Fig 2: IDD occurred gradually in the natural groups and incomplete compensation of self-repair in acute injury induced IDD(A) Quantitative polymerase chain reaction (qPCR) comparing the relative expression of LATS1, LATS2, yes-associated protein (YAP), and connective tissue growth factor (CTGF) in the 4w, 14w, P4w, and 50w groups. (B–E) Western blot analysis of Hippo pathway-related proteins using anti-LATS1, anti-LATS2, and anti-YAP (B) and anti-CTGF (D) antibodies. Histogram showing average relative protein levels normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (C and E) (* P<0.05).

Fig 3: Glucocorticoids activate YAP via FAK/Src-dependent actin cytoskeleton remodelling.(a) MDA-MB-231 cells were grown in serum-free medium and treated with betamethasone (BM) 1 μM alone or in combination with RU486 1 μM for 24 h. Representative images of immunofluorescence are shown. Experiment repeated three times. Scale bars, 15 μm. (b) MDA-MB-231 cells were treated as in a. Representative blots are shown. Experiment repeated three times. (c) Quantification of MDA-MB-231 cells with nuclear YAP by immunofluorescence. Cells were grown in serum-free medium in presence of BM 1 μM alone or in combination with dasatinib (DAS) 0.1 μM or saracatinib (SAR) 0.1 μM or PF573228 (PF) 5 μM for 24 h. Error bars represent mean±s.d., from n=3 biological replicates. (d) MDA-MB-231 cells were grown in serum-free medium in presence of BM 1 μM alone or in combination with DAS 0.1 μM or PF 5 μM for 24 h. Representative blots are shown. Experiment repeated three times. (e) MDA-MB-231 cells were trypsinized and then maintained in suspension and treated with BM 1 μM for indicated times. Representative blots are shown. (f) Quantification of MDA-MB-231 cells with nuclear YAP by immunofluorescence. Cells were transfected with control siRNA (siCTL) or a combination of siRNA targeting LATS1 and 2 (siLATS1/2). The day after cells were grown in serum-free medium in presence of BM 1 μM alone or in combination with DAS 0.1 μM for 24 h. Error bars represent mean±s.d., from n=3 biological replicates. (g) Representative images of immunofluorescence. MDA-MB-231 cells were grown in serum-free medium and treated with BM 1 μM alone or in combination with RU486 1 μM for 24 h. Experiment repeated three times. Scale bars, 15 μm. (h) Quantification of MDA-MB-231 cells with nuclear YAP by immunofluorescence. Cells were grown in serum-free medium in presence of BM 1 μM alone or in combination with latrunculin A (lat.A) 0.5 μM for 24 h. Error bars represent mean±s.d., from n=3 biological replicates. (i) Quantitative PCR with reverse transcription analysis of MDA-MB-231 cells grown in serum-free medium in presence of BM 1 μM alone or in combination with lat.A 0.5 μM for 24 h. Error bars represent mean±s.d., from n=3 biological replicates. (j) MDA-MB-231 cells were treated as in g. Representative blots are shown. Experiment repeated three times. *P<0.05, **P<0.01; two-tailed Student's t-test is used throughout.

Fig 4: Expression of TRIM65 is negatively correlated with LATS1. (a) Other proteins were differentially expressed after sh-TRIM65 expression by LC-MS. (b) Correlation expression of TRIM65 with LATS1 and LATS2 based on StarBase database. (c) Knockdown of TRIM65 increased expression of LATS1 but not LATS2 in MDA-MB-231 and MDA-MB-453 cell lines. p < 0.05, p < 0.01, and p < 0.001 were considered statistically significant.

Fig 5: TRIM65 promotes the malignant biological behavior by degrading the expression of LATS1 in TNBC cells. (a, b) Cell proliferation was assessed by the CCK-8 assay and colony-forming assay in MDA-MB-231 and MDA-MB-453 cells. (c) Representative images and analysis of cell invasion and migration assays in MDA-MB-231 and MDA-MB-453 cells. (d) Scientific hypothesis diagram. p < 0.05, p < 0.01, and p < 0.001 were considered statistically significant. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.