Supplementary Figure 1

Generation of SoNar-based NADPH sensors.

(a) The proportion(%) of charged amino acid residues around the 2'-hydroxyl group of the ligand in NADP(H) and NAD(H) binding proteins. Most NADP(H) binding proteins contain positively charged residues around the phosphate, while the NAD(H) binding proteins favor a negatively charged environment at the corresponding site. The proteins selected for analysis are given in Supplementary Table 1. (b) The NADH binding site of T-Rex (carbon atoms in yellow, PDB ID:1xcb) superimposed with the NADP binding site of glyoxylate reductase from P. horikoshii (carbon atoms in cyan, PDB ID: 2dbr). The structure of glyoxylate reductase has been selected because the geometrical arrangement of its ligand binding site is highly similar to that of T-Rex (according to CPASS), despite that it’s binding to NADPH rather than NADH. Ligands and side chains are shown as sticks, with oxygen atoms in red, nitrogen in blue, and phosphorous in orange. (c) The proportion(%) of nonpolar residues in the adenine binding pocket of protein structures containing bound NAD(H) and NADP(H), respectively. For a given protein structure, amino acid residues with at least one atom within 5 Å from any adenine atom were identified to calculate the proportion of nonpolar residues. Val, Pro, Trp, Gly, Ala, Met, Phe, Leu, Ile or Cys are counted as nonpolar residues. The proteins used for analysis are listed in Supplementary Table 1.

Nature Methods: doi:10.1038/nmeth.4306

Supplementary Figure 2

Fluorescence properties of iNap sensors.

(a) Fluorescence response of SoNar and 40 mutants listed in Supplementary Table 3 to 1.0 mM NADPH and its analogs, 4 mM ADP, 4 mM ATP, 4 mM AMP, 0.5 mM NAD

+, 50 μM NADH, and 10 μM NADP

+. The 3rd, 8th, 31st, 39th and 40th mutants were denoted iNap3,

iNap2, iNap1, iNap4 and iNapc, respectively. The ratio of fluorescence excited at 420 nm and 485 nm shown as a heatmap. (b) The initial excitation ratios of different iNap sensors at pH 7.3. (c and d) Emission spectra of purified iNap1 in the control condition (black), and after addition of 0.2 mM NADPH (orange), normalized to the peak intensity in the control condition. Excitation was fixed at 420 nm (c) and 490 nm (d), respectively. (e) Fluorescence response of iNap1 corrected by iNapc to 0.2 mM NADPH at various temperatures. pH correction is determined by dividing the fluorescence ratio (R420/485) of iNap1 by that of iNapc. (f) iNap1 fluorescence plotted against NADPH in the presence of NADH (50 μM), NAD

+ (500 μM), and NADP

+ (10 μM). (g) iNap1 fluorescence plotted against NADPH in the

presence of ADP (1 mM, 2 mM or 4 mM) and ATP (1 mM, 2 mM or 4 mM). (h) iNap1 fluorescence plotted against NADPH at the indicated ATP: ADP ratios; the total adenine nucleotide concentration was 2 or 4 mM. In supplementary Fig. 2f-h, fluorescence ratios

were normalized to the value at the saturating concentration of NADPH at pH 7.3, experiments were performed in triplicate, and the

data were from the three independent detections, error bars represent SEM.

Nature Methods: doi:10.1038/nmeth.4306

Supplementary Figure 3

The pH sensitivity of iNap1 sensors.

(a) Fluorescence ratios (R420/590) of mCherry-iNap1 plotted against NADPH at the indicated pH. The fusion construct allows ratiometric and pH-resistant measurement of NADPH. (b) Fluorescence intensities of iNap1 and iNapc with excitation at 420 nm or 485 nm, and emission at 528 nm. Data were normalized to the fluorescence at pH 7.0. (c) Fluorescence intensity of iNap1 when excited at 485 nm plotted against the NADPH concentration at the indicated pH. (d) NADPH titration curves of iNap1 corrected by iNapc when excited at 485 nm at the indicated pH. (e) NADPH titration curves of iNap1 normalized by iNapc at the indicated pH. (f and g) pH-correction of the excitation ratio 420/485 nm of iNap1 with iNapc in the absence (f) and presence (g) of 0.2 mM NADPH. Data were normalized to the fluorescence ratio at pH 7.0. (h) Fluorescence intensity of iNapc when excited at 485 nm plotted against the NADPH concentration at the indicated pH. In supplementary Fig. 3a-h, experiments were performed in triplicate and the data were from the three independent detections, error bars represent SEM.

Nature Methods: doi:10.1038/nmeth.4306

Supplementary Figure 4

Fluorescence intensity and homogeneity of iNap1 in mammalian cells.

(a) Comparison of the fluorescence intensity of different genetically encoded fluorescent sensors in HeLa cells. (b and c) The excitation spectra of HeLa expressing iNap1 cells (b) or control cells (c) with or without 160 μM diamide. (d-f) The homogeneity of iNap1 (d), iNapc (e) and mCherry-iNap1(f) fluorescence in individual cells. Quantitative data of the sensor’s fluorescence were obtained from at least 200 cells. Error bars represent SD. Scale bar 20 μm.

Nature Methods: doi:10.1038/nmeth.4306

Nature Methods: doi:10.1038/nmeth.4306

Supplementary Figure 5

Subcellular distribution and regulation of NADPH in mammalian cells.

(a and b) Quantification of iNap1 (a) and iNap3-Mit (b) fluorescence in NADK overexpressing or knockdown cells. Data are normalized to digitonin-permeabilized cell samples in the absence of NADPH. Error bars represent SEM. (c and d) NADK knockdown or overexpression (OE) in HeLa cells was assessed by Western blot (c) or activity analysis (d). (e and f) Effect of NADK knockdown or overexpression (OE) on whole-cell (e) and mitochondrial (f) NADPH levels in HeLa cells. Data obtained by a single extraction method and in vitro biochemical assay. (g and h) Ratiometric fluorescence imaging (g) and quantification by microplate reader (h) of mCherry-iNap1 or Mit-mCherry-iNap3 in wide-type and NADK-overexpressing HeLa cells. Scale bar, 10 μm. (i) Fluorescence response of cells expressing iNap1 or PercevalHR to the glycolysis inhibitor 2-DG (2 mM). Fluorescence was measured 1 hr after 2-DG treatment and normalized to the control condition. (j) G6PD knockdown or PGD knockdown in HeLa cells was assessed by Western blot. (k) Effect of G6PD knockdown or PGD knockdown on cytosolic NADPH levels assessed by iNap1 fluorescence. (l and m) NADPH detection of in resting (Ctrl) or activated (LPS plus IFN-γ stimulation) RAW264.7 cells by flow cytometry (l) or microplate reader (m). In supplementary Fig. 5 a, b, i and k, R is normalized by iNapc. In supplementary Fig. 5a, b, d, e, f, k, m, experiments were performed in triplicate and the data were from the three independent detections, unpaired t-test. *p < 0.05, **p < 0.01, and ***p < 0.001. All experiments were performed in HeLa cells; error bars represent SEM unless otherwise indicated.

Nature Methods: doi:10.1038/nmeth.4306

Supplementary Figure 6

NADPH dynamics of glucose-deprived cells in the setting of oxidative stress.

(a) Kinetics of FLII12

Pglu-700µ, SoNar, iNap1, or roGFP1 fluorescence in HeLa cells in response to glucose deprivation. (b) Kinetics of iNap1 and roGFP1 fluorescence in glucose-deprived HeLa cells in response to 80 μM H2O2. (c) Effect of the glutathione reductase and thioredoxin reductase inhibitor BCNU on diamide-induced NADPH oxidation in glucose-deprived HeLa cells. Cells were pre-treated with BCNU for 30 min. (d) Effect of DHEA on G6PD activity and PGD activity. The cell lysate was pre-incubated with DHEA for 1 hr. (e) PGD knockdown would not affect NADPH recovery under oxidation. In supplementary Fig. 6 a, b, c and e, R is normalized by iNapc. All experiments were performed in glucose-deprivation conditions with triplicate and the data were from the three independent detections. Error bars represent SEM.

Nature Methods: doi:10.1038/nmeth.4306

Supplementary Figure 7

NADPH dynamics of glucose-fed cells in the setting of oxidative stress.

(a) Quantification of iNap1 or iNapc fluorescence in HeLa cells before and 40 min after treatment with 40 μM diamide; data derived from Fig. 4a. (b) Kinetics of iNap1 or roGFP1 fluorescence in glucose-fed HeLa cells in response to 80 μM H2O2. (c) Effect of the glutathione reductase and thioredoxin reductase inhibitor BCNU on diamide-induced NADPH oxidation in glucose-fed HeLa cells. Cells were pre-treated with BCNU for 30 min. (d) Effect of different metabolites (glutamine or pyruvate, 2 mM; isocitrate, malate or glucose, 10 mM) on diamide-induced NADPH oxidation in glucose-fed HeLa cells. Data were normalized to samples in the absence of metabolite. (e) Dose-dependent fluorescence response of cytosolic iNap1 and SoNar to different concentrations of glucose in diamide-treated HeLa cells. Fluorescence was measured immediately after diamide addition. Data were normalized to diamide-treated samples in the absence of glucose. In supplementary Fig. 7 b, c, d and e, R is normalized by iNapc, experiments were performed in triplicate and the data were from the three independent detections. Error bars represent SEM.

Nature Methods: doi:10.1038/nmeth.4306

Supplementary Figure 8

NADPH metabolism in diamide-treated cells.

(a) The effect of ionomycin on diamide-induced NADPH oxidation in glucose-fed cells at 35 min. Data from Fig. 5c. R is normalized by iNapc. Unpaired t-test. *p < 0.05. (b) Effect of dorsomorphin on NADPH recovery in glucose-deprived cells under oxidative stress. (c) Working models for NADPH regulation in glucose-deprived cells under oxidative stress. In supplementary Fig. 8a, b, experiments were performed in triplicate and the data were from the three independent detections.

Nature Methods: doi:10.1038/nmeth.4306

Supplementary Figure 9

Imaging of iNapc in zebrafish larvae.

No pH variation was observed in wound margin of iNapc-expressing zebrafish larvae. Tail-fin tip amputation was performed at the 0 min time point. Scale bar, 50 μm.

Nature Methods: doi:10.1038/nmeth.4306

Supplementary Figure 10

The central nodes for cellular NADPH metabolism.

NADPH is mainly consumed through redox defense, reductive biosynthesis, and mutant isocitrate dehydrogenase (mIDH1/2) in cells. NADPH is regenerated primarily via the oxidative pentose phosphate pathway (PPP) and other redox reactions catalyzed by malic enzyme (ME1/3), IDH1/2, methylene tetrahydrofolate dehydrogenase (MTHFD1/2) and nicotinamide nucleotide transhydrogenase (NNT). NADP

+ can be generated from NAD

+ by cytosolic (NADK) or mitochondrial (NADK2) forms of NAD kinases. GSSG, oxidized

glutathione; GSH, reduced glutathione; Arg, arginine; Glu, glutamate; Pro, proline; NTP, nucleotide triphosphate; dNTP, deoxynucleoside triphosphate; α-KG, alpha-ketoglutarate; 2-HG, 2-hydroxyglutarate; G-6-P, Glucose 6-phosphate; R-5-P, Ribulose 5-phosphate; THF, tetrahydrofolate; me-THF, methenyl/methylene-THF; for-THF, formyl-THF; GR, glutathione reductase (GR); FASN, fatty acid synthase; G6PD, glucose-6-phosphate dehydrogenase; 6PGD, 6-phosphogluconate dehydrogenase.

Nature Methods: doi:10.1038/nmeth.4306

Supplementary Table 1 ∣Proteins used in comparative analysis.

NAD(H)-binding proteins:

1A5Z 1A7K 1A9Z 1AD3 1AGN 1AHH 1ARZ 1B3R 1B8U 1BDB 1BMD 1BPW 1BW9 1BXK 1BXS 1CDO 1CWU 1DHS 1DIR 1DLI 1DXY 1E3L 1E3W 1EBF 1EE9 1EVJ 1EVZ 1EZ4 1F3P 1F8F 1FDV 1FK8 1G1A 1G6K 1GAD 1GEG 1GEU 1GIQ 1GR0 1GRB 1GUZ 1GV0 1H94 1HDG 1HDY 1HKU 1HYH 1I2C 1I2Z 1I3N 1IB0 1ICI 1IE3 1IY8 1J0X 1J49 1J5P 1JQ5 1JU9 1K4M 1KAE 1KOL 1KQN 1KYQ 1LC3 1LDM 1LJ8 1LLD 1LLQ 1LLU 1LSS 1LVL 1LW7 1M76 1M8F 1M9H 1MEW 1MG5 1MP0 1MV8 1N2S 1NBO 1NFB 1NFR 1NRX 1NUU 1NVM 1O5I 1O6Z 1O9B 1O9J 1OBB 1OC2 1OG3 1OJZ 1OMO 1ORR 1P1H 1P9L 1PJ3 1PJC 1PJS 1PL6 1PSD 1PZH 1QAX 1QS2 1R37 1R66 1RKX 1RZ1 1S20 1S7G 1SB9 1SBY 1SZJ 1T24 1T2F 1T90 1TAE 1TOX 1U1I 1U28 1U7T 1U8X 1UP6 1UWK 1UXG 1UXT 1V59 1V8B 1V9L 1VBI 1VJT 1VKO 1VM6 1VRQ 1WDK 1WNB 1WWK 1WXH 1X0X 1X14 1X7D 1X87 1XAH 1XCB 1Y3I 1Y9E 1YE6 1Z0Z 1Z2I 1Z45 1ZBQ 1ZEM 1ZJZ 1ZMC 1ZNQ 1ZRQ 2A5F 2AG5 2B4T 2B69 2BKJ 2BL4 2C20 2C5E 2C8C 2CFC 2CNB 2CZC 2D1Y 2D37 2D4E 2D4V 2DC1 2DFD 2DFV 2DLD 2DPH 2DVM 2ED4 2EER 2EJV 2EKL 2EKP 2EP7 2EQ7 2EWM 2FKN 2FN7 2FOI 2G76 2G82 2G8Y 2GDZ 2GRU 2GSD 2GWL 2H4H 2HAE 2HSD 2HUN 2I29 2I65 2I9P 2IXA 2IZZ 2J5N 2JG7 2LDB 2NAD 2NPX 2NSY 2O2Z 2O4C 2OME 2ONM 2P5U 2PD4 2PD6 2PH5 2PLA 2PV7 2PZM 2Q1U 2Q1W 2Q2Q 2Q3E 2QIO 2QJO 2RAB 2RC3 2V7P 2VOJ 2VT2 2VUT 2VZJ 2VZL 2W2L 2WN7 2WYV 2X06 2X0I 2Y42 2YY7 2ZIT 2ZTV 3A28 3A9W 3ABI 3B4W 3B6J 3BTS 3C7D 3CE6 3CEA 3CGD 3CIF 3CIN 3CLH 3COS 3CZM 3D4P 3D64 3DBV 3DOC 3E18 3E5R 3EC7 3EFV 3EHE 3ENK 3EYW 3F3S 3F4B 3F8P 3FG0 3FLK 3FNE 3G1U 3GD4 3GEG 3GFB 3GGP 3GNQ 3GVH 3H9E 3H9U 3HAZ 3HJA 3HL0 3HMQ 3HQ4 3I0P 3I9K 3IDS 3IJR 3IWJ 3IWK 3JSA 3JU8 3JV7 3JYO 3JZD 3K2B 3K31 3K88 3KB6 3KEO 3KFT 3KO8 3L0D 3L98 3LDH 3LN3 3LQF 3LU1 3M2T 3M6I 3MTG 3MW9 3N58 3N7U 3NJ8 3NRC 3NTQ 3O9Z 3OA2 3OET 3OIG 3OKF 3OX4 3P2O 3PGX 3PVZ 3PXX 3Q2I 3Q2K 3Q3C 5MDH 9LDB

NADP(H)-binding proteins:

1AE1 1AFS 1AH3 1AMO 1CF2 1CIV 1CYD 1DDI 1DIG 1DQA 1DR1 1E1K 1EDO 1EQU 1EZ0 1F20 1FRB 1FXS 1GET 1GQ2 1GVE 1H5Q 1H9A 1HE4 1HYG 1I0S 1I36 1IDE 1IHI 1J1W 1JAY 1LUA 1LY4 1MXH 1N9G 1NVT 1NYT 1O2D 1OAA 1OG6 1P0F 1PIW 1PQU 1PS9 1PZ0 1PZ1 1Q13 1Q7C 1QFY 1QKI 1QOR 1RY0 1S3W 1SNY 1TDF 1TYP 1U28 1U31 1USF 1UXP 1UZN 1VBJ 1VL8 1VLJ 1VP5 1W4Z 1W73 1W87 1WNT 1XG5 1XKD 1YB5 1YBM 1YE6 1YQD 1YS4 1Z0U 1Z6Z 1ZGD 1ZH8 1ZK4 1ZUA 2A87 2AE2 2AG8 2AHR 2ANO 2AZN 2B4Q 2B5W 2BD0 2BF7 2BPO 2BRU 2C3C 2C91 2CDB 2CF6 2CVQ 2D1C 2D2I 2D38 2DBR 2DM6 2E5M 2EHQ 2EV9 2F1K 2FVL 2FZJ 2GNA 2GQ2 2GRA 2GZ1 2H63 2HK9 2HRB 2I2A 2I3G 2IKI 2IZ0 2J3J 2JKV 2NVB 2NVK 2O2Q 2O7P 2OBY 2OFP 2Q0L 2QE0 2QFV 2QTZ 2QZZ 2RAF 2RC6 2RCY 2RIR 2UVB 2UVC 2V6G 2VNH 2VQ3 2VQB 2VRB 2VUU 2VZ9 2W5L 2W98 2WMD 2WME 2X6T 2X7H 2XNJ 2Y05 2YUT 2YW9 2YYY 2ZAT 2ZCV 2ZZA 2ZZC 3AFN 3AI2 3BAZ 3BHI 3BXX 3C1O 3CH6 3CMF 3CRZ 3CV6 3D8X 3DTT 3E8X 3EEL 3F0B 3F8R 3G49 3GN2 3GQV 3GR6 3GWF 3GY0 3H7R 3H7U 3HBB 3HSK 3I52 3IAH 3ICC 3IJP 3JW5 3JZ4 3KJR 3KRB 3KVO 3LF2 3LNS 3LUT 3LZX 3MAP 3NX4 3O03 3O3R 3O4R 3OP4

Nature Methods: doi:10.1038/nmeth.4306

Supplementary Table 2∣Amino acid residues at structurally aligned sites of some NAD(H)

and NADP(H) binding proteins.

pdbid NAD(H) adenine pocket around 2' -oxygen

1xcb VAL 86 VAL 113

VAL 130

VAL 148

PRO 149

ALA 152

ALA 156

ASP 112

ASP 114

3keo VAL 90 LEU 118

ILE 137

VAL 156

PRO 157

ASP 117

ASN 121

1gr0 VAL 21 VAL 70 LEU 146

PRO 147

SER 150

ALA 153

ASP 69 ASP 71

3cin LEU 7 VAL 54 VAL 87 SER 91 ALA 159

ASP 53 ASP 55

1pjc LEU 173

ILE 198

ILE 224

VAL 238

ALA 245

ASP 197

ASN 199

1psd ILE 157

ILE 182

VAL 211

PRO 212

SER 216

ASP 181

GLU 183

1llu ILE 179

ILE 202

ILE 200

VAL 245

PRO 269

SER 246

ALA 249

ASP 201

ASP 203

9ldb VAL 27 VAL 54 VAL 52 ALA 98 ASP 53

3kb6 ILE 147

VAL 172

VAL 202

PRO 203

THR 205

ASP 171

3Q2I VAL 17 ILE 45 LEU 85 PRO 82

GLN 89

ASP 44 ASP 46

1j49 VAL 152

ILE 177

LEU 193

VAL 207

PRO 208

ALA 212

MET 216

ASP 176

3e18 VAL 9 ILE 36 THR 71

PRO 72

SER 75 LEU 79 ASP 35

3abi LEU 6 VAL 30 ASP 48 LEU 69 PRO 70

SER 50 ALA 49 ASP 29 ASN 31

2dld VAL 152

ILE 177

VAL 207

PRO 208

ALA 212

ASP 176

1ldm VAL 26 VAL 53 VAL 51 ILE 116

ILE 120

ASP 52

NADP(H) adenine pocket

2dbr ILE 156

ARG181

TYR179

VAL 212

PRO 213

THR 215

LEU 221

SER 180

1yqd HIS 259

THR 215

VAL 255

PRO 280

ALA 257

SER 214

Note: The proteins have been selected using CPASS, which performs sequence order-independent

structure alignments between ligand-binding sites. Known NAD(H) and NADP(H) binding proteins

in PDB were aligned to 1xcb as the template. Those with a CPASS similarity score of above 30 were

retained. The polar residues are shown in bold.

Nature Methods: doi:10.1038/nmeth.4306

Supplementary Table 3∣Summary of the mutation sites in all variants and the relevant affinity to NADPH.

Variant number Position 112-116 other mutations Kd’ (~μM）

Wt (SoNar) DVDPE 150

1 SRKAE 13.2

2 SRKAE V130T 28.7

3 (iNap3) SRKAE V130Y 25.2

4 SRKGE 11.8

5 SRKGE V130T 19.3

6 SRKGE V130Y 22.0

7 SRSAE 2.9

8 (iNap2) SRSAE V130T 6.4

9 SRSAE V130Y 5.6

10 SRSGE 4.3

11 SRSGE V130T 5.4

12 SRSGE V130Y 6.8

13 SRTAE 2.8

14 SRTAE V130T 5.0

15 SRTAE V130Y 4.4

16 SRTGE 4.0

17 SRTGE V130T 5.8

18 SRTGE V130Y 4.9

19 SRKPQ 7.3

20 SRKPQ V130T 16.6

21 SRKPQ V130Y 13.7

22 SRSPQ 1.6

23 SRSPQ V130T 18.1

24 SRSPQ V130Y 3.6

25 SRTPQ 1.5

26 SRTPQ V130T 2.1

27 SRTPQ V130Y 2.5

28 SRKAQ 9.7

29 SRKAQ V130T 16.5

30 SRKAQ V130Y 9.5

31 (iNap1) SRSAQ 2.0

32 SRSAQ V130T 3.8

33 SRSAQ V130Y 3.6

34 SRTAQ 1.3

35 SRTAQ V130T 2.2

36 SRTAQ V130Y 2.6

37 SRSAQ V148A 12.6

38 SRSAQ V148T 7.8

39 (iNap4) SRSAQ R90D 120.2

40 (iNapc) SVSPE R90D,V148A ∞

Note: The mutation sites are relative to wild type T-rex. The apparent dissociation constant (Kd’) of

numbered variants was calculated using the Hill equation based on the 420/485 nm ratio.

Nature Methods: doi:10.1038/nmeth.4306

Supplementary Table 4∣ Quantification of cytosolic and mitochondrial free NADPH concentrations

with iNap 1, 2, 3 sensors in HeLa cells.

Cytosol Mitochondria

NADPH

sensors

Sensor occupancy

(%)

NADPH level

(μM)

Sensor occupancy

(%)

NADPH level

(μM)

iNap1 ~60% ~3.1 μM ~ 95% N.D.

iNap2 ~43% ~4.5 μM ~86% ~41 μM

iNap3 ~18% ~5.5 μM ~55% ~37 μM

N.D., not determined.

Nature Methods: doi:10.1038/nmeth.4306