(a) Top: map of all detected transcripts at the CDKN1A promoter. Middle two tracks: Example of RNA hybridization intensity in control or 24 hour doxorubicin treated (200ng/ml) human fetal lung fibroblasts. Note, not all DNA damage inducible transcripts are observed in one single time point. Bottom track: p53 ChIP-chip signal relative to input confirmed the p53 binding site immediately upstream of the CDKN1A TSS upon DNA damage. RACE clone of upst:CDKN1A:-4845 closely matches predicted transcript on tiling array. See also: Supplementary Fig S7.
(b) Quantitative RT-PCR of lncRNAs shows coordinate induction or repression across a 24 hour time course of doxorubicin treatment. A cluster of lncRNAs transcribed from the CDKN1A locus are induced.
(c) Expression of transcripts from the CDKN1A locus over a 24 hour time course after doxorubicin-treatment of normal human fibroblasts (FL3). See also: Supplementary Fig S6.
(d) Northern blot of PANDA confirms transcript size of 1.5Kb.
(e) Doxorubicin induction of PANDA requires p53 but not CDKN1A. Mean + s.d. are shown, *p<0.05 relative to sictrl, student’s t-test.
(f) Expression of wild type p53 in p53-null H1299 cells restores DNA damage induction of CDKN1A and PANDA. p53(V272C) loss-of-function mutant fails to restore induction, whereas a gain-of-function Li-Fraumeni allele, p53(R273H), selectively retains the ability to induce PANDA.