Fig 2: Activation of calcium signaling by Dr-TrkA. a Images of HeLa cells co-expressing mCherry-Dr-TrkA and GCaMP6m. Upper row: cells under constant 660 nm light (0.5 mW cm−2) were stimulated with 20 s pulse of 780 nm light (0.5 mW cm−2). Bottom row: the same cells under constant 660 nm light (0.5 mW cm−2). Note the decrease of cellular calcium in the last images. Scale bar, 10 μm. b Changes of GCaMP6m fluorescence for Dr-TrkA-expressing cells either stimulated by 780 nm light for 20 s (black line) or kept under 660 nm light (red line). c Images of HeLa cells co-expressing TrkA-DsRed2 and GCaMP6m. Upper row: cells before and after addition of 50 ng ml−1 nerve growth factor (NGF). Bottom row: cells stimulated with NGF in the presence of 100 nM of K252a. Note the decrease of cellular calcium in the last images. Scale bar, 10 μm. d Changes of GCaMP6m fluorescence upon stimulation of the cells with NGF in the absence (black line) or presence of K252a (red line). Error bars represent s.d., n = 3 experiments

Fig 3: Light-induced activation of MAPK/ERK pathway in mice. a PC6-3 cells were co-transfected with pCMVd2-Dr-TrkA, pFA-Elk-1, and pFR-RLuc8 plasmids and injected 25 h later in mammary glands of female mice. Mice were kept in transparent cages and illuminated from below with either 660/25 nm or 780/25 nm LED arrays at 3 mW cm−2 for 19 h. Then, animals were injected with a coelenterazine substrate for RLuc8 luciferase through retro-orbital vein and immediately imaged with an IVIS Spectrum instrument. b Quantification of the RLuc8 reporter signals shown in a. Error bars represent s.d., n = 3 experiments

Fig 4: Induction of apoptosis and viability after Entrectinib and Larotrectinib treatment in hES-MP cells expressing LMNA::TRKA and ETV6::TRKC fusion proteins.A hES-MP cells from clones 1 and 1.14, 3 and 3.3 and clone G2 before and after expression of Cre recombinase were exposed for 72 h to two doses of Entrectinib (20 and 200 nM) and Larotrectinib (200 and 1000 nM) and Annexin V was quantified as a measure of apoptosis. Annexin V intensity is represented as the ratio of the percentage of untreated cells. Error bars represent standard deviation from the mean of n ≥ 3 independent experiments. Unpaired t test *p < 0.05, **p < 0.01, ***p < 0.001, if not indicated the difference is not statistically significant. B Cells expressing the LMNA::TRKA protein (clone 1.14) and the corresponding parental cells (clone 1) were treated at increasing concentration of Entrectinib and Larotrectinib for 7 days continuously, before determination of cell viability. Results are presented as the mean of three independent experiments.

Fig 5: Generation of the ETV6::NTRK3 chromosomal translocation in hES cells and differentiation to hES-MP.A Breakpoints within ETV6 on chromosome 12 and NTRK3 on chromosome 15 create the ETV6::NTRK3 t(12;15)(p13;q25). B A donor template containing a puromycin selectable marker under the control of a PGK promoter is inserted by HDR at the ETV6 and NTRK3 participating loci after induction of DSBs by sgRNA (scissors). The selectable marker is removed by Cre recombinase allowing the expression of the oncogenic fusion. C Dual color FISH analysis with telomeric (red) and centromeric (green) probes on ETV6 showing the ETV6::NTRK3 translocation on clone G2. D RT-PCR time course (days) for ETV6::NTRK3 fusion after Cre expression in hES and hES-MP cells. The fusion product is detectable in cells before Cre recombination indicating a partial splicing of PGK-Puro sequence. E Western blot analysis showing the ETV6::TRKC fusion protein and the TRKC phosphorylation in hES-MP clone G2-6 and G2 after expression of Cre recombinase. The total ETV6::TRKC protein was detected by pan-TRK antibodies and the TRKC phosphorylation by phospho TRKA/B antibodies (same used for detection of LMNA::pTRKA).