Fig 2: PAG1 inhibits c-Src and downstream effector proteins.A. Western blots of represented proteins in PAG1 overexpression (OEX), knockdown (KD) and control (C) NGP and SH-SY5Y cell lines. CyPB was used as a loading control for all blots, as it is not known and observed to be affected by PAG1 levels in NB cell lines. B. Mechanism of PAG1 action in neuroblastoma. PAG1 acts as an adaptor protein to recruit the Csk from cytoplasm at specific site Tyr-314 and bring it to close proximity with membrane bound c-SRC and other SFKs. Csk then directly inhibit c-Src and other SFks by phosphorylating the Tyr527 [5, 6]. c-Src is known to activate different cellular functions by controlling the effector proteins of different pathways (RAS, PI3K and JAK) either directly or indirectly [4]. Inhibition of c-Src by PAG1 leads to downstream inhibition of different cellular pathways that further translate in to reduction in overall proliferation and tumorigenicity, as confirmed by our in vitro and in vivo results in neuroblastoma.

Fig 3: PAG1 is a tumor suppressor in neuroblastoma in vivo.A. Tumor xenografts of different PAG1 gain and loss of function cell lines in NGP and B. SH-SY5Y were developed and tumor weights were analyzed. Data represented for individual tumors in cohorts and as mean ± SD (Mann-Whitney test, ns= non significant, **p<0.01). C. Western immunobloting analysis of tumor samples shows overexpression of PAG1 in NGP-OEX and SH-SY5Y-OEX tumor xenografts in comparison to control NGP and SH-SY5Y xenografts respectively. Protein was isolated from flash frozen tumor tissues. +C represents positive control and is the protein isolated from respective OEX cell line. OEX= PAG1-overexpression, KD= PAG1-knockdown.

Fig 4: PAG1 is a prognostic factor in neuroblastoma.Kaplan-Meier curves shows the probability of overall survival based on PAG1 expression level A. 88 patients in the Versteeg dataset, B. 649 patients in the Kocak dataset and C. 56 patients in the NCI-POB dataset. D. R2-Versteeg dataset analysis showing correlation of PAG1 expression levels to neuroblastoma recurrence or progression and overall outcome. E. R2-Kocak dataset analysis showing correlation of PAG1 expression levels to neuroblastoma disease stages.

Fig 5: PAG1 is a tumor suppressor in neuroblastoma in vitro.A. Quantitative PCR (qPCR) analysis of PAG1 mRNA expression in PAG1 overexpression and knockdown cell lines in NGP and SH-SY5Y. Experiment was performed in triplicates and repeated thrice. Data represented as mean ± SD (T-test, ***p<0.001, ****p<0.0001). PAG1 mRNA expression in control cell lines NGP and SH-SY5Y was used to quantitate relative expression. B. Cellular proliferation assay of PAG1 gain and loss of function in NGP and SH-SY5Y NB cell lines. Cell proliferation was measured using MTS assay over 120 h. Proliferation index over the time was calculated by normalizing to the 24 h time point. Experiments were repeated three times with six replicates for each condition and represented as mean ± SD. (T-test, **p<0.01, ***p<0.001). C. Representative pictures of colony formation assay for PAG1 gain and loss of function in NB cell lines. Survival index shows the quantitation of relative inhibition of colony formation in different cell lines and is normalized to control or original cell line. Data represented as mean ± SD (T-test, **p<0.01, ***p<0.001, ****p<0.0001). OEX= PAG1-overexpression, KD= PAG1-knockdown, shNC= negative/mock control knockdown.