FIG 3.

Ca²⁺ signaling in epithelial cells infected with S. aureus 6850 hla or JE2 scpA strains. (A) HeLa cells were infected with S. aureus 6850 or the 6850 hla mutant, and cytotoxicity was determined at 6 h p.i. by LDH release. (B to I) HeLa R-Geco cells were infected with S. aureus 6850 GFP, 6850 hla GFP, JE2 GFP, or JE2 scpA GFP strains, and cytosolic Ca²⁺ concentration, i.e., R-Geco fluorescence, was measured by time-lapse imaging. (B) Stills of time-lapse imaging at 5 h 6 min postinfection (p.i.) are shown (green, S. aureus; red, R-Geco; gray, brightfield). Bar, 50 μm. (C) Relative R-Geco fluorescence was quantified over the time period of infection in single uninfected cells (ni) or in cells infected with S. aureus 6850 or S. aureus 6850 hla strains. (D) The peak amplitude of the relative R-Geco fluorescence of 4 to 12 single infected cells was determined. (E) The latency of the relative R-Geco fluorescence peak after S. aureus intracellular infection was calculated in 4 to 12 single cells. (F) Representative stills from time-lapse fluorescence microscopy (green, S. aureus; red, R-Geco; gray, brightfield). Bar, 20 μm. (G) Relative quantification of cytosolic Ca²⁺ concentrations of single infected (JE2) or uninfected (ni) cells. (H) The maximum amplitude of relative R-Geco fluorescence of 10 single infected cells was determined. (I) The latency after infection until the maximum amplitude of relative R-Geco fluorescence was quantified in 10 single cells. Statistical significance was determined by unpaired t test (*, P < 0.05).