Fig 2: Regulation of ER-UPR and MT-UPR in vitro and in vivo. Western blot analysis of (A-C) HSP90, (D-F) HSP60, (G-I) GRP78 and (J-L) CHOP expression in each group of rats. *P<0.05, **P<0.01, ***P<0.001. The data are displayed as the means ± SD; n=6 for each subgroup. D-gal, D-galactose; Met, metformin.

Fig 3: ALPP interacts with BIP directly and is stabilized by BIP. (A) Immunoprecipitation (IP) with Flag-ALPP as bait followed by immunoblotting (IB) with anti-Flag or anti-BIP antibodies. WCL, whole-cell lysate. (B) Immunofluorescence confocal microscopy of U-251 MG cells stably expressing Flag-ALPP. The cells were stained with anti-ALPP antibody (red) or anti-BIP antibody (green). Scale bars, 10 µm. (C) Immunoblot analyses of the BIP protein levels (left) and sequences of mutations in BIP-/- U-251 MG clones (right). BIP was depleted using CRISPR/Cas9. (D) Immunoblot analyses of the ALPP protein levels in WT and BIP-/- U-251 MG cells transiently transfected with pcDNA3.1-ALPP for 6, 24, or 48 h. (E) RT-qPCR analyses of the ALPP mRNA levels. WT and BIP-/- U-251 MG cells were transiently transfected with pcDNA3.1-ALPP for 4 and 12 h. The data were obtained from four biological replicates (n = 4). (F) Immunoblot analyses of the ALPP, NS1, and BIP protein levels in WT and ALPP-/- JEG-3 cells transiently transfected with pCMV-NS1 for 24 h. (G) Immunoblot analyses of the NS1, ALPP, and BIP protein levels. WT and BIP-KO JEG-3 cells were transiently transfected with pCMV-NS1 for 24 h. The quantitative data in this figure are shown as means ± SEMs.

Fig 4: Determination of senescence and oxidative stress levels in cultured neurons. Optimal concentration of D-gal was selected by CCK-8 test (a), N=9 for each subgroup. Primary cultured neurons were observed after being seeded on a plate for 7 days and after D-gal treatment for 7 days (b). Senescence was evaluated by the Senescence ß-Galactosidase Staining Kit in neurons treated with graded concentrations of D-gal (c). Positive cells for ß-galactosidase staining were counted and shown in panel d, N=9 for each subgroup. The oxidative stress level was evaluated by western blotting analysis of GRP78, lamin A/C and lamin B1 in senescent cells, cells treated with NaHS and cells treated with inhibitors of PI3K, AMPK and CaMKKß (e-g), N=6 for each subgroup. Generation of ROS in the cultured neurons was determined (h), N=6 for each subgroup. Scale bar=25 µm, p<0.05, ** p<0.01, *** p<0.001. The data are displayed as mean ±SD.

Fig 5: BIP chaperone activity is essential for ALPP stability and ZIKV infection. (A) Immunofluorescence (left) and statistical (right) analyses of ZIKV and DENV infection in WT and BIP-/- U-251 MG cells. Each biological replicate (n = 3) contained 3,000 analyzed cells. MOI = 1. n.s., no significance; ****, P < 0.0001 (significantly different compared with the WT cells; two-tailed Student's t test). Scale bars, 10 µm. (B) Production of ZIKV proteins in WT and ALPP-/- JEG-3 cells infected with ZIKV for 24 h. MOI = 1. (C) Western blot analysis of ALPP expression in WT and BIP-/- U-251 MG cells. Rescue of ALPP expression by BIP reconstitution was observed with wild-type BIP but not enzymatically impaired BIP mutants (T37G and T229G). (D) Immunofluorescence (left) and statistical (right) analyses of ZIKV infection in WT and BIP-/- U-251 MG cells overexpressing wild-type BIP or enzymatically impaired BIP mutants (T37G and T229G). MOI = 0.5. Each biological replicate (n = 3) contained 3,000 analyzed cells. n.s., no significance; *, P < 0.05; **, P < 0.01 (significantly different compared with the NC-treated cells; two-tailed Student's t test). Scale bars, 10 µm. (E) Immunoprecipitation with ALPP-2×Flag as bait followed by Western blotting with anti-Flag, anti-ZIKV ENV, anti-ZIKV NS5, or anti-BIP antibodies 24 h after ZIKV infection. MOI = 0.5. The lysates were prepared in the absence or presence of 2 mM ATP to modulate BIP substrate binding.