Fig 2: Neuronally enriched endosomal proteins Nsg-1 (NEEP21) and Nsg-2 (P19) are highly expressed in Satb2-, Ctip2-, and GAD65-positive neurons, but expressed at low/undetectable levels specifically in Prox1-positive neurons.(A) Nsg-1/NEEP21 (red) is specifically enriched in Neu-N-positive neurons (green), but undetectable in GFAP-positive astrocytoes (blue) in DIV9 rat hippocampal cultures. (B) Nsg-1/NEEP21 (red) and its family member Nsg-2/P19 (green) are present in the somatic Golgi region as well as in dispersed dendritic endosomes. (C-G) Nsg-1/NEEP21 and Nsg-2/P19 were co-localized with cell-type markers Satb2 (C, C’), GAD65 (D, D’), Ctip2 (E, E’), and Prox1 (F, F’). For each combination, the cell type marker is shown with either MAP2 (blue) and P19/Nsg-2 (green) (C, D, E, F), or with Nsg-1/NEEP21 (red) and Nsg-2/P19 (green) (C’,D’, E’, F’). In each panel, cells positive for the marker and NEEP21+P19 are labeled with “1”, cells negative for the marker and positive for NEEP21+P19 with “2”, cells negative for the marker and low for NEEP21+P19 with “3”, and cells positive for the marker but low for NEEP21+P19 with “4”. (G) Quantification of MAP2-positive neurons positive for one marker (Ctip2, Prox1) expressing either high levels of Nsg-1/NEEP21 and Nsg-2/P19 (black bars; correspond to type “1” neurons in panels C-F) or low levels of NEEP21+P19 (grey bars; correspond to type “4” neurons in panels C-F). Several hundred MAP2-positive neurons were scored for each marker in each of two independent experiments.

Fig 3: Non-exclusivity in expression of transcription factors used as cell type markers.(A,B) Quadruple stainings of DIV9 rat hippocampal neurons with MAP2 (white), Ctip2 (red), Satb2 (green), and Prox1 (blue) (A), or MAP2 (white), GAD65 (green), Prox1 (blue), Ctip2 (red) (B). (A’) and (B’) show the same field with MAP2 staining omitted. (C-F) show examples of single (s) and dually (d) positive neurons. (C) Ctip2/Prox1-single (s) and dually (d) positive neurons. (D) single-positive Ctip2- or Satb2-positive neurons. (E) Gad65-positive neuron not expressing Prox1 or Ctip2. (F) GAD65/Satb2 double-positive neuron and Ctip2/Satb2 double-positive neuron. (G) Venn diagram showing the abundance of single and double-positive neurons. Two independent DIV9 hippocampal cultures were scored and normalized to MAP2-positive cells. In each experiment, 300–400 MAP2-positive neurons were counted.

Fig 4: Differential transfectability of different neuronal cell types.Hippocampal neurons were either transfected with Lipofectamine2000 at DIV6 (A) or eletroporated at DIV0 prior to plating (B) and GFP-expressing MAP2-positive neurons were scored for the presence of Satb2, Ctip2, Prox1, or GAD65 (as indicated) at DIV9 (A) or DIV5 (B). The relative abundance of each marker-positive neuron is indicated by the red bar (data from Fig 2). Four GFP-encoding plasmids were used: CMV-promoter driven GFP (black bars), CAG-promoter driven (brick bars), DCX-promoter driven (white bars), short hairpin plasmid (sh; striped bars). The transfection efficiency normalized to the relative abundance is indicated as percentages below each bar. The mean and standard deviation of 2 independent experiments is shown. Total number of MAP2-positive neurons counted was between 75 and >400 per marker and experiment, depending on the abundance of the marker.