Fig 2: Respiratory epithelial knockout of TMEM16A eliminates CFTR currents. (a) Original recordings of the transepithelial voltage Vte and the effect of ATP (100 µM) in tracheas from FOXJ1-Cre–TMEM16A wt/wt (+/+) and FOXJ1-Cre–TMEM16A flox/flox (-/-) mice. (b) Summary of the calculated ATP-induced short circuit currents (?Isc). (c,d) Original recordings of Vte and summary of CCH-induced ?Isc in +/+ and -/- tracheas. (e,f) Original recordings of Vte and summary of cAMP (IBMX 100 µM/forskolin 2 µM)-induced ?Isc in +/+ and -/- tracheas. (g) Original recordings of whole cell currents activated by ATP in primary-cultured tracheal epithelial cells from TMEM16A+/+ and TMEM16A-/- mice. Experiments were performed in the presence of an inhibitor of Ca2+ activated K+ channels, TRAM-34 (100 nM). (h) Corresponding current/voltage relationships of whole cell currents activated by ATP in TMEM16A+/+ and TMEM16A-/- cells. (i) Original recordings of whole cell currents activated by cAMP in primary cultured respiratory epithelial cells from TMEM16A+/+ and TMEM16A-/- mice. (j) Corresponding current/voltage relationships of whole cell currents activated by cAMP in TMEM16A+/+ and TMEM16A-/- cells. Mean ± SEM; *Significant activation by cAMP or ATP (paired t-test). #Significant difference between -/- and +/+ (unpaired t-test). (number of mice or cells, respectively).

Fig 3: Curcumin mitigates heat-induced inflammation by upregulating CFTR in airway epithelial cells in vitro and in vivo.(a,b) Western blotting for CFTR and COX-2 (a), and ELISA detection of PGE2and IL-8 (b) in 16HBE14o- cells before (-) and after (+) heat-treatment in the presence (+) or absence (-) of curcumin (Cur, 10 µM)or DMSO as vehicle control. Curcumin was administrated either 4 hours before (Pre), 0.5 hour after (Post, 0.5 h), or 8 hours after (Post, 8 h) the heat treatment. Tubulin was used as loading control. *P < 0.05; **P < 0.01; ***P < 0.001. n = 3, One-way ANOVA. (c) Curcumin (Cur,10 mg/kg body weight) was nasally administrated in rats in addition to heat-inhalation. H&E images show airway morphology of rats with heat-inhalation alone (Heat), treated with Cur after heat-inhalation (Heat + Cur) or without heat-inhalation (Sham). (d) Immunohistochemistry staining for MPO in airway tissues from rats of the three groups. The number of MPO-positive cells in each group was counted and is shown as mean ± SEM. ***P < 0.001 as compared to rats with heat-inhalation alone. n = 3, One-way ANOVA. (e,f)Representative images and corresponding semi-quantification of immunohistochemistry staining for CFTR (e) and COX-2 (f) in airway tissues from rats of the three groups. The intensity of CFTR and COX-2 signal was calibrated with the epithelial cell counts. **P<0.01; ***P<0.001 as compared to rats with heat-inhalation alone. n = 3, One-way ANOVA. (g) Western blotting for CFTR and COX-2 in normal rat airway tissues (Normal) or tissues with (Heat) or without (Sham) heat-inhalation administrated with curcumin (Cur, 10 mg/kg body weight) or DMSO. (h) ELISA for rat PGE2 and IL-8 using homogenates of rat airway tissues with (Heat) or without (Sham) heat-inhalation administrated with curcumin (Cur, 10 mg/kg body weight) or DMSO Bars = 100µm.

Fig 4: Heat-induced temporal changes in CFTR and COX-2 expression in airway epithelial cells in vitro and in vivo.(a,b) 16HBE14o- cells were cultured at 37 °C till 80% confluence before incubated at 52 °C for 5 min as heat-treatment and subsequently back at 37 °C for recovery. Cells were collected before (Ctrl), after heat-treatment (0 h), or after recovery for 8 hours (8 h) to 5 days (5d) for QRT-PCR (a) and western blot (b) analysis of CFTR and COX-2. Data are means ± SEM from at least three independent experiments. ***P < 0.001, **P < 0.01 as compared to Ctrl. n = 3. One-way ANOVA. (c,d) Representative images and corresponding semi-quantification of immunohistochemistry staining for CFTR (c) and COX-2 (d) in rat lung tissues 8 h-5d after inhalation of heated air. Same surgical procedures without the heat-inhalation were used in rats of the sham group. The intensity of CFTR and COX-2 positive signals was calibrated with the epithelial cell counts. ***P < 0.001, **P < 0.01 as compared to Ctrl. n = 3. One-way ANOVA. Bars = 100 µm (c,d).

Fig 5: Decrease of CFTR, ZO-1, occludin and E-cadherin in intestinal epithelia post severe burn. (a) Bacterial counts of homogenates of lymph nodes, spleen and liver of sham or burnt mice. Data are means ± SEM from at least 3 independent experiments, n = 4. (b) Western blotting of CFTR, occludin, E-cadherin and ß-actin of the ileum of C57BL/6 mice with (burn) or without (sham) 30% TBSA full-thickness cutaneous burn (left panel) and statistical analysis (right panel). Band B (160KD) and Band C (180KD) of CFTR showing different levels of glycosylation of CFTR are indicated by arrows. As band C (180KD) represents the fully glycosylated mature CFTR protein, which functions as an ion channel, the quantitative bar graph corresponds to band C. (c) Representative images and statistical analysis of immunofluorescence staining for CFTR and ZO-1 in the ilea of sham and burnt mice. Scale bar = 20 µm. To prepare the monolayer, Caco-2 cells were seeded in collagen-precoated permeable polycarbonate membrane transwell with 0.4-µm pores and used for experiments after confluence. Caco-2 monolayers were exposed to 1% O2 for 6 or 12 hours as hypoxia treatment or without hypoxia treatment (H, hypoxia). Real-time quantitative PCR (d) and western blot (e) analyses of CFTR, ZO-1, occludin and E-cadherin were performed. (f) TER measurement of Caco-2 monolayers with or without hypoxia. All TER values were normalized to the control group (normoxia). One-way ANOVA followed by Tukey’s post hoc test was used when there were more than 2 groups. The experiments were repeated at least twice. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. CFTR cystic fibrosis transmembrane conductance regulator, DAPI 4',6-Diamidino-2-phenylindole, MLN mesenteric lymph nodes, N normoxia, OCLN occludin, TER transepithelial resistance ZO-1 zonula occludens-1, TBSA total body surface area