Effect of Hls5 on erythroid differentiation. (A) Fetal liver progenitors were exposed to Hls5 or Hls5-myc retroviruses, then plated in methylcellulose in the presence of Epo (5 U/mL); CFU-E and BFU-E were determined after 2 and 7 days, respectively. CFU-M were determined separately after 7 days. Each result is the mean plus or minus SD (n = 3) and displayed as percentage of control cultures. (B) Immunoblot analysis of exogenous Hls5 in J-Hls5 / J-Hls5-myc clones, probed with antibodies against Hls5 and Erk2 (loading control). (C) J-Hls5-myc cells were exposed to Epo (5 U/mL), and 48 hours after induction Hls5 protein was visualized using anti-myc antibodies. For all confocal analysis slides were mounted in 50 mM Tris HCL pH 8.0, 50% glycerol, 2.5% 1,4-diazobicyclo-[2,2,2]-octane (DABCO; Fluka, Castle Hill, Australia) containing 0.00005% Hoescht 33258 (Calbiochem, San Diego, CA). Micrographs were acquired with a Bio-Rad MRC 1024 UV Laser scanning confocal microscope (Bio-Rad, Hemel-Hempstead, United Kingdom) using a Nikon (Tokyo, Japan) 40× Fluor, NA 1.15 water immersion objective. Images were prepared for publication using Confocal Assistant (v4.02, BioRad). (D) Cytocentrifuge preparations of J2E and J-Hls5 cells were Wright-stained and mounted in DePex Mounting Medium (Biolabs, Vic, Australia). Micrographs were acquired using an Olympus IX71 inverted microscope fitted with a 40×/0.6 NA objective, connected to an Olympus DP70 microscope digital camera (Olympus, Tokyo, Japan). Image-Pro Plus (v4.5, MediaCybernetics, Bethesda, MD) was used to process acquired images. (Bar represents 20 μm). (E) The clonogenicity of various J2E and J-Hls5 clones was determined by plating cells in soft agar. Each result is the mean plus or minus SD (n = 3). (F) Growth rate of various J2E and J-Hls5 clones was determined by MTT assays. Each result is the mean plus or minus SD (n = 3). (G) J2E (blue trace) and J-Hls5 (red trace) clones were released from a nocodazole-induced G₂/M block, and at the times indicated cell cycle progression was assayed by propidium iodide staining and visualized by flow cytometry. (H) Hemoglobin production of J2E and J-Hls5 cells after Epo induction was determined by benzidine staining. Each result is the mean plus or minus SD (n = 3). (I) Quantitative RT-PCR was performed on J2E cells expressing vector-alone, Hls5, or Hls5-myc. Transcript levels are displayed relative to β-actin (loading control). Each result is the mean (± SD) of 3 independent assays performed in triplicate. (J) Immunoblot (IB) analysis of J2E (vector alone) and J-Hls5 clones during Epo-induced erythroid differentiation, probed with antibodies against globins, GATA-1, and v-raf (loading control). GATA-1 levels (relative to v-raf) were quantitated and represented as a percentage of the GATA-1 (vector-alone) value at 0h. (K) Wright-stained cytocentrifuge preparations of J-Hls5 clones during erythroid terminal differentiation. Bar represents 20μm. Images were acquired as in panel D.