American Society of Hematology

Figure 2.

$Figure 2. CYTH1 regulates adhesion of human HSPCs through integrins. (A) CD34+ cells were transduced with CYTH1 shRNAs from a GFP-expressing vector and subjected to the adhesion assay. Representative FACS histograms showing GFP levels in adherent and nonadherent cells (left) and quantification of the ratios GFP frequencies expressing cells (right) from the adherent and nonadherent cell fractions; n = 4. (B) CYTH1 knockdown efficiency from CYTH1-sh1 and CYTH1-sh2 by quantitative polymerase chain reaction (n = 3). (C) CYTH1 knockdown efficiency from CYTH1-sh1 and CYTH1-sh2 by western blot (n = 1). (D) CD34+ cells transduced with SCR, β-galactosidase (BGAL), CYTH1-sh1, and CYTH1-sh2 were followed up with flow cytometry for maintenance of CD34 and GFP expression on day 4 after transduction (n = 4). (E) CD34+ cells transduced with SCR, BGAL, CYTH1-sh1, and CYTH1-sh2 were followed up with flow cytometry for maintenance of CD34 and GFP expression on day 3 or 4 and 7 or 8 after transduction (n = 5). (F) Nontransduced (mock), SCR, BGAL, CYTH1-sh1, and CYTH1-sh2 transduced CD34+ cells were assessed for cell cycle status (n = 2). (G) Average attachment area of transduced CD34+ cells plated onto RN and ICAM1-covered surface analyzed with IRM (n = 3). (H) Attachment dynamics (difference between maximum and minimum attachment area over time) analyzed with IRM (n = 3). (I) Representative photos from spinning disc confocal and total internal reflection fluorescence (TIRF) microscope of SCR and CYTH1-sh1 transduced CD34+ cells plated on RN (left) and summary of cell area analysis (n = 2) (right). (J) Representative FACS analysis of active ITGβ1 cell-surface expression in unstimulated (UNS) and PMA-stimulated (PMA) CD34+ cells transduced with SCR and CYTH1-sh1 (i). Table on the right summarizes the data from this experiment. “FMO” row depicts fluorescence minus 1 (FMO) control. A summary of 4 experiments on ITGβ1 activation including the representative experiment is shown on the right (ii). (K) Activation of Rap1 was analyzed in PMA-stimulated SCR and CYTH1-sh1 transduced cells. CYTH1-sh1, CYTH1-shRNA1; CYTH1-sh2, CYTH1-shRNA2; MFI, mean fluorescence intensity; PD, pull down; TL, total lysate.$

CYTH1 regulates adhesion of human HSPCs through integrins. (A) CD34⁺ cells were transduced with CYTH1 shRNAs from a GFP-expressing vector and subjected to the adhesion assay. Representative FACS histograms showing GFP levels in adherent and nonadherent cells (left) and quantification of the ratios GFP frequencies expressing cells (right) from the adherent and nonadherent cell fractions; n = 4. (B) CYTH1 knockdown efficiency from CYTH1-sh1 and CYTH1-sh2 by quantitative polymerase chain reaction (n = 3). (C) CYTH1 knockdown efficiency from CYTH1-sh1 and CYTH1-sh2 by western blot (n = 1). (D) CD34⁺ cells transduced with SCR, β-galactosidase (BGAL), CYTH1-sh1, and CYTH1-sh2 were followed up with flow cytometry for maintenance of CD34 and GFP expression on day 4 after transduction (n = 4). (E) CD34⁺ cells transduced with SCR, BGAL, CYTH1-sh1, and CYTH1-sh2 were followed up with flow cytometry for maintenance of CD34 and GFP expression on day 3 or 4 and 7 or 8 after transduction (n = 5). (F) Nontransduced (mock), SCR, BGAL, CYTH1-sh1, and CYTH1-sh2 transduced CD34⁺ cells were assessed for cell cycle status (n = 2). (G) Average attachment area of transduced CD34⁺ cells plated onto RN and ICAM1-covered surface analyzed with IRM (n = 3). (H) Attachment dynamics (difference between maximum and minimum attachment area over time) analyzed with IRM (n = 3). (I) Representative photos from spinning disc confocal and total internal reflection fluorescence (TIRF) microscope of SCR and CYTH1-sh1 transduced CD34⁺ cells plated on RN (left) and summary of cell area analysis (n = 2) (right). (J) Representative FACS analysis of active ITGβ1 cell-surface expression in unstimulated (UNS) and PMA-stimulated (PMA) CD34⁺ cells transduced with SCR and CYTH1-sh1 (i). Table on the right summarizes the data from this experiment. “FMO” row depicts fluorescence minus 1 (FMO) control. A summary of 4 experiments on ITGβ1 activation including the representative experiment is shown on the right (ii). (K) Activation of Rap1 was analyzed in PMA-stimulated SCR and CYTH1-sh1 transduced cells. CYTH1-sh1, CYTH1-shRNA1; CYTH1-sh2, CYTH1-shRNA2; MFI, mean fluorescence intensity; PD, pull down; TL, total lysate.

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