Figure 2. C/EBPδ promotes primary tumor growth and experimental metastasis in vivo.

(a) Tumor volumes of SUM159 cells with Dox-inducible shRNAs at the indicated times of treatment, started at 50mm³ (n=13, *P<0.05, **P<0.01, by exact logistic regression in the SAS logistic procedure). (b) Representative tumors from panel (a) at endpoint (day 14). Scale bar = 1 cm. (c) Western analysis of the indicated proteins in seven independent tumors per group as in panel (b). (d) SFE of cells dissociated from tumors as in panel (b), cultured for 5 days (n=5–7, **P<0.01, two-tailed unequal variance t-test). (e) Representative magnetic resonance images of mouse thoraxes 8 weeks after tail vein injection and quantification of the tumor load in lungs by ImageJ from those mice that showed lesions, i.e. 3/4 for shControl and 4/9 for shCEBPD. Data represent the mean ± S.E.M; n=3 shControl, n=4 shCEBPD, ***P<0.001, two-tailed unequal variance t-test. (f) Quantification of lung tumor load by MRI imaging before (Pre) and after four weeks (Post) of doxycycline treatment using two SUM159 cell lines with independent Doxinducible CEBPD-shRNAs or shControl (shControl, n=8; shCEBPD#5, n=5; shCEBPD#3, n=4). The indicated P values were generated with the Paired Wilcoxon rank test. Dox significantly reduced the lung tumor load in the combined shCEBPD mice compared to controls (P=0.03 by Wilcoxon rank test of log-fold changes in tumor load). (g) Tumor volume measurements of MCF-7 stable cells (shControl and shCEBPD) injected orthotopically into athymic nude mice. Data are from three independent sets of cells as described in Fig. S2i-j (Batch I, n=14; Batch II, n=4; Batch III; n=5; mean ±SEM; *P<0.05, ** P<0.01, *** P<0.001, by exact logistic regression in the SAS logistic procedure). (h) SFE of single cell suspensions from similar-sized MCF-7 xenograft tumors, cultured for 5 days (n=5, **P<0.01, two-tailed unequal variance ttest). (i) Western analysis of the indicated proteins in four independent tumors as in panel (g). (j) Tumor initiation by MCF-7 cells (3×10⁶ or 0.3×10⁶) with stable depletion of C/EBPδ (shCEBPD) or control cells (shControl) injected orthotopically. Data are from two experiments and indicate the presence of tumors from palpable stage at the indicated times after injection (n=9, *P<0.05, ***P<0.001, by exact logistic regression in the SAS logistic procedure).