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

Effect of 10 mM DCA on metabolic activity after 48 hours.

<p>In comparison to untreated control no significant reduction metabolic activity was observed in MTH53A (A) and DT14/06T (D). ZMTH3 (B) showed significantly reduced metabolic activity after 48 hours and MTH52C after 24 hours (C). Data are shown as mean ± SD, n = 3 and are presented as relative metabolic activity 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. (A) MTH53A; (B) ZMTH3; (C) MTH52C; (D) DT14/06T.</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

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

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

Effect of 10 mM DCA on protein expression in mammary cell lines after 48 hours.

<p>In comparison to negative control, significant decrease in JNK expression was observed in all cell lines except DT14/06T which showed increased JNK values. Significantly increased BAD expression was observed in all cell lines except MTH53A. Data are shown as mean ± SD, n≥3 and are presented as relative mean 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. (A) Apoptotic and dead cells in flow cytometry; (B) relative expression of JNK; (C) relative expression of BAD.</p

Impact of 10 mM DCA on proliferation of mammary cell lines after 48 hours determined with fluorescence microscopy.

<p>(A-H) Images showing fluorescence of Ki67 (red) and DAPI (blue). In comparison to non-treated control (A,C,E,G) amount of Ki67 is reduced in all DCA treated cell lines (B,D,F,H) except in MTH53A (B). Statistical significant reduction of Ki67 was observed in ZMTH3, MTH52C and DT14/06T in comparison to non-treated control and non-neoplastic tissue derived cell line MTH53A. No significant decrease in proliferation could be determined in within neoplastic cell lines (I). Data are shown as mean ± SD, n≥3 and are presented as relative proliferation 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. (A) MTH53A control; (B) MTH53A+DCA; (C) ZMTH3 control; (D) ZMTH3+DCA; (E) MTH52C control; (F) MTH52C+DCA; (G) DT14/06T control; (H) DT14/06T+DCA; (I) relative proliferation.</p

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

<p>(A-H) Fluorescence of mitochondrial derived ROS (red) and counterstained cell nuclei (DAPI, blue). (I) In comparison to untreated control significant improvement of mitochondrial activity was observed in mammary carcinoma cell line MTH52C. A visible but insignificant increase was observable in the mammary carcinoma cell line DT14/06T and non-cancerous mammary gland derived cell line MTH53A. The adenoma cell line ZMTH3 showed significantly decreased mitochondrial activity in comparison to untreated control and non-cancerous cell line MTH53A. No significant difference was evaluated between MTH53A and the other cell lines. Data are shown as mean ± SD, n≥3 and are presented as relative fluorescence (mitochondrial activity) 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. (A) MTH53A control; (B) MTH53A+DCA; (C) ZMTH3 control; (D) ZMTH3+DCA; (E) MTH52C control; (F) MTH52C+DCA; (G) DT14/06T control; (H) DT14/06T+DCA; (I) relative mitochondrial activity.</p