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 quantitative reverse transcription polymerase chain reaction (qRT-PCR). Values are mean ± standard deviation [SD] from 5 individual CD34+ cultures. Fold-change in gene expression normalized to day 7 cells. ∗P < .05; ∗∗∗P < .001; ∗∗∗∗P < .0001. (B) Representative western blots of TNS1, total actin, protein 4.1R, α1β1-spectrin, and total protein of differentiated CD34+ cells, and human U118 glioblastoma cells; 15 μg protein loaded per lane. Asterisk, full-length TNS1 in U118 cells (Mr ∼220 kDa); red arrow, immunoreactive TNS1 polypeptide in erythroid cells (Mr ∼125 kDa). (C) Quantification of immunoblots normalized to total protein. Band intensities analyzed by ImageJ. Values are mean ± SD from 6 individual CD34+ cultures. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. (D) Maximum intensity projections of Airyscan Z-stacks of cells from day-14 cultures, before and at various stages of enucleation, stained for F-actin (red), TNS1 (magenta), GPA (green), and nuclei (blue). Scale bar, 5 μm. ns, non-significant.

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 quantitative reverse transcription polymerase chain reaction (qRT-PCR). Values are mean ± standard deviation [SD] from 5 individual CD34+ cultures. Fold-change in gene expression normalized to day 7 cells. ∗P < .05; ∗∗∗P < .001; ∗∗∗∗P < .0001. (B) Representative western blots of TNS1, total actin, protein 4.1R, α1β1-spectrin, and total protein of differentiated CD34+ cells, and human U118 glioblastoma cells; 15 μg protein loaded per lane. Asterisk, full-length TNS1 in U118 cells (Mr ∼220 kDa); red arrow, immunoreactive TNS1 polypeptide in erythroid cells (Mr ∼125 kDa). (C) Quantification of immunoblots normalized to total protein. Band intensities analyzed by ImageJ. Values are mean ± SD from 6 individual CD34+ cultures. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. (D) Maximum intensity projections of Airyscan Z-stacks of cells from day-14 cultures, before and at various stages of enucleation, stained for F-actin (red), TNS1 (magenta), GPA (green), and nuclei (blue). Scale bar, 5 μm. ns, non-significant.

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