Effect of 10 mM DCA on miR expression after 48 hours.

<p>In comparison to untreated control no significant changes in miR-145 was proved. Regarding miR-375, a significant increase was observed in ZMTH3. Data are shown as mean ± SD, n = 3 and are presented as relative miR expression in comparison to untreated control. Ct-values were analyzed with Rest2009 and statistical analysis was performed with two-tailed t-test, *p<0.05, **p<0.01, ***p<0.001. (A) miR-145 expression of DT14/06T; (B) miR-375 expression.</p

Immunocytochemical staining.

<p>A: Native CT1258 cells, B: CT1258-EGFP cells, C: CT1258-EGFP-HMGA2 cells. Approximately 50% of the native CT1258 cell line and of CT1258-EGFP cells showed a HMGA2-positive nuclear labelling. In approximately 70–80% of CT1258-EGFP-HMGA2 cells, a strong and exclusively nuclear labelling for HMGA2 was detectable.</p

Effect of 10 mM DCA on PDK-1 expression after 48 hours.

<p>Western Blot analysis of PDK-1 expression in MTH53A, MTH52C, ZMTH3 and DT14/06T. All cell lines showed positivity for PDK-1 and GAPDH but no changes in PDK-1 expression was detectable between untreated and DCA exposed cells in any of the evaluated cell lines. GAPDH was used as loading control.</p

<i>HMGA2</i> real-time PCR analyses.

<p>Relative <i>HMGA2/HPRT1</i> and <i>HMGA2/GUSB</i> expression in native CT1258, CT1258-EGFP and CT1258-HMGA2-EGFP cells. Error bars are standard deviations. *p≤0.05 indicates a statistical significant expression deregulation of <i>HMGA2</i> in CT1258-HMGA2-EGFP cells when compared to native CT1258.</p

BrdU cell proliferation assay.

<p>Measured proliferation of native CT1258, CT1258-EGFP and CT1258-HMGA2-EGFP cells. A statistical significant increased proliferation was detected for CT1258 cells expressing the EGFP-HMGA2 fusion protein in comparison to native CT1258 and EGFP expressing CT1258 cells. Each bar represents a mean ± SD, *p≤0.05, ***p≤0.001.</p

Effect of 10 mM DCA on PDH phosphorylation at three different residues after 48 hours treatment.

<p>Data was assessed with Luminex Magnetic Bead technology. PDH-P at Ser²³² decreased significantly in all cell lines. Residue Ser²⁹³ showed no significant response to DCA treatment except in cell line DT14/06T. The third phosphorylation site Ser³⁰⁰ has decreased values in carcinoma cell lines but not in non-neoplastic mammary gland derived cell line MTH53A and benign cell line ZMTH3. Data are shown as mean ± SD, n≥3 and are presented as relative PDH-P values in comparison to untreated control (%). Control was set to 100%. Statistical analysis was performed with two-tailed t-test, *p<0.05, **p<0.01, ***p<0.001.</p

<i>HMGA1</i> real-time PCR analyses.

<p>Relative <i>HMGA1/HPRT1</i> and <i>HMGA1/GUSB</i> expression in native CT1258, CT1258-EGFP and CT1258-HMGA2-EGFP cells. Error bars are standard deviations. *p≤0.05 indicates a statistical significant increased expression of <i>HMGA1</i> in CT1258-HMGA2-EGFP cells when compared to native CT1258 and CT1258-EGFP.</p

<i>Let-7a</i> real-time PCR analyses.

<p>Relative <i>let-7a</i>/RNU6B expression in native CT1258, CT1258-EGFP and CT1258-HMGA2-EGFP cells. Error bars are standard deviations. No statistical significant expression deregulation of <i>let-7a</i> in CT1258-EGFP and CT1258-HMGA2-EGFP was detected when compared to native CT1258 cells. Statistical significant p value was defined as ≤0.05.</p

Influence of 10 mM DCA on mammary cell lines after 48 hours.

<p>Statistical significant reduction of cell number was observed in all cell lines. Significant difference in cell number between MTH53A and mammary carcinoma DT14/06T was observed. Data is shown as mean ± standard deviation (SD), n≥3 and are presented as relative cell numbers in comparison to the respective corresponding untreated control (%). Control values were set to 100%. Statistical analysis was performed with two-tailed t-test, *p<0.05, **p<0.01, ***p<0.001.</p

Influence of DCA on cellular ATP production in mammary cell lines after 48 hours.

<p>In comparison to untreated control significant enhancement of ATP-production was observed in all cell lines. No difference was detectable between non-neoplastic mammary gland derived cell line MTH53A and neoplastic tissue derived cell lines. Data are shown as mean ± SD, n≥3 and are presented as relative ATP-production (mitochondrial respiration) in comparison to untreated control (%). Control was set to 100%. Statistical analysis was performed with two-tailed t-test, *p<0.05, **p<0.01, ***p<0.001.</p