TF siRNA screen and validation was performed as in [2 (link)]. For RNA and protein extraction and GSH assays, 1.2 × 105 Calu-3 or 16HBE14o− cells were seeded per well in 24 well plates. Calu-3 and 16HBE14o− cells were reverse or forward transfected, respectively, with BACH1 siRNA (Ambion Silencer®Select Catalog # 4392420- S1860, or Santa Cruz sc-37064) or negative control #2 siRNA (Catalog # 4390846, or Santa Cruz control siRNA-A sc-37007) at 18 pmol/well using Lipofectamine RNAiMAX reagent or Santa Cruz sc-37064, 48 h post-transfection, cells were exposed to oxidative stress by addition of H2O2 — 800 μM for 16 h or 600 μM for 8 h for Calu-3 and 16HBE14o− cells, respectively, in DMEM without serum, before collection of samples.
Protocol detail
Oxidative Stress Response Assay
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Assays
Cells
H2o2
Lipofectamine
Oxidative stress
Protein
Samples collection
Serum
Sirna
Transfection
Corresponding Organization : Case Western Reserve University
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Variable analysis
independent variables
- BACH1 siRNA (Ambion Silencer®Select Catalog # 4392420- S1860, or Santa Cruz sc-37064)
- Negative control #2 siRNA (Catalog # 4390846, or Santa Cruz control siRNA-A sc-37007)
- RNA and protein extraction
- GSH (glutathione) assays
- Cell lines: Calu-3 and 16HBE14o- cells
- Cell seeding density: 1.2 × 10^5 cells per well in 24 well plates
- Transfection method: Reverse or forward transfection for Calu-3 and 16HBE14o- cells, respectively
- Transfection reagent: Lipofectamine RNAiMAX or Santa Cruz sc-37064
- Transfection time: 48 h post-transfection
- Oxidative stress exposure: 800 μM H2O2 for 16 h (Calu-3) or 600 μM H2O2 for 8 h (16HBE14o-) in DMEM without serum
- Negative control #2 siRNA (Catalog # 4390846, or Santa Cruz control siRNA-A sc-37007)
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