Figure S4.
Characterization of inhibitors used in the study. (A) Confocal microscopy images of MIN6 cell pseudoislets transfected with control (left) or CNGA3 (right) siRNA and immunostained for acetylated tubulin (green) and CNGA3 (magenta). (B) Quantifications of the ciliary CNGA3 intensity (line profile) from control (magenta) and CNGA3 KD (green) MIN6 cell pseudoislets (n = 78 and 81 cilia from two individual experiments). (C) Ciliary CNGA3 fluorescence intensity in control and CNGA3 KD MIN6 cell pseudoislets (means ± SEM for 78 and 81 cilia; two-tailed unpaired Student’s t test). (D) Confocal microscopy images of a mouse islet immunostained for acetylated tubulin (green) and CNGB1 (magenta). (E) Quantification of ciliary Ca2+ activity in mouse islets expressing 5HT6-GGECO1 following exposure to 100 nM ANP alone or in combination with 100 µM L-cis-diltiazem (Lc-D; means ± SEM; n = 215, 122, and 93 cilia from 7–15 islets; * P < 0.05, ** P < 0.01. Statistical significance assessed with Wilcoxon signed-rank test). (F) Mouse islets loaded with the Ca2+ indicator Fluo-4 were imaged on the stage of an epifluorescence microscope while being exposed to two consecutive 3-min pulses of 30 mM KCl. The second pulse was performed in the presence of the indicated L-type Ca2+ channel inhibitor. The data presented are the ratios between the Fluo-4 fluorescence increase during the second and first pulse. Each data point represents one cell (means ± SEM; n = 3 islets per condition; ANOVA with Dunnett’s post hoc test). (G) Confocal microscopy images of a mouse islets immunostained for acetylated tubulin (green) and CaV1.2 (magenta). Quantification of line profiles drawn along the cilium as shown in the lower figure are shown at the bottom right (n = 75 cilia; n = 2). (H) Confocal microscopy images of a mouse islets immunostained for acetylated tubulin (green) and CaV1.3 (magenta). Quantification of line profiles drawn along the cilium as shown in the lower figure are shown at the bottom right (n = 80 cilia; n = 2). (I) TIRF microscopy recordings of the FRET ratio of the cAMP sensor Epac1S188 expressed in two mouse islet β-cells during stimulation of cAMP production with 100 nM GLP-1 and inhibition via clonidine-induced activation of inhibitory G-proteins. Traces are from one control cell (black) and one cell treated with pertussis toxin (blue). (J) Quantification of the reduction in GLP-1–induced cAMP formation following addition of clonidine in control islet cells and islet cells treated with pertussis toxin. Notice that the inhibitory effect of clonidine is reduced following pertussis toxin treatment (means; n = 33 and 18 cells from 5 experiments; two-tailed unpaired Student’s t test).