Figure 1.
pATM staining of Normal Human Primary Keratinocytes +/− UVB.
Cells were UVB irradiated (10 mJ/cm2) and fixed at various times post-UV with 4% PFA. Un-irradiated cells show cytoplasmic expression of pATM with more nuclear staining observed 30–60 minutes post-UV. 2 hours following UV irradiation pATM again assumes perinuclear and cytoplasmic expression.
Figure 2.
Fluorescent pATM staining of NHPK +/− UVB.
Same cells as in Fig 1 fluorescently labelled using DAPI (blue) to stain the nucleus. pATM was fluorescently labelled with Alexa Fluor 568 (red) and cells visualised using confocal microscopy. Findings mirror those observed in Fig 1 - a very specific pattern of cytoplasmic staining is seen; at 60 minutes following UVB there is more nuclear expression of pATM which is short-lived, returning back to the cytoplasm after 2 hours.
Figure 3.
Confocal microscope image of fluorescently labelled NHPK +/− UVB with pATM and Golgi specific marker.
Fluorescently labelled NHPK +/− UVB (10 mJ/cm2) with pATM-conjugated Alexa Fluor 568 (red), Golgi-specific antibody (Giantin) conjugated Alexa Fluor 488 (green), and nuclear DAPI staining (blue). There is co-localisation of pATM to the Golgi apparatus. 30 minutes to 2 hours post-UV there is transient nuclear localisation of pATM.
Figure 4.
pATM expression in normal human skin +/− UVB.
Paraffin embedded normal skin (taken from a non-UV exposed site) was UV irradiated (10 mJ/cm2) and fixed with 4% PFA at various times post-UV, then stained with pATM antibody at various time points post-UV. pATM expression can be seen in untreated skin with upregulation 30–60 minutes following UVB irradiation. pATM expression is predominantly cytoplasmic and peri-nuclear.
Figure 5.
pATM expression in normal UV exposed skin (face).
a) Negative control – no primary pATM Ab; b) pATM staining: predominantly nuclear staining observed.
Figure 6.
Fluorescent pATM staining of PM1 (A) and Met1 (B) cells +/− UV.
PM1 and Met1 cells were UVB irradiated (10 mJ/cm2) and fixed in 4% PFA at various times post-UV. Cells were then labelled with pATM conjugated with Alexa Fluor 568 (red) and counterstained with DAPI (blue). (A) Note nuclear foci of pATM at 1 hour and 2 hours post-UV (white arrows) in PM1 cells. No nuclear expression of pATM observed in Met1 cells.
Figure 7.
pATM expression in lesional tissue – examples of scoring for nuclear (A) and cytoplasmic (B) staining.
Table 1.
pATM protein expression by immunohistochemistry – results for individual AK, CIS and SCC lesions.
Table 2.
Summary of Nuclear vs. Cytoplasmic staining in AKs, CIS and SCC.
Figure 8.
A) Scored as nuclear 5 and cytoplasm 5; B) Scored as nuclear 4 and cytoplasm 3.
Figure 9.
A) Scored as nuclear 5 and cytoplasm 5; B) Scored as nuclear 4 and cytoplasm 4.
Figure 10.
A) Scored as nuclear 3, cytoplasm 4; B) Scored as nuclear 2, cytoplasm 4.
Figure 11.
Overall pattern of pATM expression observed in spectrum of AK / CIS / SCC.
This demonstrates greater proportion and increased intensity of nuclear staining of earlier lesions i.e. AK (a) and CIS (b) compared to less extensive and weaker nuclear staining in more advanced SCC (c).