Figure 1
Reducing C/EBPδ expression by C/EBPδ siRNA treatment results in defective HC11 mammary epithelial cell growth arrest. (A) C/EBPδ mRNA levels were assessed in parental (nontransfected) HC11 cells ("-"), empty vector (pSilencer ™ 2.1) transfected HC11 controls ("vector") and C/EBPδ siRNA transfected ("siRNA") HC11 cells growth arrested by serum and growth factor withdrawal. Northern blots were probed with 32P-labelled C/EBPδ and cyclophilin (CP, loading control) cDNA probes. (B) C/EBPδ protein levels were assessed in parental (nontransfected) HC11 cells (-), empty vector transfected HC11 controls (vector) and C/EBPδ siRNA transfected (siRNA) HC11 cells growth arrested by serum and growth factor withdrawal. Western blots were probed with primary antibodies against C/EBPδ and β-actin (loading control). (C) Serum and growth factor withdrawal induced growth arrest assessed by BrdU labeling (brown). HC11 cells were cultured under exponentially growing (first panel) and 48 hr growth arrested (second panel) conditions. Cells were counterstained with hematoxylin (blue). (D) Contact inhibition (growth arrest) assessed by [3H]-thymidine incorporation. HC11 nontransfected control, HC11 vector control and HC11 C/EBPδ siRNA transfected cells were grown to ~80% confluence (0 hr) in complete growth media (CGM) and then maintained in CGM. [3H]-thymidine incorporation (cpm) was assessed at 0, 24 48 and 72 hours. (E) Contact inhibition (growth arrest) assessed by BrdU incorporation (brown). Cells were treated as described in "D" above. BrdU incorporation was assessed at the designated time points. (F) Contact inhibition (growth arrest) induction of C/EBPδ mRNA levels. Total RNA was isolated at the designated time points and Northern blots were probed with 32P-labelled C/EBPδ and cyclophilin (CP, loading control) cDNA probes. (G) Whole cell proteins were isolated at designated time points and Western blots were probed with primary antibodies against STAT3, pSTAT3, C/EBPδ, cyclin D1 and β-actin (loading control). * = significant different from C/EBPδ siRNA treated group at 72 hours at p < 0.05.