Journal: The Journal of Cell Biology
Article Title: Serpin squamous cell carcinoma antigen inhibits UV-induced apoptosis via suppression of c-JUN NH2-terminal kinase
Figure Lengend Snippet: Overexpression or down-regulation of SCCAs significantly affected UV-induced apoptosis. (A) FACS analyses of apoptotic cells using SCCA1- or SCCA2-transfected 3T3/J2 cells, 48 h after UV irradiation (30 mJ/cm 2 ). Cells were stained with FITC-conjugated Annexin V and propidium iodide. (B) Analyses of five experiments are summarized. (C) 12 clones whose expression levels of SCCA1 mRNA distributed from 1 to 2,772-fold were established. Using these clones, the effects of UV irradiation were examined. Cells were harvested 48 h after UV irradiation (50 mJ/cm 2 ) and FACS analyses were performed. The antiapoptotic activity correlated with SCCA1 expression. r = 0.734. (D) Using pSilencer vector, an siRNA construct targeted to a homologous sequence of SCCAs was stably transfected into HaCaT keratinocytes. Typical FACS analyses of nonirradiated (UV−) and UV-irradiated (UV+) siSCCA/HaCaT cells were shown. Apoptotic cells were analyzed 48 h after UV irradiation (75 mJ/cm 2 ). (E) Statistical analyses of five experiments. Error bars represent the mean of five wells ± SD.
Article Snippet: Vector-based siRNA A double-stranded oligonucleotide was designed corresponding to a common sequence of human SCCAs (5′-AAGCCAACACCAAGTTCATGT-3′) to allow formation of the hairpin structure in the expressed oligo-RNA, cloned into the pSilencer vector (Ambion), and transfected into human keratinocyte cell line HaCaT cells. siRNA against GFP mRNA (Ambion) was used as a control.
Techniques: Over Expression, FACS, Transfection, Irradiation, Staining, Clone Assay, Expressing, Activity Assay, Plasmid Preparation, Construct, Sequencing, Stable Transfection