Fig 2: KO/PyMT tumour cells have reduced cell-autonomous proliferative activity and Ras/Raf/ERK signaling compared with controls.Representative Ki-67 immunohistochemical staining (white scale bars, 50 µm) (a,c) and quantification of staining (b,d) are shown for WT/PyMT and KO/PyMT tumour sections (a,b), and for sections of tumours resulting from injection of WT/PyMT (WT) or KO,PyMT cells into WT or Col5a3-/- mice (c,d). Quantification was of six fields per tumour on sections from three different WT/PyMT and three different KO/PyMT tumours, and from three different tumours each from injection of WT/PyMT or KO,PyMT cells into WT or Col5a3-/- mice (WT?WT and KO?WT, respectively). (e) Cell proliferation levels of primary KO/PyMT and WT/PyMT tumour cells were assessed via colorimetric MTT assay. Values on the ordinate axis represent OD at 570 nm, given an arbitrary value of 1 at day 0, and then reflecting fold changes on subsequent days. (f) 3H-thymidine incorporation is compared for primary WT/PyMT and KO/PyMT tumour cells. All cell proliferation assays were performed in triplicate. Data are presented as mean±s.d. P values: **<0.01, ***<0.005. Statistical analysis was via two-tailed Student's t-test, with differences considered significant at P<0.05. (g) Representative immunoblots are shown of lysates of WT/PyMT (WT) and KO/PyMT (KO) primary tumour cells and tumours, and of normal C57BL/6 (WT) or Col5a3-/- mammary gland tissue. Blots were probed with antibodies to ß-catenin, phospho-Raf-1 (p-Raf-1), Raf-1, Ras, phospho-Erk1/2 (p-Erk1/2), Erk, phospho-FGFR2, FGFR2 or a-tubulin, the latter as a loading control. (h) Lysates were subjected to pull-downs with Raf-1 RBD domain to obtain activated Ras (GTP-Ras), which was then detected by immunoblot, using antibody to Ras. An immunoblot displaying total Ras levels in the same samples is also shown. Immunoblots were repeated three times, from three independent tumour or cell lysates. Representative blots are shown. Quantification of results for significance of differences is shown in Supplementary Fig. 3.

Fig 3: Ablation of a3(V), found at high levels juxtaposed to mammary ducts, yields decreased tumour size and increased host survival.(a) Representative immunofluorescence staining shows a3(V) chains (top panels, red); a-smooth muscle actin (aSM actin, top panels, green), which marks ductal myoepithelial cells; perilipin (adipocyte marker); and col(V) (bottom panels, green). Blue; DAPI staining. Representative immunofluorescence staining shows (b) co-localization of a3(V) (red) with marker K14 (green) in basal cells, and (c) lack of co-localization of a3(V) (green) with marker K8 (red) in luminal cells, of mammary ducts. Arrowheads and arrows denote luminal and basal cells, respectively. Col5a3 ablation Kaplan–Meier plots show significantly increased survival (P<0.0001, survival in groups was compared using the Log-Rank test) in KO/PyMT mice lacking a functional Col5a3 gene (d). (e) Volume of tumour burden is increasingly reduced, relative to WT/PyMT controls, in KO/PyMT mice at times after initial tumour appearance. All palpable tumours were measured for volume calculations. All WT/PyMT mice were killed by 8 weeks after initial tumour appearance, because of tumour burdens ?3,000 mm3. (d,e) WT/PyMT n=34, KO/PyMT n=23. Data are presented as mean±s.d. P values: *<0.05, ***<0.005. Statistical analysis was via two-tailed Student's t-test, with differences considered significant at P<0.05. Representative immunofluorescent staining shows co-localization of a3(V) (red) with marker K8 (green) (f) in the tumour cells of WT/PyMT tumours, and the lack of co-localization with marker K14 (green) in the same tumours, except in basal cells of untransformed ducts (g). White scale bars, 50 µm. In f, arrowhead and arrow denote luminal and basal cells, respectively, in an untransformed duct. In g, an arrow denotes co-localization of a3(V) and K14 only in the basal cells of an untransformed duct.

Fig 4: Non-cell-autonomous and cell-autonomous effects of Col5a3 ablation on survivability and tumour volume.WT/PyMT primary tumour cells were injected into wild type (WT?WT) or Col5a3-/- (WT?KO) mice (n=8 mice per each group), which were then characterized for survival time (a) and tumour volume (b) at time points post tumour cell injection. KO/PyMT primary tumour cells were injected into WT (KO?WT) or Col5a3-/- (KO?KO) mice, which were then characterized for survival time (c) and tumour volume (d) at time points post tumour cell injection. Injected WT/PyMT cell curves from a,b were superimposed on the injected KO/PyMT cell curves in c,d to facilitate comparison of survival ratios and tumour volumes. Survival analyses were performed using the Kaplan–Meier method. For tumour volume curves, data are presented as mean±s.d. P value: ***<0.005. Statistical analysis was via two-tailed Student's t-test, with differences considered significant at P<0.05.