FIG 3.

3OC12-mediated intracellular acidification and Ca²⁺ rise are PON2-dependent. Cells were loaded with BCECF-AM to detect cytosolic pH (A to C) or Fluo-4 AM to detect cytosolic Ca²⁺ rise (D to F); cells were then treated with test compounds, and fluorescence was measured on a microplate reader. HEK and EA.hy 926 cells were treated with 25 μM 3OC12. HBEC (C and F) were transfected with scrambled or PON2 siRNA and 3 days later treated with 50 μM 3OC12. For the experiment shown in panel F, cells were also treated with a 25 μM concentration of the phospholipase C activator m-3M3FBS. PON2 activity levels in scrambled and PON2 siRNA transfected cell lysates were 15.0 ± 2.9 U/mg and 1.6 ± 0.7 U/mg, respectively (C and F). Each trace is the mean of three separate experiments in which the background (DMSO-treated cells) values were subtracted from the values of the stimulus-treated cells. Dashed lines denote the standard deviations. Differences between the results for the groups were analyzed by a two-way repeated-measures analysis of variance. The pH drop was significantly lower in the HEK PON2 (P < 0.01), EA.hy H114Q PON2 (P < 0.05), and the scrambled siRNA (P < 0.01) groups than in the respective control groups in panels A, B and C, respectively. The Ca²⁺ rise was significantly different from that of the control group (D and E) and from that of the PON2 siRNA 3OC12-treated group (F) (P < 0.01).