Cui Y , Li H , Wu S , Zhao R , Du D , Ding Y , Nie H , Ji HL .

AHA_14GRNT20130034 None, 14GRNT20130034 American Heart Association-American Stroke Association, R01 HL087017 NHLBI NIH HHS , R03 HL095435 NHLBI NIH HHS

	Figure 1. Downregulation of mouse alveolar fluid clearance in vivo by formaldehyde.Mouse lungs were instilled with 5% bovine serum albumin dissolved in physiologic saline solution in control group (Control), and in the presence of amiloride (Amil, 1 mM), formaldehyde (FA, 200 μM), and both (FA/Amil) for exposed mice. Reabsorption rate of instillate was computed as the percentage of instilled volume after 60 min (% 60 min). Average AFC values are presented as mean ± SE, One-way ANOVA. **P < 0.01, compared with control group. N = 6.
	Figure 2. Formaldehyde reduces short-circuit current level in H441 monolayers in a dose-dependent manner.(A) Representative short-circuit current (Isc) trace showing applications of a series of concentrations of 50, 100, 200, and 500 μM formaldehyde. Amiloride-sensitive currents are defined as the difference between the total current and the amiloride-resistant current, with the basal amiloride-sensitive current set as 100%. (B) Normalized amiloride-sensitive currents at each concentration were plotted as a dose-response curve, N = 7. The raw data were calculated by fitting with the Boltzmann equation. The IC50 value was 165.6 μM.
	Figure 3. Effect of formaldehyde on α-ENaC protein level in H441 cells.(A) Representative western blot of α-ENaC protein extracted from H441 cells exposed to 200 μM formaldehyde for 0-48 h. Blots were immunostained with antibody to α-ENaC, or to β-actin as a loading control. (B) Graphical representation of data obtained from three sets of western blots and quantified using gray analysis (α-ENaC/β-actin). Data are shown as the mean ± SE, *P < 0.05, compared with control.
	Figure 4. Effects of formaldehyde on transcriptional expression of ENaC α- and γ-subunits in H441 cells.mRNA samples were isolated from H441 cells treated with 200 μM formaldehyde for various periods. Relative levels of mRNA were calculated as α- or γ-ENaC/GAPDH ratios. (A) Real-time PCR results for α-ENaC mRNA. (B) Real-time PCR results for γ-ENaC mRNA. *P < 0.05, **P < 0.01, compared with control. N = 3.
	Figure 5. Effects of formaldehyde on ERK1/2 phosphorylation in H441 cells.(A) Representative western blot of phosphorylated ERK1/2 in protein extracted from H441 cells exposed to 200 μM formaldehyde for 0–90 min. Blots were immunostained with total ERK1/2 as a loading control. (B) Graphical representation of data obtained from three sets of western blots and quantified using gray analysis (p-ERK1/2/ERK1/2). Data are shown as the mean ± SE, *P < 0.05, **P < 0.01, compared with control.
	Figure 6. Effects of PD98059 on ERK1/2 phosphorylation in H441 cells.(A) Representative western blot of phosphorylated ERK1/2 in untreated H441 cells (Control), after pretreatment with PD98059 (PD98059), after treatment with formaldehyde (FA), and after pretreatment with PD98059 for 30 min prior to the addition of formaldehyde (PD98059/FA). Lanes shown in this figure are from the same western blot. (B) Graphical representation of data obtained from three sets of western blots and quantified using gray analysis (p-ERK1/2/ERK1/2). Data are shown as the mean ± SE, *P < 0.05, compared with control.
	Figure 7. Effects of formaldehyde on oxidative stress in the H441 cells.Production of ROS was measured using the fluorogenic substrate 2′,7′-dichlorofluorescein diacetate, which is oxidized to fluorescent 2′,7′-dichlorofluorescein. (A) Air-subjected control cell culture. (B–D) Cells exposed to 200 μM formaldehyde for 2 h, 6 h, and 24 h. (E) The ROS levels displays the summary fluorescence intensity measured from the images of H441 cells (N = 25 in each group) treated with different periods of formaldehyde. Background fluorescence level was corrected. Data are shown as the mean ± SE, **P < 0.01, compared with control.
	Figure 8. Formaldehyde alters the activity of heterologous human αβγ-ENaC channels expressed in oocytes.Oocytes were continuously perfused with formaldehyde and amiloride, and whole cell currents were monitored at 10-s intervals. (A) Representative inward current traces in the presence of formaldehyde and amiloride. Currents were digitized at −120 mV. Application of drugs is indicated by solid horizontal lines. (B) Average currents at −120 mV (mean ± SE). **P < 0.01 compared with basal level. N = 10 from 4 different frogs.
	Figure 9. Formaldehyde impairs properties of the plasma membrane.Oocytes expressing human αβγ-ENaC were incubated with formaldehyde for 24 h. (A) The shape of oocytes expressing human αβγ-ENaC observed by microscopy, with and without formaldehyde exposure. (B) Average amiloride-resistant currents at −120 mV (mean ± SE), reflecting oocyte permeability. **P < 0.01 compared with control. N = 10 from 4 different frogs.

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