Panel A shows a line graph depicting the accumulation of intracellular calcium (Ca2 positive) in AR42J cells treated with increasing doses of CCK-8 for 15 minutes. The x-axis represents the concentration of CCK-8 in nanomolar, and the y-axis represents the calcium concentration in arbitrary units per milligram of protein. The graph shows an upward trend, indicating increased calcium accumulation with higher doses of CCK-8. Panel B shows a line graph depicting the accumulation of intracellular cyclic adenosine monophosphate (cAMP) in AR42J cells treated with increasing doses of CCK-8 for 15 minutes. The x-axis represents the concentration of CCK-8 in nanomolar, and the y-axis represents the cAMP concentration in picomoles per milligram of protein. The graph shows a slight increase in cAMP accumulation at lower doses, with a more significant increase at higher doses. Panel C shows confocal fluorescent images of AR42J cells transiently transfected with various constructs and incubated with 10 nanomolar CCK-8. The images are stained with -HA antibody (red) and ZG-specific AMY is detected with a monoclonal antibody (green). The images show orthogonal projections of Z-stack series. Panel D shows line scan profiles of fluorescence intensity for the analysis of colocalization of different constructs and AMY. The x-axis represents the distance in micrometers, and the y-axis represents the fluorescence intensity in arbitrary units. The red curves represent the constructs, and the green curves represent AMY. Panel E shows Western blot images of total extracts (TE) or purified ZGs from AR42J cells transfected with different constructs. The blots are detected using -HA antibody or an -RnAQP12-specific antibody, with protein loading normalized by the -AMY antibody. Panel F shows representative immunoblots of endogenous AQP12 and AMY in TE and ZG extracts from cells treated with CCK-8 in the presence of PKC inhibitor BimII or the drug vehicle. The right panels show the corresponding quantitation of the channel in ZGs normalized to AMY. Panel G shows representative immunoblots of endogenous AQP12 and AMY in TE and ZG extracts from cells treated with PMA. The right panels show the corresponding quantitation of the channel in ZGs normalized to AMY. Panel H shows representative immunoblots of RnAQP12-WT-HA, RnAQP12-S9A-HA mutant, and AMY in TE and ZG extracts using -HA and -AMY antibodies, with corresponding quantitation in ZGs normalized to AMY. Panel I shows a line graph depicting the time-course of AMY secretion into the culture medium by cells transiently transfected with different constructs and induced by CCK-8. The x-axis represents time in minutes, and the y-axis represents AMY production as a percentage of control at 60 minutes. The graph shows the mean with standard error of the mean (SEM) from three independent experiments.
PKC and YPD control the targeting of rat and human AQP12 to the ZGs in AR42J pancreatic acinar cells. (A and B) Accumulation of [Ca2+]i (A) and [cAMP]i (B) in dexamethasone-treated AR42J cells induced with increasing doses of CCK-8 for 15 min. (C) Confocal fluorescent images of dexamethasone-differentiated AR42J cells transiently transfected with an empty pcDNA3 expression vector (pcDNA, control) or a vector carrying the RnAQP12-WT-HA, RnAQP12-ΔYPD-HA, HsAQP12-WT-HA, or HsAQP12-ΔYPD-HA constructs, and incubated with 10 nM CCK-8. The channels are stained with α-HA antibody (red), whereas ZG-specific AMY is detected with a monoclonal antibody (green). The images show the compilation of Z-stack series into orthogonal projections (reconstructed 3D image). (D) Line-scan profiles (white bar in the left merge image in C) of fluorescence intensity for the analysis of colocalization of RnAQP12-WT-HA, RnAQP12-ΔYPD-HA, HsAQP12-WT-HA, or HsAQP12-ΔYPD-HA (red curves), and AMY (green curves). (E) Western blot of TE or purified ZGs from AR42J cells transfected with the empty pcDNA3 plasmid or expressing the different rat (left) or human (right) constructs as indicated. The different polypeptide constructs were detected using the α-HA antibody or an α-RnAQP12-specific antibody (to detect the endogenous channel), and protein loading was normalized by the α-AMY antibody. (F and G) Representative immunoblots of endogenous AQP12 and AMY in TE and ZG extracts from using the α-RnAQP12–specific antibody from nontransfected cells treated with 10 nM CCK-8, in the presence of 10 µM of the PKC inhibitor BimII or the drug vehicle (Veh) (F), or from cells treated only with 100 nM PMA (G). The right panels show the corresponding quantitation of the channel in ZGs normalized to AMY. (H) Representative immunoblots of RnAQP12-WT-HA, RnAQP12-S9A-HA mutant, and AMY in TE and ZG extracts using α-HA and α-AMY antibodies, and corresponding quantitation in ZGs normalized to AMY (lower panels), from cells expressing both constructs and treated with 10 nM of CCK-8. In F-H, data (n = 3–4 independent experiments) were statistically analyzed by unpaired Student’s t test (**P < 0.01; ***P < 0.001; as indicated in brackets). (I) Time course of AMY secretion into the culture medium by cells transiently transfected with empty pcDNA3 or vector carrying the different RnAQP12 constructs as indicated and induced by CCK-8. Data (mean ± SEM, n = 3 independent experiments) were statistically analyzed by one-way ANOVA at each time point, followed by Tukey’s multiple comparisons test. ***P < 0.01, with respect to empty pcDNA3-transfected cells. TE, total extracts. Source data are available for this figure: SourceData F8.