We further assessed the expression of the CDK inhibitor p21
CIP1, the principal mediator of p53-dependent cell cycle arrest in response to DNA damage.
38,39 No p21
CIP1+ cells were detected in the control retinas and MNU-treated retinas between day 1 and day 2.5 (
Fig. 8B and data not shown). However, some p21
CIP1+ Müller glia were identified at day 3, with the highest number observed at day 3.5 (
Fig. 8B). The p21
CIP1+ cells then decreased with time, and few positive cells were found at day 7 (
Fig. 8B and data not shown). The upregulation of p21
CIP1 was also analyzed by qRT-PCR. Significantly higher expression of
Cdkn1a, which encodes p21
CIP1, was observed at day 2 compared to the controls, and there was further significant increase between day 2 and day 4 (
Fig. 8C). We further performed Western blotting to confirm immunohistochemical findings. Intense immunoreactivity for γ-H2AX and p53 was detected as early as day 1 (
Fig. 8D), which likely reflects the DNA damage response in dying photoreceptor cells (
Fig. 7A). We tested two different p53 antibodies obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Cell Signaling Technology (CST; Beverly, MA, USA) with similar results (
Fig. 8D). Immunoreactivity for γ-H2AX, p53, and p21
CIP1 between days 3 and 4 was consistent with the DNA damage response in Müller glia detected by immunofluorescence (
Fig. 8D).