Research Techniques Made Simple: Identification and Characterization of Long Noncoding RNA in Dermatological Research Dario Antonini, Maria Rosaria Mollo,

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Research Techniques Made Simple: Identification and Characterization of Long Noncoding RNA in Dermatological Research Dario Antonini, Maria Rosaria Mollo, Caterina Missero Journal of Investigative Dermatology Volume 137, Issue 3, Pages e21-e26 (March 2017) DOI: 10.1016/j.jid.2017.01.006 Copyright © 2017 The Authors Terms and Conditions

Figure 1 Schematic experimental procedure for the (a) identification, (b) validation, and (c) functional characterization of lncRNAs. RNA interactome analysis with high-throughput sequencing (RIA-Seq); protein microarray analysis (PMA); RNA binding protein (RBP) pull-down. FISH, fluorescence in situ hybridization; lncRNA, long noncoding RNA; qRT-PCR, quantitative reverse transcription polymerase chain reaction. Journal of Investigative Dermatology 2017 137, e21-e26DOI: (10.1016/j.jid.2017.01.006) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Expression of the lncRNA PICSAR is specifically upregulated in cSCC cells and its depletion suppresses growth of cSCC xenografts. (a) The heatmap of whole transcriptome analysis showing significantly (P < 0.05) regulated lncRNAs in primary (Prim; n = 5) and metastatic (Met; n = 3) cSCC cell lines and in NHEKs (n = 4). (b) Expression of PICSAR in cSCC (n = 6) and in normal skin (n = 7) was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). (c) Expression of PICSAR in cSCC and NHEK was determined by RNA FISH (in red). Scale bar = 10 μm. (d) PICSAR siRNA or negative control transfected cSCC cells were injected subcutaneously into the back of immunodeficient mice. Xenografts were harvested 18 days after inoculation and weighed. Mean ± SEM is shown; *P < 0.05. (e) Histology of the tumors was analyzed by hematoxylin and eosin (H&E) staining. The proliferation marker Ki-67 was detected in xenografts by immunohistochemistry. Hematoxylin was used as a counterstain. Scale bar = 100 μm. cSCC, cutaneous squamous cell carcinoma; FISH, fluorescence in situ hybridization; lncRNA, long noncoding RNA; NHEK, normal human keratinocyte; PICSAR, p38 inhibited cutaneous squamous cell carcinoma associated lincRNA; SEM, standard error of the mean; siRNA, small interfering RNA. Figure reproduced with permission from Piipponen et al. (2016). Journal of Investigative Dermatology 2017 137, e21-e26DOI: (10.1016/j.jid.2017.01.006) Copyright © 2017 The Authors Terms and Conditions