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Fig. 3 | Cell Communication and Signaling

Fig. 3

From: Autophagy modulators influence the content of important signalling molecules in PS-positive extracellular vesicles

Fig. 3

Autophagic flux and energetic consequences of autophagy manipulation. (a) Autophagic flux in FaDu cells after 12 h-lasting effect of treatment traced with an mRFP-GFP-LC3 tandem construct; scale bar equals 10 µm. Autophagosomes and autolysosomes are labelled in yellow and TaqRFP, respectively. (b) The mean RFP signal (signal of autolysosomes) was significantly diminished after APB, CPD18, EACC, and BAFA1 and enhanced after HCQ treatment. The intensity of the yellow signal (situation where the autophagosome is positive for both red and green signal) was significantly diminished after APB, CPD18, EACC, and BAFA1 treatment indicating decreased autophagic flux. Significantly increased LysoTracker fluorescence intensity was observed for cells treated with HCQ, Torin1, and rapamycin. On the other hand, BAFA1 caused the vanishing of the fluorescent signal of acidic organelles from the cytoplasm. P-values from group comparisons based on the t-test are shown. Asterisks represent statistical significance (* p < 0.05; ** p < 0.01; *** p < 0.001). One dot represents single cell. (c) Pearson correlation. LC3II/I ratio assessed by WB was in positive correlation with LC3-mRFP signal (red = autolysosomes) (p = 0.0003) and with yellow signal (meanRFP-times-GFP) (p = 0.006). (d) and (e) ATP production from glycolysis and OXPHOS based on Seahorse parameters OCR (oxygen consumption rate) and ECAR (extracellular acidification rate) obtained from XF Real-Time ATP Rate Assay before and after autophagy inhibitors treatment; PER = Proton efflux rate, rate of protons extruded into the extracellular medium during glycolysis. OM = oligomycin; ROT = rotenone; AA = antimycin A. P-values from group comparisons based on the t-test are shown. Asterisks represent statistical significance (* p < 0.05; ** p < 0.01; *** p < 0.001). One dot represents a replicate in a single well

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