Fig. 3: GAL1-overexpressing HepG2 cells accumulate less intracellular DOX and exhibit increased P-gp protein expression. | Cell Death & Disease

Fig. 3: GAL1-overexpressing HepG2 cells accumulate less intracellular DOX and exhibit increased P-gp protein expression.

From: Galectin-1 confers resistance to doxorubicin in hepatocellular carcinoma cells through modulation of P-glycoprotein expression

Fig. 3

A HepG2-M and HepG2-GAL1 cells were loaded with DOX (20 µM) and 5-chloromethylfluorescein diacetate (CMFDA) (2.5 µM). Metabolized glutathione methylfluorescein (GS-MF) was used to visualize canalicular structures. DOX and GS-MF fluorescence was registered in the same field. Ten coverslips/cell type were photographed, and representative images are shown (40X) (n = 3). Arrows indicate pseudocanaliculi that secreted both DOX and GS-MF. B HepG2 cells with different levels of GAL1 expression were exposed to DOX (20 µM) for the indicated times, and after treatment were scraped and lysed. The amount of DOX uptake by cells was determined with a fluorescence spectrophotometer using calibration curves. Total protein content in each sample was also measured. Results are expressed as the mean of DOX concentration (pmol/µg protein) ± SEM for each cell line and time point with respect to the accumulated by HepG2 cells after 1 h (n = 5). **p < 0.01 with respect to HepG2-M cells. #p < 0.05, ##p < 0.01, with respect to HepG2-shScr. &p < 0.05, &&p < 0.01, &&&p < 0.001, with respect to HepG2 cells. C Intracellular DOX accumulation in HepG2-GAL1 cells after 6 h treatment, with or without 20 µM verapamil (P-gp inhibitor). Results are expressed as the mean ± SEM of intracellular DOX accumulated by HepG2-GAL1 cells co-incubated with verapamil, with respect to DOX-treated HepG2-GAL1 cells in the absence of inhibitor (100%) (n = 4). **p < 0.01. D Western blot and densitometric analysis showing relative P-gp expression in GAL1-overexpressing, GAL1-silenced, and the corresponding control HepG2 cells. β-tubulin was used as loading control (n = 5). *p < 0.05 with respect to HepG2-M cells. &p < 0.05 compared to HepG2 cells. E Relative P-gp protein expression in tumors derived from vehicle-treated (control) or DOX-treated mice inoculated with HepG2-M and HepG2-GAL1 cells (n = 5) (Fig. 2). &p < 0.05 with respect to control HepG2-M-derived tumors.

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