Supplementary Figure 2: PBX1 induces morphological and cytoskeletal changes consistent with differentiation in NB. A) Morphology of SHSY5Y cells. Cells.

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Supplementary Figure 2: PBX1 induces morphological and cytoskeletal changes consistent with differentiation in NB. A) Morphology of SHSY5Y cells. Cells were grown in complete media in the presence of DMSO (left column) or 10 µM 13-cis RA for 5 days, then examined under 10X magnification phase microscopy. Neurite extensions are indicated (open arrows). Black scale bar = 50 μm. B) Morphology of SK-N-BE(2) cells. Cells were grown in complete media in the presence of DMSO (left column) or 10 µM 13-cis RA for 5 days, then examined under 10X magnification phase microscopy. Neurite extensions are indicated (open arrows); white scale bar = 400 μm. C-E) Immunofluorescence of TUBB3 in SK-N-SH cells (C), SK-N- RA cells (D), and SHSY5Y cells (E). Cells were grown in complete media with DMSO or 10 µM 13-cis RA for 5 days, then passaged onto CultureSlides and grown under the same conditions for an additional 2 days. Cells were fixed and incubated with anti-TUBB3 antibody, then treated with AlexaFluor®488-conjugated secondary antibody and mounted with DAPI stain, then imaged under 40X magnification fluorescence microscopy. Complete sets of images can be found in the Supplementary Data. Green = β-3 tubulin; blue = nucleus. Total PBX1 and TUBB3 expression is measured in Figure 2.

SHSY5Y Vector SHSY5Y PBX1 Pool 1 SHSY5Y PBX1 Clone 3 SHSY5Y shPBX1 #4 SHSY5Y shPBX1 #5 DMSO13-cis RA Supplementary Figure 2A

SK-N-BE(2) Vector SK-N-BE(2) PBX1 Pool 1 SK-N-BE(2) PBX1 Pool 2 SK-N-BE(2) shPBX1 #3 SK-N-BE(2) shPBX1 #5 DMSO13-cis RA Supplementary Figure 2B

DMSO13-cis RA SK-N-SH Vector Control SK-N-SH PBX1 Clone 3 SK-N-SH PBX1 Pool 1 SK-N-SH PBX1 Pool 2 SK-N-SH shPBX1 #4 SK-N-SH shPBX1 #5 Supplementary Figure 2C

DMSO13-cis RA SHSY5Y Vector Control SHSY5Y PBX1 Pool 1 SHSY5Y PBX1 Clone 3 SHSY5Y shPBX1 #4 SHSY5Y shPBX1 #5 Supplementary Figure 2D

DMSO13-cis RA SK-N-RA Vector Control SK-N-RA PBX1 Pool 1 SK-N-RA PBX1 Clone 4 SK-N-RA shPBX1 #4 SK-N-RA shPBX1 #5 Supplementary Figure 2E