HOXB4 Can Enhance the Differentiation of Embryonic Stem Cells by Modulating the Hematopoietic Niche

Melany Jackson, Richard A. Axton, A. Helen Taylor, Julie A. Wilson, Sabrina A. M. Gordon-Keylock, Konstantinos D. Kokkaliaris, Joshua M. Brickman, Herbert Schulz, Oliver Hummel, Norbert Hubner, Lesley M. Forrester

In Figure 5C of the originally published article the labeling “Control” and “Hoxb4ERT2” were inadvertently switched. The corrected figure is published here.

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Figure 5. Frzb is upregulated by HOXB4 and requires protein synthesis for its induction. (A): Expression of Frzb by quantitative RT-PCR (qRT-PCR) in HOXB4 ERT2-expressing ESCs and wild-type controls both without and with 200 nM 4-OHT for 24 and 48 hours (days 2 + 3 of differentiation, respectively). Frzb expression is shown as fold induction relative to no 4-OHT. Experiments were performed three times, and error bars represent standard deviations (*, p < .05 by Mann--Whitney U test) unless stated otherwise. (B): Plasmids to express HOXB4, FRZB, HOXB4VP16, or homeodomain mutants: Hoxb4(N266P)VP16 (Mut1) and Hoxb4(del266)VP16 (Mut2) were cotransfected into wild-type ESCs for a dual luciferase assay to detect activity of the Frzb promoter Frzb-luc. pGL-3-luc (pGL3 basic Promega) was used as a control reporter plasmid, and eGFP was used as a control effector plasmid. (C): Induction of Frzb after activating HOXB4 ± CHX is shown as fold compared with wildtype -- 4-OHT. Control and Hoxb4ERT2 ESCs were cultured in monolayers either ± 200 mM 4-OHT and ± 100 μg/ml CHX protein synthesis inhibitor for 5 hours and Frzb expression was measured by qRT-PCR. Two replicate experiments were performed and error bars represent the range between the two experiments. (D): Expression of Hoxb4 by qRT-PCR in wild-type and three human RC7 Es cell clones stably transfected with a Hoxb4ERT2 transgene. Hoxb4 expression is shown as fold expression compared with wild type. (E): Frzb expression in the human Hoxb4-expressing ESC clones both + and --4-OHT. (F): Expression of Frzb and Hoxb4 by qRT-PCR are shown for mouse hematopoietic tissues and are expressed as fold expression compared with bone marrow. Abbreviations: AGM, aorta gonad mesonephros; CHX, cycloheximide; eGFP, enhanced green fluorescent protein; 4-OHT, 4-hydroxytamoxifen; PAS, para aortic splanchopleura; RT-PCR, reverse-transcription polymerase chain reaction.

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