Fig 2: 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.

Fig 3: Kaempferol inhibits UPR signaling (a) IMR32 cells were cultured with BFA in presence or absence of kaempferol pre-incubation. Q-RTPCR was performed to measure the expression of genes (IRE1a, PERK, ATF6, GRP78 and CHOP) involved in UPR signaling. (b) Immunoblot representing the reduction in the expression of GRP78 in BFA treated conditions upon pretreatment with kaempferol. Densitometry analysis of GRP78 was performed. Data represents the average ± SEM of two independent experiments (n = 2). (c) MDA-MB-468 cells were cultured with BFA in presence or absence of kaempferol pre-incubation. Q-RTPCR was performed to measure the expression of GRP78 and CHOP. Data represented as average ± SEM of two independent experiments performed in duplicate. *Represents the significant increase in expression of BFA treated cells with respect to control cells; #represents the reduction in gene expression in kaempferol pre-treated cells when compared to BFA alone treated cells (p = 0.05; one way ANOVA).

Fig 4: MAP4K4 and GRP78 control NFAT signaling. (a) qRT-PCR analysis of the expression of GRP78 mRNA level in neonatal rat cardiomyocytes (NRCM) treated with MAP4K4 siRNA or negative control (NC) siRNA for 48 h (n = 6 per group). (b) Western blot analysis and quantification of the expression of GRP78 protein level in NRCM treated with MAP4K4 siRNA or NC siRNA for 48 h (n = 6 per group). (c) qRT-PCR analysis the expression of MAP4K4 mRNA level in NRCM treated with GRP78 siRNA or NC siRNA for 48 h (n = 6 per group). (d) Western blot analysis and quantification of the expression of MAP4K4 protein level in NRCM treated with GRP78 siRNA or NC siRNA for 48 h (n = 6 per group). (e) Western blot analysis and quantification of the expression of NFATc3 protein level in NRCM treated with MAP4K4 siRNA or NC siRNA for 48 h (n = 6 per group). (f) Western blot analysis and quantification of the expression of NFATc3 protein level in NRCM treated with GRP78 siRNA or NC siRNA for 48 h (n = 6 per group). (g) Western blot analysis and quantification of the expression of NFATc3 protein level in NRCM treated with miR-30d mimic or NC mimic for 48 h (n = 6 per group). (h) Western blot analysis and quantification of the expression of NFATc3 protein level in miR-30d transgenic (TG) and wildtype (WT) rat injection with isoproterenol (ISO) (n = 5 per group). (i) Western blot analysis and quantification of the expression of NFATc3 protein level in NRCM treated with miR-30d inhibitor or NC inhibitor for 48 h (n = 6 per group). Data are presented as mean±SD. Statistical significance was determined by Student t test (a-g, i) and two-way ANOVA with post hoc tukey (h). **, P < 0.01 and ***, P < 0.001.

Fig 5: 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.