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Cell Biology International (2012) 36, 195–202 (Printed in Great Britain)

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Ectopic expression of Hmgn2 antagonizes mouse erythroid differentiation in vitro

Kasem Kulkeaw*, Tomoko Inoue*†, Chiyo Mizuochi*, Yuka Horio*, Yasushi Ishihama‡ and Daisuke Sugiyama*¹

*Advanced Medical Initiatives, Division of Hematopoietic Stem Cells, Department of Advanced Medical Initiatives, Faculty of Medical Sciences, Kyushu University, Fukuoka, 8128582, Japan, †Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, 8128582, Japan, and ‡Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, 6068501, Japan

¹To whom correspondence should be addressed (email ds-mons@yb3.so-net.ne.jp).

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SUPPLEMENTARY ONLINE DATA

Figure S1 Isolation of the erythroid lineage from mouse FL by flow cytometry and HSC and erythroid cell surface markers

(A) Use of flow cytometry to isolate the erythroid lineage from mouse FL at 12.5 dpc. The CD45⁺/Sca-1⁺/c-Kit⁺ fraction defines HSCs; the c-Kit⁺ (Sca-l⁻/c-Kit⁺/CD71⁻/Ter119⁻) fraction defines BFU-E; the c-Kit⁺/CD71⁺ (Sca-l⁻/c-Kit⁺/CD71⁺/Ter119⁻) fraction defines committed erythroid progenitors or CFU-E; the CD71⁺/Ter119⁺ (Sca-I⁻/c-Kit⁻/CD71⁺/Ter119⁺) fraction defines proerythroblasts; and the Ter119⁺ (Sca-l⁻/c-Kit⁻/CD71⁻/Ter119⁺) fraction defines reticulocytes and erythrocytes. (B) HSC and erythroid cell surface markers are shown in the table. HSCs were defined as CD45⁺/Sca-1⁺/c-Kit⁺ cells, although CD45 is not shown in the table.

Figure S2 Hmgn2 amino acid sequence, vector and transfection into cells

(A) Deduced amino acid sequence of mouse Hmgn2. Hmgn2 exhibits NLS1 and NLS2, and nucleosome binding and CHUD. (B) Schematic representation of the pIRES2-Hmgn2-AcGFP vector. (C) Schematic of DNA transfection into Friend erythroleukaemia cells (FS-S.fl). Plasmids with and without (mock) Hmgn2 inserts were electroporated into Friend erythroleukaemia cells. Two days later, GFP⁺ cells were isolated by cell sorting and cultured for 6 days. Cell cycle status was analysed at days 2 and 8 of culture. Surface markers were analysed at day 8 of culture.

Figure S3 Cell cycle analysis of Friend erythroleukaemia cells transfected with indicated vectors

Sorted GFP⁺ cells at day 2 of culture and all cells at day 8 of culture were fixed and stained with PI. DNA content was quantified by flow cytometry. Cell cycle status at G_l, S and G₂ phases was calculated by the Watson Pragmatic method and is shown as black, grey and black spotted areas respectively.

Figure S4 Surface phenotype of Friend erythroleukaemia cells

Friend erythroleukaemia cells were maintained in containing 10% FBS, 10 units/ml penicillin and 10 mg/ml streptomycin. Friend erythroleukaemia cells are characterized as erythroid progenitor cells expressing c-Kit and CD71 (equivalent to CFU-E).

Received 18 March 2011/25 July 2011; accepted 12 October 2011

Published as Cell Biology International Immediate Publication 12 October 2011, doi:10.1042/CBI20110169

© The Author(s) Journal compilation © 2012 Portland Press Limited