Negative effects of stable CIS3 transfection on LPS-signaling in murine M1 cells. (A) Suppression of LPS-induced differentiation in M1 cells. Parental M1 cells (M1-P) and stable transfectants expressing CIS3 (M1-CIS3) were incubated without or with 100 ng/mL LPS. After 72 hours of culture, cells were spun down on slide glass using a cytospin and examined after May-Grunwald Giemsa staining. (B) Inhibition of NO synthesis of M1 cells by CIS3. Parental M1 cells (5 × 105; parent) and stable transfectants expressing CIS3 were cultured with or without 100 ng/mL LPS for the days indicated. No synthesis was determined by the Griess method. Similar results were obtained with at least 2 independent clones of each transfectants. (C) Expression of IL-6 mRNA in M1 cells and transformants. Parental M1 cells (parent) and stable transfectants expressing CIS3 were stimulated with 100 ng/mL LPS for the days indicated. Total RNA was extracted and expression of IL-6 and G3PDH was analyzed by Northern blotting.
