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. 2021 May 6;1(3):100023. doi: 10.1016/j.xjidi.2021.100023

Figure 4.

Figure 4

High concentrations of SP and Can induce replication stress responses and apoptosis in keratinocytes in vitro. (a) HaCaT keratinocytes were treated with 1 mM SP for the indicated periods of time. Immunoblotting was performed with cell lysates with antibodies targeting the indicated proteins or -P proteins. (b) Quantitation of protein levels in which signals were normalized to the time point at which the signal was highest. The asterisk (∗) above the XPB band denotes a nonspecific band recognized by the anti-XPB antibody. (c) HaCaT cells were treated with the indicated concentrations of SP for 2 hours and were then analyzed by immunoblotting. (d) HaCaT cells were treated with vehicle (0.83% DMSO), 1 mM SP, 1 mM or Can or exposed to the indicated fluences of UVB radiation. Cell lysates were prepared 2 hours later and examined by immunoblotting. (e) Cells were pretreated with DMSO (0.1% DMSO) or 10 μM of the ATRi VE-821 for 30 minutes before exposure to DMSO or 1 mM SP and/or Can. Cells were harvested 2 hours later. (f) Cells were treated with 1 mM SP and/or Can for the indicated periods of time in which BrdU (10 μg/ml) was added to the media during the final 15 minutes before harvesting. Genomic DNA was purified and analyzed by immunoblotting with an anti-BrdU antibody. Signals were normalized to cells that were not treated with SP or Can, and the average (and SEM) relative DNA synthesis from two independent experiments was graphed. (g) Cells were treated for 12 hours with the indicated concentrations of SP or Can. (h) Cells were pretreated with DMSO (0.2% DMSO) or 20 μM of the pan-caspase inhibitor Z-VAD-FMK along with vehicle or 1 mM SP and/or Can. Cells were harvested 9 hours later. (i) Quantitation of H2AX phosphorylation from three independent experiments performed as in (g). The individual experimental values are indicated by the black squares and circles. Paired t-tests were used to compare the relative changes in H2AX phosphorylation. For all other experiments, the results are representative of at least two independent experiments. ATRi, ATR inhibitor; Can, canrenone; hr, hour; -P, phosphorylated; PCNA, proliferating cell nuclear antigen; SP, spironolactone; -ub, ubiquitin.