第1090回千葉医学会例会・第7回環境生命医学研究会

<p>HepG2 cells were incubated with BITC 30 μM, insulin 100 nM and insulin + BITC for 30 min. PathScan Signaling Array Kit was used for the detection of the phosphorylated AKT (Thr308). Data shown as mean of fold phosphorylation normalized to untreated control + SEM (n = 3) *p<0.05(Unpaired Student’s t test).</p

FOXO1 phosphorylation upon BITC treatment.

<p>HepG2 cells were incubated with 30 μM BITC for 24 h and cell lysates were prepared and subjected to Western blot analysis with antibodies for pFOXO1 Thr24 (Phospho FOXO1). Total FOXO1 was used for normalization. Data shown as mean + SEM (n = 3) ***p<0.001 (Unpaired Student’s t test).</p

CAT gene expression in HepG2 cells modulated by BITC.

<p>Dose-dependency is shown for the antioxidant enzyme CAT. Results are presented as fold mRNA expression, normalized to the housekeeping gene RPL32 and the control. Data shown as mean value ± SEM *(p<0.05), **(p<0.01) and ***(p<0.001). (Oneway ANOVA and posthoc Bonferroni).</p

Gluconeogenic enzymes protein expression.

<p>HepG2 cells were incubated with 30 μM BITC for 24 h and cell lysates were prepared and subjected to Western immuno-blot analysis with primary antibodies for PEPCK and G6Pase. Results are normalized with GAPDH. Data shown as + SEM (n = 3) **p<0.01, ***p<0.001 (Unpaired Student’s <i>t</i> test).</p

HepG2 cells siRNA knock-down analyses and G6Pase gene expression.

<p>BITC 30 μM influence after starvation on G6Pase gene expression upon FOXO1, AKT, NRF2 and SIRT knock-down conditions. *p<0.05, **p<0.01 (Unpaired Student’s t test) vs control cells treated with NT “*”or NT+BITC “#”, normalized with RPL32. Data shown as mean of fold mRNA + SEM (n = 4).</p

U-2 OS-FOXO1-GFP cells treated with BITC 1–100 μM for 1h and addition of 100 nM insulin after 30 min.

<p>The curves show the relative nuclei/cytoplasm intensity of FOXO1-GFP for BITC without (black curve) and with insulin addition (red curve). Results are presented as mean values + standard error (SEM) (n = 4). Significant differences vs control (0 μM) are labelled for *(p<0.05) and **(p<0.01) (Oneway ANOVA and posthoc Dunnett T3).</p

CAT time dependency analyses in HepG2 cells modulated by BITC.

<p>Time dependent analyses for CAT. Results are presented as fold mRNA expression, normalized to the housekeeping gene RPL32 and the control. Data shown as mean value ± SEM *(p<0.05). (Oneway ANOVA and posthoc Bonferroni).</p

Primer sequences for qRT-PCR.

<p>Primer sequences for qRT-PCR.</p

U-2 OS-FOXO1-GFP cells treated with BITC at selected concentrations.

<p>Nuclear accumulation was estimated by measuring the ratio of FOXO1 nuclear/FOXO1 cytosolic (FOXO1 Nuc/Cyt) after 1 h treatment normalized to untreated control cells. Ratios higher than 1 showed a nuclear accumulation of FOXO1. Results are presented as mean values + standard error (SEM) (n = 4). Significant differences vs control (0 μM) are labelled for *(p<i><</i>0.05) and **(p<i><</i>0.01) (Oneway ANOVA and posthoc Bonferroni multiple comparisons).</p

FOXO1 translocation in stably transfected U-2 OS (human osteosarcoma cells).

<p>Fig 1A–1D show FOXO1-GFP expressing cells and nuclear DAPI-staining (red) changing to orange-yellow in overlays with GFP after incubation with BITC at concentrations of 0 (1A-A’), 1 (1B-B’), 10 (1C-C’) and 100 μM (1D-D’), respectively. Following 1 h of starvation, stimulation of cells was performed with BITC for 1 h. Cells were fixed and stained with DAPI for identification of nuclear areas by fluorescence microscopic detection, for segmentation of cells and calculation of GFP-intensity ratios in nuclei and cytoplasm (Nuc/Cyt).</p