Fig 2: Deletion of miR-200c/141 cluster increases mammary tumor lung metastasis and induces EMT. (A) Images of lungs from the same cohort of mice as shown in Figure 1B. Red arrows represent metastatic nodules in lungs. (B) HE staining of lung sections from (A) and the numbers of metastatic lesions per lung section were counted. ***p < 0.001. (C) The representative images for Ki67 IHC staining in lung sections from WT or cKO mice and quantification of Ki67 positive cells. ***p < 0.001. (D) FACS was performed to detect the effect of miR-200c/141 cluster on breast cancer stem cells (BCSCs) with CD24/CD29 and ALDEFLUOR assay. (E) E-cadherin (E-cad) and Vimentin (Vim) expression was verified by Immuno- fluorescence staining in tumor sections with indicated genotypes. (F) E-cadherin (E-cad), Vimentin (Vim) and Zeb1 protein immunoblotting in freshly lytic tumors and primary cells from the mice with indicated genotypes.

Fig 3: Inhibition of miR-200c and miR-141 results in reduced cell proliferation and increased cell invasion in vitro by regulating BCSC plasticity. SUM149 and T47D cells were treated with 200nM miR-200c and miR-141 inhibitors (miR200c- 141inh) or negative control inhibitor (NC) for 48h. (A) Treated SUM149 or T47D cells were accessed for proliferation ability which was performed with MTT assay as described in methods. **p<0.01. (B-C) The plate colony formation assay was carried out with 500 treated SUM149 (B) or T47D(C) colonies were counted in the whole field for statistics. **p<0.01. (D-E) The invasive ability of treated SUM149 (D) or T47D (E) cells was investigated with the transwell assay according to the manufacturer's protocol. Quantitative analysis was accessed for the total invasive cells from three independent experiments. *p<0.05, **p<0.01. (F-G) Flow cytometry analysis of BCSCs was carried out for the CD24/CD44 and ALDEFLUOR assay in treated SUM149 (F) and T47D (G) cells. (H) After being treated for 48 hours, cells were harvested to detect the protein expression levels of E-cadherin (E-cad), Snail, Vimentin (VIM) and ZEB1 by Western Blotting.

Fig 4: Relationship between LMNA and the EMT-related gene between HCC827 and HCC827/ER cells. (A) Heatmap illustrating the expression of genes related to LMNA and EMT between HCC827 and HCC827/ER cells. (B) An interaction network using the STRING online database and Cytoscape software for 22 common gene signatures involved in resistance to erlotinib and EMT mechanisms. (C) A total of 10 genes were identified as hub genes from 22 candidate genes using the CytoHubba tool in Cytoscape: LMNA, EGFR, PTEN, CDH1, CDH2, VIM, SNAI2, TWIST1, AKT1, and AKT2. (D) Relative expression levels of genes related to LMNA and EMT in HCC827 and HCC827/ER cells were confirmed by qPCR. Data are presented as mean ± SD. ** p< 0.001, *** p < 0.001. (E) The expression of laminA/C, laminB1, E-cadherin, N-cadherin, vimentin, and snail was analyzed by Western blot in HCC827 and HCC827/ER cells. GAPDH was used as a loading control.

Fig 5: Twist1 is also expressed and can modulate the outcome of WIHN in spiny mice.a Gene expression heatmap of spiny mouse epidermis at different post-wound days. b Proliferation of epithelial cells and cell movement associated genes in the spiny mouse wound epidermis. c Twist1 related transcription factors in the spiny mouse wound dermis. d ECM remodeling in the spiny mouse wound dermis. e IHC of Twist1, Zeb2, MMP9, E-cad and P-cad in PWD14 the regenerating hair placodes of the spiny mice. White arrows point to the downregulation of E-cad at the tip of hair placode. f The effects of Twist1 inhibitor Harmine and pan-MMP inhibitor GM6001 treatment on hair neogenesis in spiny mouse. Red: wound border; yellow arrow: hair follicles. Observed on PWD35. g Dot graph of the number of regenerated hair follicles (HF) observed in spiny mouse wounds under different treatments. n = 6 biologically independent animals. Data are presented as mean values ± SEM. *p = 0.0265; **p = 0.0027; unpaired two-sided t-test. Ctl: control. The images in f represent six out of six experiments performed. The images in e represent 4 out of 4 experiments performed.