ATG5 siRNA (m)

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	Multidrug-resistant Ras-NIH 3T3/Mdr was derived from v-Haras transformed NIH 3T3 cell line (Ras-NIH 3T3), which show morphologically transformed foci of cells with the characteristics of crisscrossed margins, piling up properties and invasiveness (Lee et al., 2009), by stepwise increased concentrations of paclitaxel. The Ras-NIH 3T3 cells and its drug-resistant counterpart were maintained at 37°C in DMEM supplemented with 10% FCS, penicillin-streptomycin, and glutamine. Before experimental use, Ras-NIH 3T3/Mdr cells were maintained in a paclitaxel-free culture medium and subcultured at least three times. MEFs WT and Atg5-/- were kindly given by Dr. Young Sang Kim (Chungnam National University, Korea) with Dr. Noboru Mizushima’s (Tokyo Medical and Dental University, Japan) permission (Kuma et al., 2004). Where indicated the cells were transiently transfected with vector expressing the fulllength Beclin 1 (OriGene Technologies, Inc., USA) or pmCherryAtg5 (Addgene, USA), using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. After 24 h, the transfected cells were serum-deprived overnight prior to treatment with chemical. For Atg5 knockdown, Atg5 siRNA and a nontargeting siRNA were from Santa Cruz Biotechnology (USA). RT-PCR primer for detecting Atg5 knockdown was also from Santa Cruz Biotechnology.

Protocol tips

Multidrug-resistant Ras-NIH 3T3/Mdr was derived from v-Haras transformed NIH 3T3 cell line (Ras-NIH 3T3), which show morphologically transformed foci of cells with the characteristics of crisscrossed margins, piling up properties and invasiveness (Lee et al., 2009), by stepwise increased concentrations of paclitaxel. The Ras-NIH 3T3 cells and its drug-resistant counterpart were maintained at 37°C in DMEM supplemented with 10% FCS, penicillin-streptomycin, and glutamine. Before experimental use, Ras-NIH 3T3/Mdr cells were maintained in a paclitaxel-free culture medium and subcultured at least three times. MEFs WT and Atg5-/- were kindly given by Dr. Young Sang Kim (Chungnam National University, Korea) with Dr. Noboru Mizushima’s (Tokyo Medical and Dental University, Japan) permission (Kuma et al., 2004). Where indicated the cells were transiently transfected with vector expressing the fulllength Beclin 1 (OriGene Technologies, Inc., USA) or pmCherryAtg5 (Addgene, USA), using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. After 24 h, the transfected cells were serum-deprived overnight prior to treatment with chemical. For Atg5 knockdown, Atg5 siRNA and a nontargeting siRNA were from Santa Cruz Biotechnology (USA). RT-PCR primer for detecting Atg5 knockdown was also from Santa Cruz Biotechnology.

Publication protocol

Multidrug-resistant Ras-NIH 3T3/Mdr was derived from v-Haras transformed NIH 3T3 cell line (Ras-NIH 3T3), which show morphologically transformed foci of cells with the characteristics of crisscrossed margins, piling up properties and invasiveness (Lee et al., 2009), by stepwise increased concentrations of paclitaxel. The Ras-NIH 3T3 cells and its drug-resistant counterpart were maintained at 37°C in DMEM supplemented with 10% FCS, penicillin-streptomycin, and glutamine. Before experimental use, Ras-NIH 3T3/Mdr cells were maintained in a paclitaxel-free culture medium and subcultured at least three times. MEFs WT and Atg5-/- were kindly given by Dr. Young Sang Kim (Chungnam National University, Korea) with Dr. Noboru Mizushima’s (Tokyo Medical and Dental University, Japan) permission (Kuma et al., 2004). Where indicated the cells were transiently transfected with vector expressing the fulllength Beclin 1 (OriGene Technologies, Inc., USA) or pmCherryAtg5 (Addgene, USA), using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. After 24 h, the transfected cells were serum-deprived overnight prior to treatment with chemical. For Atg5 knockdown, Atg5 siRNA and a nontargeting siRNA were from Santa Cruz Biotechnology (USA). RT-PCR primer for detecting Atg5 knockdown was also from Santa Cruz Biotechnology.

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