(C) Glucose starvation-resistant cells (Hs578t.LG) from Hs578t cells were obtained in low glucose (0.5mM with 5% FBS) for 4 days. 0.05).Supplemental Physique S2: SIRT6 expression in Hs578t knockdown clones. (A) Comparison of SIRT6 expression in parental (Hs578t), control knockdown 54.5, or Runx2 knockdown (55.5) Hs578t cells. Cells were alpha-Bisabolol culture in full media made up of 10%FBS and 25mM glucose (FM) or in glucose starvation media made up of 2%FBS for 8hr (S). Nuclear extracts were examined for RUNX2 and SIRT6 expression by Western blot. Relative SIRT6 protein expression normalized to actin (NIH Image-J) from scanned blots is usually indicated under alpha-Bisabolol each lane. (B) Overexpression of SIRT6 in breast malignancy cells. Hs578t cells (RUNX2+) were transfected with vacant vector (Vector) or cDNA expression vector encoding human SIRT6. After a brief selection (1 week), nuclear extracts were isolated and analyzed for Rabbit Polyclonal to DUSP6 SIRT6 and RUNX2 expression by Western blotting. SIRT6 relative density in arbitrary models (AU) normalized to actin was calculated from three determinations using NIH Image-J; * indicates p 0.05 relative to Vector. (C) Glucose starvation-resistant cells (Hs578t.LG) from Hs578t cells were obtained in low glucose (0.5mM with 5% FBS) for 4 days. Surviving cells were expanded in standard cell culture media (DMEM+10% FBS) for 10C14 days. Cells were analyzed for RUNX2 and GLUT1 expression. Significant differences in RUNX2 (p 0.01; t-test) and GLUT1 (p 0.06; t-test) expression for LG clones compared to parental cells were calculated by Image-J. (D) Hs578t parental and LG2 cells were treated with different concentrations of glucose as indicated and analyzed for SIRT6 expression after 16hr. Relative alpha-Bisabolol SIRT6 protein expression normalized to actin (NIH Image-J) from scanned blots is usually indicated. Supplemental Physique S3: Mitochondrial OCR in MCF7 and Hs578t cells . (A) MCF7 RUNX2 + or RUNX2 ? cells were compared for OCR using the Seahorse metabolic flux analyzer. FCCP was used to depolarize the inner mitochondrial membrane and inhibitors of mitochondrial ETC were used as in Physique 5. Treatments include: FCCP + pyruvate (Pyr) ( 0.4M + 10mM), FCCP (0.1M), FCCP (0.1M), and antimycin-A (1M). Cell protein was extracted after determination of OCR and was comparable for RUNX2+ (10.68 0.17 g) and RUNX2? (10.10 2.07 g) from n = 11 wells per sample (p 0.05) (B) Hs578tP (Parental) or control knockdown (Hs578t/54.5) cells were compared for OCR using the Seahorse metabolic flux analyzer. As in Physique 5, FCCP (0.75M), pyruvate (10mM), and antimycin-A (1M) were used to treat cells. Oligomycin (2.5nM) inhibition indicates that in these cells 95% of the oxygen consumption was linked to mitochondrial ATP synthesis. Inset shows RUNX2 expression in Hs578t/54.5 (control knockdown) and Hs578t/55.5 (RUNX2 knockdown) cells compared to parental cells. (C) Knockdown of SIRT6 in breast malignancy cells. Hs578t/55.5 (low RUNX2 expression) cells were transfected with scrambled (Control) siRNA or three different SIRT6-specific siRNA oligonucleotides (siRNA A, siRNA B, and siRNA C) from Origene. Levels of SIRT6 were determined by Western blot with specific SIRT6 antibody. Relative density in arbitrary models (AU) represents the mean from three determinations normalized to actin (NIH Image-J); * indicates p 0.05 relative to Control. (D) Hs578t/55.5 cells were transfected with control or siRNA C targeting SIRT6. SIRT6 and RUNX2 protein levels are shown and compared relative to actin. The Western blot was overexposed to visualize the low levels of RUNX2 in 55.5 knockdown cells. Band density relative to control is usually indicated. Supplemental Physique S4: Hif1 and SIRT6 expression in response to RUNX2. (A) MCF7 cells cultured in the presence (+Dox; RUNX2-) or absence of doxycycline (?Dox; RUNX2+) were starved in the absence of glucose for 16hr to lower Hif1 levels and then treated with 5mM glucose for the indicated occasions. Some cells were treated with the SIRT inhibitor, sirtinol (Sirt; 10M) and 5mM glucose for 8hr. Cell lysates were prepared and the expression of Hif1, SIRT6, or RUNX2 was measured by Western blotting. Gels were stripped and reprobed for -actin (loading control). Relative density of scanned Western blot (NIH Image-J) was normalized to actin and expressed as arbitrary models (AU). (B) MCF7 cells cultured in +Dox or ?Dox to induce RUNX2 were cultured in 5mM glucose, placed in a hypoxia chamber, and exposed to 1%O2 for the indicated occasions. Cells were lysed and the expression of Hif1 was measured by Western blotting. Relative band.