ON-TARGETplus Rat Atg7 (312647) siRNA - SMARTpool

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	The rat UMR-106 osteosarcoma cell line75 was maintained in Dulbecco modified Eagle's medium (Lonza, BE12-604) supplemented with 10% Hyclone fetal calf serum (Thermo Scientific, SH30071.03). The UMR-106 GFP-LC3 clone 4 cells correspond to the UMR-106 cell line stably transfected with the pGFP-LC3 construct. These cells are maintained in the same medium in the presence of G418 (800 μg/ml; Sigma-Aldrich, G8168). For mineralization, the cells were cultured in α-MEM (Lonza, BE02-002) supplemented with 10% Hyclone fetal calf serum, CaCl2 (1.4 mM; Merck 2382), ascorbic acid (50 μg/mL; Sigma-Aldrich A4034) and dexamethasone (20 μg/mL; Sigma-Aldrich, D8893) for 3 d, and then for 3 d in the same medium in the presence of β-glycerophosphate (50 mg/mL; Sigma-Aldrich G9891). ON-TARGET plus SMARTpool siRNA (Dharmacon RNAi Technologies, Abgene Ltd) for rat Atg7 (L095596-01) and Becn1 (L-099237-01) were transfected in the UMR-106 cell line using nucleofection and Amaxa solution V (Lonza, VCA-1003).

Protocol tips

The rat UMR-106 osteosarcoma cell line75 was maintained in Dulbecco modified Eagle's medium (Lonza, BE12-604) supplemented with 10% Hyclone fetal calf serum (Thermo Scientific, SH30071.03). The UMR-106 GFP-LC3 clone 4 cells correspond to the UMR-106 cell line stably transfected with the pGFP-LC3 construct. These cells are maintained in the same medium in the presence of G418 (800 μg/ml; Sigma-Aldrich, G8168). For mineralization, the cells were cultured in α-MEM (Lonza, BE02-002) supplemented with 10% Hyclone fetal calf serum, CaCl2 (1.4 mM; Merck 2382), ascorbic acid (50 μg/mL; Sigma-Aldrich A4034) and dexamethasone (20 μg/mL; Sigma-Aldrich, D8893) for 3 d, and then for 3 d in the same medium in the presence of β-glycerophosphate (50 mg/mL; Sigma-Aldrich G9891). ON-TARGET plus SMARTpool siRNA (Dharmacon RNAi Technologies, Abgene Ltd) for rat Atg7 (L095596-01) and Becn1 (L-099237-01) were transfected in the UMR-106 cell line using nucleofection and Amaxa solution V (Lonza, VCA-1003).

Publication protocol

The rat UMR-106 osteosarcoma cell line75 was maintained in Dulbecco modified Eagle's medium (Lonza, BE12-604) supplemented with 10% Hyclone fetal calf serum (Thermo Scientific, SH30071.03). The UMR-106 GFP-LC3 clone 4 cells correspond to the UMR-106 cell line stably transfected with the pGFP-LC3 construct. These cells are maintained in the same medium in the presence of G418 (800 μg/ml; Sigma-Aldrich, G8168). For mineralization, the cells were cultured in α-MEM (Lonza, BE02-002) supplemented with 10% Hyclone fetal calf serum, CaCl2 (1.4 mM; Merck 2382), ascorbic acid (50 μg/mL; Sigma-Aldrich A4034) and dexamethasone (20 μg/mL; Sigma-Aldrich, D8893) for 3 d, and then for 3 d in the same medium in the presence of β-glycerophosphate (50 mg/mL; Sigma-Aldrich G9891). ON-TARGET plus SMARTpool siRNA (Dharmacon RNAi Technologies, Abgene Ltd) for rat Atg7 (L095596-01) and Becn1 (L-099237-01) were transfected in the UMR-106 cell line using nucleofection and Amaxa solution V (Lonza, VCA-1003).

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