*3.2. Fungal Material*

The strain DHS-8 of *Phomopsis asparagi* was isolated from a healthy tree root of the mangrove plant *Rhizophora mangle*, which was collected in the Dong Zhai Gang-Mangrove Garden (110◦320-–110◦37- E, 19◦51-–20◦01- N) in Hainan Province in October 2015. The strain was isolated under sterile conditions from the inner tissue of the root, following an isolation protocol described previously [36] and identified using a molecular biological protocol by DNA amplification and sequencing of the ITS region (GenBank Accession no. MT126606). A voucher strain was preserved on potato dextrose agar slants stored at 4 ◦C at one of the authors' laboratory (J. X.).

#### *3.3. Fermentation and Extraction*

The fungus was fermented onto autoclaved rice solid-substrate medium (thirty 1000 mL Erlenmeyer flasks, each containing 100 g of rice and 100 mL of 0.3% saline water) and incubated for 28 days at 28 ◦C. In total, 140 flasks of culture were extracted three times with EtOAc, and the filtrate was evaporated under reduced pressure to yield crude extract (65 g).

## *3.4. Purification and Identification*

The crude extract was partitioned with petroleum ether (PE), dichloromethane, ethyl acetate (EA), and *n*-butyl alcohol (BA). The dichloromethane fraction and ethyl acetate fraction were combined (30 g) and then subjected to silica gel column chromatography using gradient elution with CH2Cl2–MeOH mixtures of increasing polarity (100:0–0:100, *v*/*v*) to afford 8 fractions (Fr. 1–Fr. 8). Fr. 2 was subjected to open silica gel CC using gradient elution with CH2Cl2-EtOAc (4:1–1:1, *v*/*v*) to yield fractions Fr. 2.1–2.6. Then Fr. 2.3 and Fr. 2.4 were purified by semipreparative reversed-phase HPLC using MeOH–H2O (50:50, *v*/*v*) to afford **3** (5.0 mg) (Figures S19–S27). Purification of Fr. 2.5 was conducted by CC over RP-18 with a MeOH–H2O gradient (50:50–90:10, *v*/*v*) to yield **2** (8.0 mg) (Figures S10–S18) and **6** (119.2 mg) (Figures S34–S36). Fr. 2.6 was separated using Sephadex LH-20 CC with CHCl3–MeOH (1:1, *v*/*v*) to afford **1** (105 mg) (Figures S1–S9). Fr. 4 was subjected to CH2Cl2-EtOAc (1:1, *v*/*v*) and further fractionated by RP C-18 CC eluted with CH3OH-H2O (70:30, *v*/*v*) to produce the major component **7** (818.3 mg) (Figures S37–S39). Fr. 5 was subjected to silica gel CC using CH2Cl2-CH3OH as the eluent (100:4–100:6, *v*/*v*). The promising subfraction Fr. 5.1 was separated by semipreparative reversed-phase HPLC using MeOH–H2O (60:40, *v*/*v*) to obtain **8** (2.7 mg) (Figures S40–S42). Separations of Fr. 5.4 following a procedure similar to that used for Fr. 5.1 gave **4** (semiprep. HPLC, MeOH–H2O = 60:40, *<sup>v</sup>*/*<sup>v</sup>*, 120 mg) (Figures S28–S30). Fr. 6.2, collected from Fr. 6 was subjected to silica gel CC with CH2Cl2-EtOAc (100:10, *v*/*v*), followed by Sephadex LH-20 CC using CHCl3–MeOH (1:1, *v*/*v*) as the eluent to yield **5** (6.1 mg) (Figures S31–S33).

**Phomoparagin A** (**1**): colorless amorphous residue (MeOH); [α]20D -13 (*c* 0.001, MeOH); UV (MeOH) λmax 215 nm; 1H and 13C NMR data, see Table 1; HRESIMS *m/z* 434.2644 [M + H]+ (calcd for C28H36NO3, 434.2695).

**Phomoparagin B** (**2**): colorless amorphous residue (MeOH); [α]20D +54 (*c* 0.001, MeOH); UV (MeOH) λmax 212 nm; 1H NMR data, see Table 1; HRESIMS *m/z* 516.2733 [M + Na]+ (calcd for C30H39NO5Na, 516.2726).

**Phomoparagin C** (**3**): colorless amorphous residue (MeOH); [α]20D -14 (*c* 0.001, MeOH); UV (MeOH) λmax 212 nm; 1H and 13C NMR data, see Table 1; HRESIMS *m/z* 434.2678 [M + H]+ (calcd for C28H36NO3, 434.2695).

#### *3.5. Electron Circular Dichroism Calculation*

Monte Carlo conformational searches were run by employing Spartan's 14 software using the Merck Molecular Force Field (MMFF). Conformers with a Boltzmann population of over 0.4% were chosen for ECD calculations (Tables S1 and S2). Then, the conformers were initially optimized at the B3LYP/6-31 g level in gas using the PCM polarizable conductor calculation model. The theoretical calculation of ECD was conducted in MeOH using time-dependent density functional theory (TD-DFT) at the B3LYP/6-31+g (d, p) level for all conformers of Compounds **1**–**3**. Rotatory strengths for a total of 30 excited states were calculated. ECD spectra were generated using the program SpecDis 1.6 (University of Würzburg, Würzburg, Germany) and GraphPad Prism 5 (University of California San Diego, USA) from dipole-length rotational strengths by applying Gaussian band shapes with sigma = 0.3 eV.

## *3.6. Splenocyte Proliferation Assay*

Spleen cells were collected from BALB/c mice under aseptic conditions, plated in a 96-well plate at a density of 1.5 × 10<sup>6</sup> cells/well and activated by Con A (5 μg/mL) or LPS (10 μg/mL) in the presence of various concentrations of compounds or cyclosporine A (CsA) at 37 ◦C and 5% CO2 for 48 h. Then, 20 μL CCK-8 was added to each well, 4 h before the end of the incubation. Absorbance at OD450 was measured on an ELISA reader, and the IC50 value was calculated from the correlation curve between the compound concentration and the OD450.

## *3.7. Cell Viability Assessment*

Cell viability was evaluated using the CCK-8 method. Spleen cells were plated in a 96- well plate at a density of 1.5 × 10<sup>6</sup> cells/well. Then, the cells were incubated with various concentrations of compounds or 0.1% DMSO at 37 ◦C and 5% CO2 for 72 h. Proliferation was measured using the CCK-8 assay, as described above. Cyclosporin A (CsA) and cytochalasin D were used as positive controls.

## *3.8. CN Phosphatase Assay*

A spectrophotometric assay was used to determine the activities of CN with 20 mM *p*-nitrophenyl phosphate (*p*-NPP) as the substrate in 1 mM CaCl2, 0.5 mM MnCl2, 2 mM CaM, 2 mM CNB, 1 mM DTT, 0.1 mg/mL bovine serum albumin (BSA), and 50 mM Tris-HCl (pH 7.4) at 4 ◦C. The reaction was initiated by the addition of CNA, various concentrations of compounds (6.25, 12.5, 25, 50 or 100 μM) were added, and the solution was preincubated for 10 min at 4 ◦C. The OD410 value was measured, and the inhibitory concentration (IC50) was calculated. DMSO (2%) was used as a vehicle control. CsA was used as a positive control.

## *3.9. Western Blot Analysis*

Splenocytes were washed with PBS and lysed in PMSF lysate. After centrifugation at 12,000 rpm for 5 min, the protein concentration was determined using a BSA Protein Assay Kit. Protein lysates were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking, the membranes were incubated overnight with the respective primary antibodies against NFAT1, NFAT-P, and β-actin in 5% BSA, and the secondary antibody was horseradish peroxidase (HRP)-conjugated goa<sup>t</sup> anti-rabbit IgG. Immunoreactive bands were visualized by incubation with luminescent liquid and exposed to lightsensitive film.
