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SpiD3 induces the unfolded protein response and modulates CLL survival factors independent of TME stimuli. A, HG-3 and OSU-CLL cells were treated for 4 hours with SpiD3 (5, 10 µmol/L), thapsigargin (Thaps; 10 µmol/L), or equivalent DMSO vehicle (VEH) and then incubated with TPE-NMI dye to probe for unfolded proteins. Data are represented as fold change in TPE-NMI MFI compared with VEH (n = 3–5 independent experiments/cell line). B, Representative immunoblot analyses of IRE1α, XBP1, PERK, ATF4, CHOP, p-eIF2α (Ser51), and total eIF2α in HG-3 and OSU-CLL cells treated with VEH, SpiD3 (0.5–2 µmol/L), or Thaps (2 µmol/L) for 4 hours (n = 4–5 independent experiments/cell line). β-ACTIN served as the loading control. Blue arrow: spliced XBP1, green arrow: PERK shift. C, HG-3 cells were treated with VEH (24 hours), SpiD3 (0.5–2 µmol/L; 24 hours), or cycloheximide (CHX; 50 µg/mL; 30 minutes) and then incubated with OPP for 30 minutes. Data are represented as fold change in OPP MFI compared with VEH (n = 3–4 independent experiments). D, Representative immunoblot analyses of PDCD4, eIF4A1, p-4E-BP1 (Ser65), total 4E-BP1, eIF4E, and eIF4G1 levels in HG-3 and OSU-CLL cells treated with VEH or SpiD3 (0.5–2 µmol/L) for 4 hours (n = 4 independent experiments/cell line). β-ACTIN served as the loading control. Black arrows indicate the three isoforms of 4E-BP1. E, OSU-CLL cells were incubated with the alkyne-tagged analog 19 (10 µmol/L) for 2 hours. Cell lysates were clicked with TAMRA-biotin and biotin-tagged-19-bound proteins were isolated using streptavidin agarose beads, trypsinized, and then evaluated via mass spectrometry. Top: Pathway enrichment (EnrichR) analysis of similar proteins found in at least two of the three biological replicates. Bottom: Representative immunoblot analysis of biotin-alkyne-tagged proteins, with their corresponding input lysates for IKKα, IKKβ, p65, RELB, BTK, and GAPDH. The blue arrow indicates IKKα and IKKβ. Immunoblot analysis of the indicated proteins in whole-cell lysates of HG-3 and OSU-CLL cells treated with SpiD3 (1, 2 µmol/L) for 4 hours and cocurrently stimulated with α-IgM (10 µg/mL; F) or rhBAFF ligand (50 ng/mL; G). α-TUBULIN served as the loading control (n = 4 independent experiments/cell line). Asterisks denote significance versus VEH: *, P P P
Eiken et al. (Wed,) studied this question.