Fig 2: Functional role of TRADD/p75NTRinteraction in NF-?B signaling.A, representative micrographs of p75NTR knockout (KO) CGNs transfected with expression plasmids containing HA-tagged p75WT (wild-type) and HA-tagged p75K349A mutant. After 2 days in vitro, cultures were immunostained with anti-P65 NF-?B (green) and anti-HA (magenta) antibodies, and counterstained with DAPI (blue). Transfected cells expressing HA constructs are indicated with white arrows. Scale bar, 20 µm. B, quantification of nuclear p65 NF-?B (expressed as nuclear p65 fluorescence intensity/cytoplasmic p65 fluorescence intensity) in p75NTR wild-type (WT) and knockout (KO) CGNs transfected with HA-p75WTand HA-p75K349A plasmids as indicated. Results are expressed as mean ± SEM from three independent cultures (*p < 0.05 and **p < 0.01, one-way ANOVA followed by Turkey's multiple comparison test).

Fig 3: Induction of differentiation using the Ex.1 method. (A) Colony of human iris-derived iPS cells. (B-D) Cell colony in differentiation culture: (B) 6th day in STEP-1 medium, (C) 18th day in STEP-2 medium, and (D) 25th day in STEP-3 medium. Scale bar in (A-D), 100 µm. (E) Fold-increase in p75NTR mRNA expression level at different steps relative to STEP-0: *P<0.05. (F) Cell morphology on 35th day of differentiation in STEP-3 medium. (G) Immunostaining for aA-crystallin in the same area as indicated in (F). (H) Immunostaining for SOX2 in the same area as in (G). (I) Merged image of aA-crystallin and SOX2 immunostaining. Scale bar in (F-I), 100 µm. Ex, experiment; iPS cells, induced pluripotent stem cells.

Fig 4: Solution structure of the complex between the p75NTR DD and RhoGDI.(A) Superposition of backbone heavy atoms of the 10 lowest-energy structures of the human p75NTR DD:RhoGDI complex. N- and C-termini are indicated. (B) Ribbon drawing of the lowest-energy conformer. Light brown, p75NTR DD; Cyan, RhoGDI. N- and C-termini, as well as DD helices H1 and H6 are indicated. (C) Details of binding interface in the complex viewed in the same orientations as panel B, respectively. Key residues at the binding interface are labeled and depicted as stick models. Red labels denote interface residues functionally validated in our earlier mutagenesis study (Charalampopoulos et al., 2012). (D) Co-immunoprecipitation of wild type (WT) and DD point mutants of human p75NTR with Myc-tagged RhoGDI in transfected HEK 293 cells. Antibodies used for immunoprecipitation (IP) and Western blotting (WB) are indicated. WCE, whole cell lysate. The immunoblots shown are representative of three independent experiments. (E) Co-immunoprecipitation of WT and point mutants of Myc-tagged human RhoGDI with p75NTR in transfected HEK 293 cells. Antibodies used for immunoprecipitation (IP) and Western blotting (WB) are indicated. WCE, whole cell extract. The immunoblots shown are representative of three independent experiments.DOI: http://dx.doi.org/10.7554/eLife.11692.004

Fig 5: Characterization of ENCDC cultures. Flow cytometry of ENCDC cultures stained with antibodies against the immature neuronal marker, p75NTR+-FITC demonstrated 95.1% p75NTR+-positive, 97.3% viability in subculture 3, and 96.1% p75NTR+-positive, 98.2% viability in subculture 4 (a). Proportion of ENCDC cultures characterized for expression of p75NTR+, pH3, 5-HT, nNOS, and TH. Scale bar = 20 µm (b). There was a significant increase in the proportion of serotonergic neurons and nitrergic neurons in subculture 5 (gray) compared to subculture 4 (black), but no significant difference in proliferating cells or dopaminergic neurons across the cultures. *p = .05. Values are presented as mean ± SEM (c)