Activation of ATR itself promotes squamous differentiation in human keratinocytes. (a) IF for γH2AX (red) in primary cells expressing TopBP1^ER, after 48 h in the absence or presence of OHT, as indicated. Nuclear DNA by DAPI. Scale bar, 50 µm. (b) Immunodetection of TopBP1^ER by an anti-estrogen receptor antibody (ER) or γH2AX by WB in keratinocytes expressing control pMXPIE empty vector (CT) or TopBP1^ER, OHT as indicated. GAPDH as loading control. A DOXO 24-h treatment was used as positive control in b–d. (c) Percentage of keratinocytes positive for γH2AX relative to CT + OHT (100%), as determined by FC. OHT as indicated. (d) Quantitation of DNA breaks by comet tail lengths, in pixels, of keratinocytes infected with CT or TopBP1^ER, OHT treatment as in a. Plot: Black small bars indicate the tail average length, n = 168 cells of eight fields per slide. Photos: Representative average comets for each treatment. (e) Representative FC for Inv expression in cells infected with CT or TopBP1^ER, after 4 d OHT. Positive (+) keratinocytes according to negative isotype antibody control (red broken line). (f) Percentage of keratinocytes in e displaying HS typical of differentiation or Inv expression, relative to CT + OHT. (g) Clonogenicity assays of keratinocytes infected with CT or TopBP1^ER vectors and released after 4 d in OHT. Representative images of triplicate samples of representative experiments. (h) Percentage of large proliferative colonies in g, relative to CT + OHT. Data are mean ± SEM of duplicate (c and d) or triplicate (f and h) samples. See also Fig. S4. Datasets were compared by an unpaired t test (two-sided). *, P < 0.05; **, P < 0.01.