FVIII forms amyloid-like fibrils in the ER. (A) Increased FVIII synthesis activates the UPR. H9 cells were treated with 5 mM NaB for increasing times to induce FVIII expression and cell lysates prepared for immunoblotting with indicated antibodies. (B) FVIII aggregates within the ER lumen. H9 cells were cultured in: (i) complete media; (ii) NaB (5 mM) for 24 hours; (iii) Glc-free media containing 20 mM 2DG and 10 mM NaN₃ for 2 hours; or (iv) 2DG and NaN₃ treatment of 2 hours and then recovered in Glc-containing media for 4 hours. Cells were fixed and stained with Thio-S (green) or antibodies for FVIII (red) and KDEL (blue); original magnification ×40. (C) FVIII colocalizes with PDIA6. CHO-K1 cells and H9 cells were treated without/with 5 µM SAHA for 24 hours, and ultrathin sections were prepared for immunogold labeling and TEM by first staining with FVIII primary antibody using secondary antibody coupled to 12-nm gold particles. Subsequently, grids were stained with PDIA6 antibody using a secondary coupled to 18-nm gold particles: (i) SAHA-treated H9 cells stained without primary antibody; (ii, v) untreated H9 cells; (iii, vi) SAHA-treated H9 cells (insets [panels on the right] show regions in subpanels iii and vi in greater detail; scale bars, iii and vi, 50 nm); and (iv) SAHA-treated control CHO cells. Red and black arrows indicate FVIII and PDIA6 particles, respectively. Scale bars: i, ii, iv-vi: 200 nm; iii, 100 nm. (D-E) FVIII forms amyloid fibrils. H9 cells were treated with 5 mM NaB for 24 hours; lysates were prepared and analyzed by sucrose gradient sedimentation. FVIII in each fraction was immunoprecipitated with FVIII-monoclonal antibody-conjugated Sepharose beads. After IP, FVIII was eluted and analyzed by negative-stain TEM (D) and cryo-EM (E). Two images are shown for each. (D) Arrows indicate FVIII fibrils in the HMW sucrose fractions. Scale bars: top, 50 nm; bottom, 100 nm. (E) Cryo-EM images of FVIII reveal dense networks composed of ∼5-nm wide fibrils. Scale bar, 100 nm. CHOP, C/EBP homologous protein; eIF2α-P, phosphorylated eukaryotic initiation factor 2 α subunit; UT, untreated.