Related Article from The Origins and Implications of Intratumor Heterogeneity

Related Article from The Origins and Implications of Intratumor Heterogeneit

Psoriasin and CD24 demonstrate a similar staining pattern in DCIS.

<p>The expression patterns of psoriasin and CD24 in ductal carcinoma <i>in situ</i> (DCIS) were analyzed by immunohistochemisty. Psoriasin and CD24 showed an extremely intense staining and a similar staining pattern in DCIS. <b>A</b> Psoriasin staining was observed in the cytoplasm and the nucleus of the cells. <b>B</b> CD24 staining was observed in the cytoplasm, the nucleus and in the membranes of the mammary epithelial cells. The figures illustrate representative examples of psoriasin and CD24 expression in the same DCIS tumor.</p

Confluence- and suspension-cultured mammary epithelial cells demonstrate increased psoriasin and CD24 expression.

<p>MCF10A cells were cultured in confluence for 5 and 10 days or in suspension for 3 days. <b>A</b> Psoriasin expression, analyzed by Western blotting, was induced in MCF10A cells cultured in confluence and suspension, compared with exponentially growing cells. Equal loading was confirmed by GAPDH. The figure illustrates a representative example (n = 3). <b>B</b> In confluence- and suspension-cultured cells, psoriasin and CD24 expression were increased, whereas CD44 expression was decreased compared with exponentially growing cells. The expression level of CD24 and CD44 was measured using flow cytometry and the expression level of psoriasin was quantified from Western blots. The data are presented as the mean ± SD of relative expression (n = 3). The p-values (*<0.05, **<0.01, ***<0.001) were calculated using the Student's t-test.</p

Psoriasin and MUC1 expression is elevated in CD24⁺ mammary epithelial cells.

<p>MCF10A cells were cultured in confluence for 10 days and separated for CD24⁺ cells, using magnetic activated cell sorting. <b>A</b> Psoriasin expression, analyzed by Western blotting, was confined to the CD24⁺ cell fraction, compared with negative selection and controls. Equal loading was confirmed by GAPDH. The figure illustrates a representative example (n = 3). <b>B</b> Separated CD24⁺ cells showed an increase in the expression of CD24 and a decrease in the expression of CD44, compared with negative selection. The expression of MUC1 was increased in the same level as CD24 expression. The figures illustrate representative examples (n = 4).</p

Endogenous psoriasin causes increased CD24 expression.

<p>MCF10A cells were transfected with a psoriasin-targeting shRNA (Pso-shRNA) or siRNA (Pso-siRNA), and their corresponding controls,(C-shRNA and C-siRNA). Transfected MCF10A cells were cultured in confluence for 10 days or in suspension for 3 days. The expression level of psoriasin was detected by Western blot. CD24 and CD44 expression were measured using flow cytometry. <b>A</b> No induction of psoriasin expression was observed in Pso-shRNA, compared with C-shRNA, in confluence or suspension. <b>B</b> Pso-shRNA showed a decrease in the expression of CD24 and an increase in the expression of CD44, compared with C-shRNA, during confluence and suspension. <b>C</b> The expression of psoriasin in Pso-siRNA was dramatically downregulated, compared with C-siRNA. <b>D</b> Pso-siRNA in suspension culture showed a reduced CD24 expression, compared with C-siRNA. Equal loading was confirmed by GAPDH. The figures illustrate representative examples (n = 3). The data are presented as the mean ± SD of relative expression (n = 3). The p-values (*<0.05) were calculated using the Student's t-test.</p

Inhibition of ROS and the NF-κB signaling pathway suppresses psoriasin and CD24 expression.

<p>MCF10A cells were cultured in confluence for 10 days. The inhibition of ROS using NAC (<b>A</b>) and NF-κB using CAPE (<b>B</b>) and dnIKKB (<b>C</b>) led to a significantly reduction in CD24 and psoriasin expression in confluence-cultured MCF10A, measured by flow cytometry and Western blot, respectively. No reduction of CD24 expression was seen by the inhibition of PLC-IP3 using U73122 (<b>D</b>). The reduced CD24 expression by Tyrphostin (<b>E</b>) did not reach statistical significance. The inhibition of PI3-K by Wortmannin (<b>F</b>) showed no decrease in psoriasin or CD24 expression. The inhibitors showed no effect on CD44 expression, except for treatment with NAC (<b>A</b>), which increased the expression of CD44. Western blot inserts illustrate representative examples (n = 4) of the psoriasin expression. Equal loading was confirmed by GAPDH. The data are presented as the mean ± SD of relative expression (n = 4). The p-values (*<0.05, **<0.01, ***<0.001) were calculated using the Student's t-test.</p

S100A7 downregulation decreases osteolytic lesions <i>in vivo.</i>

<p>Cells were directly introduced into the tibias of 4 week-old mice, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001741#s4" target="_blank">Materials and Methods</a>. Micro-CT scanning of live mice was done using a GE eXplore Micro-CT scanner (GE Healthcare Ltd, UK) at 93 µm resolution, 6 weeks after injection. The tibias were dissected out and scout view radiographs were taken with a micro-CT40 scanner at 12 µm resolution obtained from SCANCO Medical AG (Switzerland). There were osteolytic lesions, destruction of the cortex and extension of the tumor into the soft tissue in the mice injected with the control S100A7 cells (Panels A1 and A2). The cortex of the mice injected with the S100A7-downregulated cells was intact (Panels B1 and B2). Tibias were excised and processed for conventional histological examination. S100A7 control mice showed tumor-induced osteolysis (Panel C1), and the osteolysis was evident in all three injected mice as compared to mice injected with the S100A7-downregulated cells (Panel C2) (H & E staining, 20×). The letter ‘T’ in panel C1 represents tumor. The experiments were repeated twice with identical results.</p

S100A7 downregulation decreases the phosphorylation of EGFR/HER2, Src and SHP2 in MDA-MB-468 cell lines.

<p>Vector control and S100A7-shRNA stably-transfected MDA-MB-468 cell lines were starved overnight and stimulated with various concentrations of EGF (0 to 100 ng/ml) for 30 minutes at 37°C. The cells were then lysed and the lysates were analyzed by Western blotting with p-EGFR Tyr1173/p-HER2 Tyr1248 (A, upper panel) or p-EGFR Tyr1173 (B, upper panel) antibodies. The protein levels were monitored by stripping the blots with anti-EGFR antibody (A and B, lower panels). Vector-transfected controls and S100A7 shRNA-transfected stable MDA-MB-468 cell lines were starved overnight for 17 hours and stimulated with various concentrations of EGF (0 to 100 ng/ml) for 15 minutes at 37°C. The cells were then lysed and the lysates were analyzed by Western blotting with p-Src Tyr416 (C, upper panel) or p-SHP2 Tyr542 antibody (D, upper panel). The protein levels were monitored by stripping the blots with total Src and SHP2 antibodies, as indicated (C and D, lower panels).</p

EGF stimulation enhances S100A7 expression in breast cancer cells.

<p>70–80% confluent MCF-7 cells (A) and MDA-MB-468 cells (B) were washed twice and rinsed with PBS followed by 18 hrs of serum starvation. The cells were then stimulated with various concentrations of epidermal growth factor (EGF) for 30 minutes at 37°C. The cells were lysed and S100A7 expression was analyzed by Western blotting with anti-S100A7 antibody, as indicated. Equal protein loading in each lane was checked by stripping the blots and probing with β-Actin antibody (A and B, lower panels). The experiments were repeated thrice with identical results.</p

Supplementary Data from Heterogeneity for Stem Cell–Related Markers According to Tumor Subtype and Histologic Stage in Breast Cancer

Supplementary Data from Heterogeneity for Stem Cell–Related Markers According to Tumor Subtype and Histologic Stage in Breast Cance