Fig 1.
Accelerated photorepair of CPDs in HaCaT cells transfected with CPD-PL Ψ-mRNA.
HaCaT cells were transfected with lipofectamine-complexed Ψ-mRNA encoding CPD-photolyase. Twelve hours later, cells were subjected to 20 mJ/cm2 UVB and immediately exposed to photoreactivating light (photoreactivated) or left in the dark (non-photoreactivated) for 1 h and then maintained at 37°C for 5 and 23 hs. After incubation at 5 and 23 h, genomic DNA was isolated at the indicated times after UVB irradiation and the amount of CPDs was measured by ELISA. The values were calculated relative to those obtained with cells that were not UVB-irradiated. Significance was assessed by unpaired, two-sample t-test, p<0.05. Error bars represent the standard error of the mean from three experiments performed independently.
Table 1.
Summarized fold change values by microarray results of the selected 9 CPD-dependent genes.
Fig 2.
Experimental verification of microarray results for 9 selected genes.
HaCaT cells were exposed to 20 mJ/cm2 UVB at 12 h after delivery of CPD-PL Ψ-mRNA. Immediately thereafter, cells were either subjected to photoreactivating light (active CPD-photolyase) or left in the dark (inactive CPD-photolyase) for 1 h. Following incubation total RNA was extracted at 5 and 23 h, then real-time RT-qPCR was performed to validate the CPD-dependent expression of ATF3, CCNE1, CDKN2B, EGR1, ID2, PTGS2, RUNX1, SNAI1 and SNAI2. Values measured in UVB irradiated cells with or without photoreactivation were related to those measured in non-UVB irradiated cells that were transfected control Ψ-mRNA, (pecked lines). Asterisks indicate significant differences (two-tailed, unpaired t-test; p<0.05) between photoreactivated (active CPD-photolyase) and non-photoreactivated (inactive CPD-photolyase) samples. The results of RT-qPCR are means ± SEM from three independent experiments in triplicate.
Fig 3.
Photorepair of CPDs prevents altered expression of cyclin E1 and p15INK4b protein in UVB irradiated HaCaT cells.
Cells were transfected with lipofectamine-complexed CPD-PL Ψ-mRNA, 12 hs later irradiated with 20 mJ/cm2 UVB and immediately exposed to photoreactivating light (active CPD-photolyase) or kept in the dark (inactive CPD-photolyase) for 1 h. Subsequently, cells were cultured at 37°C until harvested at the indicated time after UVB irradiation. (A) The expression of cyclin E1 and p15INK4b were analyzed by Western blot. (B) Quantitation of western blots displays relative changes in protein expression normalized to β-actin. Pixel densities were calculated relative to those obtained with cells that were not UVB irradiated (pecked lines). Significance was assessed by two-tailed, unpaired t-test (asterisk, p<0.05) showing differences between photoreactivated and non-photoreactivated samples. Error bars represent the standard error of the mean. The results are means of three independent experiments.
Fig 4.
Induction of cyclin E1 and p15INK4b protein expression upon UVB exposure is regulated through the JNK signalling pathway in HaCaT cells.
Keratinocytes transfected with CPD-PL Ψ-mRNA were incubated in serum-free medium supplemented with JNK inhibitor (SP600125) for 1 h. Immediately thereafter, cells were irradiated with a physiological dose of UVB or left untreated followed by exposure to photoreactivating light (or not) for 1 h. The cells were cultured further in serum-free medium supplemented with the inhibitor. Cells were harvested for western blot assay at the indicated time. Protein levels of cyclin E1, p15INK4b, and β-actin are noted. The figure shows representative results from three independent experiments.