Figure 4.

ESC autophagy upregulates expression of HCK by activating STAT3 signaling pathway. (a,b) The proteomic microarray was performed to evaluate the different proteins of cell lysate (A, left) and supernatant (B, left) between Ctrl-ESC and 3-MA-ESC. KEGG database-derived bioinformatics analysis was used to look for the differential expressions of proteins (non-cytokines) (A and B, right), which has the relationship with NK function molecules (FCGR3, KIR2DL1, KIR3DL1, NCR3, NCR2, NCR1, KLRK1, IFNG, PRF1 and GZMB). (c) The expression of HCK in normESC (n = 6), eutoESC (n = 6) and ectoESC (n = 6) was analyzed by western blotting. (d) The expression of HCK in Ctrl-ESC (n = 6), 3-MA-ESC (n = 6) or Rap-ESC (n = 6) was analyzed by western blotting. (e) The C57BL/6 EMS mice were treated with vehicle, 3-MA or Rap (n = 6 mice/group) on day 3 and day 10 after surgery. Then the transcriptional level of Map1lc3b, Becn1 and Hck in EMS-like lesions was detected by RT-PCR (one-way ANOVA). (f,g) NC-ESC and siATG5-ESC were stimulated with Rap (1 mM) for 4 h, and then cultured for another 12, 24, or 48 h, and the protein level of ATG5, BECN1, SQSTM1, LC3B and HCK, and the mRNA level of HCK was detected by western blotting and real-time PCR. (h,i) NC-ESC and siATG5-ESC were stimulated with Rap (1 mM) for 4 h, and then cultured for another 6, 12, or 24 h, and the level of phosphorylated and total STAT3 was analyzed by FCM. (j) Ctrl-ESC was treated with Rap (1 mM) for 4 h, and incubated with the selective inhibitor of STAT3 (HO-3867, 10 μM) for another 4 h, and then the mRNA level of HCK was analyzed by real-time PCR. eutoESC, ESCs of eutopic endometrium from women with EMS. Data are expressed as the mean± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.001.