Journal: Cell Death and Differentiation

Article Title: Selective elimination of senescent cells by mitochondrial targeting is regulated by ANT2

doi: 10.1038/s41418-018-0118-3

Figure Lengend Snippet: a MCF7, 4T1 and MCF10a cells were exposed to MitoTam for 3 days at the concentrations as indicated and cell survival was evaluated as annexin V/Hoechst negativity by flow cytometry. b MCF7, 4T1 and MCF10a cells were treated for 3 days with MitoTam (1 μM) and assessed for β-gal positivity. MCF7 cells treated for 8 days with 0.75 μM doxorubicine were used as a positive control of staining. Scale bar represents 10 μm. Balb-c mice were injected s.c. with 1 × 10 6 4T1 cells per animal and treated i.p. with MitoTam (0.25 μmol/mouse/dose) dissolved in 4% ethanol in corn oil or with the excipient twice per week for 2 weeks; the level of p16 Ink4a , p21 waf1 and PAI mRNA was estimated by qRT-PCR ( c ) and % of β-gal-positive tissue was detected ( d ). e Transgenic FVB/N c-neu mice with spontaneous tumours were treated twice per week i.p. with MitoTam (0.54 μmol/mouse/dose) dissolved in 4% ethanol in corn oil or with the excipient for 2 weeks. Mice treated with doxorubicin (1.5 mg/kg in 0.9% NaCl) for the same time were used as positive control; the level of p16 Ink4a , p21 waf1 and PAI mRNA was estimated by qRT-PCR. NSG mice with PDX tumours were treated with MitoTam (0.375 μmol/mouse/dose) dissolved in 4% ethanol in corn oil or with the excipient twice per week for 2 weeks. The level of p16 Ink4a , p21 waf1 and PAI mRNA was estimated by qRT-PCR ( f ) and % of β-gal-positive tissue was detected ( g ). h Control and senescent 4T1 cells (treated with 0.75 μM doxorubicin for 4 days) were exposed to 1 μM MitoTam for 3 days and cell survival was evaluated using the annexin V/Hoechst assay and flow cytometry. MCF10a cells were used as a control of toxicity. i 4T1 cells were treated with 0.75 μM doxorubicin for 4 days and then treated with 1 μM MitoTam for 72 h, and assessed for β-gal positivity. Scale bar represents 10 μm. RPE-1 ( j ) and HFP-1 ( k ) control and senescent (BrdU, 100 μM BrdU for 8 days; RS replicative senescence) cells were exposed to MitoTam for 48 h at the concentrations as indicated, and their survival was evaluated based on annexin V/Hoechst negativity using flow cytometry. The asterisk represents p < 0.05

Article Snippet: Human breast carcinoma cell line MCF7, telomerase-immortalized human RPE-1, foreskin fibroblast cell line (BJ) and lung-derived fibroblast cell line (HFP-1) were cultivated in DMEM containing 4.5 g/l glucose (Biochrom, Berlin, Germany).

Techniques: Flow Cytometry, Positive Control, Staining, Injection, Quantitative RT-PCR, Transgenic Assay, Control