Fig 2: Proteome dynamics induced by NGF−mediated NTRK1 activation in IMR5 cells. (A) Volcano plots illustrating the differential expression of proteins for the time points indicated. Significantly differentially abundant proteins highlighted in red (p-value ≤ 0.05; FC ≤ −1.5 or ≥ 1.5), selected proteins highlighted in green. (B) Heatmaps representing hierarchical cluster analysis (Pearson correlation, complete linkage) of significantly regulated proteins. Magnifications show clusters of proteins with late and early increasing abundance, respectively. (C) Regulation profiles of selected protein candidates in NGF-treated cells (blue) compared to untreated controls (green).

Fig 3: Validation of inducible expression and activation of NTRK1 in four neuroblastoma cell lines. (A) NTRK1 expression was verified in the NTRK1-negative cell lines NGP, IMR5, SKNAS and SH-SY5Y using qPCR at time points indicated. (B) NTRK1 activation was validated for time points t = 1 h, 4 h and 24 h upon treatment with NGF using immunostaining of phosphorylated NTRK1 (pTrkA Y674/675). Numbers indicate the intensity ratio of the TrkA/pTrkA bands and Gapdh. Uncropped blots were added in supplemental materials.

Fig 4: Changes in nuclear localization of LMNA upon NTRK1 expression and activation. Representative immunofluorescence stainings of LMNA (green) illustrating accumulation of LMNA within foci inside the nuclei upon NTRK1 expression and activation for IMR5 (A) and SH-SY5Y cells (B) (actin = red, DAPI = blue). In the merged pictures, zoom-ins are marked to highlight the foci formation in individual cells. (C,D) Area-corrected count of LMNA foci in IMR5 (C) and SH-SY5Y cells (D). Individual data points correspond to analyzed pictures. Significance (ANOVA with subsequent Tukey’s post hoc analysis **** p < 0.0005, p-values Bonferroni-corrected) is indicated by asterisks.

Fig 5: YM155 and lapatinib show high degree of synergy in neuroblastoma regardless of the MYCN and TRKA status. (A) Overview of the drug library used in the concentration matrix-based combination screen. (B) Chemical structures of lapatinib and YM155. (C) Microarray-based gene expression profiling. Bar graphs are showing the number of significantly upregulated and downregulated genes in each condition. (D) Colony formation assay; YM155 (black border) was used at 1/16 IC50 concentration (15 nM), lapatinib (gray border) at 1/8 IC50 (1 µM). Combination treatment (blue border): 15 nM YM155 and 1 µM lapatinib. DMSO (no border) treated cells were used as a control. Data shown as mean ± s.e.m. and images are representative of three individual experiments (ANOVA, **P < 0.01). (E) Heatmap of CI values in the YM155 and lapatinib combination matrix, indicating a strong synergistic interaction. Data represent the mean of triplicates. (F,G) Synergistic effect is preserved in NTRK1 and MYCN highly expressed state. (F) Left, overexpression and activation of TRKA signaling in SH-SY5Y; right, heatmap of CI values in the combination matrix; data represent the mean of triplicates. (G) Left, overexpression of MYCN in SH-SY5Y and levels of MYCN protein in the IMR5–75 MYCN amplified cell line; right, heatmap of CI values in the combination matrix; data represent the mean of triplicates.