Fig. 2. LPS decreases the cellular concentration of H₂S while increasing CSE abundance and H₂S production capacity in RAW264.7 cells.

(A) RAW264.7 cells loaded with the H₂S indicator SF7-AM were pretreated with 500 µM GYY4137 or vehicle before being incubated for 4 hours with either vehicle (control) or LPS (1 µg/ml). Cells were then analyzed by fluorescence microscopy. Images are representative of six independent experiments. When used, GYY4137 was present throughout the entire incubation period. Scale bar, 50 µm. (B) Analysis of SF7-AM fluorescence data. Data are means ± SEM of the SF7-AM fluorescence intensities of individual cells expressed as a percentage of control, untreated cells. Data are from single cells and were pooled from six independent samples. *P = 0.017, **P = 0.012, ^#P = 0.016 by one-way analysis of variance (ANOVA). (C) RAW264.7 cells were pretreated with vehicle or 500 µM GYY4137 before being incubated for 4 hours with vehicle or LPS (1 µg/ml). Cells were then analyzed by Western blotting with antibodies against the indicated proteins. Western blots are representative of three independent experiments. For quantification of band intensities, see fig. S1. (D) RAW264.7 cells were incubated for 4 hours with vehicle or LPS (1 µg/ml). Cell lysates were then analyzed by the methylene blue technique to detect H₂S. Data are expressed as a percentage of the H₂S measured in untreated cell lysates and are means ± SEM of five experiments. *P < 0.001 by unpaired t test. (E) RAW264.7 cell cultures were left untreated or were treated overnight with 50 µM GYY4137 in the absence or presence of LPS (1 µg/ml). Cell culture medium was then analyzed by the methylene blue technique to determine the concentration of H₂S. Data were normalized to the amount of H₂S measured in cell-free medium. Data are expressed as a percentage of the H₂S found in the culture medium of untreated cells and are means ± SEM of five experiments.