*4.1. Compounds* **1***–***6**

In the present study, compounds **1**–**6** (Figure 1) were isolated from Formosan soft coral *Clavularia flava* (wet weight of 1.1 kg), which was collected at Green Island, Taiwan. Briefly, corals were minced and exhaustively extracted with acetone. The organic extract was partitioned between ethyl acetate and water, and the ethyl acetate layer was separated further over a normal phase silica gel by column chromatography eluted with *n*-hexane, ethyl acetate, and methanol to yield 15 fractions. Fraction 14 (116.5 mg) eluted with *n*-hexane–EtOAc (1:5) was subjected to a RP-18 gravity column (MeOH/H2O, 70:30 to 100% MeOH) to separate 6 subfractions. Subsequently, a subfraction 14-3 (23 mg) was purified by RP-18 HPLC (50% acetonitrile in H2O) to obtain 1 (10.2 mg) and 2 (3.2 mg). Fraction 13 (96.7 mg) eluted with *n*-hexane–EtOAc (1:3) was subjected to column chromatography on silica gel using *n*-hexane–EtOAc gradient (10:1 to 1:10) for elution to give 11 subfractions. Subsequently, a subfraction 13-6 (22 mg) was purified by RP-18 HPLC (60% MeOH in H2O) to obtain 3 (5.2 mg). Fraction 15 (86.5 mg) eluted with *n*-hexane–EtOAc (1:10) was subjected to a RP-18 gravity column (MeOH/H2O, 70:30 to 100% MeOH) to separate 7 subfractions. Subsequently, a subfraction 15-4 (20 mg) was purified by RP-18 HPLC (50% ACN in H2O) to obtain 4 (2.4 mg), **5** (3.2 mg), and **6** (3.0 mg).

### *4.2. High-Content Screening (HCS) Assay for Proteasome Inhibition*

We followed the HCS assay routinely conducted in the lab. In brief, the complete assay includes a series of experiments [4]. Experiments were sequentially performed: (1) the DNA transfection into cell culture with compound treatment, (2) cells fixation, (3) image acquisition using an ImageXpress Micro Widefield HCS system (Molecular Device, San Jose, CA, USA), (4) high-content measurements analyzed by modules of MetaExpress, Cell Scoring and Multi-Wavelength Cell Scoring, (5) Western blotting imaging and densitometric analysis, and (6) RNA purification and quantitative PCR.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1660-3397/18/1/39/s1, Figure S1. HRESIMS of **1**; Figure S2. IR spectrum of **1**; Figure S3. 1H NMR spectrum (400 MHz) of **1** in CDCl3; Figure S4. 13C NMR spectrum (100 MHz) of **1** in CDCl3; Figure S5 HSQC spectrum (400 MHz) of **1** in CDCl3; Figure S6 COSY spectrum (400 MHz) of **1** in CDCl3; Figure S7 HMBC spectrum (400 MHz) of **1** in CDCl3; Figure S8 NOESY spectrum (400 MHz) of **1** in CDCl3; Figure S9. HRESIMS of **5** Figure S10. IR spectrum of **5** Figure S11. 1H NMR spectrum (400 MHz) of **5** in C6D6; Figure S12. 13C NMR spectrum (100 MHz) of **5** in C6D6; Figure S13. HSQC spectrum (400 MHz) of **5** in C6D6; Figure S14. COSY spectrum (400 MHz) of **5** in C6D6; Figure S15. HMBC spectrum (400 MHz) of **5** in C6D6; Figure S16. NOESY spectrum (400 MHz) of **5** in C6D6; Figure S17. HRESIMS of **6** Figure S18. IR spectrum of **6** Figure S19. 1H NMR spectrum (400 MHz) of **6** in C6D6; Figure S20. 13C NMR spectrum (100 MHz) of **6** in C6D6; Figure S21. HSQC spectrum (400 MHz) of **6** in C6D6; Figure S22. COSY spectrum (400 MHz) of **6** in C6D6; Figure S23. HMBC spectrum (400 MHz) of **6** in C6D6; Figure S24. NOESY spectrum (400 MHz) of **6** in C6D6, Table S1: Evaluation of ED50cytotoxicity of tested compounds.

**Author Contributions:** Conceptualization, S.-K.W. and C.-Y.D.; data curation, S.-K.W.; formal analysis, X.-H.L. and C.-Y.C.; funding acquisition, S.-K.W. and C.-Y.D.; investigation, X.-H.L. and C.-Y.C.; writing—original draft, S.-K.W.; writing—review and editing, C.-Y.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by grants from the Ministry of Science and Technology, Taiwan, Republic of China (MOST108-2320-B110-005) and from Kaohsiung Medical University (NSYSU-KMU 108-P018) awarded to C.-Y.D. and S.-K.W.

**Conflicts of Interest:** The authors declare no conflict of interest.
