Journal: Cell Death & Disease

Article Title: Sublethal UV irradiation induces squamous differentiation via a p53-independent, DNA damage-mitosis checkpoint

doi: 10.1038/s41419-018-1130-8

Figure Lengend Snippet: Primary human keratinocytes 6 days after infections with the empty vector (CT) or with shRNA specific to p53 (shp53) and 24 h after lethal UV (150–300 mJ/cm 2 ; lthUV) or sublethal UV doses (sbUV), as indicated. a Expression of p53, its target p21 or γH2AX by western blotting 24 h after sbUV. GAPDH as loading control. b Expression of p53 mRNA as measured by RT-qPCR. c Percent of cells in the sub-G1 (apoptotic) fraction of the cell cycle. d Representative flow-cytometry analyses for DNA content as in Fig. . Inset histograms show DNA content in logarithmic units. NI: non-irradiated. Irradiation units: /cm 2 . * p < 0.05. Data are representative or mean ± s.e.m. of triplicate samples

Article Snippet: For gene delivery in primary keratinocytes, the following lentiviral constructs driven by constitutive promoters were used: control GFP pLVTHM and a construct expressing shRNA against p53: pLVUH-shp53-GFP (shp53; a gift from Patrick Aebischer and Didier Trono ; Addgene plasmid 11653); control pLVX (CT-pLVX) and pLVX-FoxM1 (FOXM1; kindly provided by S. Stoll, University of Michigan, Ann Arbor, USA); control plKO1 (Sigma-Aldrich, Inc) and a construct expressing shRNA against Wee1 (shwee1).

Techniques: Plasmid Preparation, shRNA, Expressing, Western Blot, Quantitative RT-PCR, Flow Cytometry, Irradiation

Journal: Cell Death & Disease

Article Title: Sublethal UV irradiation induces squamous differentiation via a p53-independent, DNA damage-mitosis checkpoint

doi: 10.1038/s41419-018-1130-8

Figure Lengend Snippet: Primary human keratinocytes analysed 9 days after infections with the empty vector (CT) or with shRNA specific to p53 (shp53) 24 h ( b , c ) or 48 h ( a ) after a sbUV dose. a Representative flow cytometry light scatter analyses. Black boxes gate cells with high light scatter. Bar histogram displays the percent of cells with high scatter as indicated. b Expression of squamous differentiation markers keratin K1, keratin K10, filaggrin or involucrin (Invol) as measured by RT-qPCR 24 h after irradiation. c Western blotting of cells as in ( b ) on insoluble (Invol, K13 and K16; same number of cells per lane) or soluble (Filaggrin and GAPDH as its loading control) cellular protein fractions. NI: non-irradiated. * p < 0.05. Data are representative or mean ± s.e.m. of duplicate samples

Article Snippet: For gene delivery in primary keratinocytes, the following lentiviral constructs driven by constitutive promoters were used: control GFP pLVTHM and a construct expressing shRNA against p53: pLVUH-shp53-GFP (shp53; a gift from Patrick Aebischer and Didier Trono ; Addgene plasmid 11653); control pLVX (CT-pLVX) and pLVX-FoxM1 (FOXM1; kindly provided by S. Stoll, University of Michigan, Ann Arbor, USA); control plKO1 (Sigma-Aldrich, Inc) and a construct expressing shRNA against Wee1 (shwee1).

Techniques: Plasmid Preparation, shRNA, Flow Cytometry, Expressing, Quantitative RT-PCR, Irradiation, Western Blot