Journal: Advanced Science

Article Title: IncRNA‐ ZFAS1 , an Emerging Gate‐Keeper in DNA Damage‐Dependent Transcriptional Regulation

doi: 10.1002/advs.202412385

Figure Lengend Snippet: Zfas1 ‐deficient mice are defective in NER. A) Schematic representation of the mouse Zfas1 gene locus and sgRNAs sequences. Zfas1 exons are indicated in grey solid box. The sgRNA‐targeting sequences are underlined, with the PAM sequences highlighted in red. B) left : representative images of clonogenic survival assays of Zfas1 ‐/‐ mouse fibroblasts and WT controls under the indicated UV dose. right : percentage of surviving cells (logarithmic scale) plotted against UV‐C dose. Error bars indicate the standard error of the mean from three independent experiments. * p < 0.05 compared to the WT mice (Student's t ‐Test). NIH3T3 cells lacking either CSB or XPC were treated as positive controls. C) Representative images of primary cells showing repair of CPDs using a specific antibody to CPDs (green signal) for the WT and Zfas1 ‐/‐ mice. DAPI‐stained nuclei in blue. Scale bar = 50 µm. D) top : representative images of primary fibroblast cells showing nuclear γH2A.X foci using a specific antibody to H2A.X phosphorylation (green signal) for the WT and Zfas1 ‐/‐ mice. Nuclear DNA was counterstained with DAPI in blue. Scale bar = 50 µm. bottom : quantification of percent (%) γH2A.X‐positive (≥ 5 foci/nucleus) nuclei for each genotype, treatment and time point. A minimum of 500 cells per cell line per condition were analyzed. Each data point is presented as the means ± SD, n = 3. * p < 0.05; ** p < 0.01 (Student's t ‐Test). E) Nascent mRNA production in two distinct regions of the mouse Kifap3 gene following UV‐C damage in the WT and Zfas1 ‐/‐ fibroblasts. Means ± SD are shown from three independent experiments. * p < 0.05 (Student's t ‐Test). F) Representative images of UV‐induced skin burn of Zfas1 ‐/‐ mice. Shaven animals were exposed to UV‐B (500 J m −2 /day) for 4 consecutive days, photographs were taken on the 7th day after the first exposure. Sample size for each genotype: n = 3 females. G) Hematoxylin and eosin staining (HE) of the skin sections of shaven Zfas1 ‐/‐ and WT mice after exposure to UV‐B (500 J m −2 /day) for 4 consecutive days. The wild‐type skin appears normal, the Zfas1 ‐/‐ skin shows hyperplasia of epidermis (indicated by dashed boxes). Scale bar = 50 µm.

Article Snippet: The following cell lines were used in this study: A549, human lung carcinoma cell line (ATCC, CCL‐185); IMR‐90, normal primary human lung fibroblast cells (ATCC, CCL‐186); MRC5_VA, SV40‐immortalized normal human fetal lung fibroblast cells.

Techniques: Staining, Phospho-proteomics