Egfp lc3 plasmid

MCF-7/ADR and K562/ADR cells were transfected with EGFP-LC3 plasmid (Addgene, USA) by means of Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's protocol. The transfected cells were incubated with CQ (10 µg/mL) for 24 h. Afterwards, cells were washed with precooled PBS, then the cell nuclei and lysosomes were labeled with Hoechst 33342 (Invitrogen, USA) and LysoTracker^® Red DND-99 (Invitrogen, USA), respectively, for confocal laser scanning microscope (CLSM, Olympus, Japan) observation. In addition, some transfected cells were incubated with various formulations for 24 h at the final DOX·HCl and CQ concentration of 5 µg/mL and 10 µg/mL, respectively. Then, these cells were processed with the same operation as described above and cell nuclei were labeled with Hoechst 33342. The untreated or treated cells were observed with CLSM after being fixed. Furthermore, confocal microscopy analysis was used to measure the fluorescent dots representing EGFP-LC3 translocation per cell.
Moreover, the expression of autophagy related marker protein LC3 and autophagy substrate protein p62 was detected using western blot. The cellular autophagy was also investigated by bio-TEM. The details were described in Supplementary Material (Supplementary section 1).

DMEM (Gibco, Thermo Fisher Scientific, MA, USA, 4.5 g/L D-glucose, L-glutamine, 110 mg/L sodium bicarbonate, 10% FBS, 1% penicillin and streptomycin) was used to culture JG cells. After being passaged, cells were confluent at approximately 60% and then treated with 40 mM glucose, 400 μm palmitate (PA) and 200 μm oleate (OA) for 48 h as a cellular model to mimic T2DM for subsequent experiments.
EGFP-LC3 plasmid (Addgene, Watertown, MA, USA, plasmid 11546) was transfected into JG cells usingLipofectamin 3000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instruction.

The autophagic flux experiment was performed in HEK 293 cells transfected with an eGFP-LC3 plasmid (Addgene, Cambridge, MA). Twenty-four hours after transfection, cells were starved in DMEM without serum for 12 h. 3 methyladenine (Sigma) and chloroquine (Sigma) were added at the time of starvation to inhibit autophagy induction and autophagosome–lysosome fusion at a concentration of 2 mmol/L and100 μmol/L, respectively. HEK 293 cells cultured in DMEM with 10% FBS were used as control (CTL). Images were acquired 12 h after starvation using fluorescent microscope, Olympus 1 × 71. For the ROS measurement, 24 h after starvation, cells were treated with 10 μmol/L dihydroethidium (DHE) (Sigma) for 30 min as previously described (Wojtala et al. 2014 (link)), and images were obtained using fluorescent microscope, Olympus 1 × 71. Cells cultured in DMEM with 10% FBS were used as CTL. This experiment was performed in two different cell lines, HEK 293, and AC-16 cells. DHE staining, in vivo, was performed by obtaining thin sections from frozen cardiac tissue, which were treated with 1 mmol/L dihydroethidium in PBS for 30 min at room temperature. Thereafter the sections were washed in PBS for 3 min × 3 and then mounted with DAPI. Images were taken immediately using fluorescent microscope, Olympus 1 × 71. Image J was used for the quantification of the fluorescent intensity.

150,000 cells were seeded in a 12 well-plate. Once attached, transfection of EGFP-LC3 plasmid (Addgene, 11546) was performed using Lipofectamine 3000 (Invitrogen, L3000015) according to the manufacturer’s instructions. 1.4 μg/μL of plasmid DNA were used and incubated overnight. Subsequently, cells were trypsinized and plated on coverslips. After 24 h of treatment, cells were fixed with 4% formaldehyde for 15 min and stained with Hoechst (10 μg/mL) for 5 min. Coverslips were mounted on slides using 50% glycerol in PBS as mounting media. Cytoplasmic EGFP-LC3 positive puncta indicated the formation of autophagosomes. Fluorescent images were obtained with an Axio Imager Zeiss microscope with a 63x objective. 30 cells were counted per experiment and graphs show the data from three independent experiments.

To monitor microglial autophagy activity, BV2 cells were transiently transfected with eGFP‐LC3 plasmid (Addgene) using lipofectamine 2000. At 48 h post transfection, cells were subjected to indicated treatments for 6 h. Cells were fixed in 4% PFA for 10 min, stained with DAPI, and observed under a confocal microscope (LSM700; Carl Zeiss).