RAP and LRP-1 silencing by siRNA decreased the level and density of lysosome in MCF-7R cells.

MCF-7S, MCF-7R cells that incubated with or without 500 nM RAP for 12 hours were used in this experiment. The endocytic organelles were isolated by density gradient centrifugation as detailed in Materials and Methods. sucrose gradient was analysed using invertase enzyme assay as described in Materials and Methods. Detection of P-gp and Lamp-1 were evaluated by Western-blot in all collected aliquots (A-D). The intensity of the bands was quantified by densitometry using quantity one program. Student’s t-test was used for the statistical significance of different values. ** p < 0.01, *** p < 0.001 for MCF-7R cells versus MCF-7S cells, ## p < 0.01, ### p < 0.001 for MCF-7R treated cells versus MCF-7R untreated cells (E,F,G).</p

Schematic diagram of P-gp internalization by LRP-1 receptor and its implication in MCF-7 cells drug resistance.

Schematic diagram of P-gp internalization by LRP-1 receptor and its implication in MCF-7 cells drug resistance.</p

RAP sensitized MCF-7R cells to Dox cytotoxic effects and reduced IC₅₀.

A and B, MCF-7S and MCF-7R cells were treated with different Dox concentrations (from 0 to 10 μM) with or without 500 nM RAP. After 48 and 72h, cell viability was measured by UptiBlue Viable Cell Counting Assay. The results were presented as percentage of control and represented with standard deviation (S.D.) of at least three independent experiments. Student’s t-test was used for the statistical significance of different values.° NS, *** pC, MCF-7R cells were pretreated with Verapamil (5 μM) for 6h and incubated with Dox (1 μM). After 48 and 72h, cell viability was measured using UptiBlue Viable Cell Assay. The results obtained from three independent experiments (% of control) were represented with standard deviation (S.D.). Student’s t-test was used for the statistical significance of different values. **** pD, summary table of IC50 and RI (resistance Index). IC50 represents the mean half maximal inhibitory concentration. RI was assessed using the quotient of the IC50 values (IC50 MCF-7R/IC50 MCF-7) in each treatment conditions. Student’s t-test was used for the statistical significance of different values. *** p<0.001 for RAP-MCF-7R cells compared to untreated MCF-7R cells.</p

RAP and LRP-1 silencing by siRNA promoted the caspase-7 activation in Dox-treated MCF-7R cells.

MCF-7S, MCF-7R, scRNA-MCF-7R and siRNA-MCF-7R cells were incubated with 5 μM Dox with or without 500 nM RAP for 12 hours. A, Procaspase-7 and cleaved caspase-7 were detected by Western blotting. β-actin antibody was used as a control. The results were represented by three independent experiments. Quantity one software was used to quantify the intensity of the bands. Student’s t-test was used for the statistical significance of different values. *** pB, Caspase-7 activity was measured by caspACE assay kit. The results obtained from three independent experiments were represented with standard deviation (S.D.). Student’s t-test was used for the statistical significance of different values. ** p<0.01 and **** p<0.0001 compared to control cells. ¥¥¥ p<0.001 compared to Dox-treated cells.</p

Overexpression of P-gp and LRP-1 in MCF-7R.

MCF-7S and MCF-7R cells were cultured for 24 hours. Detection of P-gp, Lamp-1 and LRP-1/β chain was evaluated by Western-blot. β-actin antibody was used as a control. The results were represented by three independent experiments. Quantity one software was used to quantify the intensity of the bands. Student’s t-test was used for the statistical significance of different values. *** p<0.001 for MCF-7R cells compared to MCF-7S cells. The ratio was calculated with densitometry value of the protein of interest/ densitometry value of the β-actin.</p

ERK1/2 inhibition reduced Dox cytotoxic effects on MCF-7 cells.

MCF-7S and MCF-7R cells were incubated with 1 μM Dox with or without 500 nM RAP and 1 μM U0126 48 hours. A, Cell viability was measured using UptiBlue Viable Cell Assay. B, Caspase-7 activity was measured by caspACE assay kit. The results obtained from three independent experiments (% of control) were represented with standard deviation (S.D.). Student’s t-test was used for the statistical significance of different values. *** p<0.001 compared to Dox-treated cells and ¥¥¥ p<0.001, ¥¥¥¥ p<0.0001 compared to Dox- and RAP-treated cells.° NS, U0126-treated cells versus Dox-treated cells.</p

LRP-1 silencing by siRNA reduced P-gp expression and sensitized MCF-7R cells to Dox.

A, scRNA-MCF-7R and siRNA-MCF-7R cells were cultured for 24 hours. Detection of P-gp and LRP-1/β chain was evaluated by Western-blot. β-actin antibody was used as a control. The intensity of the bands was quantified by densitometry using quantity one program. Student’s t-test was used for the statistical significance of different values. *** pB, scRNA-MCF-7 and siRNA-MCF-7R cells were incubated with Dox at concentrations ranging from 0 to 10 μM. After 48 and 72h, cell viability was measured using UptiBlue Viable Cell Assay. The results obtained from three independent experiments (% of control) were represented with standard deviation (S.D.). Student’s t-test was used for the statistical significance of different values. *** pC, summary table of IC50. IC50 represents the mean half maximal inhibitory concentration. Student’s t-test was used for the statistical significance of different values. **** p<0.0001 for siRNA-MCF-7R cells compared to scRNA-MCF-7R cells.</p

RAP and LRP-1 silencing by siRNA promoted the ERK1/2 phosphorylation in Dox-treated MCF-7R cells.

A, MCF-7S cells were incubated with 1 μM Dox for 0.5, 1 and 2 hours. Detection of pERK, ERK, pAKT, AKT, Phospho-p38 and p-38 were evaluated by Western-blot. A representative blot of three independent experiments was shown. The intensity of the bands was quantified by densitometry using quantity one program. The ratio was calculated with densitometry value of the pERK/ densitometry value of ERK. Student’s t-test was used for the statistical significance of different values. *** p B, MCF-7S and MCF-7R cells were incubated with 1 μM Dox with or without 500 nM RAP for 0.5, 1 and 2 hours. Detection of pERK and ERK were evaluated by Western-blot. A representative blot of three independent experiments was shown. The intensity of the bands was quantified by densitometry using quantity one program. The ratio was calculated with densitometry value of the pERK/ densitometry value of ERK. Student’s t-test was used for the statistical significance of different values.° NS, ** p C, MCF-7S cells were incubated with or without 500 nM RAP for 0.5, 1 and 2 hours. Detection of pERK and ERK were evaluated by Western-blot. A representative blot of three independent experiments was shown. The intensity of the bands was quantified by densitometry using quantity one program. The ratio was calculated with densitometry value of the pERK/ densitometry value of ERK. Student’s t-test was used for the statistical significance of different values.° NS, RAP treated cells versus untreated cells.</p

RAP reduced the P-gp expression in MCF-7R cells.

A, MCF-7S and MCF-7R cells were incubated with or without 5 μM Dox for 4 and 8 hours. Detection of LRP-1/β chain was evaluated by Western-blot. β-actin antibody was used as a control. A representative blot of three independent experiments was shown. The intensity of the bands was quantified by densitometry using quantity one program. Student’s t-test was used for the statistical significance of different values. *** p B, MCF-7R cells were incubated with or without 500 nM RAP for 4 and 8 hours. Detection of P-gp was evaluated by Western-blot. β-actin antibody was used as a control. A representative blot of three independent experiments was shown. The intensity of the bands was quantified by densitometry using quantity one program. Student’s t-test was used for the statistical significance of different values. *** p < 0.001 for RAP-treated cells versus MCF-7S cells,° NS for Dox-treated cells versus untreated cells. The ratio was calculated with densitometry value of the protein of interest/ densitometry value of the β-actin.</p

RAP and LRP-1 silencing by siRNA increased Dox nuclear uptake in MCF-7R cells.

MCF-7S, MCF-7R, scRNA-MCF-7R and siRNA-MCF-7R cells were placed in petri dishes, pretreated with Verapamil (1 μM) for 6h and with 4 μM Dox with or without 500 nM RAP for 5h. The cells were washed with PBS free of drugs at 4°C. The nuclear Dox was followed through its fluorescence emission spectra by confocal laser microspectrofluorometry. A nuclear spectrum of treated cells was obtained over the wavelength range 500–700 nm [44]. The semi-quantification of nuclear Dox incorporation was evaluated by the fluorescence emission intensity of the band at 590 nm. The results represent mean ± standard deviation (S.D.) of at least three independent experiments. The data was analysed by Labspec software (Horiba). Student’s t-test was used for the statistical significance of different values. **** p < 0.0001 versus Dox-treated MCF-7S cells, ### p < 0.001 versus Dox-treated MCF-7R cells and ¥¥¥ p<0.001 compared to scRNA-cells.</p