Effect of IL-6 on XBP-1 expression and its functional significance.

MM.1S cells were treated with IL-6 (10 ng) for the indicated times. (A) Total cellular RNA was subjected to Northern blot analysis. Data shown is representative of 3 independent experiments with similar results. (B) Total cell lysates were separated by 12.5% SDS-PAGE and analyzed by immunoblotting (IB) with anti–XBP-1 (upper panel) or anti-SHP2 (lower panel) Abs. (C) Total cell lysates from Dex-, 2ME2-, Dex + IL-6, or 2ME2 + IL-6–treated MM.1S cells were separated by 12.5% SDS-PAGE and analyzed by immunoblotting (IB) with anti–XBP-1 (upper panel) or anti-SHP2 (lower panel) Abs. (D) Effect of overexpression of dominant-negative XBP-1 (DN-XBP-1) in MM.1S cells. Cells were transiently transfected with cDNA expression construct containing green fluorescence protein (GFP) alone or with dominant-negative XBP-1 (DN-XBP-1). Following transfections, GFP-positive cells were selected by flow cytometry, treated with Dex (5 μM) or Dex + IL-6 (10 ng) for 48 hours, and analyzed for cell viability by MTT assay. Median viability was 53% for Dex in GFP-transfected cells and 32% for GFP + DN-XBP-1–transfected cells (P < .005, n = 3; mean ± SD of 3 independent experiments. Error bars indicate SD.) (E) Schematic representation of the role of XBP-1 in 2ME2-induced apoptosis and overcoming Dex resistance, as well as in IL-6–mediated protective effect against Dex. Arrows indicate the flow of signal; ×, no effects.