Fig. 3

Real-time tracking NADH/NAD⁺ and NADPH dynamics during the cell cycle. Fluorescence images A, C, E and quantification B, D, F of NADPH (iNap1) A-B, NADH (SoNar) C-D, and iNapc E-F dynamics during cell division. The entire coding sequences of iNap1, SoNar, iNapc were subcloned into the vector pLVX-IRES-Puro vector for the generation of recombinant lentivirus. HeLa cells with lentiviral infection and stable expression of sensors were then seeded into glass-bottom plates. After about 24 h, the culture medium was changed into phenol-red free medium for fluorescence imaging. The fluorescence microscopy system was equipped with a Nikon Eclipse Ti-E automatic microscope, monochrome-cooled digital microscope camera (model no. DS-Qi1 Mc-U2), PFS, A Plan Apo 40 × 0.95 NA objective, a highly stable Sutter Lambda XL light source, and an on-stage CO₂ incubator (Tokai Hit). For dual-excitation ratio imaging, 407-BP 17-nm or 482-BP 35-nm excitation filters (Semrock) and a 535-BP 40-nm emission filter altered by a Lambda 10-3 filter wheel (Sutter Instruments) were used. A perfect focus system was applied to stabilize the focal plane and high-sensitivity detectors were utilized to shorten the exposure time. The NADPH level exhibits a moderate and robust increase in the mitotic phase A-B, while the NADH/NAD⁺ level remains almost constant C-D, as well as the iNapc fluorescence E-F. All fluorescence images were pseudocolored by F₄₀₇/F₄₈₂ (R_407/482). Scale bar, 10 μm. For A-F, adapted from the reference (Zou et al. 2018)