Mrfp gfp lc3 plasmid

Cells were transfected with the mRFP-GFP-LC3 plasmid (Addgene, # 21074) and were subsequently treated with DMSO or H89/tetrandrine. Autophagy flux was assessed by counting cells that were mRFP⁺GFP⁺LC3 (yellow puncta), which represents autophagosomes, and mRFP⁺GFP⁻LC3 (red puncta), which represents autolysosomes.

Harvest the exponential growth phase cells of CGA-T1-OE, shL3MBTL2 and corresponding control cells. Mix 2×10⁵ cells with 1 μg ptfLC3 plasmid (mRFP-GFP-LC3 plasmid, Addgene, USA) and applied to the routine nucleofection process. After the program is finished, cells were transferred to the 6-well plate with 1.5 mL culture medium for 36 h cell culture. Afterwards, cells were starved with EBSS medium (Gibco:14155063, USA) for 3 h and then the GFP and mRFP fluorescence in cells were observed using a confocal microscope (Leica DMI6000B-TCS-SP5 confocal system, USA). The numbers of red puncta (RFP⁺GFP⁻) versus yellow puncta (RFP⁺GFP⁺) per cell in each cell line were quantified. At least 30 cells in triplicate per condition were counted.

Tandem fluorescent mRFP-GFP-LC3 plasmids were used to label and track LC3 and measure the autophagic flux. In brief, cultured primary microglia were transfected with mRFP-GFP-LC3 plasmids (Addgene, Watertown, MA, USA), according to the manufacturer’s protocol, and incubated for 48 h. The microglia were then treated with PFF or capsaicin for 24 h before fixing and staining with DAPI. The locations of mRFP and GFP were tracked using a TCS SP8 confocal laser scanning microscope. The autophagic flux was evaluated by counting the puncta of different colors with ImageJ software.

The autophagy levels were measured as previously reported (Lu et al., 2021 (link)). In brief, the BV2 cells and primary microglia were seeded at a density of 1 × 10⁵ cells/well and cultured overnight, after which cells were transfected with mRFP-GFP-LC3 plasmids (Addgene, Inc., Watertown, MA, United States) for 48 h according to the manufacturer’s protocol (Guangzhou RiboBio Co., Ltd, Guangzhou, China). Media was aspirated and then cells were incubated with 10 μM capsaicin or 2 μM rapamycin for 24 h. Cells were fixed with 4% paraformaldehyde and the digital images of LC3-positive puncta were captured by TCS SP8 confocal laser scanning microscope (Leica Microsystems GmbH). The numbers of LC3-positive puncta per cell in each experimental group were counted with ImageJ software.

Autophagic flow was determined using mRFP-GFP-LC3 plasmids (Addgene). Pan 02 cells were transfected with plasmid expressing eGFP-LC3 using Lipofectamine 2000 (Thermo Fisher, USA). Pan 02-eGFP-LC3 cells were seeded in 6-well plates (2 ×10⁵ cells/well) and incubated overnight. The cells were treated with CAP/CQ, PDGL-GEM + CQ, PDGL-GEM@CAP/CQ or HEPES at 6.5 or 7.4 for 4 h, fixed with 4% paraformaldehyde, then observed for the presence of eGFP-LC3 puncta (autophagosomes) using confocal laser scanning microscopy (LSM 800, Zeiss, Germany). Autophagosomes were also observed by transmission electron microscopy (JEM-1400 FLASH, JEOL, Japan) in ultrathin sections of Pan 02 cells treated with PDGL-GEM@CAP/CQ at pH 6.5 or 7.4.
In other experiments, Pan 02 cells were treated with a simple mixture of GEM + CQ, PDGL-GEM + CQ, PDGL-GEM@CAP, CAP@CQ or PDGL-GEM@CAP/CQ at pH 6.5, then collected and lysed in the presence of the protease inhibitor PMSF. Western blotting was used to assess the ratio of LC3 Ⅰ to LC3 Ⅱ and the ratio of p62 to GAPDH. Semi-quantitation was performed using Image J (National Institutes of Health, USA).