Fig 1: Effect of ERK inhibition and retinoic acid on growth factor activity. (A) – Distributions of ERK activity (violin plots) 30 min after addition of the growth factors EGF, IGF-1, NGF, and BDNF (100 ng/mL each) to SH-SY5Y, U-251, and TE-671 cells pretreated with 10 μM retinoic acid (ATRA) or 250 nM of the ERK inhibitor SCH772982 (SCH) for 72 h. DMSO-treated cells were used as the control. A non-treated control (NTC) is a cell added with the culture medium without growth factors. Dots indicate median values for each of 6 analyzed independent, randomly selected fields. Standard deviations (SDs) are shown in violin plots. Statistical significance is determined using the nonparametric Mann–Whitney U-test. (B) – Expression of the EGFR, IGF-1R, NTRK1 (TrkA), and NTRK2 (TrkB) genes by real-time PCR 72 h after the addition of agents. PCR data are normalized to the expression of the GAPDH gene in each sample; the results are presented as a logarithm of the change in gene expression relative to the control (DMSO-treated cells). Gene expression measurements were performed in triplicate. Plots show the mean expression change and a 95% confidence interval. Statistical significance is determined using the Student’s t-test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001
Fig 2: Head and neck cancer patient derived exosomes induce neurite outgrowth. Quantified neurite outgrowth of PC12 cells following plasma (a) or tissue (b) exosome stimulation. NGF (100 ng/ml) serves as a positive control. Nl1- Nl3 non-cancer control blood, Pt1-Pt8 cancer patients, TL1,2 normal tonsils. n = 3 technical replicates/sample; assay repeated at least n = 2 times with similar results. The exosome yield from TL1 was low and insufficient for multiple replicates. For statistical analysis, one-way ANOVA with post-hoc Fisher’s Least Significant Difference (LSD) test was used; LSD p values reported; *p ≤ 0.03; ns, not significant, center value used was the mean. Error bars, standard deviation. The variance between groups compared is similar. All comparisons and LSD p values found in Supplementary Tables 1-3
Fig 3: Compromised exosome release leads to sparsely innervated tumors. a Neurite outgrowth of PC12 cells following exosome stimulation from the indicated sources. Stimulation with recombinant NGF (100 ng/ml) serves as a positive control. n = 4 technical replicates/condition; experiment repeated at least three times with biological replicates. Statistical analysis by one-way ANOVA with post hoc Fisher’s Least Significant Difference (LSD) test. LSD p values reported; *p ≤ 0.02. The variance between groups compared is similar. Central value used is the mean. All comparisons and p values found in Supplementary Table 4. b Western blot analysis of whole tumor lysate from mice bearing mEERL parental or Rab27A−/+Rab27B−/− tumors. n = 3 technical replicates. n = 4 biological replicates. Western blots have been cropped for clarity and conciseness. Western blot quantification by densitometry for c β-III tubulin, *p<0.05; d Tau, *p<0.0001; and e TRPV1, *p<0.001. Statistical analysis by unpaired, two-tailed Student’s t-test. Central value used was the mean. The variance between groups compared is similar. f Nanoparticle tracking analysis of exosomes purified from the plasma of tumor bearing mice treated with GW4869 or vehicle. Statistical analysis by two-tailed Student’s t-test. Central value used was the mean. The variance between groups compared is similar; *p<0.0001. g Quantification of β-III tubulin IHC staining of tumors from mice (n = 7/group) treated with GW4869 or vehicle; Statistical analysis by unpaired, two-tailed Student’s t-test; *p<0.05. Central value used was the mean. Experiment performed once. The variance between groups compared is similar. h En face bright field representative images of IHC for β-III tubulin of mEERL tumors treated with GW4869 or vehicle. n = 7/group. Scale bar, 100 µm. All error bars are standard deviation
Fig 4: mEERL exosomes induce neurite outgrowth without NGF. a PC12 cells were stimulated with 100 ng/ml of recombinant NGF (NGF), mEERL parental (parental) or mEERL EphrinB1 (EphrinB1) conditioned media with or without neutralizing anti-NGF antibody (+Ab); 24 h later, cells were fixed and stained for β-III tubulin expression which was then quantified. Statistical analysis by two-way ANOVA with post hoc Fisher’s Least Significant Difference (LSD) test. LSD p values reported; *p<0.006. ns, not significant. The variance between groups compared is similar. n = 3 technical replicates/condition; experiment repeated at least two times with biological replicates. Central value used was the mean. All comparisons and LSD p values found in Supplementary Table 4. b Western blot analysis of exosomes (Exo) or whole cell lysate (WCL) from the indicated mEERL cell lines. Western blot repeated at least n = 2 times with biological replicates. c Neurite outgrowth quantification of PC12 cells following exosome treatment from the indicated sources. Statistical analysis by one-way ANOVA with post hoc Fisher’s Least Significant Difference (LSD) test. LSD p values reported; *p<0.02; ns, not significant. n = 5 technical replicates/condition; experiment repeated at least twice with biological replicates. Central value used was the mean. The variance between groups compared is similar. All comparisons and LSD p values found in Supplementary Table 5. d Western blot analysis of exosomes purified from the indicated mEERL cell lines. EphrinB1-Ex, EphrinB1 extracellular epitope antibody. EphrinB1-In, EphrinB1 intracellular epitope antibody. Western blot repeated at least n = 2 times with biological replicates. e Western blot analysis of PC12 lysate following stimulation for 5 min with PBS, NGF (50 µg/ml) or exosomes purified from mEERL parental, EphrinB1, or Null2 conditioned media. Experiment repeated at least three times with biological replicates. All error bars are standard deviation. Western blots have been cropped for clarity and conciseness
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