Fig. 2: Constitutive MYCN expression sustains alt-NHEJ components during NCSC differentiation | Cell Death & Disease

Fig. 2: Constitutive MYCN expression sustains alt-NHEJ components during NCSC differentiation

From: Alternative NHEJ pathway proteins as components of MYCN oncogenic activity in human neural crest stem cell differentiation: implications for neuroblastoma initiation

Fig. 2

NCSC were double-sorted (p75, HNK-1) and plated as described. For forced MYCN expression, a doxycycline inducible pCL6TRRIP-MYCN vector was generated from a pCL6 lentiviral vector. Doxycycline was added to NCSC-MYCN to induce expression of MYCN. a and b Protein and mRNA levels of MYCN induction were confirmed using immunocytochemistry, western blot and RT-PCR. Representative immunocytochemistry images are shown for MYCN- (NCSC-MYCN without doxycycline) and MYCN+ (NCSC-MYCN with doxycycline) at 24 h post MYCN induction. MYCN+ cells had significant MYCN overexpression compared to MYCN- cells in all three methods. c Representative images of immunocytochemistry and western blot analysis for p75, an early NCSC marker are shown in MYCN- and MYCN+ at D14 in differentiation media. p75 expression levels in MYCN+ at D14 were similar to early normal NCSC at D1. d A quantitative color map was generated from western blot analysis (Supplemental Fig S1b) of protein level expression of alt-NHEJ components (Lig1, Lig3, PARP1). All three alt-NHEJ components tested were downregulated by D14 as normal NCSC differentiated in culture. Significantly, MYCN+ cells maintained significantly higher expression of alt-NHEJ proteins at D7 that did not decrease by D14. Bars represent mean ± 2SD. Statistical analyses were performed with ANOVA (n = 3), all experiments repeated in three times, (**p < 0.01)

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