Fig. 2: SLC38A2 undergoes re-localisation from the Trans–Golgi network under diverse stress in different breast cancer cell lines.

Co-localisation study of SLC38A2 at TGN in different cell lines and after diverse treatments. a Representative confocal images of MCF7 (top row), T47D (second row), SKBR3 (third row), HCC1806 (fourth row). Cells were fixed and stained with SLC38A2 (green) and with TGN46 (red) in normoxia (left column) and after 24 h of: amino acid deprivation (EBSS medium, no AA, 10% dialysed FBS; second column), PP242 treatment (20 μm; third column) and thapsigargin treatment (8 h, fourth column, Scale bars 5 μm). b Pearson’s test co-localisation analysis of SLC38A2 at TGN46 during treatments. Scheme of the analysis procedure: Circular patches surrounding a cell or a group of cell or manual evaluation SLC38A2 levels (ROI) and non- SLC38A2 signal (random ROI) were selected. Co-localisation values were calculated using a pixel-wise Pearson’s test. Frequency quantification of Pearson’s test values (−1: opposing, 0: no and 1: maximum co-localisation) for SLC38A2 vs. TGN in MCF7 (top row), SLC38A2 vs. TGN in T47D (second row), SLC38A2 vs. TGN in SKBR3 (third row) and SLC38A2 vs. TGN in HCC1806 (fourth row). N = 3, one-way ANOVA, *p < 0.05, **p < 0.05. c Western blot analysis of protein expression in the same four cell lines after previously mentioned treatments. β-actin served as loading control. N = 3. Exposure time (HCC1806: 90 s; MCF7: 3 min; T47D: 5 min; SKBR3: 9 min. Arrows indicate predicted SLC38A2 protein) d The relative expression of SLC38A2 total mRNA, in MCF7, T47D, SKBR3 and HCC1806 after different treatment as above, was analysed. Results were obtained by using the mean of the Ct values of SLC38A2 transcript after normalisation to housekeeping genes (β-actin and RPL11). Error bars, SD; ANOVA t-test. *p < 0.05, ***p < 0.001; N = 3.