Figure 2: Expression of TNS1 is upregulated during in vitro human terminal erythroid differentiation.
(A) mRNA levels of TNS1, β-actin (ACTB), α1-spectrin (SPTA1), and protein 4.1R (EPB41) in CD34+ cell cultures on days 7, 11, and 14 of differentiation, measured by TaqMan qRT-PCR. Values are mean ± SD from 5 individual CD34+ cultures. Fold-change in gene expression normalized to day 7 cells. *p<0.05; ***p<0.001; ****p<0.0001. (B) Representative Western blots of TNS1, total actin, protein 4.1R, (α1β1)-spectrin, and total protein of differentiated CD34+ cells, and U118 glioblastoma cells; 15 μg protein loaded per lane. Asterisk, full-length TNS1 in U118 cells (Mr ~220kDa); Red arrow, immunoreactive TNS1 polypeptide in erythroid cells (Mr ~125kDa). (C) Quantification of immunoblots normalized to total protein. Band intensities analyzed by ImageJ. Values are mean ± SD from 6 individual CD34+ cultures. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. (D) Maximum intensity projections of Airyscan Z-stacks of cells from day 14 cultures, prior to and at various stages of enucleation, stained for F-actin (red), TNS1 (magenta), GPA (green), and nuclei (blue). Scale bar, 5μm.