1. PARP Determines the Mode of Cell Death in Skin Fibroblasts, but not Keratinocytes, Exposed to Sulfur Mustard 
Dana Anderson, Betty Benton, Zhao-Qi Wang, William Smith, Radharaman Ray 
Journal of Investigative Dermatology 
Volume 117, Issue 6, Pages (December 2001)
DOI: /j x x
Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Exposure of primary dermal fibroblast cells derived from PARP−/− but not PARP+/+ mice results in caspase-3 activation. Primary dermal fibroblasts were derived from newborn mice as described in Materials and Methods. Cells were incubated for 24 h (A, B) with the indicated concentrations of SM in keratinocyte growth medium, or for the indicated times with 100 µM SM (C), after which whole cell extracts were prepared and assayed for the presence of PARP by immunoblot analysis (A), or caspase-3 activity with the specific substrate DEVD-AMC (B, C). All the data in (B) and (C) are presented as means ± SD of three replicates of a representative experiment; essentially the same results were obtained in three independent experiments.
Journal of Investigative Dermatology  , DOI: ( /j x x)
Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Exposure of primary dermal fibroblast cells derived from PARP−/− but not PARP+/+ mice results in proteolytic processing of procaspase-3 and internucleosomal DNA fragmentation. Primary dermal fibroblasts were derived from newborn mice as described in Materials and Methods. Cells were incubated for 24 h with the indicated concentrations of SM in keratinocyte growth medium, after which whole cell extracts (A), or DNA (B), were prepared and assayed for proteolytic cleavage of caspase-3 by immunoblot analysis (A) or DNA fragmentation by agarose gel electrophoresis (B).
Journal of Investigative Dermatology  , DOI: ( /j x x)
Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Exposure of primary dermal fibroblast cells derived from PARP−/− mice results in a dose-dependent increase in annexin-V-positive cells dependent upon caspase-3 activity. Primary dermal fibroblasts were derived from newborn mice as described in Materials and Methods. Cells were incubated for 24 h with the indicated concentrations of SM in keratinocyte growth medium, after which cells were prepared and assayed for annexin V binding plus PI staining at the doses indicated (B), or at 500 µM SM with or without the indicated caspase inhibitors (C) by FACS analysis. Dot plots of the results show viable (B, C, lower left quadrants), early apoptotic (annexin-V-FITC-positive; lower right quadrant), late apoptotic (upper right quadrant), and necrotic (PI-positive; upper left quadrant). (A) Percentage of cells exhibiting annexin V binding (A, left) or PI staining (A, right) as determined by FACS analysis. All the data in the left and right panels of (A) are presented as means ± SD of three replicates of a representative experiment; essentially the same results were obtained in three independent experiments.
Journal of Investigative Dermatology  , DOI: ( /j x x)
Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Stable expression of PARP in immortalized PARP−/− fibroblasts eliminates SM-induced in vitro PARP cleavage activity and inhibits processing of procaspase-3 to its active form. Cultures of fibroblasts were analyzed for expression of PARP by immunoblot analysis (A), and treated with the indicated concentrations of SM (B, C). Extracts were then derived after 24 h and assayed for caspase-3 activity, using [35S]PARP as a substrate (B), or analyzed for caspase-3 processing by immunoblot analysis (C).
Journal of Investigative Dermatology  , DOI: ( /j x x)
Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Stable expression of PARP in immortalized PARP−/− fibroblasts results in the elimination of a dose-dependent increase in annexin-V-positive cells. Immortalized PARP+/+, PARP−/−, or PARP−/− (+PARP) fibroblasts were incubated for 24 h with the indicated concentrations of SM in keratinocyte growth medium, after which cells were prepared and assayed for annexin V binding (A) or PI staining (B) by FACS analysis. All the data in panels A and B are presented as means ± SD of three replicates of a representative experiment; essentially the same results were obtained in three independent experiments.
Journal of Investigative Dermatology  , DOI: ( /j x x)
Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
SM induces caspase-3 activity in different clones of both PARP−/− and PARP+/+ cells. Primary keratinocytes were derived from PARP+/+ or PARP−/− newborn mice and immortalized with the E6 and E7 genes of HPV 16 as described in Materials and Methods. Cell extracts were subjected to immunoblot analysis using antibodies specific for PARP (A, top), vimentin (A, middle), or cytokeratin (A, bottom). Representative clones of PARP−/− or PARP+/+ keratinocytes were incubated for 24 h with the indicated concentrations of SM in keratinocyte growth medium, after which whole cell extracts were subjected to caspase-3 activity assays with the specific substrate DEVD-AMC (B), or immunoblot analysis using antibodies specific for caspase-3 (C), or PARP (D). Bars in (B) represent the range of average activities for two different clones.
Journal of Investigative Dermatology  , DOI: ( /j x x)
Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions