Journal: Journal of Veterinary Internal Medicine

Article Title: Effects of the potassium‐sparing diuretic amiloride on chemotherapy response in canine osteosarcoma cells

doi: 10.1111/jvim.15382

Figure Lengend Snippet: Combination treatment with calculations of the combination index (CI) in D17, Abrams, and Dharma treated with amiloride and doxorubicin, or amiloride and carboplatin. Decreased cell viability in each cell line after treatment with increasing doses of (A) amiloride and doxorubicin or (B) amiloride and carboplatin for 72 hours. X‐axis shows dose pairings for amiloride (Am, μM), doxorubicin (Dox, nM) and carboplatin (Carb, μM), which were chosen from IC50 curves. Relative absorbance values at 590 nm (A 590 ) were presented with median ± range (N = 9). (C) Mean CI values from 3 independent experiments, stratified by dose pairing. CI < 1 was synergistic, C = 1 additive, and C > 1 antagonistic. Significance was recorded at * P < .05. (D) Mean CI values stratified by cell line independent of drug pairings. N = 15 with N = 3/drug pairing. IC50, half‐maximal inhibitory concentration

Article Snippet: D17 was obtained from Sigma‐Aldrich/European Collection of Cell Cultures (ECACC; Salisbury, United Kingdom).

Techniques: Concentration Assay

Journal: Journal of Veterinary Internal Medicine

Article Title: Effects of the potassium‐sparing diuretic amiloride on chemotherapy response in canine osteosarcoma cells

doi: 10.1111/jvim.15382

Figure Lengend Snippet: Western blot of proteins involved in p53 and Akt signaling in (A) D17, (B) Abrams, and (C) Dharma cells. (D) Viability after pharmacological inhibition of p53 translocation to the mitochondria. Cells were treated with pifithrin‐μ (3 nM) alone or in combination with 300 μM amiloride for 24 hours. Cell viability was measured with the crystal violet assay. Means were presented with SD, and significance reported at * P < .05

Article Snippet: D17 was obtained from Sigma‐Aldrich/European Collection of Cell Cultures (ECACC; Salisbury, United Kingdom).

Techniques: Western Blot, Inhibition, Translocation Assay, Crystal Violet Assay

Journal: Journal of Veterinary Internal Medicine

Article Title: Effects of the potassium‐sparing diuretic amiloride on chemotherapy response in canine osteosarcoma cells

doi: 10.1111/jvim.15382

Figure Lengend Snippet: Immunofluorescence of p53 localization in canine OSA cells after amiloride treatment. (A) Representative images of p53 localization in D17 cells after treatment with high‐dose amiloride (300 μM) or vehicle for 24 hours before p53 staining. Cells were imaged with the Leica DMLB fluorescent microscope. Scale bar 10 μm. (B) Mean fluorescence intensity (MFI) of p53 staining in D17, Abrams, and Dharma cells as calculated by ImageJ/Fiji image analysis. Native images were taken at 10x/0.7 aperture, the regions of interest (ROI) were defined and quantified using ImageJ. N = 3 per group, with mean ± SD. Significance was reported at * P < .05 and ** P < .01. OSA, osteosarcoma

Article Snippet: D17 was obtained from Sigma‐Aldrich/European Collection of Cell Cultures (ECACC; Salisbury, United Kingdom).

Techniques: Immunofluorescence, Staining, Microscopy, Fluorescence

Journal: Journal of Veterinary Internal Medicine

Article Title: Effects of the potassium‐sparing diuretic amiloride on chemotherapy response in canine osteosarcoma cells

doi: 10.1111/jvim.15382

Figure Lengend Snippet: Seahorse metabolic tests of glycolytic and mitochondrial function in D17, Abrams, and Dharma cells. Cells were equilibrated in low‐buffered XF medium for basal readings of oxygen consumption rates (OCR) and extracellular acidification rates (ECAR). (A) Glycolytic test measuring ECAR in each cell line after amiloride (300 μM) or vehicle injection in real time, before the injection of glucose (10 mM), oligomycin (1 μM) and 2‐Deoxy‐D‐glucose (2‐DG) (100 mM). (B) Mitochondrial test measuring OCR in each cell line after amiloride or vehicle injection in real time, before the injection of oligomycin (1 μM), FCCP (0.5 μM), and rotenone/AA (0.5 μM). N = 3 per time point with mean ± SD. AA, antimycin A; FCCP, carbonyl cyanide‐4‐(trifluoromethoxy)phenylhydrazone

Article Snippet: D17 was obtained from Sigma‐Aldrich/European Collection of Cell Cultures (ECACC; Salisbury, United Kingdom).

Techniques: Injection

Journal: Journal of Veterinary Internal Medicine

Article Title: Effects of the potassium‐sparing diuretic amiloride on chemotherapy response in canine osteosarcoma cells

doi: 10.1111/jvim.15382

Figure Lengend Snippet: Combined energy phenotype analysis reveals a dose‐dependent effect of amiloride on ECAR and OCR in D17 cells. (A) Linear regression model of ECAR during the first 50 minutes, representing readings after amiloride injection. (B) Fold changes in ECAR expressed across all treatment groups tested after amiloride injection. Coupling efficiency (C) and maximal respiration (D) as measured using the mitochondrial stress test. (E‐F) Energy phenotype analysis after a 24‐hour pretreatment with amiloride (100 μM). ECAR and OCR were normalized to cell density (4 x 10 −4 cells), and vehicle‐treated cells were compared to amiloride‐treated cells after oligomycin injection. A metabolic switch is indicated by the black arrow between the phenotype markers. Median ± range values were presented in box plots. Mean ± SD values were presented in energy phenograms. Significance was recorded at * P < .05, ** P < .01, **** P < .0001. ECAR, extracellular acidification rates; OCR, oxygen consumption rates

Article Snippet: D17 was obtained from Sigma‐Aldrich/European Collection of Cell Cultures (ECACC; Salisbury, United Kingdom).

Techniques: Injection