Hematoxilyn and eosin stain of xenograft tumors generated by injection of mock (A) and transfected (B) PE/CA-PJ15 cells.

<p>(<b>A</b>) Histological examination of tumor revealed nests of cohesive neoplastic epithelial cells, with connective tissue and a band of inflammatory infiltrate. (<b>B</b>) Histological examination of tumor revealed nests of neoplastic epithelial cells with ialine connective tissue and focal inflammatory infiltrate. Original magnification ×20.</p

NNMT enzyme assay.

<p>NNMT specific activity in 7 human cancer cell lines was tested by measuring the amount of N¹-methylnicotinamide produced.</p

<i>In vivo</i> tumor formation of mock and silenced cells.

<p>Representative photo showing BALB/c nude mice subcutaneously injected, in the right and left flank, with mock (A) and transfected PE/CA-PJ15 cells (B), respectively.</p

Effects of NNMT knockdown on the cell proliferation and colony formation <i>in vitro</i>.

<p>Cell proliferation was assessed by MTT analysis (A) and by soft-agar colony-forming assay (B). Cell growth and colony formation were evaluated in mock and NNMT silenced cells after 24–48–72 hours and 30 days of incubation, respectively. Three of the four plasmids were able to significantly reduce cell proliferation (*, p<0.05; **, p<0.005).</p

Detection of NNMT knockdown.

<p>PE/CA-PJ15 cells were treated with four shRNA plasmids against NNMT (pLKO. 1-330, 1-448, 1-164, 1-711), or with transfection reagent only (mock). (A) Real-Time PCR was used to analyze the amount of NNMT mRNA in transfected compared to mock cells. (B) Protein lysates, obtained from NNMT silenced and mock cells, were analyzed for NNMT expression by Western blot.</p

Real-Time PCR analysis of NNMT.

<p>Relative NNMT expression levels were determined in 7 human cancer cell lines. ΔCt values indicate the difference in the Ct values between NNMT and β-actin (housekeeping gene).</p