman Chemical, Ann Arbor, MI, USA). Figure 1 illustrates the strategy used for plasmid construction. Plasmids were rescued from yeasts as described by Singh and Weil [60] (link) and then amplified in E. coli according to the standard procedures [3] . Transformants were selected in 100 µg mL -1 ampicillin (MIDSCI, Valley Park, MO, USA). The correctness of the constructions was checked by PCR; restriction analysis using KpnI, BamHI, and SpeI enzymes; sequencing.
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