Zhang J , Chen S , Liu H , Zhang B , Zhao Y , Ma K , Zhao D , Wang Q , Ma H .

5R01-DK067110 NIDDK NIH HHS

	Figure 2. H2S ameliorates H2O2-elicited oxidative stress in A6 cells.All experiments were assessed in the presence of 50 µM 2,2′-dipyridyl. The images represent the level of intracellular ROS detected by the membrane-permeable fluorescent probe, carboxy-H2DCFDA, under indicated conditions in A6 cells. (A) The left image shows that there was a certain residual level of intracellular ROS under control condition; middle image shows a significant elevation of intracellular ROS level upon application of 3 mM H2O2; right image shows that this H2O2-induced increase in intracellular ROS was abolished by additional 0.1 mM NaHS. (B) The images represent that the intracellular ROS levels under control (left), treatment of A6 cells with 0.1 mM NaHS for 30 min (middle) followed by additional 0.1 mM H2O2 for 30 min (right). (C) and (D) Summarized fluorescent intensity of (A) and (B), respectively. Data were from three independent paired experiments. **P<0.01 compared with control group, ##P<0.01 compared with H2O2 treatment group.
	Figure 3. H2S inhibits H2O2-induced elevation of PI(3,4,5)P3 near the apical compartment of A6 cells.Left images show confocal microscopy XY sections of apical, lateral, and basal membranes of A6 cells as indicated. Right images show XZ sections of A6 cells. (A) Control; (B) the cells were treated with 3 mM H2O2 for 30 min; (C) the cells were treated with 0.1 mM NaHS for 30 min; (D) the cells were co-treated with 3 mM H2O2+0.1 mM NaHS for 30 min. (E) Summarized mean fluorescent intensities measured in and in the vicinity of the apical membrane from three independent experiments, which represent the levels of PI(3,4,5)P3 near the apical region of the membrane. **P<0.01 compared with control group, ##P<0.01 compared with H2O2 treatment group.
	Figure 4. H2O2 oxidizes PTEN and H2S protects PTEN to be oxidized by H2O2.All experiments were assessed in the presence of 50 µM 2,2′-dipyridyl. (A) and (B) Western blot analysis demonstrates that the total PTEN protein expression was not altered by treatment of A6 cells with 1 mM, 3 mM and 5 mM H2O2. (C) and (D) Western blot analysis shows that the magnitude of reduced (active) PTEN was dramatically decreased upon treatment of A6 cells with 0.3 mM, 1 mM and 3 mM H2O2 for 30 min; the intensity of the bands corresponding to reduced PTEN in the top panels of (C) was determined and presented as a percentage of the sum of the intensities of the bands corresponding to the oxidized and reduced proteins. (E) and (F) show that H2S protected 3 mM H2O2 induced PTEN oxidation (n = 3 in each group) **P<0.01 compared with control group; P<0.05 compared with 0.3 mM H2O2 treatment group; ##P<0.01 compared with H2O2 treatment group.
	Figure 5. Inhibition of PTEN by BPV(pic) results in accumulation of PI(3,4,5)P3 near the apical compartment of A6 cells.The experiments were performed under different conditions as follows: (A) control; (B) the cells were treated with 3 mM H2O2 for 30 min; (C) the cells were treated with 30 nM BPV(pic) for 30 min; (D) the cells were co-treated with 30 nM BPV(pic) +3 mM for 30 min. (E) Summarized mean fluorescent intensities measured as ascribed in Figure 3 from three independent experiments, which represent the level of PI(3,4,5)P3 in the apical membrane. **P<0.01 compared with control group, ##P<0.01 compared with BPV(pic) treatment group.
	Figure 6. Inhibition of PTEN leads to an aberrant activation of ENaC in A6 cells.Representative single-channel ENaC trace recorded from an A6 cell before and after addition of 30 nM BPV(pic) and 3 mM H2O2 to the basolateral bath. Two breaks between the traces indicate the 20 min omitted recording periods. (B) Summarized Po of ENaC before and post application of different reagents. n = 5 paired experiments. **P<0.01 compared with control group, #P<0.05 compared with BPV(pic) treatment group.
	Figure 1. H2S inhibits H2O2-induced enhancement of ENaC activity in A6 cells.(A) and (B) Representative single-channel ENaC traces respectively recorded from two A6 cells before and after addition of 3 mM H2O2 and 3 mM H2O2+0.1 mM NaHS to the basolateral bath; the breaks between the traces indicate omitted 20 min of the recording periods; downward events represent channel openings (the dashed gray line); the currents were monitored for at least 30 min before and after chemical application for both in (A) and (B). (C) and (D) Summarized Po of ENaC before and after application of different reagents as indicated (n = 6 paired experiments). **P<0.01 compared with control group, ## P<0.01 compared with H2O2 treatment group.

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