MOESM1 of Cyclic helix B peptide inhibits ischemia reperfusion-induced renal fibrosis via the PI3K/Akt/FoxO3a pathway

Additional file 1: Figure S1. Validation of FoxO3a siRNA and Wortmannin. The p-FoxO3a proteins (A) and mRNA (B) expression of HK-2 cells were significantly down-regulated by FoxO3a shRNA treatment. The expression of p-Akt was also significantly reduced by Wortmannin in the HK-2 cells (C)

Effect of LJK-11 on tyrosine phosphorylation of signaling proteins.

<p>A549 cells were treated with 50 µM LJK-11 for 6, 12, 24, or 36 hours. The cell lysates were resolved by SDS-PAGE and analyzed by Western bolt analysis using antibodies as indicated. Antibodies specific to the phosphorylated forms of the indicated proteins are labeled with (-P).</p

Chemical structure of LJK-11.

<p>Chemical structure of LJK-11.</p

Reversibility of LJK-11 treatment.

<p>Hela cells were incubated with 50 µM LJK-11 or 20 nM colchicine for 20 hours (A) and then the compound-containing media were removed, the cells were washed with fresh media, and the cells were incubated in new compound-free media for additional 18 hours (B). The pictures were taken using a light microscope.</p

Synergistic effect of LJK-11 and colchicine on blocking mitosis.

<p><b>A</b>. Flow cytometry analysis of the effects of LJK-11 (10 µM), colchicines (20 nM), or the combination of the two on cell cycle distribution. A549 cells were incubated with 10 µM LJK-11, 20 nM colchicine, or combination of 10 µM LJK-11 and 20 nM colchicine for 24 hours. The cells were then fixed and stained with PI, and analyzed by flow cytometry. <b>B</b>. Percentage of cells in G2/M phase after 24 hours treatment with 10 µM LJK-11, 20 nM colchicine, or combination of 10 µM LJK-11 and 20 nM colchicine. <b>C</b>. Concentration dependent G2/M arrest by treatment of colchicines or LJK-11 for 24 h. Also indicated in the figures are the percentages of G2/M arrest induced by the combination of 10 µM LJK-11 and 20 nM colchicine. Data are the means of triplicates ± SD.</p

Effects of LJK-11 on mitotic spindle formation.

<p>A549 cells were incubated on glass coverslips with different reagents for 16 hours, and then fixed and stained with α-tubulin antibody to visualize microtubules (green) and with DAPI to visualize chromosomes (blue). The cells were visualized by indirect immunofluorescent microscopy. A: control cells treated with equal volume of DMSO (0.1%). B: cells treated with 100 µM LJK-11. C: cells treated with 5 nM nocodazole. D: cells treated with 100 nM colchicine. E: cells treated with 50 nM Taxol.</p

Effect of LJK-11 on tubulin polymerization.

<p>Effects of LJK-11 (250 µM), colchicines (10 µM), nocodazole (10 µM), or Taxol (10 µM) on bovine brain tubulin polymerization were measured turbidimetrically(A). Effects of 1 µM, 5 µM, 25 µM, 100 µM, 200 µM LJK-11 on bovine brain tubulin polymerization were also measured. Changes in absorbance at 340 nm (A₃₄₀) were measured and plotted as a function of time(B).</p

Effects of LJK-11 on cell cycle distribution.

<p>A. Flow cytometry analysis of LJK-11-treated A549 tumor cells. A549 cells were incubated with different concentrations of LJK-11 for 24 hours. The cells were then fixed and stained with PI, and analyzed by flow cytometry. B. Percentage of cells in G2/M phase after 24 hours treatment with different concentrations of LJK-11. The data are the means of triplicates ±SD. C. Flow cytometry analysis of LJK-11-treated MDA-MB-453 tumor cells. MDA-MB-453 cells were incubated with different concentrations of LJK-11 for 24 hours. The cells were then fixed and stained with PI, and analyzed by flow cytometry. D. Percentage of cells in G2/M phase after 24 hours treatment with different concentrations of LJK-11. The data are the representative of three independent experiments.</p

Effect of LJK-11 on the growth and death of different tumor cells.

<p>A549, Hela, HGC-27, or MDA-MB-453 cells were incubated with the indicated concentrations of LJK-11 for 48 hours. The effect of LJK-11 on cell growth and death was evaluated by MTT assay. The cell viability is expressed as a percentage of the compound-treated viable cells divided by the viable cells of the untreated control. The data are the means of triplicates ±SD.</p