Journal: Biochimica et biophysica acta
Article Title: Lovastatin protects keratinocytes from DNA damage-related pro-apoptotic stress responses stimulated by anticancer therapeutics.
doi: 10.1016/j.bbamcr.2016.02.009
Figure Lengend Snippet: Fig. 5. Effect of lovastatin on the activation of DDR mechanisms following irradiation and treatment with doxorubicin or hydroxyurea. A: Logarithmically growing HaCaT cells were pretreated with 30 μM lovastatin (Lova) or were left untreated. Protein extracts were collected 30 min or 8 h after irradiation (IR) with 10 Gy. For doxorubicin (Doxo) treatment cells were collected immediately after 2 h pulse treatment (0 h postDoxo) or 6 h after the end of the incubation time (6 h postDoxo) for Western Blot analysis. Shown are the protein levels of Ser1981 phosphorylated ATM (pATM), Ser428 phosphorylated ATR (p-ATR), Ser345 phosphorylated checkpoint kinase-1 (pChk1), Thr68 phosphorylated checkpoint kinase-2 (pChk2), Ser4/Ser8 phosphorylated replication protein A (pRPA32), Ser15 phosphorylated protein 53 (pp53), Ser824 phosphorylated KRAB-associated protein-1 (pKap1) and Ser139 phosphorylated histone 2AX (γH2AX). Expression of β-actin and Talin-1 was used as loading controls. B: Logarithmically growing HaCaT cells were pretreated with 30 μM lovastatin (Lova) for 24 h or were left untreated. Protein extracts were collected after 2 h pulse treatment with 20 mM hydroxyurea (HU). Shown are the protein levels of Ser1981 phosphorylated ATM (pATM), Ser428 phosphorylated ATR (p-ATR), Ser345 phosphorylated checkpoint kinase-1 (pChk1), Thr68 phosphorylated checkpoint kinase-2 (pChk2), Ser4/ Ser8 phosphorylated replication protein A (pRPA32), Ser15 phosphorylated protein 53 (pp53), Ser824 phosphorylated KRAB-associated protein-1 (pKap1) and Ser139 phosphorylated histone 2AX (γH2AX). Expression of β-actin and Talin-1 was used as loading controls. Shown are representative data from two independent experiments. C: Logarithmically growing HaCaT cells were pretreated with 30 μM lovastatin (Lova) for 24 h or were left untreated. Afterwards, cells were irradiated with 10 Gy (IR), or incubated with 1 μM doxorubicin (Doxo) or 20 mM hydroxyurea (HU) for 2 h. Protein extracts were collected 24 h after the beginning of the respective treatment. Ser345 phosphorylated checkpoint kinase-1 (pChk1), Ser428 phosphorylated Ataxia telangiectasia and Rad3-related (Ser428 pATR), Thr1989 phosphorylated Ataxia telangiectasia and Rad3-related (Thr1989 pATR). Expression of β-actin was used as loading control. D: Logarithmically growing HaCaT cells were pretreated with 30 μM lovastatin (Lova) or were left untreated. Protein extracts were collected 0.5 h–24 h after irradiation (IR) (10 Gy). For doxorubicin (Doxo) treatment cells were collected immediately after 2 h pulse treatment (0 h post-Doxo) or up to 24 h later for analyzing the expression of protein phosphatase 2A, subunit C (PP2A). Expression of β-actin was used as loading control.
Article Snippet: Antibodies directed against caspase 3, activated caspase 7, Ser15 phosphorylated protein 53 (pp53), Ser345phosphorylated checkpoint kinase-1 (pChk1), Ser1981phosphorylated ataxia telangiectasia mutated (pATM), Ser428 phosphorylated ataxia telangiectasia and Rad-3 related (pATR), 53BP1 and Talin-1 were obtained from Cell Signaling (Beverly, MA, USA).
Techniques: Activation Assay, Irradiation, Incubation, Western Blot, Expressing, Control
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![Standard curve: p53 ELISA (OD [450 nm] vs Concentration [ng/mL]).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0774/pmc09170774/pmc09170774__gr1.jpg)