HeLa cells were grown in F12:DMEM 50:50 (Hyclone) with 10% FBS, 2mM L-glutamine and antibiotics in 5% CO2 at 37 °C. Cells were synchronized in G1/S by treatment with 2 mM thymidine (Sigma-Aldrich) for 18-hours. The following siRNAs were used for siRNA transfections: ThermoFisher Silencer Select 4390843 (control non-targeting siRNA) and S9187 and S9188 (M1 and M2 siRNAs targeting MYL5); Dharmacon ON-TARGETplus D-001810-10 (control non-targeting siRNA) and J-011739-03 and J-011739-04 (M3 and M4 siRNAs targeting MYL5) were used as described previously (Torres et al. 2010 (link)). See Table S1 for a list of key reagents and resources used in this study and their pertinent information.
On targetplus d 001810 10
Manufactured by Horizon Discovery
ON-TARGETplus D-001810-10 is a small interfering RNA (siRNA) product from Horizon Discovery. It is a pool of four individual siRNA duplexes designed to target a specific gene. The core function of this product is to provide a tool for gene knockdown experiments in cell-based assays.
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Lab products found in correlation
3 protocols using on targetplus d 001810 10
1
HeLa Cell Synchronization and siRNA Knockdown
2
Lentiviral Knockdown of p53 and p21
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Lentiviral expression plasmids pLKO.1-sh-Scr, sh5-MDM2, sh1-p53, sh2-p53, sh1-p21 and sh2-p21 were obtained from Open Biosystems and Biomedicum Functional Genomics Unit (Helsinki, Finland). Lentivirus stocks were produced as described [56 (link)]. To establish BC-3 cell lines stably expressing sh-p53, cells were selected with medium containing 3.5 μg/ml puromycin (Sigma) and to establish TREx BCBL1-Rta (here referred as BCBL1RTA) and BC-3 cell lines stably expressing sh-p21, cells were selected with medium containing 2 μg/ml puromycin (Sigma).
The nonspecific siRNAs control (ON-TARGETplus, D-001810-10) and siRNA against p21 (ON-TARGETplus, L-003471-00) were from Dharmacon (SMARTpool siRNA). Cells were reverse-transfected (2000 cells per well) for 48 h in 96-well view plates (Perkinelmer) using RNAiMax (Invitrogen), and reactivated by indicated treatments for 24 h. For more details see supplemental information.
The nonspecific siRNAs control (ON-TARGETplus, D-001810-10) and siRNA against p21 (ON-TARGETplus, L-003471-00) were from Dharmacon (SMARTpool siRNA). Cells were reverse-transfected (2000 cells per well) for 48 h in 96-well view plates (Perkinelmer) using RNAiMax (Invitrogen), and reactivated by indicated treatments for 24 h. For more details see supplemental information.
3
HeLa Cell Synchronization and siRNA Treatment
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HeLa cells were grown in F12:DMEM 50:50 (Hyclone) with 10% FBS, 2mM Lglutamine and antibiotics in 5% CO2 at 37 o C. Cells were synchronized in G1/S by treatment with 2 mM thymidine (Sigma-Aldrich) for 18-hours. The following siRNAs were used for siRNA treatments: ThermoFisher Silencer Select 4390843 (control non-targeting siRNA) and S9187 and S9188 (M1 and M2 siRNAs targeting MYL5); Dharmacon ON-TARGETplus D-001810-10 (control non-targeting siRNA) and J-011739-03 and J-011739-04 (M3 and M4 siRNAs targeting MYL5) were used as described previously. 39 Please see Table S1 for a list of key reagents and resources used in this study and their pertinent information.
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