Association of Vemurafenib and Pipobroman Enhances BRAF-CRAF Dimerization in Squamous Cell Carcinoma  Charles Cassius, Cécile Pages, Jennifer Roux, Raphael Lhote, Romain Lavocat, Delphine Réa, Martine Bagot, Samia Mourah, Maxime Battistella, Céleste Lebbé, Nicolas Dumaz  Journal of Investigative Dermatology  Volume 136, Issue 6, Pages 1302-1305 (June 2016) DOI: 10.1016/j.jid.2015.12.047 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Illustration of lesions and course of the treatment. (a) Preoperative view of the squamous cell carcinoma of the left leg. (b) Diagram explaining the course of the treatment over time. Journal of Investigative Dermatology 2016 136, 1302-1305DOI: (10.1016/j.jid.2015.12.047) Copyright © 2016 The Authors Terms and Conditions

Figure 2 BRAF-CRAF dimers in tumoral material revealed by Duolink. (a) Interactions between BRAF and CRAF were detected by in situ proximity ligation assay (PLA) in tumor biopsies from patients treated with pipobroman and vemurafenib, scale 400 μm for a1 and 100 μm for a2 (a1, a2), vemurafenib alone, scale 400 μm (a3) or untreated, scale 400 μm (a4). Following dewaxing and rehydration of tissue sections, antigen retrieval was performed by heating the slides for 30 minutes at 95 °C in Tris-EDTA buffer, pH 9. The PLA protocol was followed according to the manufacturer’s instructions (Olink Bioscience, Uppsala, Sweden), with incubation of the primary antibodies at 4 °C overnight. After blocking, the antibodies were used at 1/50 dilution: BRAF (mouse, clone F7, Santa Cruz Biotechnology, Dallas, TX, 1:50); CRAF (rabbit, clone C12; Santa Cruz Biotechnology, 1:50). PLA minus and PLA plus probes (containing the secondary antibodies conjugated to oligonucleotides) were added and incubated for 1 hour at 37 °C. Oligonucleotides were then added and allowed to hybridize to the PLA probes. Ligase was used to join the two hybridized oligonucleotides into a closed circle. The DNA was then amplified (with rolling circle amplification), and detection of the amplicons was carried out using the orange detection kit for fluorescence. Cell nuclei were stained with DAPI. The sections were mounted with Olink Mounting Medium. The results were visualized by fluorescent microscopy and the number of PLA signals per cell was counted (more than three fields) by the Duolink Image Tool. (b) The number of PLA dots in three tumor biopsies from patients treated with pipobroman and vemurafenib (Vemu+Pipo), three tumor biopsies from patients treated with vemurafenib alone (Vemu), and two tumor biopsies from untreated patients (UV) are shown. Student’s t-test results are given above the plots when significant. (c) NHEK were treated with DMSO (control) or with pipobroman (10 μM) and vemurafenib (3 μM). After 2 weeks of treatment, cells were lysed in RIPA, and CRAF was immunoprecipitated and probed for BRAF (clone F7; Santa Cruz Biotechnology). Lysates were probed for pEGFR (Y1068, Cell Signaling), BRAF, CRAF, phosphorylated ERK (ppERK, Sigma), and total ERK (Millipore). DAPI, 4′,6-Diamidine-2′-phenylindole dihydrochloride; DMSO, dimethyl sulfoxide; ERK, extracellular signal-regulated kinase; NHEK, normal human epidermal keratinocytes; pEGFR, phosphorylated EGFR; RIPA, radioimmunoprecipitation assay. Journal of Investigative Dermatology 2016 136, 1302-1305DOI: (10.1016/j.jid.2015.12.047) Copyright © 2016 The Authors Terms and Conditions