Nitric oxide may regulate focal adhesion turnover and cell migration in MDA-MB-231 breast cancer cells by modulating early endosome trafficking

Al-Fahad, Dhurgham; Alharbi, Bandar Fahad; Ibeas Bih, Clementino; Dash, Philip Richard

doi:10.2478/acb-2021-0010

Figure 1

Effect of NO inhibitors or NO donors on cell migration. cells were treated with the vehicle, for 24 hours. (A) Representative wound-healing assay images for MDA-MB-231 cells treated with the vehicle (0.5% PBS), L-NAME (5 mM), 1400W (2 mM) or PAPA NONOate (50 μM). Images were taken at 0 and 24 hours to wound closure (the lines show wound edges). (B) Bar plot representing quantitative analyses of total covered area at 24 hours after scratching. (C) Representative images of migration assay showing cell tracking after a 24-hour period using a time-lapse microscope. (D) Bar plot representing quantitative analyses of migration speed. The data shown here are representative of three independent experiments; 40 cells were analysed in each experiment. Dunnett’s test comparison test was performed following a confirmed statistical difference using one-way ANOVA. *p<0.05, **p<0.01 and ***p<0.01. Results are presented as mean ± SEM

Figure 2

Effect of NO inhibitors or NO donors on FA turnover duration. MDA-MB-231 cells transfected with zyxin-mCherry were treated with the vehicle (0.5% PBS), L-NAME (5 mM), 1400W (2 mM) or PAPA NONOate (50 μM) for 10 minutes and were subsequently imaged using live-cell imaging. (A) Representative images showing FA assembly and disassembly. (B) Bar plot showing quantitative analyses of FA turnover duration. White arrows indicate FA. The data include three biological independent experiments; 100 zyxin-containing FA were used to assess the duration of FA turnover across 24 cells (per experiment). Dunnett’s test comparison test was performed following a confirmed statistical difference using one-way ANOVA. *p<0.05 and **p<0.01. Results are presented as mean ± SEM. The scale bar represents 10 μm

Figure 3

Effect of NO inhibitors on transferrin and dextran uptake. (A) Representative images of MDA-MB-231 cells treated with the vehicle (0.5% DMSO), the micropinocytosis inhibitor Amilorid (50 μM) or with endocytotic inhibitors Pitstop 2 (25 μM) and Dynasore (20 μM). After 48 hours, a transferrin/dextran uptake assay was performed using Alexa-Fluor-546-conjugated transferrin and fluorescein-conjugated dextran for 30 minutes. The scale bar represents 20 μm. Red arrows indicate nuclei. Yellow arrows indicate vesicles (B) Graphs representing quantitative analysis of fluorescent intensity, number of vesicles and vesicle size containing transferrin and dextran. (C) Representative images showing a transferrin and dextran uptake assay on MDA-MB-231 cells treated with the vehicle (0.5% PBS), L-NAME (5 mM) or 1400W (2 mM) for 48 hours. All conditions were performed as mentioned above using Alexa-Fluor-546-conjugated transferrin and fluorescein-conjugated dextran for 30 minutes. The scale bar represents 50 μm. (D) Bar plots representing quantitative analysis of intracellular fluorescent intensity, the number and the size of dextran/transferrin-containing vesicles following L-NAME and 1400W treatment. Results are presented as mean ± SEM. Dunnett’s test comparison test was performed following a confirmed statistical difference using one-way ANOVA. *p<0.05, **p<0.01 and ***p<0.01

Figure 4

The effect of NOS inhibitors on EEA1-containing endosomes and Rab5-containing vesicles in MDA-MB-231 cells. Cells were treated with vehicle or different nitric oxide inhibitors (L-NAME, 1400W) and PAPA NONOate for 48 hours, fixed and were stained with anti-Rab5 and anti-EEA1 antibodies. (A) Effect of L-NAME, 1400W and PAPA NONOate on the number and size of EEA1-positive endosomes. (B) Effect of L-NAME, 1400W and PAPA NONOate on the number and size of Rab5-positive vesicles. Yellow arrows indicate EEA1-positive endosomes and Rab5-positive vesicles. The scale bar represents 50 μm. One-way ANOVA with Dunnett’s multiple comparison test were used to compare each treatment with the control. Results are presented as mean ± SEM. The data include three independent experiments, in each experiment consisted of at least 80 cells that were analysed. Statistical significance differences were accepted at *p<0.05 and **p<0.01

Figure 5

Cellular localisation of eNOS and iNOS with EEA1. (A) Confocal images of the localisation between eNOS and EEA1 and between iNOS and EEA1. Immunocytochemistry was performed on fixed cells. The correlation between the localisation of eNOS (green) and EEA1 (red) and between the localisation of iNOS (green) and EEA1 (red) was determined with the Spearman’s (rho) correlation coefficient. Yellow arrows indicate regions of co-localisation. Three independent experiments were performed. (B) S-nitrosylation of early endosomal proteins. Representative immoblotting images showing S-nitrosylation in early endosomal proteins (EEA1, Rab5A and APPL1). No S-nitrosylation was found in nuclear proteins (H2B) and ubiquitin. MDA-MB-231 cell lysate treated with 100 μM HgCl2 displayed no S-nitrosylated proteins. Three independent experiments were performed. List of proteins with cysteine residues (highlighted in red) with predicted S-nitrosylation

Figure 6

A three-dimensional model of the human EEA1 structure. (A) depicts the location of all cysteine residues (highlighted in red) in EEA1. (B) shows the location of Cys-1102 (highlighted in red). (C) shows the locations of Cys-894 and Cys-46, both highlighted in red (D) shows the location of Cys-255 (highlighted in red)

Figure 7

A three-dimensional model of the human Rab5. (A) depicts the location of all cysteine residues (highlighted in red) in human Rab5. (B) shows the location of Cys-212 (highlighted in red)

Figure 8

A three-dimensional model of the human APPL1. (A) depicts the position of all cysteine residues (highlighted in red) in human APPL1. (B) shows the locations of Cys-99 and Cys-603, both highlighted in red