Fig. 6

Suppression of the TBK1/IKK?-PDE3 signaling axis promotes ?-cell proliferation by increasing cAMP levels and mTOR activity. (A) Schematic of the TBK1/IKK?-PDE3 signaling that modulates cAMP-PKA-mTOR pathway. The sites of inhibition by PIAA and cilostamide are shown in red. (B) Quantification of cAMP levels (mean?±?SD) at 48 hpa (0.4?±?0.1 pmol/larva (DMSO) and 0.9?±?0.0 pmol/larva (PIAA)). (C) Representative Western blot showing increased pS6K1 levels in PIAA-treated recovering larvae. (D-I?) Confocal images of [Tg(ins:CFP-NTR)^s892; Tg(ins:Kaede)^jh6] larvae at 48 hpa, concurrently treated with EdU and DMSO (D-D?), PIAA (E-E?), cilostamide (F-F?), a combination of PIAA and cilostamide (G-G?), a combination of PIAA and rapamycin (H-H?), or a combination of cilostamide and rapamycin (I-I?), respectively, from 0?48 hpa, stained for pRPS6 (blue). The number of EdU-incorporated (white arrows) and pRPS6-positive (white arrowheads) ?-cells was increased in recovering larvae treated with both PIAA and cilostamide (G,G?) compared to individual compound-treated larvae (E-E? and F-F?). Rapamycin substantially suppressed the PIAA- and cilostamide-dependent increases in the number of EdU-incorporated and pRPS6-positive ?-cells (H-I?). (J) Quantification of the number (mean?±?SD) of total regenerated ?-cells (green bars) and regenerated ?-cells that incorporated EdU with pRPS6 immunoreactivity (white bars) at 48 hpa (in D-I?; 5.0?±?1.3 total regenerated ?-cells, of which 0.3?±?0.5 (DMSO), 17.8?±?2.8, of which 8.4?±?1.7 (PIAA), 12.7?±?1.9, of which 5.0?±?1.5 (cilostamide), 24.7?±?1.2, of which 13.3?±?0.6 (PIAA and cilostamide), 6.0?±?2.0, of which 1.0?±?1.0 (PIAA and rapamycin), and 5.2?±?1.1, of which 0.8?±?0.8 (cilostamide and rapamycin) incorporated EdU with pRPS6 immunoreactivity). (K) The percentage (mean?±?SD) of regenerated ?-cells that incorporated EdU with pRPS6 immunoreactivity at 48 hpa (in D-I?; 6.1?±?9.5% (DMSO), 47.3?±?7.5% (PIAA), 50.4?±?8.8% (cilostamide), 54.2?±?4.2% (PIAA and cilostamide), 13.9?±?12.7% (PIAA and rapamycin), and 13.7?±?13.0% (cilostamide and rapamycin)). Cells in 20 planes of confocal images from 10 individual larvae were counted per condition. ***P?<?0.001.