HPRP-A2-induced BGC-823 and SGC-7901 cell death.

<p>The cells were treated with different concentrations of HPRP-A2 for 1 hour. Cell viability was determined using the MTT method. Results are expressed as percentage of the control ± SD of three independent experiments. Statistical analysis compared the HPRP-A2 treatment group with the control groups (*P < 0.005; **P <0.001).</p

HPRP-A2-induced mitochondrial membrane potential.

<p>(A) BGC-823 cells were treated for 1 h and the generation of reactive oxygen species (ROS) was analyzed by FACS to quantitatively compare the fluorescence intensity (in Geomean) at various concentrations. (B) After 1 h treatment, the ratio of FL2-H/FL1-H decreased, indicating a reduction of MMP. (C) BGC-823 cells were treated with HPRP-A2 (10 and 15 μM) for 24 h and then levels of caspase activities were measured. Data are expressed as mean ± SD of three independent experiments. Statistical analysis compared the HPRP-A2 treatment group with the control groups (*P < 0.005; **P <0.001).</p

Membrane permeability changes of BGC-823 cells by monitoring PI and LDH.

<p>The cells were incubated with increasing peptide concentrations for 1 h at 37 oC. (A) Quantitative comparisons of fluorescence intensity (in Geomean) at various concentrations were analyzed by flow cytometry. (B) LDH in the supernatant was measured with a microplate reader at 450 nm. Cells without treatment or lysed with triton X-100 was used as negative and positive controls, respectively. LDH activity was calculated as the percentage of experimental group and positive control, after subtraction of negative control respectively (*P<0.005). Data are the mean ± SD of three independent experiments.</p

Cell viability and combination index of BGC-823 and SGC-7901 treated with a drug combination.

<p>Panel (A, B and C) represents growth inhibition in BGC-823 and SGC-7901 cells with a combination of HPRP-A2 (6 μM) and DOX (1.6 μg/ml) after incubation for 4, 24 and 48 hours, respectively. Results are expressed as the percentage of the control ± SD of three independent experiments. Panel D shows combination index (Q) of the combination treatment of HPRP-A2 and DOX, where Q<0.85, Q>1.15 and 0.85</p

HPRP-A2 triggered cell cycle arrest.

<p>Cell-cycle distributions after treatment with 5, 10 and 15 μΜ HPRP-A2 for 24 h separately were detected by flow cytometry. Cell-cycle distributions were assessed by PI staining. The results were showed from one of three experiments with similar results.</p

Hemolytic activity of HPRP-A2 against hRBCs.

<p>Data points present mean ± S.D. of three independent experiments. PBS and dH2O were used as negative and positive controls, respectively. Statistical analysis compared the HPRP-A2 treatment group with the positive control groups (*P < 0.005; **P <0.001).</p

Peptide sequence and the helical wheel of HPRP-A2.

<p>Peptide sequence and the helical wheel of HPRP-A2.</p

Effect of GST-irisin on lipolysis in 3T3-L1 mature adipocytes.

<p>A: Effect of GST-irisin on the release of glycerol in 3T3-L1 mature adipocytes. The adipocytes were cultured with various GST-irisin concentrations (0, 50, 100, and 200 nM) for 2 (a), 4 (b), 6 (c) and 8 (d) days. B: The mRNA expression of FABP4, ATGL, and HSL was determined by qPCR. C: The protein levels of FABP4, ATGL, FNDC5, and β-actin were determined by western blotting. The adipocytes were cultured with various GST-irisin concentrations, and the concentration was 0 (a), 50 (b), 100 (c), and 200 (d) nM. Values are expressed as the mean ± SD of three independent experiments. *<i>P</i> < 0.05 versus control; **<i>P</i> < 0.01 versus control.</p

Circular dichroism spectra of peptides.

<p>(<b>A</b>) In benign medium (50 mM KH₂PO₄/K₂HPO₄ containing 100 mM KCl, pH 7.4) at 25°C and (<b>B</b>) in the presence of 50% TFE at 25°C. The symbols used are as follows: ■, HPRP-A1 peptide; ▲, HPRP-A1-TAT peptide.</p

Peptide-induced cell apoptosis.

<p>(<b>A</b>) Mitochondrial membrane potential measured by JC-1, an indicator of mitochondrial function. HeLa cells were treated with various concentrations of HPRP-A1 or HPRP-A1-TAT for 24 h. (<b>B</b>) Percentage of early apoptotic cells, as assessed by flow cytometry. HeLa cells were treated with various concentrations of HPRP-A1 or HPRP-A1-TAT for 1 or 24 h. (<b>C</b>) Caspase-3, -8, and -9 activity. HeLa cells were treated with HPRP-A1 (4 μM) or HPRP-A1-TAT (4 μM) for 24 h before measuring caspase activity levels. Data are presented as the mean ± SD of three independent experiments.</p