American Society of Hematology

Figure 3

$Figure 3. Hoxa9 colocalizes with Smad4 in the cytoplasm of primitive hematopoietic cells immortalized by HOXA9 or NUP98-HOXA9. (A) Transmission microscopy showing the colocalization of Smad4 (green) and Hoxa9 (red) in the cytoplasmic fraction of Wt primitive hematopoietic cells immortalized with HOXA9 (*), while the colocalization is not observed in more differentiated cells (δ). Experiment was performed using total hematopoietic cells immortalized at serial passage 2 or 3 using CFU assay. (B) Confocal microscopy in sequential scanning mode, using a 63× oil-immersion objective showing the colocalization of Smad4 (green) and Hoxa9 (red) in the cytoplasmic fraction of Wt HSPCs immortalized by HOXA9. (C) Immunostaining showing accumulation of Smad4 and Hoxa9 in the cytoplasmic fraction of Wt HSPCs immortalized by HOXA9 or NUP98-HOXA9 (left panel) and cytoplasmic/nuclear localization of Hoxa9 in Smad4−/− HSPCs immortalized by HOXA9 or NUP98-HOXA9 (right panel; representative of 3 individual experiments). (D) Representative Western blot showing increased expression of the Hoxa9 protein in the nucleus of Smad4−/− HSPCs immortalized by HOXA9 (light blue dots) or NUP98-HOXA9 (pink dots) and presentation of the relative increase compared with control samples (Mig vector, blue dark dots), representative of 3 individual experiments. (E) Activation of specific Hoxa9 targets in Smad4−/− HSPCs immortalized by HOXA9 or NUP98-HOXA9 (relative fold change in mRNA expression compared with Wt Mig HSPCs, data show mean ± SEM, n = 4, *P < .01 measured by Student unpaired t test). (B-E) HSPCs (Lin− cells) were isolated from hematopoietic cells generated at serial passage 2 or 3 using CFU assay (HSPCs immortalized by HOXA9 or NUP98-HOXA9). Control HSPCs (Lin− cells) were isolated from hematopoietic cells generated from first passage (HSPCs not immortalized, transduced with Mig).$

Hoxa9 colocalizes with Smad4 in the cytoplasm of primitive hematopoietic cells immortalized by HOXA9 or NUP98-HOXA9. (A) Transmission microscopy showing the colocalization of Smad4 (green) and Hoxa9 (red) in the cytoplasmic fraction of Wt primitive hematopoietic cells immortalized with HOXA9 (*), while the colocalization is not observed in more differentiated cells (δ). Experiment was performed using total hematopoietic cells immortalized at serial passage 2 or 3 using CFU assay. (B) Confocal microscopy in sequential scanning mode, using a 63× oil-immersion objective showing the colocalization of Smad4 (green) and Hoxa9 (red) in the cytoplasmic fraction of Wt HSPCs immortalized by HOXA9. (C) Immunostaining showing accumulation of Smad4 and Hoxa9 in the cytoplasmic fraction of Wt HSPCs immortalized by HOXA9 or NUP98-HOXA9 (left panel) and cytoplasmic/nuclear localization of Hoxa9 in Smad4^−/− HSPCs immortalized by HOXA9 or NUP98-HOXA9 (right panel; representative of 3 individual experiments). (D) Representative Western blot showing increased expression of the Hoxa9 protein in the nucleus of Smad4^−/− HSPCs immortalized by HOXA9 (light blue dots) or NUP98-HOXA9 (pink dots) and presentation of the relative increase compared with control samples (Mig vector, blue dark dots), representative of 3 individual experiments. (E) Activation of specific Hoxa9 targets in Smad4^−/− HSPCs immortalized by HOXA9 or NUP98-HOXA9 (relative fold change in mRNA expression compared with Wt Mig HSPCs, data show mean ± SEM, n = 4, *P < .01 measured by Student unpaired t test). (B-E) HSPCs (Lin⁻ cells) were isolated from hematopoietic cells generated at serial passage 2 or 3 using CFU assay (HSPCs immortalized by HOXA9 or NUP98-HOXA9). Control HSPCs (Lin⁻ cells) were isolated from hematopoietic cells generated from first passage (HSPCs not immortalized, transduced with Mig).

This Feature Is Available To Subscribers Only