FIGURE 3.

LMW agonists of FSHR induce a greater exocytic rate than FSH. (A) Maximum intensity projections from SEP-FSHR expressing HEK 293 cells imaged live by TIR-FM before and after 5-min stimulation with either DMSO, FSH (10 nM), B3, T1 or T2 (10 μM). Yellow squares indicate exocytic events detected in the corresponding TIR-FM movies (Supplementary Movies S1–S5). Scale bar = 10 μm. (B) Quantification of the number of FSHR exocytic events from (A); n = 7–11 cells/condition collected across at least three independent experiments. One-way ANOVA: *p < 0.05, ****p < 0.0001. (C) Images (top) and kymographs (bottom) of representative FSHR exocytic events (yellow squares) detected after 5-min stimulation with either FSH (10 nM), B3 or T1 (10 μM). (D) Fluorescence intensity of the representative FSHR exocytic events shown in (C). Data represent average fluorescence values within an ROI drawn around each event measured 3 s (30 frames) before and after event burst (t0) minus background fluorescence (= fluorescence measured in the ROI during the 30 frames before event burst (t0) was averaged and subtracted from fluorescence values at each frame); values were normalized considering FSH fluorescence at t0 = 100%. (E) Fluorescence intensity at time of burst of n = 212, 326, and 328 exocytic events from 11, 9 and 9 cells treated with FSH, B3 and T1, respectively, from (B); values represent maximum intensity fluorescence minus background fluorescence in each ROI at time of event burst; values were normalized considering FSH = 100%; Kruskal-Wallis test: ***p < 0.001, ****p < 0.0001.