Skip to main content
. 2017 Jan 10;8:13989. doi: 10.1038/ncomms13989

Figure 5. Fibroblasts secrete matrix proteins that enhance epithelial cell survival under starvation.

Figure 5

(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.