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. 2020 Mar 23;21(6):2207. doi: 10.3390/ijms21062207

Figure 5.

Figure 5

Confocal analyses: ER morhology and extension and its relationship with lysosomes. (A) Single confocal optical sections of ER Tracker (in green) and LTDR (in red) of ATCC33291 lysate treated U937 cells and U937 cells preincubated with lysate and treated by RM, after 72 h. Co-localization is shown in yellow-orange and white arrows indicate the positioning of lysosomes, from a perinuclear area (ATCC 33291 RM-) to a peripheral distribution (ATCC 33291 RM+). Bars: 10 µM. In (B), Pearson’s coefficient, able to quantitate co-localization, specifically induced by RM in ATCC 33291 lysate treated cells. Asterisks denote a statistically significant difference (* = p < 0.05) between strains. (C) Lysosome distance from surface calculated by ImageJ. Calculation performed on 30 cells for each experiment (n = 3). Only lysosomes with a puncta and definite morphology were counted, from a minimum of 10/cell to a maximum of 30/cell. Mitochondria membrane potential and ER–mitochondria interactions. Asterisks denote a statistically significant difference (*** = p < 0.001) between strains. (D) Single confocal optical sections of ER Tracker (in green) and TMRE (in red) of ATCC33291 lysate treated U937 cells and U937 cells preincubated with lysate and treated by RM, after 72 h. Red fluorescence from mitochondria and their puncta are clearly increased in RM+ cells. Bars: 10 µM. (E) TMRE MFI values of RM- and RM+ lysate-treated U937 cells. Asterisks denote a statistically significant difference (*** = p < 0.001) between strains. (F) Pearson’s coefficient, able to quantitate TMRE/ER-tracker co-localization, specifically induced by RM in ATCC 33291 lysate treated cells. Total extracellular vesicles (EVs) and EVs from lysosome exocytosis. Asterisks denote a statistically significant difference (* = p < 0.05) between strains. (G) Flow cytometric quantitation of CD107a + EVs with (RM ATCC 33291) or without (ATCC 33291). Asterisks denote a statistically significant difference (* = p < 0.05) between strains. (H) Flow cytometric quantitation of lipid dye + EVs with (RM ATCC 33291) or without (ATCC 33291).