pLKO.1 lentiviral vectors expressing small hairpin (sh)RNAs targeting WIPI-1, WIPI-2, PI3KC3 and BECN1 or a non-targeting shRNA control (SHCOO2) were purchased from Sigma-Aldrich. A lentiviral vector expressing EGFP-Cherry-LC3 was provided by Dr. MS Soengas. Lentiviral production and transduction of NB4 cells was done as described.51 (link) EGFP-Cherry-LC3 NB4 cells with WIPI-1, WIPI-2 and BECN1 knockdown were generated by serial transduction with both lentiviral vectors described above. Transduced cell populations were selected for 4 days using 1.5 μg/ml puromycin. qPCR was performed to evaluate knockdown efficiency in NB4 WIPI-1, WIPI-2 and BECN1 knockdown cells, whereas western blotting was employed for demonstration for beclin 1 inhibition. To generate PU.1-inducible cells, the pCl10A1 retroviral packaging plasmid and the pBabe-PUER-puro (kindly provided by Dr. H Yoshida) were co-transfected into 293T cells. Cell culture medium was removed after 1 day and cells were incubated for 2 min with PBS/15% glycerol. PBS/15% glycerol was replaced by fresh medium. Viral supernatants were harvested 48 h later. NB4 cells were transduced for 24 h in the presence of 8 μg/ml polybrene. Transduced NB4 cell populations were selected with 1.5 μg/ml puromycin for 4 days.
Shcoo2
Manufactured by Merck Group
SHCOO2 is a laboratory equipment product designed for scientific research and analysis. It serves as a core functional tool for various experimental and analytical procedures. The product's specifications and technical details are maintained by the Merck Group, the manufacturer.
Automatically generated - may contain errors
Lab products found in correlation
Plko 1 lentiviral vector, by Merck Group (2 mentions)
Williams medium e, by Thermo Fisher Scientific (1 mentions)
Rosiglitazone, by Cayman Chemical (1 mentions)
Wortmannin, by Merck Group (1 mentions)
Bovine insulin, by Merck Group (1 mentions)
Akt1 2 inhibitor, by Merck Group (1 mentions)
Gw9662, by Merck Group (1 mentions)
Mission shrna, by Merck Group (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Axiophot 2, by Zeiss (1 mentions)
3 protocols using shcoo2
1
Lentiviral Transduction of NB4 Cells
2
Silencing ANGPTL3 in Immortalized Hepatocytes
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Liver IHHs immortalized by SV40 large T-Antigen (IHH, ATCC® PTA-5565™) were transduced with MISSION™ shRNA (small hairpin RNA) Lentiviral Vector particles (TRCN0000242782, Sigma Aldrich) targeting ANGPTL3 (NM_014495.2) or with non-target shRNA (SHCOO2, Sigma Aldrich) [MOI (multiplicity of infection) 1]. Positive cells were selected against 5 μg/ml puromycin for 12 days. The transduction was repeated once after the first selection. Cells were cultured in Williams medium E (Gibco by Life Technologies, 22551-022) with added 10% (v/v) FBS and glutamine 0.2 mg/ml and incubated +37 °C. FBS was removed during experiments and total protein from cell lysates was used to normalize the data. Cells were washed with PBS (pH 7.4) and lysed in RIPA buffer. Protein concentration was measured with Bradford protein assay (Bio-Rad). Following compounds were used: insulin (bovine, Sigma-Aldrich), Wortmannin (Sigma-Aldrich), Akt1/2 inhibitor (Sigma-Aldrich), rosiglitazone (Cayman Chemical) and GW9662 (Sigma-Aldrich).
3
Lentiviral Knockdown of DAXX and Micronuclei Quantification
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For virus infection, 6 × 10 5 cells were seeded in 6-well plates the day before. pLKO.1 lentiviral vectors expressing small hairpin (sh) RNAs targeting DAXX (Sh-DAXX_H2410: and Sh-DAXX_2503) or a nontargeting shRNA control (SHCOO2) were purchased from Sigma-Aldrich. Lentiviral production and transduction were done as described in Tschan et al. (2003) . Transduced cells were selected by puromycin treatment (1.5 µg/mL, Invitrogen). For micronuclei counting, cells were seeded in 24-well plates on coverslips coated with FBS, 1 × 10 4 cells per well. Cells were fixed with 4% paraformaldehyde for 30 min at room temperature. DAPI was used to stain nuclei. Micronuclei were counted under a fluorescent microscope (Axiophot2, Zeiss).
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