Anti gal 3 m3 38

Cells were seeded at 6 × 10⁴ in 24-well plates with cover glass for overnight culture and infected with GAS for 30 min. Extracellular bacteria were killed by the use of 100 μg/ml gentamicin. At various time points postinfection, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and stained with anti-LC3 (pM036; MBL), anti-Gal-3 (M3/38; Santa Cruz), anti-Gal-8 (D-18; Santa Cruz), anti-ubiquitin (ab7780; Abcam, Inc.), and anti-parkin (PRK8; Santa Cruz) antibodies at room temperature for 1 h. After the cells were washed with PBS, they were stained with Alexa Fluor-conjugated secondary antibodies and DAPI (4′,6-diamidino-2-phenylindole) for 1 h, and the samples were then analyzed by confocal microscopy (FV1000; Olympus).

Cells seeded at 4 × 10⁴ per well in 24-well plates with cover glasses were cultured for 2 d with or without VEGF treatment and infected with GAS according to the bacterial infection protocol. Cover glasses were coated in advance with cellular matrix (Cellmatrix type I-C, 100 μg/mL, 37°C, 30 min). At various time points postinfection, the cells were fixed with 4% paraformaldehyde (PFA), permeabilized with 50 μg/mL digitonin, blocked with 0.2% gelatin in PBS, and stained with anti-Gal-3 (M3/38, Santa Cruz), anti-LC3 (PM036, MBL), anti-LAMP-1 (H4A3, Santa Cruz), anti-FIP200 (17250-1-AP, Proteintech), or anti-Rab7 (EPR7589, Abcam) antibodies, followed by staining with secondary antibodies conjugated with Alexa Fluor 488, 568, or 647. LysoTracker (Red DND-99, Invitrogen) at 100 nM was used to stain cells for 30 min before fixation. Hoechst (33342, #639, ImmunoChemistry Technologies) at 1 μg/mL was used for cell nucleus and bacterial DNA staining. Images were obtained using a confocal microscope (TCS SP8, Leica).