Figure 4.

sAC‐derived cAMP promoted BSIA, while tmAC‐derived cAMP inhibited BSIA. (A) Control and inducible sAC knockdown H69 cholangiocytes were treated with 0.2 μg/mL doxycycline for the indicated periods of time. sAC knockdown was examined by immunoblotting. Immunoblot was quantified by ImageJ. Results are normalized to β‐actin. (B) Short hairpin control and sAC knockdown H69 cholangiocytes were induced with or without 0.2 μg/mL doxycycline for 48 hours and subsequently incubated with 750 μM NaCDC or vehicle for 1 hour. Caspase 3/7 activity was determined. One‐way ANOVA, P < 0.0001. The figure shows a representative experiment from a series of three independent experiments with similar results. (C) H69 cholangiocytes were incubated with or without 750 μM NaCDC in the presence of dimethyl sulfoxide (vehicle control), 50 μM KH7 (sAC‐specific inhibitor), 20 μM forskolin (tmAC‐specific activator), or 1 mM dibutyryl‐cAMP (membrane‐permeant cAMP analogue) for 4 hours in a 5% CO₂, 37°C incubator. Caspase 3/7 activity was determined. One‐way ANOVA, P < 0.0001. (D) Primary mouse cholangiocytes were incubated with or without 750 μM NaCDC in the presence of dimethyl sulfoxide (vehicle control) or 100 μM KH7 for 3 hours in a 5% CO₂, 37°C incubator. One‐way ANOVA, P < 0.0001. ***P < 0.001. Abbreviations: db‐cAMP, dibutyryl‐cAMP; DMSO, dimethyl sulfoxide; KD, knockdown; n.s., not significant; SHC, short hairpin control, TO, Tet‐operator.