Casais et al: Transgene expression in multicellular spheroids
Figure 5. Effect of EBNA1/oriP persistence elements on !gal expression. (a-c) Time course of specific !gal reporter activity following lipofection with pCMV! (#,"), pEBCMV! ($,"), pEB2CMV! (",#) or pCMV!+pCMVGM (pCMV!/2) (%,&) plasmids in LM05e (a), LM3 (b) and HEp-2 (c) cells cultured as spheroids (main plot, black symbols) or monolayers (inserted plot, open symbols). At the indicated times, cells were homogenized and assayed for !gal activity as described in Materials and Methods. Results were expressed as mU of !gal activity/mg protein ± s.e.m. of (n) independent assays after correction for background (pCMV!: n=14; pEBCMV!: n=9; pEB2CMV!: n=8). Showing the P-values obtained by ANOVA test PLASMID \ CELLS pCMV! vs. pEBCMV! pEB2CMV! pCMV!/2 pCMV!/2 vs. pEBCMV! pEB2CMV!
Spheroids n.s.
LM05e Monolayer n.s.
p<0.05 (days 4-30) p<0.01 (days 4-45) p<0.01 (days 4-15) p<0.01 (days 4-15)
n.s.
Spheroids n.s
LM3 Monolayer n.s.
p<0.05 (days 4-8)
p<0.05 (days 4-45) p<0.01 (days 4-45)
n.s. p<0.05 (days 4-8)
p<0.05 (days 4-8) p<0.05 (day 8)
p<0.01 (days 4-15) p<0.05 (day 8)
p<0.01 (days 4-8) p<0.05 (days 4-8)
HEp-2 Spheroids Monolayer p<0.05 p<0.05 (days 8-60) (day 15) n.s p<0.05 (day 4) p<0.01 p<0.01 (days 4-8) p<0.01
p<0.01
p<0.01
n.s.
(d) Effect of EBNA1/oriP cassette on gene transfer efficiency: LM05e (gray bars), LM3 (white bars) and HEp-2 (light gray bars) cells transfected with pCMV!, pEBCMV! or pEB2CMV! lipoplexes were stained with X-Gal 48 h later and counted as described in Materials and Methods. The results were expressed as % of X-Gal blue staining cells ± s.e.m. of (n) independent experiments (pCMV!: n=16; pEBCMV!: n=9; pEB2CMV!: n=8). + p < 0.05 and ++ p<0.01: with respect to pCMV! in the same cell line. o p < 0.05 and oo p<0.01: with respect to LM05e/S respective plasmid.
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