Tf lc3

HT-29 cells seeded in a 24-well plate reached 70% confluent at the time of transfection. tfLC3 (Addgene plasmid 21074, Cambridge, MA, USA) plasmids are tandem fluorescent (mRFP and EGFP) tagged LC3B that are able to detect different stages of autophagy. The EGFP tag is acid sensitive whereas the mRFP tag is acid resistant. The double-tagged LC3B can be used to label both autophagosomes and autolysosomes. In the beginning, autophagosomes are tagged by both mRFP and EGFP, resulting in a yellow fluorescence. When fused with lysosomes in late stage, the acidic autolysosomes degrade EGFP and emit red fluorescence.^{20 (link)} The tfLC3 plasmids were transfected by using X-tremeGENE HP DNA Transfection Reagent (Roche, Mannheim, Germany) following the company’s manual.
Transfected cells were incubated at 37 °C in a CO₂ incubator for 24 h. The transfected cells were washed with PBS and infected with E. coli O157:H7 EDL933 WT strain as described above. Cells were fixed with 4% paraformaldehyde for 30 min at room temperature and mounted with Fluoro-gel with DAPI (Electron Microscopy Sciences, Hatfield, PA, USA). Red and yellow puncta were visualized by using inverted EVOS FL fluorescence microscope (Life Technologies) by counting a total of >20 cells for each treatment.^{20 (link)}