Table 1.
Western Blot-Based Quantification of PEDF Secretion by PEDF-Transfected Bovine IPE and Human RPE Cells
| Signal Intensities (Mean ± SD) | pFAR-PEDF versus pFAR-PEDFo | ||
|---|---|---|---|
| 1 × 104 bIPE cells | Control 1.00 ± 0.03 (n = 19) | Control 1.00 ± 0.00 (n = 9) | |
| pFAR-PEDF 17.96 ± 35.28 (n = 211) *p = 0.0376 | pFAR-PEDFo 8.84 ± 7.65 (n = 106) **p = 0.0028 | **p = 0.0090 | |
| 5 × 103 bIPE cells | Control 1.00 ± 0.11 (n = 18) | Control 1.00 ± 0.00 (n = 11) | |
| pFAR-PEDF 5.25 ± 5.20 (n = 200) ***p = 0.0007 | pFAR-PEDFo 8.42 ± 9.95 (n = 132) *p = 0.0149 | ***p = 0.0002 | |
| 1 × 104 hRPE cells | Control 1.00 ± 0.18 (n = 52) | Control 1.00 ± 0.17 (n = 26) | |
| pFAR-PEDF 2.54 ± 0.99 (n = 312) ****p < 0.0001 | pFAR-PEDFo 1.93 ± 0.76 (n = 156) ****p < 0.0001 | ****p < 0.0001 | |
| 5 × 103 hRPE cells | Control 1.00 ± 0.13 (n = 48) | Control 1.00 ± 0.19 (n = 26) | |
| pFAR-PEDF 2.02 ± 1.09 (n = 288) ****p < 0.0001 | pFAR-PEDFo 2.25 ± 1.14 (n = 156) ****p < 0.0001 | *p = 0.0378 | |
For each sample, one or two control transfections without the addition of miniplasmid DNA and ten to 12 transfections using 0.03 μg pFAR4-CMV SB100X SV40 transposase and 0.47 μg pFAR4-ITRs CMV PEDF BGH or pFAR4-ITRs CMV PEDFoptimized BGH miniplasmid DNA were carried out. Culture supernatants were analyzed for total PEDF secretion 21 days after transfection (Figures S2–S5). Signal intensities of PEDF-transfected and PEDFo-transfected cells were normalized to the signal intensities of control cells. Statistical analysis showed a significant difference in secretion between PEDF-transfected and PEDFo-transfected cells.