Figure 3.

The large vacuoles that engulf the LGP⁺ membranous aggregates are autophagosomes. (A) Live-cell imaging microscopy of a stably transfected NRK-49F fibroblast expressing CD63-GFP, showing an LGP⁺ aggregate (green) and 2 SCV (red) that are engulfed simultaneously and digested within a large vacuole. (B-D), Confocal microscopy images of stably transfected CD63-GFP NRK-49F fibroblasts showing: (B) large vacuoles with acidic pH (LysoTracker Deep Red-positive; Lyso; magenta); (C) large vacuoles containing CTSD (red); and, (D) large vacuoles positive for the autophagosome markers SQSTM1 (red) and LC3B (magenta). Images were acquired at 16 hpi. Bacteria are identified by DsRed expression (B); recognition of S. Typhimurium LPS (blue) (C); or DAPI (blue) (D). Scale bars: 5 μm.