MCF-7 Cells Overexpressing GABARAPL1

MCF-7 control cells (C) and MCF-7 Flag:GABARAPL1:6His (GABARAPL1) cells were obtained previously in our laboratory following transfection with pcDNA3.1 control, pcDNA3.1-Flag-GABARAPL1-(His)6 [47 (link)]. MCF-7-Flag:GABARAPL1-G116A:6his (GABARAPL1 G116A c1 and c2) cells were obtained in the same way following transfection with pcDNA3.1-Flag-GABARAPL1-G116A-(His)6 vectors. The cells were cultured in Dulbecco's minimum essential medium (DMEM) (PAA, E15-891) supplemented with 100 μg/ml penicillin/streptomycin (PAA, P11-010) and 10% fetal bovine serum (FBS) (PAA, A15-101) in a 5 % CO₂ incubator at 37°C. To inhibit autophagosome/lysosome fusion, cells were incubated for 2 h in complete medium supplemented with 100 nM bafilomycin A1, 40 μM chloroquine or 50 mM NH₄Cl. To induce autophagy, cells were incubated in EBSS for 4h at 37°C. To inhibit proteasome degradation, cells were cultured in complete medium supplemented with 2 μM MG132 for 16 h.
For transient transfection, 2 μg of pGFP, pGABARAPL1-GFP, pGABARAPL1-G116A-GFP, 200 μl Jetprime Buffer and 4 μl Jetprime reagent (Polyplus transfection, 114-07) were used per reaction according to the manufacturer's protocol.