Fig 2: CAF-derived exosomes promote YKL-40 expression in HEC-1A and RL95-2 cells. (A) Immunofluorescence assay for detection of a-SMA and vimentin expression in NFs and CAFs. Scale bar, 100 µm. (B) The morphology of exosomes was observed by transmission electron microscopy. (C) Flow cytometry for evaluating CD63 expression in exosomes. (D) Western blot analysis of TFIIB, LaminA/C, CD63, CD81, Hsp70, and TSG101 protein levels in CAF lysate and exosomes. (E) The green exosome fluorescence in HEC-1A and RL95-2 cells after incubation with Dil-labeled exosomes (10 µM) for 24 h was observed under a laser confocal microscopy. (F) RT-qPCR analysis of NEAT1 expression in the exosomes derived from three paired NFs and CAFs. HEC-1A and RL95-2 cells were incubated with PBS, or exosomes isolated from three paired NFs and CAFs for 48 h. (G) NEAT1 expression in EC cells was detected by RT-qPCR. (H) & (I) RT-qPCR and Western blot assays for determining YKL-40 expression in HEC-1A and RL95-2 cells. All data from three independent experiments were shown as mean ± SD (n = 6). **, P < 0.01; ***, P < 0.001.

Fig 3: Western blot analysis of human cartilage tissue, A: Western blot band map of each protein, B-F: quantitative histogram of protein bands for the expression of Cbfß, MMP-13, IL-1ß, COMP, and YKL-40 (***: p < 0.001)

Fig 4: Expression of NEAT1, YKL-40, and miR-26a/b-5p in human endometrial cancer. RT-qPCR analysis of NEAT1(A), YKL-40(B), and miR-26a/b-5p (C, D) levels in endometrial cancer and normal endometrial tissues. All data were shown as mean ± SD (n = 30). **, P < 0.01; ***, P < 0.001.

Fig 5: Exosomal NEAT1 sponges miR-26a/b-5p to facilitate STAT3 and YKL-40 expression in EC cells. (A) RT-qPCR for miR-26a/b-5p level in exosomes derived from CAFs, NEAT1-overexpressed CAFs, or NEAT1-silenced CAFs. (B) HEC-1A and RL95-2 cells were co-cultured with NEAT1-overexpressed or silenced CAFs, and miR-26a/b-5p level was detected by RT-qPCR. (C) Expression of miR-26a/b-5p in HEC-1A and RL95-2 cells infected with lentivirus containing miR-26a/b-5p mimics or inhibitor was detected by RT-qPCR. (D) Expression of YKL-40 in HEC-1A and RL95-2 cells after overexpression or silencing of miR-26a/b-5p was assessed by Western blot. (E) The binding sites between NEAT1 and miR-26a/b-5p. Luciferase analysis (F) and RIP assay (G) was performed to determine interaction between NEAT1 and miR-26a/b-5p. The expression of STAT3 and YKL-40 in HEC-1A and RL95-2 cells received various treatments was assessed by RT-qPCR (H) and Western blot (I). All data from three independent experiments were shown as mean ± SD (n = 6). *, P < 0.05; **, P < 0.01; ***, P < 0.001, ns = no significant.