Figure 6.
Effects of spautin-1 treatment in combination with metformin. (a) Representative OCR plot of Rb1cc1F/+ BPK primary cells treated with vehicle, 2.5 mM metformin, 2.5 μM spautin-1, or 2.5 mM metformin + 2.5 µM spautin-1 for 24 h and measured using a Seahorse XF24 extracellular flux analyzer. (b) Quantification of basal and spare capacity of Rb1cc1F/+ BPK primary cells treated with vehicle, 2.5 mM metformin, 2.5 μM spautin-1, or 2.5 mM metformin + 2.5 μM spautin-1(n = 4 independent experiments). Statistical significance was determined by two-tailed t-test, * denotes p ≤ 0.05, ** denotes p ≤ 0.01 and *** denotes p ≤ 0.001. (c) Representative image and (d) quantification of colony-forming assays with Rb1cc1F/+ BPK primary cells treated with vehicle, 2.5 mM metformin, 2.5 μM spautin-1, or 2.5 mM metformin + 2.5 μM spautin-1 for a week (n = 9, triplicates from 3 independent experiments). Statistical significance was determined by two-tailed t-test, *** denotes p ≤ 0.001. (e) Tumor growth curves of Rb1cc1F/+ BPK primary cells transplanted into Nude mice and treated daily with vehicle, 100 mg/kg metformin, 40 mg/kg spautin-1 or the combination of metformin and spautin-1 (n = 10 tumors each). Statistical significance for growth curves was determined via ANCOVA. *** denotes p ≤ 0.001. (f) Quantification of the percentage of MKI67-positive cells and cleaved-CASP3-positive cells in Rb1cc1F/+ BPK primary cells transplanted into Nude mice and treated daily with vehicle, 100 mg/kg metformin, 40 mg/kg spautin-1 or the combination of metformin and spautin-1 (n = 6 tumors each). Representative images of (g) MKI67-positive cells and (h) cleaved-CASP3-positive cells in Rb1cc1F/+ BPK primary cells transplanted into Nude mice and treated daily with vehicle, 100 mg/kg metformin, 40 mg/kg spautin-1 or the combination of metformin and spautin-1.