FIGURES AND TABLES

Figure 1.

Effects of Pluronics on luciferase gene expression in Luc^CMV-NIH3T3 cells. The cells were exposed for 3 h (A) to P85 at 0.03, 0.1, or 0.3% or (B) P85 at 1% or L64 at 0.1%. After treatment the cells were washed and incubated for an additional 24 h and then lysed to measure the luciferase activity. Data are means SD (n = 3). The statistical comparisons were made for treated versus untreated cells: ^* P < 0.05, ^** P < 0.005.

Figure 2.

Effects of Pluronics on GFP expression. (A) Mean fluorescence values for GFP^CMV-Cl66 cells treated with P85 for 3, 6, and 9 h. (B) Mean fluorescence values for GFP^CMV-NIH3T3 cells treated with 1% P85 or 0.1% L64 for 3 h. (A, B) Data are reported as means SD (n = 3). The statistical comparisons were made for treated versus untreated cells: ^* P < 0.05; ^** P < 0.005. (C, D) Confocal fluorescence micrographs of (C) untreated GFP^CMV-C2C12 cells and (D) GFP^CMV-C2C12 cells treated with 0.1% L64 for 3 h. The micrographs superimpose nonconfocal differential interference contrast and confocal images collected simultaneously. A similar increase in gene expression by 0.1% L64 with no changes in cell morphology was observed in GFP^CMV-NIH3T3 cells (not shown).

Figure 3.

Effects of Pluronics on mRNA levels of luciferase (Luc) and hsp68 genes in Luc^CMV-NIH3T3 cells. Cells were treated with 0.1% L64 or 1% P85 for 3 h, washed, and incubated in complete medium for an additional 24 h. (A) The RT-PCR products of Luc (230 bp) and hsp68 (664 bp) were run by electrophoresis on a 2% agarose gel containing ethidium bromide. The image was acquired using an Alpha Imager and analyzed using ImageQuant. (B) mRNA levels for hsp68 and luciferase normalized with respect to GAPDH and expressed as arbitrary units. (C) mRNA levels for luciferase relative to GAPDH quantitated by RT2-PCR. Data are reported as means SD (n = 3). Differences in treated versus control groups were considered significant at ^* P 0.05, ^** P 0.005.

Figure 4.

Effects of Pluronics on expression of GFP driven by different promoters in (A) mouse fibroblast cells GFP^NF-B-NIH3T3 (filled bars) and GFP^AP-1-NIH3T3 (striped bars) and (B) mouse myoblast cells GFP^NF-B-C2C12 (filled bars) and GFP^AP-1-C2C12 (striped bars). Cells were treated with Pluronics P85 (0.3, 0.7, and 1%) and L64 (0.03, 0.07, and 0.1%) for 3 h and the mean fluorescence values for (A) NIH3T3 and (B) C2C12 are presented. Data are reported as means SD (n = 3). Differences in treated versus control groups were considered significant at ^* P 0.05, ^** P 0.005.

Figure 5.

Effects of P85 on IB phosphorylation. (A) A representative Western blot of phospho-IB- protein. C2C12 cells were exposed to P85 (2% w/v) for 2, 5, or 10 min and then lysed to obtain cellular protein. Western blots were obtained using 50 g total protein. (B) The signal band intensities were quantified using Scion Image software and are illustrated in the bar graph. The results are expressed as the relative intensity of the target, phospho-IB-, vs that of the control, -actin. Data represent means SEM (n = 3). Statistical significance for the treatment groups compared to the untreated controls (0 min) is shown as follows: ^* P < 0.05, ^*** P < 0.001.

Figure 6.

(A) Effects of 0.03% P123 added during transfection of PC-3 cells using gWIZ-Luc plasmid formulated with PEI (25 kDa) or Superfect. (B) Effects of 1% P85 or 0.08% L64 on gene expression in C2C12 cells transiently transfected with gWIZ-Luc using ExGen 500. Data are means SD (n = 3). Differences in treated versus control groups were considered significant at ^* P 0.05 (A) or ^** P 0.005 (B).

Table 1 - Transfected cells used in this study.