Fig 1: Combination of TNO155 and ribociclib additively inhibits cell cycle and induces apoptosis.(A) Upset matrix plot derived from RNAseq analysis, showing the overlapping numbers of significant genes (P adj< 0.05 and |FC|> 1.5) after 24-hour treatment with 0.3 µmol/L TNO155, 1 µmol/L ribociclib and their combination, normalized to DMSO control. (Rows=the sets; Columns=intersections between these sets). (B and C) Heatmaps demonstrating the more potent transcriptional inhibition of mitotic prometaphase (B) and cell cycle checkpoints (C) by combined TNO155+ribociclib relative to either single agent alone, highlighting BIRC5, PLK1, CLSPN and CCNA2. + = significance. (D) Five NF1-MPNST cell lines were treated with DMSO, 0.3 µmol/L TNO155 and/or 1 µmol/L ribociclib for 48 hours, following overnight starvation in 0.1% FBS-containing culture medium to synchronize cells. Cells were fixed in ice cold 70% ethanol and stained with propidium iodide/ RNase staining solution (Cell Signaling Technology, #4087), and then analyzed by flow cytometry. (E) ST8814 and JH-2–079c were treated with DMSO, 0.3 µmol/L TNO155 and/or 1 µmol/L ribociclib for 48 hours, and then protein lysates were assessed using human apoptosis antibody array (R&D systems, #ARY009). Signal intensity from technical duplicates was quantified by densitometry analysis using image J and normalized to DMSO. Data represent mean ± SEM. (F) Nine NF1-MPNST cell lines were treated as in E. and the indicated proteins involved in apoptosis and ERK signaling were detected using immunoblot.
Fig 2: Combination of TNO155 and ribociclib additively inhibits cell cycle and induces apoptosis.(A) Upset matrix plot derived from RNA-seq analysis, showing the overlapping numbers of significant genes (P adj < 0.05 and |fold change| > 1.5) after 24-hour treatment with 0.3 μM TNO155, 1 μM ribociclib, and their combination, and normalized to DMSO control. Rows, the sets; columns, intersections between these sets. (B and C) Heatmaps demonstrating the more potent transcriptional inhibition of mitotic prometaphase (B) and cell cycle checkpoints (C) by combined TNO155 and ribociclib relative to either single agent alone, highlighting BIRC5, PLK1, CLSPN, and CCNA2 (P adj < 0.05 marked with + and LFC). (D) Five NF1-MPNST cell lines were treated with DMSO, 0.3 μM TNO155, and/or 1 μM ribociclib for 48 hours, following overnight starvation in 0.1% FBS-containing culture medium to synchronize cells. Cells were fixed in ice cold 70% ethanol and stained with propidium iodide/ribonuclease staining solution (Cell Signaling Technology, no. 4087) and then analyzed by flow cytometry. (E) ST8814 and JH-2-079c were treated with DMSO, 0.3 μM TNO155, and/or 1 μM ribociclib for 48 hours, and, then, protein lysates were assessed using human apoptosis antibody array (R&D Systems, no. ARY009). Signal intensity from technical duplicates was quantified by densitometry analysis using ImageJ and normalized to DMSO. Data represent means ± SEM. (F) Nine NF1-MPNST cell lines were treated as in (E), and the indicated proteins involved in apoptosis and ERK signaling were detected using immunoblot.
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