Fig 2: Treatment of starved mammary epithelial cells with laminin results in mTORC1 activation.(a,b) Confocal images (a) and quantification of immunofluorescence (b) of p-S6 (S240/244, red) in 24-h starved MCF10A cells, or starved/non-starved MCF10A cells treated with laminin (2.5 μg ml−1, 1 h); nuclei were counterstained with DAPI (blue); scale bar, 50 μm. Fluorescence intensity in ∼300 cells per condition was quantified by ImageJ; one-way analysis of variance (ANOVA), Tukey test, ***P<0.001. (c) Levels of total proteins and S6K1 phosphorylation (T389) in lysates of 24-h starved MCF10A cells treated with laminin (2.5 μg ml−1) for the indicated times. (d,e) Confocal images (d) of MCF10A cells treated as in a, showing localization by immunofluorescent staining, of ITGB4, LAMP1 (green), mTOR (red) and overlay of LAMP1 and mTOR; scale bar, 5 μm; co-localization (e) was analysed and quantified from confocal sections in ∼50 cells per condition using ImageJ; one-way ANOVA, Tukey test, *P=0.05; **P<0.01. (f) Levels of total proteins and phosphorylated S6 (S240/244) in lysates of MCF10A cells treated as in a. Where indicated, the cells were first incubated for 10 min with concanamycin A (8 μM), followed by laminin/concanamycin A co-incubation for 1 h and then lysed. DMSO was used as a vehicle control. (g) Levels of total proteins and phosphorylated S6K1 (T389) in lysates of MCF10A cells treated as in a. Where indicated, the cells were treated for 30 min with the dynamin inhibitor Iminodyn-22 (2 μM), followed by laminin/Iminodyn-22 co-incubation for 1 h. (h) Top, confocal images of 24-h-starved MCF10A cells treated with iminodyn-22 for 30 min, before addition of Alexa-647-laminin (red, 2.5 μg ml−1, 1 h). The cells were fixed and immunostained for ITGB4 (green) and nuclei counterstained with DAPI (blue). Bottom, three-dimensional projection of cells in the top panel imaged with confocal sectioning, that shows accumulation of extracellular laminin post-iminodyn treatment; scale bar, 10 μm. (i,j) Confocal images of 24-h starved MCF10A cells incubated with ITGB4-blocking antibody for 3 h, before laminin addition (2.5 μg ml−1, 1 h). The cells were immunostained for phosphorylated S6 (S240/244; i) and fluorescence intensity-quantified (j) as described in b; scale bar, 100 μm; Student's t-test, ***P<0.001. In b,e,j, data represent the mean±s.e.m.

Fig 3: miR-302d-3p/ITGB4 axis modulates LPS-treated CHON-001 cells viability and apoptosis. (A,B) CCK-8 assay was utilized to evaluate cell viability of LPS-treated CHON-001 cells. *p < 0.05, # p < 0.05, & p < 0.05. (C,D) Flow cytometry assay was used to measure the apoptosis rate of each group. **p < 0.01, ## p < 0.01, && p < 0.01. Results are the average of three individual experiments in triplicate with error bars indicating mean ± SD (ANOVA followed by Tukey’s post hoc test).

Fig 4: Fibroblasts secrete matrix proteins that enhance epithelial cell survival under starvation.(a) Levels of matrix proteins in total cell lysates and TCA-precipitated secreted proteins in the media of primary human fibroblasts or MCF10A cells starved for 7 days. Loading was normalized to cell number. (b) Fold proliferation of MCF10A cells that were first starved for 24 h and then treated daily with non-conditioned, or conditioned medium from starved primary human fibroblasts for 12 days; n=3; Student's t-test, *P<0.05. (c) Phase contrast image of MCF10A cells treated as in b for 48 h; scale bar, 100 µm. (d) Levels of total proteins, phosphorylated S6K1 (T389) and AKT (S473) in lysates from 24-h starved MCF10A cells treated for 1 h with conditioned or non-conditioned media of starved (S) or non-starved (NS) fibroblasts. (e) Levels of proteins as in d and phosphorylated S6 (S235/236) from MCF10A cells treated with siRNAs for GFP, ITGB1 or ITGB4, starved for 24 h, and then incubated with conditioned (C) or non-conditioned (NC) starved fibroblast media. (f) Fold proliferation of MCF10A cells with stable knockdown of GFP, ITGB1 or ITGB4. The cells were starved for 24 h, and then fed conditioned starved fibroblast medium every 24 h for 8 days; n=3; one-way ANOVA, Tukey test, *P<0.05; **P=0.01; ***P<0.001. (g) Total laminin levels in TCA-precipitated proteins in the media of 24-h starved primary human fibroblasts, before or following laminin depletion using a laminin antibody affinity column. Loading was normalized to cell number. (h) Levels of proteins as in e in lysates from 24-h starved MCF10A cells treated for 1 h with NC, C or conditioned laminin-depleted (C-Lam)-starved fibroblast media. (i) Fold change in cell number of 24-h starved MCF10A cells fed daily with fibroblast media as in h for 10 days; n=3; one-way ANOVA, Tukey test, **P<0.01. (j) Levels of proteins in MCF10A cells treated as in h, or treated with laminin-5 (2.5 µg, C-lam+lam) for 1 h after culture for 24 h in laminin-depleted conditioned medium. In b,f,i, each value represents the mean±s.e.m.

Fig 5: Starved mammary epithelial cells induce ß4-integrin expression and internalize laminin.(a) Levels of total proteins and S6 phosphorylation (S235/236) in lysates from mouse mammary glands of AL and DR mice (n=4). (b) Levels of total proteins, phosphorylated S6K1 (T389), S6 (S235/236) and AKT (S473) in lysates from non-starved (NS), 24-h starved (S), confluent (confl.) or subconfluent (sub-confl.) MCF10A cells; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (c,d) Confocal images of immunofluorescently stained ITGB4 in non-starved or starved MCF10A cells (c), with line scan quantification (d) of integrin localization from cell edge towards cell interior, representing the fluorescence intensity average (38 cells per condition); P values were measured by Student's t-test along the length of the line scan (0.017), or at the cell edge, indicated by the arrow (0.023); scale bar, 30 µm. (e,f) Confocal images (e) and quantification of fluorescence intensity (f) of laminin-Alexa-647 (red) added (2.5 µg ml-1, 30 min) to non-starved or starved MCF10A cells. The cells were immunostained for ITGB4 (green), and the nuclei counterstained with DAPI (blue); intracellular laminin fluorescence intensity was measured in 28 cells per condition using the confocal mid-sections by ImageJ; Student's t-test, **P<0.01; scale bar, 30 µm. (g) Top, laminin internalization (intern.) assay in starved MCF10A cells demonstrating increased uptake of biotinylated laminin at the 15 min (compared with 0 min) time point. Total surface bitotinylation is shown as positive control. The cells were lysed and probed for biotin by western blot (wb) after laminin immunoprecipitation (ip). The blot was then stripped and re-probed for total laminin as a loading control; bottom, quantification of biotinylated laminin normalized to total immunoprecipitated laminin. (h) Confocal images of non-starved or 24-h starved MCF10A cells that were fed laminin (2.5 µg ml-1, 1 h). The cells were stained for ITGB4 (green), laminin (red) or LAMP1 (yellow). z, x and y planes are shown from images reconstructed from serial sections obtained throughout whole cells and demonstrate intracellular staining; scale bar, 20 µm. (i,j) Co-localization of laminin and LAMP1 (i) or ITGB4 and laminin in LAMP1-positive regions (j) as analysed in 150 cells per condition, using ImageJ; Student's t-test, *P<0.05 and ***P<0.001. Data in (d,f,i,j) represent the mean±s.e.m.