LC-MS results of MEL-A.

(a), Mass spectra of different homologs isolated from the cultivation medium optimized by LC-MS. The units of CH2 or 2H molecular weight differences which are resulted from the different lengths of fat acid chain and the degree of unsaturation present in these compounds. (b), Loss of the erythritol, fatty acids and acetic acid was shown in the MS2 spectra.</p

Effects of MEL-A on cell cycle distributions of B16 cells.

<p>Cells were treated with different concentrations of MEL-A for 24 h. (a) ~ (e) refer to the cell cycle distributions of B16 cells treated by 0, 9.0, 15.0, 20.0, 25.0 μg/mL MEL-A, respectively. G1/G0, S, G2/M and Sub-G1 indicate the different cell phases. Mean ± SD, n = 3. The results in (f) summarized the relative ratios of each cell cycle, indicating that MEL-A caused cell cycle arrest at the S phase. The cell population of Sub-G1 phase was slightly increased with exposure to above 15.0 μg/mL of MEL-A.</p

¹H NMR spectrum and ¹³C NMR spectrum of MEL-A.

<p>(a), ¹H signals at 0~7.0 ppm and (b), ¹³C signals at 0~180.0 ppm. (c), Structure of MELs, MEL-A: R₁ = R₂ = Ac; MEL-B: R₁ = Ac, R₂ = H; MEL-C: R₁ = H, R₂ = Ac; MEL-D: R₁ = R₂ = H, n = 6–16.</p

Effects of MEL-A on the protein expression and mRNA expression in B16 cells.

(A) and (B), protein expression analysis of Caspase-3, cleaved Caspase-3, Caspase-12, Bcl-2, CHOP and GRP78 in the B16 cells treated with 15.0 μg/mL MEL-A for 24 h. The untreated cells were used as control. (C) and (D), fold changes of IRE1 and ATF6 protein expression. (E), mRNA expression analysis of Bcl-2, Caspase-3, CHOP, GRP78 and Caspase-12 in the B16 cells treated with 15.0 μg/mL MEL-A for 24 h. The untreated cells were used as control. Mean ± SD, n = 3.</p

Detected MEL-A homologs with various masses and fatty acids chain combinations.

<p>Detected MEL-A homologs with various masses and fatty acids chain combinations.</p

The solution state of MEL-A in water.

<p>(a), mean particle sizes of MEL-A solution at the concentrations of 12.0 mg/L. (b), mean particle sizes of MEL-A solution at the concentrations of 12.0 μg/L. (c), the surface tension of MEL-A changed with various concentrations. The concentration at the curve break records the critical micelle concentration (CMC).</p

Effects of MEL-A on apoptosis of B16 cells.

<p>Cells were treated with different concentrations of MEL-A for 24 h. (a) ~ (f) refer to the cell population changes of B16 treated by 0, 6.0, 9.0, 12.0, 15.0, 25.0 μg/mL MEL-A, respectively. Most MEL-A-induced cells were evident in B2 fraction, and the tendency of the induced cells apoptosis was in a dose-dependent manner. Mean ± SD, n = 3.</p

The viabilities of B16 cells and NIH3T3 cells in response to MEL-A treatments.

<p>(a), B16 cells treated with increasing doses of MEL-A had obvious changes. (b), the viability of the normal NIH3T3 cells treated by 15.0 μg/mL MEL-A for 72 h. (c, d, e, f) refers to the photographs of untreated B16 cells, and cells treated with MEL-A for 24 h, 48 h and 72 h. (g, h, m, n) refers to the photographs of untreated NIH3T3 cells and cells treated with MEL-A for 24 h, 48 h, and 72 h, respectively.</p