Fig 2: Cartoon illustration of macrophages promoting the proliferation and migration of ESCs via CCL20/CCR6/TFEB/autophagy pathway (created in BioRender.com)

Fig 3: CCL20 promotes ESC proliferation and migration via the CCR6/TFEB/Autophagy pathway. A Proliferative capability of ESCs, incubated with rCCL20 in response to TFEB-OE with and without 3-MA (inhibitor of autophagy), assessed by CCK8 assay. B Proliferative capability of ESCs in different groups assessed by Edu assay. (original magnification 200 ×) C Quantification of Edu incorporation rate. D Migration capability of ESCs in different groups assessed by wound healing assay. (original magnification 40 ×) E Quantification of wound closure rate. F Migration capability of ESCs in different groups assessed by Transwell assay. (original magnification 200 ×) G Quantification of migration cells per field at 200 × magnification. H ELISA analysis of CCL20 in macrophages culture supernatant with or without co-cultured with ESCs. I The mRNA level of CCL20 in macrophages with or without co-cultured with ESCs by qRT-PCR. J The mRNA level of IL-6 in ESCs with or without co-cultured with macrophages by qRT-PCR. K The mRNA level of TNF-a in ESCs with or without co-cultured with macrophages by qRT-PCR. Data are presented as the mean ± SD of n = 3 independent experiments. Statistical analysis was performed using one-way ANOVA with Tukey post hoc (A, C, E, G), Mann–Whitney test (I, J, K) or Student’s t test (H). ***p < 0.001, **p < 0.01, *p < 0.05, ns, non-significant

Fig 4: Macrophages promote the proliferation and migration of ESCs via the CCL20/CCR6 axis in EMs. A ELISA analysis of CCL20 in ESC culture supernatant with or without co-culture treatment. B The mRNA level of CCL20 in ESCs with or without co-culture treatment by qRT-PCR. C The protein level of CCR6 in ESCs in different groups by western blot. D Quantification of the gray value of western blot bands. E Representative immunofluorescence images of CCR6 (red) in ESCs in different groups, Nuclei were stained with DAPI (blue). (original magnification 200 ×) F Quantitative fluorescence intensity of CCR6. G Proliferative capability of ESCs in different groups assessed by CCK8 assay. H Proliferative capability of ESCs in different groups assessed by Edu assay. (original magnification 200 ×) I Quantification of Edu incorporation, Edu incorporation rate (%) = Edu positive cells (green)/Hoechst positive cells (blue). J Migration capability of ESCs in different groups assessed by wound healing assay. (original magnification 40 ×) K Quantification of wound closure rate, wound closure rate (%) = (Scratch distance 0 h—Scratch distance 24 h) / Scratch distance 0 h. L Migration capability of ESCs in different groups assessed by Transwell assay. (original magnification 200 ×) M Quantification of migrating cells per field at 200 × magnification. Data are presented as the mean ± SD of n = 3 independent experiments. Statistical analysis was performed using Mann–Whitney test (A, B) or one-way ANOVA with Tukey post hoc (D, F, G, I, K, M). ****p < 0.0001, ***p < 0.001, **p < 0.01, ns, non-significant vs. control group. ####p < 0.0001, ####p < 0.001, ##p < 0.01 vs. co-culture group

Fig 5: 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