Additional file 2: of Targeting WEE1 to enhance conventional therapies for acute lymphoblastic leukemia
Creators: name: Rorà, Andrea Ghelli Luserna Di
name: Beeharry, Neil
name: Imbrogno, Enrica
name: Ferrari, Anna
name: Robustelli, Valentina
name: Righi, Simona
name: Sabattini, Elena
name: Falzacappa, Maria Verga
name: Ronchini, Chiara
name: Testoni, Nicoletta
name: Baldazzi, Carmen
name: Papayannidis, Cristina
name: Abbenante, Maria
name: Marconi, Giovanni
name: Paolini, Stefania
name: Parisi, Sarah
name: Sartor, Chiara
name: Fontana, Maria
name: Matteis, Serena De
name: Iacobucci, Ilaria
name: Pelicci, Pier
name: Cavo, Michele
name: Yen, Timothy
name: Martinelli, Giovanni
Publisher: name: figshare
Year: 2018
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Molecular Biology
subject: Pharmacology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: FOS: Chemical sciences
subject: Immunology
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Hematology

description: Figure S1. Efficacy of AZD-1775 used as single agent. A) The graph shows the IC50 values of B/T-ALL cell lines treated with AZD-1775 for 24, 48, and 72 h. B) Cell viability analysis on CCRF-CEM cell lines showing the effect of high doses of AZD-1775. The percentage of viable cells is depicted relative to untreated controls. C) Immunoblot analysis on BV-173 treated with AZD-1775 (IC50) for 12 h. D) Cell cycle analysis in BV-173 and CCRF-CEM cell lines treated with AZD-1775 (IC50) for 12 h. E) Immunofluorescence analysis of BV-173 cells treated with AZD-1775 (IC50) for 12 h and, then, stained with DAPI and phospho-MPM2. In the picture, a cell dying in mitosis is reported with apoptotic bodies strongly positive for phospho-MPM2 antibody. Representative images are shown at × 100 magnification. F) Viability of mononuclear cells isolated from the peripheral blood of 5 healthy donors incubated with increasing concentration of AZD-1775 (2.5, 5, and 10 uM) for 24 h. G) MYT1 transcript levels in samples isolated from adult BCR-ABL1-positive ALL at diagnosis (n = 17), adult BCR-ABL1-negative ALL at diagnosis (n = 27), adult BCR-ABL1-positive ALL at relapse (unpaired, n = 8), adult BCR-ABL1-negative ALL at relapse (unpaired, n = 6), and in MNCs (n = 7) from the peripheral blood of healthy donors. One-way ANOVA test was performed to assess statistical significance. Results are expressed as Log10 2 exp.[−(ΔΔCt). Figure S2. AZD-1775 in combination with chemotherapy agents and tyrosine kinase inhibitors. A) Growth curve of BV-173 and REH cell lines treated for 4 days with AZD-1775 (185 nM) and doxorubicin (25 nM). B) Viability analyses in ALL cell lines incubated for 24 h with Bos or Bos-I (6 to 5000 nM). The percentage of viable cells is depicted relative to untreated controls. C) Cell viability analysis of BV-173 cell line treated with AZD-1775 (6 to 5000 nM, dilution rate 1:3) and with ponatinib (25, 50, 100 nM) or imatinib (250, 500, and 1000 nM) for 24 h. The percentage of viable cells is depicted relative to untreated controls, 0.1%. Figure S3. Wee1 mRNA expression across different cancer types from the Cancer Cell Line Encyclopedia (CCLE) database. A) Box plots showing the level of expression of Wee1 mRNA in different tumor samples, extracted from CCLE [63]. The red arrows point to B/T-ALL samples. Boxes define the 25th and the 75th percentiles, horizontal line within the boxes indicates the median, and whiskers define the 10th and the 90th percentiles. (PDF 1918 kb)