Cloning and expression of S100A8/A9 derivatives

For S100A8-Flag or S100A9-Flag, C-terminal 1× Flag-tagged S100A8 or S100A9 open reading frame was cloned from the cDNA library of MCF-10A–ER-Src cells and then were subcloned into pLHCX retroviral vector (Clontech, #631511). For NLS-GFP, GFP with triplicated NLS (sequence copied from Invitrogen’s pCMV/myc/nuc) at both the N- and C-terminal sides was cloned from pEGFP-N1 (provided by R. Pomerantz from Temple University School of Medicine) using ultramer primers and then was subcloned into pLHCX. For NLS-GFP-A8 or NLS-GFP-A9, S100A8 or S100A9 open reading frame was subcloned to the C-terminal side of NLS-GFP in pLHCX.

Retrovirus was produced by transfection of corresponding retroviral vector in HEK293T cells carrying pCL-Ampho (provided by V. Sukhatme). Retroviral supernatant from HEK293T cells was collected at 48 and 72 hours after transfection, filtered through 0.45-μm filter, and used to infect exponentially growing target cells in the presence of polybrene (8 μg/ml; Millipore). Cells were passaged after 72 hours of infection, after which drug selection was performed with hygromycin B (500 μg/ml; Santa Cruz Biotechnology). For nuclear-specific expression of S100A8 or S100A9, cells expressing NLS-GFP, NLS-GFP-A8, or NLS-GFP-A9 were further selected by a cell sorting flow cytometer (SH800Z, Sony) based on GFP signals, and GFP levels for all groups of cells were kept similar on average during the selection.

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