Fig 2: In cellulo sensor characterization.Intracellular labeling efficiencies. (a) Representative in-gel fluorescence detection of intracellular and in vitro (control) sensor protein labeling. The sensor protein is labeled intracellularly (U2OS cells) with CP-TMR-SMX, CP-TMR(6) or SiR-Halo (samples 1–4) with or without the presence of the efflux pump inhibitor verapamil (10 µM), overnight. The cells were washed and lysed with an excess BG-Alexa(488) and SiR-Halo or Halo-TMR to quantify the unlabeled fraction of SNAP-tag and Halo-tag. As control the purified sensor was labeled in vitro with CP-TMR-SMX/CP-TMR(6)/BG-Alexa(488) and SiR-Halo (samples 5–7). For the quantification of TMR or SiR labeling, the ratio of Alexa(488)/SiR and TMR/SiR of the intracellular samples is calculated relative to the in vitro samples. The results of the labeling efficiency and the description of the samples run on the SDS-PAGE gel can be found in Table b. (c) Comparison of the endogenous SPR level of different cell lines by Western Blot. Western blot of SPR (28 kDa) and ß-tubulin (50 kDa) as loading control with different cell lysates revealed by ECL. For each cell lysates, 20 µg total protein were loaded in each well. (d) Representation of the relative expression level of SPR in the different cell lines determined as integrated band intensity normalized to ß-tubulin integrated intensity using the displayed blot. (e) The sensor dynamic range is maintained in lysate or in cells. The purified NADP-Snifit is added to a freshly prepared U2OS lysate (0.5 mg/mL protein) to a concentration of 50 nM. The measured TMR/SiR ratio of 1.6 corresponds to a NADPH/NADP+ ratio of 11 in the whole-cell lysate (black line). The sensor was fully open by adding a saturating concentration of free ligand (2.5 mM sulfapyridine) and displays a TMR/SiR ratio of 4.5 (red line). To obtain the fully closed sensor in lysate, 10 mM NADP+ was spiked to the lysate, resulting in a TMR/SiR ratio of 0.5 (blue line). A similar FRET ratio change can be observed for closed sensor in buffer. (f) Semi-stable U2OS cells expressing the nuclear localized NADP-Snifit were used to performed an intracellular sensor calibration. The cells plated on a 12-well plate poly-L-lysine coated coverslip were imaged in HBSS with a widefield microscope. After 2 min, 10 mM NADP+ and 0.001% (w/w) digitonin prepared in HBSS was added to reach the sensor closed state. At 17 min, sulfapyridine was added to a saturating concentration (2 mM) to reach the sensor open state. The dynamic range measured with this widefield microscope was approximately of 8-fold similarly to lysate and buffer measurements.10.7554/eLife.32638.031Appendix 1—figure 3—source data 1.10.7554/eLife.32638.032Appendix 1—figure 3—source data 2.

Fig 3: SPR depletion inhibits HCC growth in vivo.a The effects of SPR depletion on the tumor volumes of xenografts in nude mice. b Loss of SPR reduced the tumor growth rate. SMMC-7721 cells (1 × 106 cells per mouse) were subcutaneously inoculated into the right flank of mice. When the tumor volume reached 100 mm3, siRNA (10 µg per tumor) was injected into tumors once every other day. c SPR knockdown had no effect on the body weight of mice. d, e The levels of SPR and Bim in different groups were detected by western blots and immunohistochemistry. Scale bar: 50 µm. f Schematic representation of the underlying mechanism of SPR based on this study. SPR regulates apoptosis of HCC cells via the FoxO3a/Bim-signaling pathway independently of its enzymatic activity. Each point represents the mean ± SD (n = 6). The p-values < 0.05 were considered statistically significant.

Fig 4: In vitro sensors characterization.(a) NADP+ binding is a prerequisite for sensor closing. Comparative emission spectra of NADP-Snifit normalized to its isosbestic point (645 nm) in absence of NADP+ (black line), in presence of 100 µM NADP+ (red line) and in presence of 100 µM NADP+ and 1 mM sulfamethoxazole (SMX) (blue line). (b) The intramolecular ligand does not bind to the sensor saturated with NADPH. Emission spectra of NADP-Snifit without NADP+ (black line), after the addition of 1 mM glucose-6-phosphate and 100 µM NADP+ (red line) and finally after a 30 min incubation in presence of 1 nM glucose-6-phosphate dehydrogenase (G6PD). The conversion of NADP+ into NADPH is not fully complete as G6PD is inhibited at high NADPH/NADP+ ratios. However, obtaining pure NADPH is difficult since most commercial stock of NADPH were found to have ~2–3% NADP+ as impurity. (c) Titrations of NAD-Snifit with NAD+ at various pH ranging from 6.8 to 8.0. (d) Titrations of NAD-Snifit with NAD+ in presence of a fixed concentration of one of the listed different metabolites and structurally close molecules and the substrate sepiapterin. (e) Titrations of NADP-Snifit with NADP+ at 25°C, 30°C and 37°C (c50 varies from 35 ± 3 nM to 88 ± 7 nM, from 25°C to 37°C) (f) Titrations of NAD-Snifit with NAD+ at 25°C, 30°C and 37°C (c50 varies from 63 ± 12 µM to 130 ± 14 µM, from 25°C to 37°C). (g) Titrations of NADP-Snifit with varying NADPH/NADP+ ratios at 25°C and 37°C. The r50 of the fitted curves do not change significantly between the two temperatures (r50 is 32 and 33, respectively for 25°C and 37°C). (h) Kinetics of sensor opening. The experiment is conducted by injection of 5 mM NADPH at time zero to the closed sensor saturated with NADP+ (100 nM sensor, 10 µM NADP+). The measured t1/2 fitted with a single-exponential decay is 25 s. (i) Time course of the sensor closing following the injection of 1 mM NADP+ at the zero time point. The experimental set-up does not resolve the closing kinetic for the unsaturated sensor. (j) Chemical structures of BG-TMR-SMX (1) and BG-TMR-SPDZ (2). (k) Titrations of NADP-Snifit labeled either with BG-TMR-SMX or BG-TMR-SPDZ with NADP+. The determined c50 values of the sensor for NADP+ are of 29 ± 7 nM for sulfamethoxazole (SMX) and 1.9 ± 0.3 µM for sulfachloropyridazine (SPDZ) as intramolecular ligand. (l) Emission spectra of the EGFP sensor version SPR(WT)-EGFP-p30-SNAP titrated with NADP+. (m) Titration of SPR-EGFP-p30-SNAP with NADP+ and NAD+. Similarly to NADP-Snifit, the fitted c50 is of 45 nM and ~2 mM (extrapolated), respectively for NADP+ and NAD+. (n) Titration of SPR(D41W42)-EGFP-p30-SNAP with NADP+ and NAD+. The sensor is specific for NAD+ with a fitted c50 of 63 ± 12 µM. (p) NADP-Snifit was titrated up to 1 mM H2O2 with a fixed concentration of NADP+. Unless indicated, the measurements were performed in 50 mM HEPES, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.4 at 25°C. Data represent the mean ± s.d. of titrations performed in triplicate.10.7554/eLife.32638.019Appendix 1—figure 1—source data 110.7554/eLife.32638.020Appendix 1—figure 1—source data 2.10.7554/eLife.32638.021Appendix 1—figure 1—source data 3.10.7554/eLife.32638.022Appendix 1—figure 1—source data 4.10.7554/eLife.32638.023Appendix 1—figure 1—source data 5.10.7554/eLife.32638.024Appendix 1—figure 1—source data 6.10.7554/eLife.32638.025Appendix 1—figure 1—source data 7.10.7554/eLife.32638.026Appendix 1—figure 1—source data 8.10.7554/eLife.32638.027Appendix 1—figure 1—source data 9.10.7554/eLife.32638.028Appendix 1—figure 1—source data 10.

Fig 5: SPR regulates Bim expression via the nuclear translocation of FoxO3a.a Two bioinformatic prediction tools, humanTFDB and JASPAR, were used to find the transcription factors that could modulate Bim expression. b Bim mRNA expression in HCC tissues was significantly correlated with FoxO3a in TCGA datasets. c The expression levels of FoxO3a and p-FoxO3a were analyzed by western blots. SPR knockdown promoted the nuclear translocation of FoxO3a in HCC cells by separation of the cytoplasm and nucleus, as revealed by the d immunofluorescence assay and e western blots. Scale bar: 20 µm. f The knockout efficiency of CRISPR/Cas9 for FoxO3a was determined by qPCR. g FoxO3a suppression abolished the upregulation of Bim expression in SPR-depleted HCC cells. h Gene silencing of FoxO3a rescued the apoptosis induced by SPR depletion in HCC cells. Data are represented as the mean ± SD of three independent experiments. The p-values < 0.05 were considered statistically significant for all tests. NC negative control.