Fig 2: Silencing TLN1 reduces triple-negative breast cancer (TNBC) cell motility by blocking epithelial-mesenchymal transformation (EMT).(A) TLN1 silencing decreased the adhesion of MDA-MB-231 cells (n = 3, p < 0.01). (B) Transwell assay was used to evaluate the migration and invasion of MDA-MB-231/NC and shTLN1 cells (n = 3, p < 0.01; scale bar, 100 µm). (C and D) The relative expression level of CK18, E-cadherin, N-cadherin, and vimentin relative to those of GAPDH in MDA-MB-231/NC and shTLN1 cells, using western blotting (n = 3, p < 0.01, respectively). (E) Haematoxylin and eosin (H&E) staining of mitigated lung metastasis derived from MDA-MB-231/NC and shTLN1 tumours in NOD/SCID mice (n = 5–8 per group; scale bar, 50 µm). (F) Western blot of integrin ß1 overexpression in MDA-MB-231/shTLN1 cells. (G) Transwell analysis of invasion and migration following integrin ß1 overexpression. (H) Adhesion assay after integrin ß1 overexpression. Data are representative images or expressed as the mean ± standard error of the mean (SEM) of each group from three separate experiments for in vitro and 5–8 per group for in vivo studies. *p < 0.05, **p < 0.01 vs. the control group. Figure 3—source data 1.Silencing TLN1 reduces TNBC cell motility by blocking EMT.

Fig 3: Hypoxia increases the expression of HIF-1a and ZEB1 in T24-P cells, and promotes the process of epithelial-mesenchymal transition. Induced hypoxia increased the expression of (A) HIF-1a and (B) ZEB1 in T24-P cells. (C) Western blotting revealed that HIF-1a, ZEB1, N-cadherin and vimentin were upregulated when T24-P cells were grown in hypoxic conditions, but that CK-18 was downregulated. ***P<0.001. HIF-1a, hypoxia inducible factor-1a; ZEB1, zinc-finger E-box-binding homeobox 1; T24-P, T24 parental cells; CK-18, cytokeratin-18.

Fig 4: SOX11 overexpression inhibits epidermal differentiation. a Constructs used to generate transgenic mice expressing epithelial-specific Sox11 under the control of tetracycline and its derivatives. b Immunofluorescence analysis of expression of FLAG-tagged SOX11. Four-day old K14-rtTA;TRE-Sox11-FLAG mice were injected intraperitoneally with Dox for the indicated time prior to skin isolation. Dashed line demarks the border between the epidermis and dermis. c Images of embryos of specified genotypes in X-gal exclusion assay. The pregnant females were on Dox-containing diet until the embryos were collected at E18.5. d Immunofluorescence analysis of differentiation markers in postnatal epidermis expressing SOX11. After birth, the mother was on Dox-containing diet for 5 days and skin sections from K14-rtTA (control) and K14-rtTA;TRE-Sox11-FLAG pups were immunostained with the indicated antibodies. KRT5 (keratin5); KRT1 (keratin 1); LOR (loricrin); FLG (filaggrin). e Immunochemical analysis of embryonic makers in control and SOX11-FLAG expressing epidermis with the indicated antibodies. KRT18 (keratin 18); TCF7L1 (transcription factor 7 like 1); TCF7L2 (transcription factor 7 like 2). (f–j) Effect of long-term SOX11 overexpression in postnatal skin. Dorsal skins from K14-rtTA (control) and K14-rtTA;TRE-Sox11-FLAG newborns were grafted in pair onto Nude mice, which were put on a diet containing Dox day 10 post grafting. n = 2. f Image of engrafted mouse 28-day post grafting with engrafted area demarcated by yellow dashed lines. g H&E staining of the sectioned pair of skins showing stunted hair follicles (*) in SOX11-induced skin. f Oil red O staining marks lipids, enriched in sebaceous glands. Immunofluorescence analysis of differentiation markers LOR (i) and FIL (j) in grafted skin. White dashed lines mark the border between epidermis and dermis. HF, hair follicles; Es, eschar; SG, sebaceous glands. Scale bars, 50 µm (b, e) 20 µm (d, i–j), 100 µm (g, h). All images reflect the same result seen across the entire embedded skin section from 2 (b, c and f–j) or 3 (d and e) biological replicates

Fig 5: Optimization of the CFC Assay(A) Schematic showing the experimental design used to optimize conditions to detect CFCs. Single cells obtained by enzymatic dissociation of normal tonsil specimens were cultured with or without irradiated fibroblasts (i3T3) or ROCK inhibitor (ROCKi), in either 20% or 5% O2, and colonies were scored in dishes fixed 7–9 days (d) later.(B) Enhancing effects of the use of feeders, a ROCK inhibitor, and a reduced O2 level on colony growth, calculated by comparing the increased numbers of colonies obtained from a given number of cells under each test condition relative to the control condition (no added i3T3 cells or ROCKi). Values shown are the mean ± SEM, derived from ten experiments.(C) Demonstration of a linear relationship between the number of input cells plated per well and colony yields under optimized CFC assay conditions. Values shown are the mean ± SEM, derived from three experiments.(D) IHC characterization of the different types of colonies produced. Holoclones expressed high levels of CK19 and p63 (markers of basal cells) but infrequently or rarely expressed involucrin, CK8/18, and CK13 (markers of differentiated tonsillar epithelial cells). Many of the cells in mero/paraclones expressed differentiation markers but rarely expressed any basal markers. Scale bars, 1 mm.