Fluoro gel with dapi

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)}

Cell culture, quercetin treatment, and infection procedure were conducted as described above. Post-infection, the cell monolayers were washed 3 times with ice cold PBS and fixed in fresh prepared 4% paraformaldehyde for 30 min at room temperature. The fixed cells were then permeabilized with 0.5% Triton X-100 for 10 min, washed with PBS, and blocked with 5% normal goat serum for 60 min at room temperature (RT). Then the cells were incubated with anti-integrin β1 antibody (rat monoclonal IgG1, DSHB), vinculin antibody (Santa Cruz) or phalloidin (Sigma) overnight at 4°C. The cells were rinsed with PBS and stained with Alexa Fluor 555 goat anti-rat IgG or Alexa Fluor 488 goat anti-mouse IgG (Cell Signaling) for 60 min at RT. These stained cells were washed 3 times with PBS and mounted with Fluoro-gel with DAPI (Electron Microscopy Sciences, Hatfield, PA). Fluorescence signal was visualized with EVOS FL fluorescence microscope (Life Technologies, Grand Island, NY).

Cells were grown on 8 well chambers (Ibidi) coated with laminin, fixed and permeabilized with 100% cold methanol (−20 °C).The slides were incubated with the following primary antibodies p-c-Src (Y416) (9A6) (Santa Cruz) 1:50 and pcMet (Tyr1234/1235) (cell signaling) 1:50 overnight at 4 °C. We used secondary antibodies conjugated to Alexa dyes (647 and 488) or to Cy3- (Jackson Immune Research) 1:100 for 45 min at RT. Mounting was performed with FLUORO-GEL (with DAPI) (Electron microscopy science). Images were acquired using Nikon A1R confocal microscope, with an ×40 lens, 1.3 oil Plan-fluor. The analysis was done using NIS element (Nikon) to obtain the intensity of protein expression per cell.