Fig. 5.

Fyn knockdown decreases sphingomyelin trafficking to the chlamydial inclusion. (A) HeLa cells were transfected with nontargeting or Fyn siRNA and then infected with C. trachomatis L2. Cells were labeled with C₆-NBD-ceramide and, after 8 h of back-exchange, were examined by fluorescence microscopy. Bar = 5 μm. The fluorescence associated with at least 20 individual inclusions was measured by microphotometry. (B) The average relative fluorescence is plotted in lux (n = 3; error bars indicate SEM). The difference was significant (P < 0.01). (C) HeLa cells were transfected with nontargeting or Fyn siRNA and then infected with C. trachomatis L2. Cells were labeled with C₆-NBD-ceramide, and, after 8 h of back-exchange, lipids were extracted and the amount of sphingomyelin was determined by TLC and densitometry. Sphingomyelin in nontargeting siRNA treated cells was defined as 100%. (n = 3; error bars indicate SEM). The difference was significant (P < 0.0001).