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  • br Cell viability assay br Cells were

    2020-08-07


    2.8. Cell viability assay
    Cells were seeded in 96-well plates at a density of 3000 cells/well in DMEM media. On the next day, the media were aspirated, Sotrastaurin (AEB071) were washed with PBS, and serine-replete or -deplete media containing compound (0, 50 μM) or DMEM media containing compound (0–200 μM) were added and incubated for 3–5 days, followed by adding 100 μL FBS-free medium containing 10% CCK8 into each well and in-cubated for an additional 30 min at 37 °C. The serine-replete or serine-deplete media were made from serine/glycine-free DMEM supple-mented with either serine (400 μM) or PBS. And the serine/glycine-free DMEM were purchased from BOSTER (Wuhan, China). The absorbance was measured at 450 nm using an enzyme-linked immunosorbent assay reader. IC50 was calculated by comparing the absorbance of cells un-treated and compound-treated groups using GraphPad Prism software. The standard deviation for each compound against each cell line was obtained by at least three independent experiments [5,13].
    2.9. Cell apoptosis analysis
    Fig. 4. Cancer cells with high expression level of PHGDH were more sensitive to azacoccones C and E. (A) PHGDH expression level in three cancer cell lines MDA-MB-231, MDA-MB-468 and Hela were determined by Western blot analysis. (B and C) Cancer cells were exposed to varying concentrations of azacoccones C and E for 72 h to investigate its cytotoxic activity. Proliferation assay for lines treated with azacoccone compounds in (D) serine-replete media (+SER) or (E) serine-deplete media (−SER). Azacoccones C and E functioned at the concentration of 50 μM. The number of cells was determined and normalized to that of DMSO group. *P < 0.05, **P < 0.01, ***P < 0.001.
    cells were examined by flow cytometry (BD Biosciences, FACSCalibur) [16].
    Hela cells were seeded in 6-well plates at a density of 2 × 104 cells/ well and treated with azacoccone E at 50 and 100 μM for 72 h. After treatment, cells were fixed with 4% paraformaldehyde for 15 min and then incubated with Hoechst 33258 at 37 °C for 30 min. The apoptotic morphological changes were observed by Hoechst 33258 under a fluorescence microscope (Zeiss, OBSERVER D1/AX10 cam HRC) [17].
    2.11. Western blot analysis
    The supernatants and intact cells in a 6-well plate after treatment with or without compound were collected and the expression level of PHGDH was detected by western blot. The cells were cleaved with RIPA lysate and the protein concentration was determined by BCA kit and equalized before loading. Equal amounts of protein from each group were denatured and subjected to electrophoresis in 12% SDS-PAGE gels followed by transfered to PVDF membrane and probed with PHGDH andβ-actin antibodies. Blot bands were visualized using the horseradish 
    peroxidase-conjugated secondary antibodies combined with chemilu-minescent substrate [18].
    3. Results and discussion
    3.1. Inhibitory effects of azacoccones C and E on PHGDH
    To explore the inhibitory activity of azacoccones C and E on PHGDH enzyme in vitro, recombinant human PHGDH and PSAT1 protein were expressed and purified (Fig. S1). And the enzymatic activity was eval-uated by detecting the increase of NADH monitored by fluorescence (340/460 nm; Ex/Em). Azacoccones C and E showed inhibitory effects against PHGDH with IC50 values of 11.71 ± 2.65 and 9.76 ± 4.32 μM, which were better effect than the positive control CBR-5884 (31.58 ± 7.18 μM) (Fig. 2A). And at the same inhibitor concentrations, azacoccones C and E had no effect on another NAD(P)+-dependent dehydrogenases, isocitrate dehydrogenase (IDH1) (Figs. 2B, 2C and Table 1).
    3.2. Specific binding of azacoccones C and E with PHGDH in vitro
    To further validate the effects of azacoccones C and E binding to
    Fig. 5. Apoptotic-driven effects of azacoccone E against Hela cell line. (A) Apoptosis ratio of azacoccone E detected by flow cytometry. Cells were treated with compound azacoccone E for 72 h. (B) Effect of azacoccone E on the nuclear morphological changes of Hela cells. Images were acquired using a fluorescence microscope (200× magnification). Bar = 20 μm.
    Fig. 6. The inhibition mechanism of azacoccone E. (A) 3-PG saturation profiles for PHGDH at a range of concentrations of azacoccone E. (B) Line weaver–Burk double-reciprocal representation of the 3-PG saturation profiles for PHGDH at a range of concentrations of azacoccone E. (C) Time-dependent inhibition was measured by preincubating azacoccone E and PHGDH for 0.5, 1, or 4 h.
    Table 2
    Kinetic parameters with azacoccone E treatment on PHGDH.a
    a Km and Vmax values were obtained from Lineweaver-Burk plots. The values were the means of results of three experiments.
    azacoccone E to PHGDH.
    To quantitatively measure the interaction between PHGDH and azacoccone E, the cellular thermal shift assay (CETSA), a recently de-veloped method that allows rapid and simple assessment of target binding of compounds in a cellular context was employed. The thermal stability of PHGDH in Hela cells was tested at the temperature range of 54–62 °C. The results implied that azacoccone E specifically targeted PHGDH proteins in cells, showing the high sensitivity of azacoccone E in Hela cancer cells (Fig. 3C).