1. Base Treatment Corrects Defects Due to Misfolding of Mutant Cystic Fibrosis Transmembrane Conductance Regulator 
Wan Namkung, Kyung Hwan Kim, Min Goo Lee 
Gastroenterology 
Volume 129, Issue 6, Pages (December 2005)
DOI: /j.gastro
Copyright © 2005 American Gastroenterological Association Terms and Conditions

2. Figure 1
Cl−/NO3− exchange in CFPAC-1 cells. Cyclic AMP–dependent Cl− channel activities were estimated by measuring [Cl−]i changes with the Cl−-quenching fluorescent probe MQAE in coverslip-grown CFPAC-1 cells. (A) Cells grown in 2% carbon dioxide for 12 hours (red trace) showed a cAMP-activated Cl− channel activity. (B) Bafilomycin A1 (BA) treatment induced a cAMP-activated Cl− channel activity in a dose-dependent manner. (C) Increases in MQAE fluorescence 10 seconds after the application of Cl−-free NO3−-containing solution with cAMP cocktail (forskolin and IBMX) are averaged. *P < .05, difference from 5% carbon dioxide with 0 μmol/L BA.
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
Luminal Cl−/HCO3− exchange in CFPAC-1 monolayers. Monolayers of CFPAC-1 cells were loaded with BCECF, and pHi was measured in a double perfusion chamber. The activities of luminal Cl−/HCO3− were measured in high-K+ (100 mmol/L), HCO3−-containing (25 mmol/L; gassed with 5% carbon dioxide; pH 7.4) solutions. (A) In 5% carbon dioxide–grown CFPAC-1 cells, cAMP treatment did not induce significant changes in luminal Cl−/HCO3− exchange. (B) In 2% carbon dioxide–grown cells, cAMP treatment increased luminal Cl−/HCO3− exchange by an average of 74%. (C) The results shown are the means ± SEM of 6 and 5 experiments. **P < .01, difference from basal.
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
Expression of CFTR in CHO cells after alkaline treatments. Immunoblottings were performed in cells transiently transfected with (A) WT or (B) ΔF508 CFTR plasmids by using M3A7 anti-CFTR antibody. The molecular sizes of the mature complex–glycosylated band C, the core-glycosylated band B, and the nascent polypeptide band A CFTR proteins were marked in between (A) and (B). In (A), parts of protein samples were digested with peptide-N-glycosidase F (PNG) to confirm each CFTR band. In (B, right), ΔF508 CFTR–transfected cells were incubated with the protein synthesis inhibitor cycloheximide (CHX) for the designated time before sample preparation. kD, kilodalton.
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
CFTR Cl− currents in CHO cells transfected with ΔF508 CFTR plasmid. CFTR Cl− currents were measured in the whole-cell configuration with cAMP stimulation (forskolin + IBMX). When incubated in 2% carbon dioxide for 12 hours, cells transfected with ΔF508-CFTR plasmid showed a cAMP-activated current (A), which had a linear I-V relationship and was inhibited by DPC (B). (C) Normalized currents as current densities are averaged. *P < .05, difference from 5% carbon dioxide.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
Golgi and cytosolic pH changes induced by 2% carbon dioxide and bafilomycin A1 (BA). (A–C) Golgi pH was measured by using pEYFP-Golgi plasmids in CHO cells. Incubation in 2% carbon dioxide or in BA increased the Golgi pH from 6.2 to approximately 7.0. (D–E) Cytosolic pH (pHi) was measured by using BCECF. The pHi was increased by 2% carbon dioxide treatment, but not by BA treatment.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
pH measurements and CFTR immunoblottings with a prolonged 2% carbon dioxide incubation. (A) Cytosolic pHi measurements in ΔF508 CFTR–transfected cells. For pHi measurements in 2% carbon dioxide for >1 hour, all procedures, including BCECF loading, were performed under 2% carbon dioxide–equilibrated conditions. The cells retained an alkaline cytosolic pH for up to 12 hours of incubation with 2% carbon dioxide. (B) Immunoblotting of CFTR. The amount of ΔF508 CFTR protein reached a peak after 4 hours of treatment with 2% carbon dioxide, and the increased protein levels were retained up to 12 hours. (C and D) Golgi pH was measured with prolonged incubations with 2% carbon dioxide or BA. #Peak pH values within 5 minutes of application of 2% carbon dioxide or BA.
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
Low carbon dioxide incubation and BA treatments do not affect CFTR gene expression or ubiquitin–proteasome pathways. (A) Real-time PCR analysis was performed according to the procedures described in Methods. The results are an average of 3 separate experiments. (B) Proteasomal activities of CHO cells were measured with UbG76V-GFP constructs.25 CHO cells grown in 5% carbon dioxide minimally expressed UbG76V-GFP because of extensive degradation by proteasome (left upper panel). Cells treated with the proteasomal inhibitor lactacystin (10 μmol/L for 12 hours; right upper panel) expressed significant amounts of UbG76V-GFP protein. Treatments with 2% carbon dioxide or with BA for 12 hours did not increase the fluorescence of UbG76V-GFP. All images were taken under the same laser intensity and image acquisition set up with a confocal microscope (Zeiss LSM 510).
Gastroenterology  , DOI: ( /j.gastro )
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9. Figure 8
Effects of chronic NaHCO3 treatment in homozygous ΔF508 mice. Mice were divided into 4 groups and treated with water (pure tap water), sucrose (tap water containing 2% sucrose), NaCl (tap water containing 150 mmol/L NaCl), or NaHCO3 (tap water containing 2% sucrose and 280 mmol/L NaHCO3). (A) The survival rate of homozygous ΔF508 mice was analyzed. Treatment with NaHCO3 significantly increased the survival of ΔF508 mice as compared with the water-treated group (P < .01), the sucrose-treated group (P < .01), and the NaCl-treated groups (P < .05). (B) Surviving animals were killed, and duodenal Isc, which was activated by cAMP (10 μmol/L forskolin and 100 μmol/L IBMX) and inhibited by DPC (100 μmol/L), was measured. The CFTR-dependent Isc of NaHCO3-treated ΔF508 mice reached 21% of that of WT mice. **P < .01; difference from water-, sucrose-, and NaCl-treated ΔF508 groups.
Gastroenterology  , DOI: ( /j.gastro )
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10. Figure 9
CFTR expression in the mouse colon. CFTR immunostainings were performed as described in Methods. In WT mice, CFTR is highly expressed in the luminal membrane of colonic crypt cells (left). This was abolished in samples obtained from water-treated ΔF508 mice (middle). Chronic NaHCO3 treatment for 15 weeks resulted in a partial resumption of the luminal expression of CFTR protein in ΔF508 mice (right).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions