An antisense cDNA for mMgcRacGAP inhibits IL-6–induced macrophage differentiation of M1 cells.

M1 cells were transduced with the antisense mMgcRacGAP (asMgcRacGAP) using a bicistronic retrovirus vector pMX-asMgcRacGAP-IRES-EGFP (M1/pMX-asMgcRacGAP-IRES-EGFP). As a control, M1 cells were also transduced with the blank vector pMX-IRES-EGFP (M1/pMX-IRES-EGFP). (A) Differentiation of parental M1, M1/pMX-IRES-EGFP, and M1/pMX-asMgcRacGAP-IRES-EGFP cells was evaluated by cell profiles in flow cytometry in the absence (i) or the presence (iii) of murine IL-6. The x-axis indicates forward scatter. The y-axis indicates side scatter. Undifferentiated and differentiated M1 cells were dotted in region R1 and region R2, respectively. GFP expression in parental M1, M1/pMX-IRES-EGFP, and M1/pMX-asMgcRacGAP-IRES-EGFP cells was also evaluated in flow cytometry to confirm the efficiency of expression of EGFP by bicistronic vector pMX-IRES-EGFP (ii). The x-axis indicates fluorescence intensity as a log scale ranging from 10⁰ to 10⁴. The y-axis indicates the number of cells. (B) Morphologic analysis of IL-6–induced differentiation in M1/pMX-IRES-EGFP and M1/pMX-asMgcRacGAP-IRES-EGFP cells. M1/pMX-IRES-EGFP and M1/pMX-asMgcRacGAP-IRES-EGFP cells were cultured in the presence of 5-ng/mL mIL-6 for 4 days, and cells were centrifuged onto glass slides and stained with May-Grunwald-Giemsa stain. Photographs were taken at 400 × magnification. The morphology of parental M1 cells is also shown as a control.