Fig 2: Cytosolic calcium influx regulates AMPK-STIM2 colocalization. HeLa cells transiently expressing AMPK-a–mCherry and STIM2-YFP were incubated with medium containing ionomycin (1 µM) or thapsigargin (1 µM) for 30 min and processed for immunofluorescence imaging. Represented images from each condition are presented. Overlap coefficients for the STIM2 and AMPK signal are presented in the upper right corner of the merge panels (n = 12 for DMSO- or ionomycin-treated cells; n = 15 for thapsigargin-treated cells). In all images, insets display the selected area along with the overlays. Scale bars, 5 µm. P < 0.0001.

Fig 3: STIM2 regulates calcium-induced AMPK activation. A, B) Cytosolic calcium influx promotes STIM2–AMPK interaction. HEK 293T (A) and HeLa (B) cells stably expressing STIM2-SFB were incubated with medium containing ionomycin (1 µM) or thapsigargin (1 µM) for 30 min. Cell lysates were subjected to immunoprecipitation using S-protein beads. Coprecipitates were analyzed by Western blotting. C–F) STIM2 modulates calcium-induced AMPK activation. STIM2-deficient 786-O (C, D) or HeLa (E, F) cells were incubated with medium containing ionomycin (1 µM) or thapsigargin (1 µM) for the indicated time points for 786-O cells or for 30 min for HeLa cells. Cell lysates were then analyzed by immunoblotting for the phosphorylated and total levels of indicated proteins. G, H) Thapsigargin-induced Ca2+ influx in control and STIM2-depleted 786-O (G) or HeLa cells (H) was measured with Fura-2 AM. Error bars denote sem [n = 20 (control), n = 20 (STIM2 sg1), n = 20 (STIM2 sg2) (G); n = 50 (control), n = 83 (STIM2 sg1), n = 67 (STIM2 sg2)] (H).

Fig 4: PRKAB1 or PRKAB2 is required for the effect of MßCD on cholesterol reduction, autophagy induction, and increase of autophagy flux. (A, B) Filipin staining of NPC1 fibroblasts treated with MßCD. (A) PRKAB1 or PRKAB2 expression was silenced by shRNA and the subunits re-expressed with transient transfection of either PRKAB1 or PRKAB2 activation vectors. (B) ATG12 expression was silenced by shRNA. All of the cells were treated with 100 µM MßCD or DMSO control for 4 d followed by the filipin staining assay. (C) NPC1 fibroblasts transfected with ATG12 shRNA or control shRNA were treated with 100 µM MßCD or DMSO control for 24 h, followed by western blot analysis with the indicated antibodies. LC3B-II, SQSTM1 and ATG12 expression were normalized to GAPDH expression. (D-G) MßCD effects on SNARE proteins interactions. (D) Immunoprecipitation and western blot analysis of 3 SNARE proteins (VAMP8, STX17 and SNAP29). WT fibroblasts with the PRKAB/AMPK ß-subunit silenced or with AMPK ß-subunit re-expression was treated with MßCD or DMSO for 24 h. Cells were lysed and directly immunoprecipitated with anti-VAMP8 antibody followed by western blot analysis with the indicated antibodies. (E) NPC1 and WT fibroblasts were treated with MßCD for the indicated times, followed by immunoprecipitation with anti-SNAP29 and western blot analysis with the indicated antibodies. (F) Immunofluorescence staining and colocalization of LC3 with VAMP8. Indicated WT fibroblasts, transiently transfected with TagRFP-LC3 lentiviral particles, were treated with 100 µM MßCD or DMSO for 24 h and stained with anti-VAMP8 antibody. The punctate structures of VAMP8 were colocalized with RFP-LC3 (yellow color in the merged images). Data represent mean ± SEM of 10 images. (G) NPC1 and WT fibroblasts, transiently transfected with TagRFP-LC3 lentiviral particles were treated with MßCD for the indicated times, followed by staining with anti-VAMP8 antibody. The colocalization of VAMP8 and RFP-LC3 puncta was analyzed as above. Abbreviations: PRKAB1 or PRKAB2 act., PRKAB1 or PRKAB2 activation vectors; Mock vec., mock vector; LY, lysosome; AP, autophagosome. Scale bar: 10 µm (yellow) and 1 µm (white).

Fig 5: The working model of STIM2 regulation of calcium-stimulated AMPK activation.