3.4.1. Preparation of Human Neutrophils

Human neutrophils from the venous blood [21] of healthy, adult volunteers (20–35 years old) were isolated using a standard method of dextran sedimentation, prior to centrifugation in a Ficoll Hypaque gradient and hypotonic lysis of erythrocytes, as previously described [29]. Purified neutrophils containing >98% viable cells, as determined by the trypan blue exclusion method, were resuspended in HBSS buffer at pH 7.4 and were maintained at 4 ◦C, prior to use [30].

#### 3.4.2. Measurement of O2 •– Generation

The assay for measurement of O2 •– generation was based on the SOD-inhibitable reduction of ferricytochrome c [31]. In brief, neutrophils (1 × <sup>10</sup><sup>6</sup> cells/mL) pretreated with the various test agents at 37 ◦C for 5 min were stimulated with fMLP (1 μmol/L) in the presence of ferricytochrome c (0.5 mg/mL). Extracellular O2 •– production was assessed with a UV spectrophotometer at 550 nm (Hitachi U-3010, Tokyo, Japan). The percentage of superoxide inhibition of the test compound was calculated as the percentage of inhibition = {(control − resting) − (compound − resting)}/(control − resting) × 100. The software SigmaPlot was used for determining the IC50 values [30].

#### 3.4.3. Chemicals and Antibodies

Fluorouracil (5-FU) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The antibodies against Bcl-2, Bax, and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Caspase-3 was obtained from GeneTex International Corporation (Hsinchu, Taiwan).

#### 3.4.4. Cells and Culture Medium

A549 (human lung carcinoma) and HT-29 (human colon carcinoma) cells were kindly provided by Prof. T. M. Hu and Prof. Y. Su, respectively, of National Yang-Ming University, Taipei, Taiwan.

All cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, 2 μM L-glutamine, and 1 mM sodium pyruvate. The cells were incubated in an atmosphere of 37 ◦C and 5% CO2 and passaged twice a week. Cells were stored in liquid nitrogen at −155 ◦C. After the cells were thawed, the experiment was completed before 30 generations. The purpose was to minimize experimental errors. The compound stock solution was stored in DMSO at a concentration of 10 mM and stored at −20 ◦C, and finally melted immediately before use.

#### 3.4.5. Cytotoxicity Assay

A MTT colorimetric assay was used to determine cell viability. The assay was modified from that of Mosmann [32]. MTT reagent (0.5 mg/mL) was added onto the attached cells mentioned above (100 μL per 100 μL culture) and incubated at 37 ◦C for 3 h. Then, DMSO was added and the amount of colored formazan metabolite formed was measured by absorbance at 570 nm, using an ELISA plate reader (μ Quant). The optical density of formazan formed in control (untreated) cells was taken as 100% viability.

#### 3.4.6. Clonogenic Assay

The clonogenic assay followed as previously described with slight changes [33]. For the clonogenic assay, cells at a density of 3000 cells/well were seeded in 6-well plates for 24 h. Next, the cells were treated with compound **3** or vehicle (DMSO) and allowed to form colonies for 14 days. Colonies were washed with PBS, and the cells attached to the plastic surface were fixed in 99% MeOH for 30 min and stained with 0.2% crystal violet for 15 min. The stained cells were quantified using the ImageJ software (BioTechniques, NY, USA).

#### 3.4.7. Western Blotting Analysis

Western blot analysis was performed according to the method previously reported [34]. In brief, A549 (1 × 105 cells) was seeded into 6 wells plate and grown until 85–90% confluent. Then, different concentrations (3.125, 6.25, 12.5, 25, and 50 μM) of compounds **3** and **4** were added. Cells were collected and lysed by radioimmunoprecipitation assay (RIPA) buffer. Lysates of total protein were separated by 12.5% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking, the membranes were incubated with anti-Bax, anti-Bcl-2 (Cell Signaling Inc., Danvers, MA, USA), anti-caspase-3, and anti-β-actin (GeneTex Inc., Irvine, CA, USA) primary antibodies at 4 ◦C overnight. Then, each membrane was washed with Tris-buffered saline containing 0.1% Tween 20 (TBST) and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature, for 1 h, while shaking. Finally, each membrane was developed using an enhanced chemiluminescence (ECL) detection kit, and the images were visualized by ImageQuant LAS 4000 Mini biomolecular imager (GE Healthcare, MA, USA). The band densities were quantified using the ImageJ software (BioTechniques, NY, USA).

## 3.4.8. Statistical Analysis

All data are expressed as mean ± SEM. Statistical analysis was carried out using Student's t-test. A probability of 0.05 or less was considered to be statistically significant. Microsoft Excel 2019 was used for the statistical and graphical evaluation. All experiments were performed at least 3 times.

#### **4. Conclusions**

Three novel (**1**–**3**) and four known compounds were isolated and identified from *Penicillium citrinum*. Among the isolated compounds, compounds **2**–**5** could significantly inhibit fMLP-induced O2 •− generation, with IC50 values ≤ 8.28 μM. These isolated compounds are worth further research, as promising new leads for developing anti-inflammatory agents. Furthermore, compounds **3** and **4** markedly induced apoptosis of A549 cells through the mitochondrial- and caspase 3-dependent pathways (Figure 11). This suggests that compounds **3** and **4** are worth further investigation and might be expectantly developed as the candidates for the treatment or prevention of non-small cell lung cancer and liver cancer.

**Figure 11.** Schematic diagram for cancer cell apoptosis mechanism of compounds **3** and **4** in A549 cells.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/1660-339 7/19/1/25/s1, Figures S1–S10: HRESIMS, CD, 1D, and 2D NMR spectra for epiremisporine C (**1**). Figures S11–S20: HRESIMS, CD, 1D, and 2D NMR spectra for epiremisporine D (**2**). Figures S21–S30: HRESIMS, CD, 1D, and 2D NMR spectra for epiremisporine E (**3**). Figures S31–S34: HRESIMS, CD, 1H and 13C NMR spectra for (**4**). Figures S35 and S36: HRESIMS, 1H NMR spectra for penicitrinone A (**5**). Figures S37 and S38: HRESIMS, 1H NMR spectra for 8-hydroxy-1-methoxycarbonyl-6 methylxanthone (**6**). Figures S39 and S40: HRESIMS, 1H NMR spectra for isoconiochaetone C (**7**). Figure S41: CD and ECD spectra of epiremisporine B. Tables S1 and S2: 1H and 13C NMR spectrum for **4** and epiremisporine B. Table S3: The ROESY correlations for compounds **1**–**3**.

**Author Contributions:** Y.-C.C. performed the isolation and structure elucidation of the constituents, and prepared the manuscript. C.-H.C. and H.-R.L. conducted the bioassay and analyzed the data. M.-J.C. and M.-D.W. performed the cultivation and preparation of the fungal strain. S.-L.F. analyzed bioassay data. J.-J.C. planned, designed, and organized all of the research of this study and reviewed the manuscript. All authors read and approved the final version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in the main text and the supplementary materials of this article.

**Acknowledgments:** This research was supported by grants from the Ministry of Science and Technology (MOST), Taiwan (No. MOST 109-2320-B-010-029-MY3 and MOST 106-2320-B-010-033-MY3), awarded to J.-J. Chen. We gratefully thank Shou-Ling Huang and Iren Wang for the assistance in NMR experiments of the Instrumentation Center at NTU which is supported by the Ministry of Science and Technology, Taiwan.

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

#### **References**


**Sample Availability:** Samples of the compounds are available from the authors.

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