Fig 2: Conditioned medium and exosomes of platelets promoted Lewis lung carcinoma (LLC) cell survival. Blood was withdrawn from C57BL/6 mice and subjected to platelet isolation. (A) The isolated platelets were subjected to flowcytometric analysis for the measurement of CD41 and CD61 positivity. (B) Platelets and medium alone were added onto the Transwell inserts, and LLC cells were grown at the lower wells of Transwell apparatus for 24 h. Cell viability of LLC cells was measured by the MTS reduction assay. Platelets were treated with vehicle, dipyridamole (10 µM), or aspirin (100 µg/mL) for 24 h. Equal amounts of cultured media were subjected to exosome isolation. (C) The obtained exosomes were subjected to Western blot analysis with indicated antibodies. Representative blots of four independent experiments are shown. (D) Various concentrations of exosomes based on protein contents were added to LLC cells (96-well) for 24 h. Cell viability was measured by the MTS reduction assay. (E) The cultured media were harvested and centrifuged, and the resultant supernatants were named platelet-conditioned medium (CM). Equal amounts of CM and fresh medium were mixed and added to LLC cells (96-well) in the absence or presence of doxorubicin (0.5 µM) for 24 h. Cell viability was measured by the MTS reduction assay. (F) LLC cells were treated with doxorubicin (0 and 1 µM) in the absence or presence of the obtained exosomes (10 µg/mL) for 24 h. Cell viability was measured by the MTS reduction assay. (G) LLC cells were treated with doxorubicin (0 and 1 µM) in the absence or presence of HMGB1 (1 µg/mL) for 24 h. Cell viability was measured by the MTS reduction assay. (H) Cell migration of LLC cells in the absence or presence of HMGB1 (1 µg/mL) was evaluated using Transwell migration assay for 24 h. * p < 0.05 vs. untreated group, # p < 0.05 vs. exosome (vehicle, 10 µg/mL)/doxorubicin, and % p < 0.05 vs. doxorubicin (CM or Exosome), n = 4.

Fig 3: Lrp5 expression (acquired post-transcriptionally) correlates with the appearance of Axin2 in luminal cells.(A) mRNA extracted from purified cell fractions was analyzed for relative expression of Lrp5 by quantitative RT-PCR analysis as described for Fig. 1. (Lrp6 mRNA expression is broadly similar to Lrp5, shown in Fig. S4) (B) Tumor cells were incubated with anti-Lrp5 antibody, and analyzed by flow cytometry, using an isotype-matched negative control to define Lrp5+ cells. These samples were also stained with EpCAM and CD49f antibodies, and the cell surface phenotypes combined to show the luminal (L) or basal (B) cell identity of Lrp5+ and Lrp5- cells. (C) Another example of this assay is shown in histogram form, to reveal the overall level of expression for Lrp5 in luminal and basal cells in normal and tumor cells. (D) Paraffin sections from normal and Wnt-induced mammary glands were stained to illustrate the relative rates of division (green, Ki67) of basal (blue, K5) and luminal (red, K8) cells. Note that the green color stain in the lumens is a common artifact associated with non-specific binding to sticky luminal proteins. Scale bar = 50 µm. (E) Axin2lacZ [MMTV-Wnt1] (and control Wnt1) glands were stained in whole mounts for the lacZ reporter, followed by embedding, sectioning and immunocytochemical assay of basal (K5+) and luminal (K8+) cells. The panel on the LHS shows there was no background when the Axin2lacZ reporter was not present. The samples were incubated in x-gal substrate (B, basal; L, luminal; as indicated). The pattern of staining of AxinlacZ was heterogeneous, in some areas, stain was basal-specific, in others, there was also light staining throughout the luminal population, in others, focalized clusters of luminal cells showed high expression. (F) Lrp5+ cells were assayed for their expression of CD61, because CD61+ cells have been shown to be enriched for TIC activity in this tumor model (Vaillant et al 2008). These markers showed high co-expression.

Fig 4: Aspirin impairs the platelet–leukocyte aggregation induced by a high-salt (HS) diet in Dahl salt-sensitive (DSS) rats. A and B, Peripheral blood lymphocytes enriched from fresh blood in DSS rats fed a normal-salt (NS; 0.4%, n=8) diet, an HS (8%, n=10) diet, or HS with aspirin gavage (HS+ASA; 10 mg/kg per day, n=10) were analyzed by flow cytometry using the leucocyte surface marker CD45+. Values are expressed as mean±SEM, 1-way ANOVA, Tukey test, DSS rat: *P=0.006 vs NS, # P=0.037 vs HS, salt-resistant rats (SS13BN): # P=0.041 vs HS, HS+ASA. C and D, CD45+CD61+/ALL indicates the ratio of leukocytes aggregated to platelets. Values are expressed as mean±SEM, 1-way ANOVA, Tukey test, *P=0.004 vs NS. # P=0.031 vs HS. E and F, Percentages of CD11b+CD61+ and CD3+CD61+ gate were circled within the CD45+CD61+gate; CD4+CD61+ gate was obtained from the CD3+CD61+ gate; CD25+CD61+ was obtained from the CD4+CD61+ gate. Values are expressed as mean±SEM, 1-way ANOVA, Tukey test, CD11b+CD61+/ALL: *P=0.006 vs NS. # P=0.029 vs HS, CD3e+CD61+/ALL: *P=0.006 vs NS. # P=0.031 vs HS, CD4+CD61+/ALL: *P=0.004 vs NS, # P=0.034 vs HS, CD25+CD61+: *P=0.009 vs NS, # P=0.043 vs HS. G, Scanning electron microscopy of aggregated platelets and leukocytes (×4000, scan bar 5 µm).