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Fig. 3 | Stem Cell Research & Therapy

Fig. 3

From: CCL20/CCR6 axis mediates macrophages to promote proliferation and migration of ESCs by blocking autophagic flux in endometriosis

Fig. 3

Macrophages block autophagic flux of ESCs through the CCL20/CCR6 axis. A The protein level of LC3 and P62 in ESCs with different treatments by western blot. B Quantification of the gray value of LC3 and P62 western blot bands. C Measurement of autophagic flux with transfecting mRFP-GFP-LC3 double fluorescence adenovirus (Ad‐LC3), which is used to distinguish autophagosomes (mRFP+/GFP+ fluorescence, yellow puncta) and autolysosomes (mRFP+/GFP fluorescence, red puncta). ESCs with different treatments were transfected with Ad‐LC3 in the presence or absence of 100 nM Baf-A1 treatment to suppress the fusion of autolysosomes. Confocal images of representative cells were shown. (original magnification 1000 ×) D Quantification of LC3 puncta number of representative cells. E Co-localization coefficient data show changes in co-localization levels of LC3 and LAMP2 after treatment. F Representative immunofluorescence images of Gal3, a specific biomarker for lysosomal membrane damage, in different treated ESCs. (original magnification 200 ×) G Quantitative fluorescence intensity of Gals. H Representative immunofluorescence images of Lyso-Tracker Green, a lysosomal probe for detecting the acidation of lysosome, in different treated ESCs. (original magnification 200 ×) I Quantitative fluorescence intensity of Lyso-Tracker. J Quantification of acid phosphatase activity in different treated ESCs. Data are presented as the mean ± SD of n = 3 independent experiments. Statistical analysis was performed using one-way ANOVA with Tukey post hoc. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns, non-significant vs. control group. ####p < 0.0001, ####p < 0.001, ##p < 0.01, #p < 0.05 vs. co-culture group. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns, non-significant vs, -Baf-A1 group

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