Reduced β-catenin expression and activity in D-DT-deficient cells A, HT-29 cells were plated in 6-well plates for 24 hours followed by oligotransfection with 50 nM scrambled shRNA oligos or 50 nM D-DT-specific shRNA oligos for an additional 48 hours. Lysates were prepared and equal amounts of protein were analyzed by immunoblotting for COX-2, β-catenin and β-actin (loading control). B, 24 hours after transfection with Scr or D-DT shRNA, cells were co-transfected with the β-catenin/TCF reporter plasmids pTOP-flash/pRL-null (Renilla) alone or together with vector control (pCDNA3.1), wildtype (pCDNA3.1/β-catenin) or phosphorylation mutant β-catenin (pCDNA3.1/β-catenin^mut) expression constructs. 24 hrs later, Firefly and Renilla luciferase activities were measured by the Dual Luciferase Reporter Assay System. Results are expressed as relative light units (RLUs) after normalizing ratios of luciferase/Renilla luciferase from triplicate samples. Parallel transfections with the β-catenin/TCF non-responsive reporter control construct, pFOP-flash, revealed equally negligible RLUs in all cells regardless of D-DT or β-catenin status (not shown). C, Cells were treated as in A) and 24 hours after shRNA transfection, D-DT adenovirus (+ Ad-D-DT) was used to infect D-DT shRNA transfected cells and GFP adenovirus was used for the remainder of the cells (− Ad-D-DT) for an additional 48 hours. Lysates were prepared and equal amounts of protein were analyzed by Western blot for COX-2, β-catenin and D-DT. D, Cells were treated with 20 μg/ml cycloheximide for the indicated times 48 hours after transfection with Scr or D-DT shRNA (top panel) or 24 hours after infection with Ad-GFP (− Ad-D-DT) or Ad-D-DT (+ Ad-D-DT). At the indicated times following treatment with cycloheximide, cells were lysed and equal amounts of protein from each sample were analyzed by immunoblotting. All results are representative of at least 3 independent experiments.

JNK/c-Jun are necessary for COX-2 expression in CRC cells and D-DT contributes to JNK/c-Jun activation pathway A, Cells were oligo-transfected with Scr and D-DT shRNA oligos for 48 hours, lysed and immunoblotted for phospho-c-Jun, total c-jun and α-tubulin. B, Cells were infected with Ad-GFP or Ad-D-DT adenovirus for 24 hours and then treated with 40 μM SP600125 for an additional 6 hours. Cells were harvested, lysed and immunoblotted for phospho-c-Jun, COX-2 and β-actin. Densitometry of COX-2/β-actin is shown below. C, HT-29 cells were transfected with Scr or D-DT shRNA and 24 hours later cells were infected with Ad-GFP or constitutively active MKK7 adenovirus (Ad-CA-MKK7) as indicated. After 48 hours, cells were harvested, lysed and immunoblotted for phospho-c-Jun, COX-2, MKK7, D-DT and α-tubulin. Densitometries of COX-2 and tubulin were determined from two independent experiments and graphed as COX-2/Tubulin.

Proposed scheme of D-DT-mediated COX-2 expression in CRC cell lines.

Regulation of COX-2 expression by D-DT A, Human colorectal carcinoma cell lines, HCT116 and HT-29 were plated in 6-well plates for 24 hrs followed by oligotransfection of 50 nM scrambled, non-specific shRNA oligos, 50 nM D-DT-specific shRNA oligos or 50 nM MIF-specific shRNA oligos as described (19, 21). After 48 hours, lysates were prepared and equal amounts of protein were analyzed by immunoblotting for COX-2 and α-Tubulin (loading control). Scion Image was used for densitometry and COX-2/Tubulin densitometry values are shown. Data is representative of 4 independent experiments. B, HT-29 cells were transfected with a D-DT overexpression construct in a dose-dependent manner (Lane 1, 4 μg pCDNA3.1 vector alone; Lane 2, 1 μg pCDNA3.1/D-DT; Lane 3, 2 μg pCDNA3.1/D-DT; lane 4, 4 μg pCDNA3.1/D-DT. 36 hours later, equal concentrations of protein lysates were analyzed by immunoblotting for COX-2, D-DT, V5-epitope tag and β-actin. Data is representative of 2 independent experiments. C, Different concentrations of pCDNA3.1/D-DT expression construct (0 ug, 1 ug or 2 ug pCDNA3.1/D-DT and 2 μg, 1 μg or 0 μg pCDNA3.1 vector alone to control for plasmid concentration) were transfected into HT-29 cells. 48 hr later total RNA was isolated and reverse transcribed to cDNA. Real time PCR was used to determine the relative quantities of mRNA in the indicated samples. Data shown represents the ΔC_t of the average of duplicate reactions for each condition between target mRNA (D-DT and COX-2) and β-actin and is representative of two independent experiments.

D-DT-mediated β-catenin stabilization contributes to, but is not wholly responsible for, D-DT-dependent COX-2 expression A, Cells were plated in 6-well plates and were transfected the following day with pCDNA3.1/D-DT or pCDNA3.1/β-catenin expression constructs, alone or in combination. For all plasmids, one “+” correlates to 1 μg construct transfected into cells. The total amount of DNA transfected into cells was equalized by co-transfecting vector control, pcDNA3.1. 48 hours post-transfection, cell lysates were assessed by immunoblotting for COX-2 and β-catenin. Densitometry of COX-2 and Tubulin were determined and graphed as COX-2/Tubulin. B, Cells were transfected with Scr or β-catenin shRNA oligos and were infected with Ad-GFP or Ad-D-DT 24 hours later as indicated. 48 hours post-infection, cells were lysed and assessed by immunoblotting for COX-2, β-catenin, D-DT and α-tubulin (loading control). Densitometry of COX-2 western blots from 2 independent experiments was determined and fold COX-2/Tubulin expression is shown.

CSN5 participates in the regulation of COX-2 and β-catenin. A, Nonspecific control (Scr) and CSN5-specific shRNA-transfected cells were harvested, lysed and immunoblotted for COX-2, CSN5 and α-tubulin. B, 24 hours after transfection with Scr or CSN5 shRNA oligos, cells were infected by either Ad-GFP or Ad-D-DT for an additional 48 hours. Cells were then harvested, lysed and immunoblotted for phospho-JNK, phospho-c-Jun, COX-2, CSN5, D-DT and α-tubulin. C, Cells were treated as in B) but were infected with Ad-GFP and Ad-CA-MKK7 adenoviruses. 48 hours after infection, cells were harvested, lysed and immunoblotted for phospho-c-Jun, COX-2, CSN5, MKK7 and α-tubulin. D, Cells were oligo-transfected with Scr or D-DT shRNA oligos and 24 hours later, cells were infected with Ad-GFP or Ad-COX-2 adenovirus as indicated (for 0 μl Ad-COX-2 cells were treated with Ad-GFP at 15 μl and Ad-GFP was co-infected with Ad-COX-2 at 5 and 10 μl to give 15 μl total adenovirus for each sample infected). 48 hrs after infection, cells were lysed and immunoblotted for β-catenin, COX-2, D-DT and α-tubulin. Densitometry of COX-2/Tubulin is shown. All data is representative of 2 independent experiments.