NKG2D-L, ERp5, and ADAM10 expression in the RS773 cell line. (A) Double staining of the RS773 cell line, derived from one case of MC cHL, with PE–anti-CD30 and FITC–anti-CD15 mAbs. Samples were run on a CyAnADP flow cytometer, and results are expressed as log red fluorescence intensity versus log green fluorescence intensity (percentages of single- or double-positive cells in each quadrant). Quadrants were set on the negative control stained with unrelated mAb (not shown). (B) Surface (Bi) or cytoplasmic (Bii) staining of RS773 for MIC-A, ULBP1, ULBP2, ULBP3, and ULBP4, with the specific mAbs followed by PE-GAM. For cytoplasmic staining, cells were fixed in 3% paraformaldehyde and permeabilized with1% Nonidet-P40 in PBS, before mAb incubation. Samples were run on a CyAnADP flow cytometer, and results are expressed as log MFI (a.u.) versus number of cells. (C) Surface (Ci) or cytoplasmic (Cii) staining of RS773 cells for ERp5 and ADAM10, with the rabbit polyclonal anti-ERp5 antiserum or the anti–ADAM10 mAb, followed by FITC-GAR or anti–isotype GAM antisera. For cytoplasmic staining, cells were fixed and permeabilized as in panel B, before Ab incubation. Results are shown as overlay histograms between the negative control (thin gray line) and the sample (black line). (D) MIC-A and ULBP1 to ULBP4 surface staining performed as in panel B, before (Di,iii) or after in vitro exposure of RS773 cells to 2.5mM VPA for 48 hours (Dii,iii). Results are expressed as log MFI (a.u.) versus number of cells (Di,ii) or as MFI ratio (ie, MFI of the sample/MFI of the negative control; Diii). (Div) Soluble MIC-A and ULBP3 were measured in the supernatants of cultured RS773, either untreated (CTR) or treated with 2.5mM VPA, by ELISA, referred to a standard curve and expressed as nanograms per milliliter.