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. 2020 Nov 30;11:593492. doi: 10.3389/fphar.2020.593492

FIGURE 6.

FIGURE 6

LMW compounds differentially affect APPL1-regulated FSHR recycling and cAMP signaling. (A) Western blot showing total cellular levels of APPL1 from cells treated with scramble (CTL) or APPL1 siRNA (siAPPL1). GAPDH was used as a loading control. (B) Quantification of APPL1 protein levels normalized to GAPDH protein levels from (A), and expressed as % of scramble, n = 3 independent experiments, t-test: **p < 0.01. (C) Maximum intensity projections from SEP-FSHR expressing cells imaged live by TIR-FM following transfection with either scramble (CTL) or APPL1 siRNA (siAPPL1) and stimulated with either FSH (10 nM), B3 or T1 (10 μM) for 5–20 min. Scale bar = 5 μm. (D–E) Quantification of the number of FSHR recycling events. n = 21–24 cells per condition collected across four independent experiments for (D) and n = 13–18 cells per condition collected across three independent experiments for (E). Two-way ANOVA: *p > 0.05, **p < 0.01, ***p < 0.001. (F,G) Intracellular levels of cAMP measured in cells stably expressing FLAG-FSHR following transfection with either scramble (CTL) or APPL1 siRNA (siAPPL1). Cells were stimulated with either DMSO, FSH (10 nM) or B3 (10 μM) (F) or DMSO, FSH (10 nM) or T1 (10 μM) (G) for 5 min. n = 5 (F) or 3 (G) independent experiments. Two-way ANOVA: *p < 0.05, **p < 0.001 ****p < 0.0001.