Fig. 4
From: Nrf3 promotes UV-induced keratinocyte apoptosis through suppression of cell adhesion
The effect of Nrf3 on UV-induced apoptosis is cell autonomous and independent of DNA damage. Primary (a) or spontaneously immortalized keratinocytes from Nrf3-ko mice or their wt littermates (b–d) were irradiated with 10 mJ/cm2 (a–c) or 20 mJ/cm2 (d) UVB. The percentage of cleaved caspase-3 or annexin V positive cells among all cells was determined by immunofluorescence staining and counting of 3–6 independent microscopic fields in three different dishes (a) or by flow cytometry (b, c). d Cell viability was determined using MTT assay 24 h post irradiation. Values were normalized to the signal obtained prior to UV irradiation and are shown as arbitrary units. e–g Immortalized keratinocytes from Nrf3-ko or wt mice were irradiated with 10 mJ/cm2 UVB (e, f, g) or treated with 0.1 µM camptothecin (CPT) or 3 mM hydroxyurea (HU) (g) and analyzed at different time points after irradiation or addition of the compound by flow cytometry for γH2AX (e, g) or by immunofluorescence staining for p53 (f). Scatter plots show mean and SD. Data points represent results from individual immortalized cell lines derived from different mice. h, i Primary human keratinocytes were transfected with siRNAs against NRF3 or caspase-5 (control) and irradiated with 10 mJ/cm2 UVB. The knockdown was verified by qRT-PCR for NRF3 relative to RPL27 (h). Apoptotic cells were identified by flow cytometry for cleaved caspase 3 (i). j HaCaT keratinocytes were irradiated with 20 mJ/cm2 UVB and cultured in the presence of absence of MG132 as indicated. Prior to and at different time points following irradiation they were co-stained with an NRF3 antibody, Hoechst and Alexa Fluor 488-coupled phalloidin. All results shown are representatives of at least two independent experiments
