Fig. 5: D-Mannose rapidly and persistently activates AMPK via the Ca²⁺-CAMKK2 and lysosomal AXIN-LKB1-v-ATPase pathway to promote nuclear ACSS2-mediated TPH2 transcription.

A HEK293T cells expressing TPH2 with its native promoter were incubated by 500 μM AMPK agonist AICAR for 5 h to activate AMPK. Immunoblotting analyses were performed with the indicated antibodies. B HEK293T cells expressing TPH2 with its native promoter were incubated by 10 μM AMPK antagonist Compound C for 60 min to inhibit AMPK. Immunoblotting analyses were performed with the indicated antibodies. C The lysates of SH-SY5Y at different times with or without 1 mM D-Mannose stimulation were subjected to western blot with indicated antibodies. D Representative fluorescent images of pAMPK and DAPI in SH-SY5Y with or without 1 mM D-Mannose stimulation for 30 min. E ATP content in SH-SY5Y lysates with or without 1 mM D-Mannose stimulation at different times. F Lysosome PH were evaluated by flow cytometry with LysoSensor Green DND-189 (1 uM, 45 min) staining in response to 1 mM D-Mannose for 30 min in SH-SY5Y. G The lysates and lysosomal lysates of SH-SY5Y stimulated with or without 1 mM D-Mannose for 30 min were western blotted with the indicated antibodies. H CoIP of FLAG-tagged p18 with AXIN in HEK293T cells with or without 1 mM D-Mannose stimulation for 24 h. I CoIP of FLAG-tagged p18 with HA-tagged V0C or V1B2 in HEK293T cells with or without 1 mM D-Mannose stimulation for 24 h. J The lysates of SH-SY5Y stimulated with or without 1 mM D-Mannose for 30 min after 48H interference with siNC or siAXIN were subjected to western blot with indicated antibodies. K Ca2⁺ was assessed by flow cytometry with Fluo-4, AM (5 μM, 30 min) staining in response to 1 mM D-Mannose 30 min in SH-SY5Y. L The lysates of SH-SY5Y co-stimulated with or without 1 mM D-Mannose for 30 min following 30 min stimulation with DMSO or 25uM STO-609 were subjected to western blot with indicated antibodies. M The lysates of Hela stimulated with or without 1 mM D-Mannose for 30 min were subjected to western blot with indicated antibodies. N Representative fluorescent images of pAMPK and DAPI in Hela with or without 1 mM D-Mannose stimulation for 30 min. O ATP content in Hela lysates with or without 1 mM D-Mannose stimulation for 30 min. P Ca2⁺ was assessed by flow cytometry with Fluo-4, AM (5 μM, 30 min) staining in response to 1 mM D-Mannose for 30 min in Hela. Q The lysates of Hela co-stimulated with or without 1 mM D-Mannose for 30 min following 30 min stimulation with DMSO or 25uM STO-609 were subjected to western blot with indicated antibodies. R The lysates of Hela stimulated with or without 1 mM D-Mannose for 30 min after 48H interference with siNC or siCAMKK2 were subjected to western blot with indicated antibodies.