Figure 4.

Endocytosis induced by starvation via NAD⁺-CD38-cADPR-Ca²⁺ signaling in U87 cells. (A) After starvation treatment for different time, the extracellular NAD⁺ was detected using commercial kits. (B) The expression of CD38 was measured by western blot. (C) The concentration of cADPR was measured in U87 cells by HLPC-MS. (D) The FKBP12.6 in ER protein extract was measured by western blot. (E) U87 cells were loaded with Fluo-3 AM, the cytosolic Ca²⁺ release in two spatial dimensions plus time (XYT) were acquired using confocal microscope. White arrow: the enrichment of Ca²⁺ to cell membrane. (F) The image of cytoskeleton remodeling in mCherry-actin expressing U87 cells was taken by confocal microscope. White arrow: actin-dependent endocytosis. White asterisk: endocytic vesicle. Scale bar: 20 μm. (G) Effects of CD38 siRNA pretreatment followed by starvation on colocalization of isolated mitochondria with the endosomes in U87 cells. Ch2: pHrodo; Ch5: MitoTracker Red; BF: brightfield; BDS Ch2-5: bright detail similarity Ch2-Ch5. (H) Schematic diagram of endocytosis-mediated mitochondrial transplantation via NAD⁺-CD38-cADPR-Ca²⁺ signaling.