Fig 2: Schematic representation of mechanism by which CuB regulates the GRP78–FOXM1–KIF20A pathway.

Fig 3: Binding of WT σ1R and σ1R E102Q to proteins that regulate NMDAR function and to the cytosolic domains of TRPA1, TRPV1, and TRPM8. Recombinant cytosolic C0-C1-C2 region of the NMDAR NR1 subunit, the C-terminal cytosolic region of MOR, the HINT1 protein, BiP, and the N- and C-terminal cytosolic domains of TRPA1, TRPV1, and TRPM8 channels were covalently attached to agarose and incubated with human WT σ1R or its mutant in the presence of 2.5 mM CaCl2 (details as in Figure 2). The assays were repeated at least twice, and comparable results were obtained. Representative blots are shown. For each interaction between σ1R WT or the σ1R E102Q mutant and a given protein, * indicates a significant difference compared to the σ1R WT group (assigned an arbitrary value of 1); all data were analyzed by ANOVA followed by the pairwise Holm–Sidak multiple comparison test; p < 0.05; σ1R, sigma receptor type 1; NR1-C1, cytosolic region the C0-C1-C2 domains of the NR1 subunit of NMDARs; BiP, binding immunoglobulin protein; HINT1, histidine triad nucleotide binding protein 1; MOR, mu-opioid receptor; TRP, cytosolic transient receptor potential types A1, V1, and M8; Ct, C-terminal domain; Nt, cytosolic N-terminal domain; WT, wild-type.

Fig 4: Target validation using different assays. (A) Labeling of recombinant GRP78 with BP-2 (different concentrations of BP-2 or different amounts of GRP78). Left: in gel fluorescence (FL). Right: Coomassie brilliant blue staining (CBB). (B) Cellular Thermal shift binding assay of CuB and CuⅡA with GRP78-overexpressing 293 T cells (n = 3). (C) MST analysis of the binding affinity between CuB and human recombinant GRP78 protein (WT and mutant). The measured Kd value has been shown. (D) Imaging of CRMM2 cells with BP-2 and immunofluorescence against GRP78 (n = 5). Scale bar: 25 μm.

Fig 5: PSTi8 competes with PST on GRP78 receptor binding. Molecular docking (a) PST (yellow) is docked in the active site of human GRP78 (blue): the residues of PST in red are showing hydrogen bond with the residues of human GRP78 (green). (b) The superimposed image of PSTi8 (purple) and PST in the active site of human GRP78. (c) PSTi8 is docked in the active site of human GRP78: the residues of PSTi8 in red are showing hydrogen bond with the residues of human GRP78. (d) Competitive binding between different concentrations of PST and Sulphorhodamine labelled PSTi8 (150 nM) in HepG2 cells. (e) Competitive binding between different concentrations of PSTi8 and Sulphorhodamine labelled PST (25 nM) in HepG2 cells. (f) GRP78 ATPase activity in presences of PST (1 µM) with different dose of PSTi8 (0, 1, 2.5, 5 µM). Western blot analysis (g) Effect of PSTi8 (800 nM) on PST (100 nM) inhibited tunicamycin (5 mg/ml) stimulated GRP78 expression in HepG2 cells. α, control vs tunicamycin; β, Tunicamycin vs Tunca. + PST; γ, Tunica. PST vs Tunica. +PST + PSTi8 and δ, control vs PSTi8. *P < 0.05; **P < 0.01; ***P < 0.001, NS, Non-significant. Error bar indicate mean ± s.e.m.