Fig 1: YAP O-GlcNAcylation promotes its activation and nuclear translocation. (A) Nuclear-cytoplasmic separation assays revealed the cellular sub-localization of YAP in the different groups. (B and C) Immunofluorescence staining revealed the cellular sub-localization of YAP in the different groups. Scale bars, 10 µm. (D and E) mRNA expression of ANKRD1, CTGF, CYR61 and GLUT3 was detected in endometrial cancer using reverse transcription-quantitative PCR in EC cells. (F) Western blot analysis was used to measure CTGF expression in endometrial cancer cells. Data represent the mean ± SD (n=3). *P<0.05 and **P<0.01. ns, not significant; YAP, Yes-associated protein; OGT, O-GlcNAc transferase; H3, Histone-H3; ANKRD1, ankyrin repeat domain 1; CTGF, connective tissue growth factor; CYR61, cysteine-rich angiogenic inducer 61; GLUT3, glucose transporter 3.
Fig 2: Effect of CM/Gel-MA on IUA cell model. The IUA model was induced by adding TGF-ß1 into endometrial stromal cells. After treating the model with CM/Gel-MA, the fibrosis was inhibited with decreased the expression of a-SMA, collagen I, CTGF, E-cadherin, and IL-6. At the same time, the cell vitality and proliferation ability were enhanced after treatment.
Fig 3: YAP1 reverses the effect of NAP1L1 in CFs. (A and B) The overexpression efficiency of YAP1 in CFs; n = 5, ** p < 0.01 vs. control. (C and D) Quantification of scratch wound healing assays; bar = 200 µm; n = 6. * p < 0.05, ** p < 0.01. (E) EdU detected the proliferation of CFs; bar = 50 µm; n = 3. (F) Knockdown NAP1L1 inhibited the viability of TGF-ß1-induced CFs and this effect was abolished after overexpression of YAP1; n = 5, ** p < 0.01 vs. TGF-ß1+siNC, ## p < 0.01 vs. TGF-ß1+siNAP1L1. (G) Immunofluorescence staining of a-SMA showed silencing of NAP1L1 slower YAP1 induced the fibroblast–myofibroblast transition after TGF-ß1 induction; bar = 20 µm, n = 6. (H and I) Western blot detected the expression levels of FN1, YAP1, and CTGF in CFs stimulated with TGF-ß1; n = 6, * p < 0.05 vs. TGF-ß1+siNC, ## p < 0.01 vs. TGF-ß1+siNAP1L1. (J–L) Analysis of the relative mRNA expression of Collagen 1a1, Collagen 3a1, and YAP1 in TGF-ß1-induced CFs cotransfected with YAP1 overexpression plasmid and NAP1L1 siRNA; n = 3, ** p < 0.01 vs. TGF-ß1+siNC. ## p < 0.01 vs. TGF-ß1+siNAP1L1. (M) Under conditions of fibrotic stimulus, knockdown of NAP1L1 reverses cardiac fibrosis through the regulation of YAP1 ubiquitination and degradation.
Fig 4: Expression level of some bio-factors. (A) Relative mRNA level of a-SMA. (B) Relative mRNA level of collagen I. (C) Relative mRNA level of CTGF. (D) Relative mRNA level of E-cadherin. (E) Relative mRNA level of IL-6. (F) The results of WB.
Fig 5: NAP1L1 promotes CFs proliferation, migration, and transition. (A and B) qRT-PCR and Western blotting analysis of relative NAP1L1 expression in CFs treated with NAP1L1 overexpression plasmid or vector; n = 6, * p < 0.05, ** p < 0.01. (C and D) The EdU assay showed the cell proliferation rate of CFs; bar = 50 µm; n = 4, * p < 0.05 vs. vector. (E) Cell viability by MTT assay; n = 6, ** p < 0.01 vs. vector. (F and G) Cell migration assessed by wound healing assay; bar = 200 µm, n = 5, ** p < 0.01, * p < 0.05 vs. vector. (H) The relative mRNA expression levels of Collagen 1a1, Collagen 3a1, Fn1, and CTGF after overexpressing NAP1L1; n = 6, ** p < 0.01, * p < 0.05 vs. vector. (I and J) The protein level of Collagen I and FN1 after simultaneous overexpression of NAP1L1; n = 6, ** p < 0.01 vs. vector. (K) Immunofluorescence assay was used to detect the expression level of a-SMA; bar = 20 µm.
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