Figure 4
Cadaverine treatment attenuate movement, invasion ability, mitochondrial oxidation and stem-ness of 4T1 cells. (A) 4T1 cells were treated with cadaverine in the concentrations indicated for 48 hours after scratching a 4T1 cell layer. Subsequent closure of the wound was assessed by the JULI-Br live cell analyzer system (n = 2). Significance was calculated using two-sample student t-test (two-tailed). (B) 4T1 cells were treated with cadaverine for 48 hours and subsequently invasion capacity of the cells was measured using the Corning Matrigel invasion chamber. Cells were counted using the Opera HCS system and invasion index was calculated (p = 0.003 both between control and 0.1 µM cadaverine, and control and 0.3 µM cadaverine, p = 4.14 × 10−4 between control and 0.8 µM cadaverine). (C) 4T1 cells were treated with vehicle and cadaverine for 48 hours, then cells were stained for MMP9 and nucleus (DAPI) and sections were analyzed by confocal microscopy using a Leica SP8 confocal system. MMP9 content was calculated from the total cellular fluorescence measured by the Image J software (p = 0.001 between both control and 0.3 µM cadaverine and control and 0.8 µM cadaverine). (D) 4T1 cells were treated with vehicle and cadaverine for 48 hours (n = 3 in 24 replicates), then cells were subjected to a Seahorse XF96 analysis. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured and plotted (pOCR = 0.006 between control and 0.1 µM cadaverine, pOCR = 1.14 × 10−6 between control and 0.3 µM cadaverine, pOCR = 1.8 × 10−5 between control and 0.8 µM cadaverine). (E) 4T1 cells were treated with vehicle and cadaverine for 48 hours (n = 3 in triplicates), then cells were subjected to an Aldefluor assay (p = 0.035 between control and 0.3 µM cadaverine). Data is plotted as mean ± SEM. Significance was calculated using One-way Anova. *,** and *** indicate statistically significant difference between control and treated groups at p < 0.05, p < 0.01 or p < 0.001 respectively. CAD – cadaverine, other abbreviations are in the text.
