Fig. 1

The effect of cobalt chloride on CaCo-2 BBe transepithelial electrical resistance and transepithelial flux of ¹⁴C–D-mannitol. a Western immunoblot data showing the significant increase of HIF-1α levels in whole cell lysates of 4 individual, 7-day post-confluent CaCo-2 BBe cell layers exposed to 125 and 250 μM CoCl₂ (or vehicle control) for 48 h. b The effect of Co⁺⁺ on barrier function. Seven-day post-confluent CaCo-2 BBe cell layers were refed in control medium or medium containing 125 μM CoCl₂ on the apical and basal-lateral sides 48 h prior to electrical measurements. Data shown represent the percent of control resistance expressed as the mean ± SEM of 20 cell layers per condition. After electrical measurements, radiotracer flux studies with 0.1 mmol/L, 0.1 μCi/mL ¹⁴C–D mannitol were performed on the same CaCo-2 BBe cell layers, as described in Materials and Methods. Data shown represent the percent of control mannitol flux rate and are expressed as the mean ± SEM of 20 cell layers per condition. ***P < 0.001 vs control. (Student’s t test, two-tailed). c The time course of changes in transepithelial electrical resistance and transepithelial mannitol flux resulting from CoCl₂ exposure. Seven-day post-seeding CaCo-2 BBe cell layers on Millicell PCF filters were refed in control medium or medium containing 125 μM CoCl₂ (apical and basal-lateral compartments) and transepithelial electrical resistance was recorded at 2, 4, 12, 24, 48, and 72 h (different cell layers were used for each designated time point). Data shown represent the percentage of time-matched resistance (relative to vehicle-treated control cell layers) and are expressed as the mean ± SEM of 4 cell layers per condition. After electrical measurements, radiotracer flux studies with 0.1 mmol/L, 0.1 μCi/mL ¹⁴C–D mannitol were performed on the same CaCo-2 BBe cell layers. **P < 0.01 (two tailed Student’s t test vs time-zero cell layers). d The effect of increasing concentrations of CoCl₂ on CaCo-2 BBe transepithelial electrical resistance and transepithelial flux of ¹⁴C–D-mannitol. Seven-day post-seeding CaCo-2 BBe cell layers on Millicell PCF filters were refed in control medium or medium containing 50 μM, 125 μM, or 250 μM CoCl₂ 48 h prior to electrical measurements (apical and basal-lateral compartments). Data shown represent the mean ± SEM of 4 cell layers per condition. After electrical measurements, radiotracer flux studies with 0.1 mmol/L, 0.1 μCi/mL ¹⁴C–D mannitol were performed on the same CaCo-2 BBe cell layers used for resistance studies. NS indicates non significance vs control. #P < 0.05 vs 50 μM CoCl₂; ###P < 0.001 vs 50 μM CoCl₂; ***P < 0.001 vs control. (one-way ANOVA followed by Tukey’s post hoc testing)