Fig 2: pSTAT1701 binding of KPNA1 post EV71 infection was reduced in STAT3 knockdown cells. (A) STAT3 knockdown increased the interaction of pSTAT1701 and KPNA1. Cell lysates of EV71 infected STAT3 KD cells were immunoprecipitated with an KPNA1-specific antibody and with a normal rabbit IgG as an isotype control. KPNA1-bound pSTAT1701 was determined by western blot with a pSTAT1701-specific monoclonal antibody. An equal quantity of KPNA1 from siNC+EV71 and siSTAT3+EV71 was loaded and the density of the co-precipitated pSTAT1701 protein was analyzed. The value in the siNC+EV71 group was set as 1.00 (100%). (B) Binding of pSTAT1701, pSTAT3705, pSTAT3727 or unSTAT3 to KPNA1 12 hpi was determined by IP assay and western blot. (C) Schematic diagram of the flag-tagged STAT3 NLS point mutant (STAT3 NLS mut). (D) STAT3 knockdown efficiency in T98G cells treated with lentivirus-shSTAT3 (lenti-shSTAT3) was determined by IF assay. (E) Nuclear translocation of STAT3 was blocked in STAT3 NLS mut transfected cells. Lenti-shSTAT3-infected T98G cells were transfected with STAT3 NLS mut or a flag-tagged wild type (WT) STAT3 plasmid. After treatment with 50 ng/mL IL-6 for 1h, cells were fixed and STAT3 location was detected by the anti-flag antibody in IF assay. (F) Increased STAT3 expression level led to decreased binding of KPNA1 and pSTAT1701. Lenti-shSTAT3 infected T98G cells were transfected with 0 ug, 5 ug, 15 ug or 25 ug WT STAT3 plasmid and were infected with EV71 36 h post transfection. Cell lysates were prepared at 24 hpi and subjected to IP assay with the KPNA1-specific antibody. Binding of STAT3 to KPNA1 was determined by the anti-flag antibody via western blot. Cells transfected with STAT3 NLS mut were used as the negative control. Relative protein levels were calculated as described, ** p < 0.01, *** p < 0.001.

Fig 3: Ivermectin decreased the expression of importin a in the nucleus. (A) The gene expression of importin a/ß in HepG2-hNTCP-C4 cells and PXB hepatocytes at 48 h after seeding. The genes for importin ß and importin a family members were represented by karyopherin ß1 (KPNB1) and karyopherin a1–7 (KPNA1–7), respectively. The gene expression of KPNB1 in HepG2-hNTCP-C4 cells was defined as 1. (B) Protein expression of nuclear KPNA1–6 and KPNB1 after treatment of HepG2-hNTCP-C4 cells with 10 µM ivermectin for 48 h. Expression was corrected for that of lamin B1 (a nuclear marker) for each sample. In the graphs, the protein expression in the vehicle control group (Ctrl; 3% DMSO) is defined as 1. IVM, ivermectin. Data are presented as the mean ± SD ((A) HepG2-hNTCP-C4 cells, n = 4; PXB hepatocytes, n = 3; (B), n = 3). * p < 0.05.

Fig 4: Rhythmic nuclear expression of class 3 PI3K subunits.a, Immunoblot analysis, using the indicated antibodies, of the soluble nuclear and cytosolic fractions of HEK293T cells. GAPDH and LAMIN A/C served as purity controls for the cytosolic and soluble nuclear fractions. Experiment repeated five times; representative repeat shown. b,c, Immunoblot analysis of soluble nuclear (b) and euchromatin (c) fractions from the livers of eight-week-old male WT mice. Densitometric analysis of Vps15 and Vps34 normalized to total protein (TGX gels) are presented as the fold change over ZT0. Data are the mean ± s.e.m. (n = 4 mice). Rhythmicity pattern was tested using JTK_CYCLE (Supplementary Table 1). d, Mean normalized Vps15 and Vps34 protein levels in soluble nuclear (as in b) and euchromatin (as in c) fractions during the light (ZT0–ZT8) and dark (ZT12–ZT20) phase. Data are the mean ± s.e.m. (n = 8 mice in dark and n = 16 mice in light). #P < 0.05 versus light; two-tailed unpaired Student’s t-test. e, Relative transcript levels of the indicated genes in the livers of mice treated as in b. Data are presented as the mean ± s.e.m. (n = 4 mice). Rhythmicity was determined using JTK_CYCLE (Supplementary Table 1). f, Immunoblot analysis, using the indicated antibodies, of the soluble nuclear protein extracts of MEF cells treated with ivermectin for the indicated times. Densitometric analyses of Vps15 and Vps34 normalized to lamin B1 presented as the fold change over vehicle-treated cells. Data are the mean ± s.e.m. (independent repeats: n = 7 ivermectin (4 h) and n = 8 for all other groups). #P < 0.05 versus DMSO; two-tailed unpaired Student’s t-test. g, Immunoblot analyses of IPOA5 co-immunoprecipitated with ectopically expressed VPS15 and VPS34 from HEK293T cells using anti-Flag (ev, empty vector). Immunoprecipitation was performed three times; representative blots shown. h, Proximity ligation assay between endogenous VPS15 and ectopic Flag–VPS34 protein in HEK293T cells (co-transfection with GFP-expressing vector visualized transfected cells). The ‘no antibody’ condition served as a control for the non-specific signal. Scale bar, 10 µm. Data are the mean ± s.e.m. of proximity puncta per cell (n = 8 fields (no antibody) and n = 14 fields (Flag + VPS15) with over 300 cells collected in three independent experiments for each condition is presented; no antibody condition shared with Fig. 4e). #P = 0.00005 versus no antibody; two-tailed unpaired Student’s t-test. Source numerical data and unprocessed blots are provided. Source data

Fig 5: Effect of KPNA1 knockdown on nuclear expression of ?Np63 in Ca9-22 cells. (A) Co-occurrence between TP63 and each KPNA mRNA expression in the TCGA HNSCC cohort was analyzed through cBioportal. (B) Correlation between TP63 and each of KPNA1 and KPNA4 mRNA in HNSCC from TCGA is shown. (C) KPNA1 protein expression in Ca9-22 cells transfected with control siRNA or siRNA against KPNA1 was analyzed by Western blotting. Actin was used as a loading control. (D) ?Np63 protein expression in the cytoplasmic and nuclear fractions of KPNA1 knockdown Ca9-22 cells was analyzed by Western blotting. a-Tubulin and Lamin B1 were used as loading controls for the cytoplasmic and nuclear fractions, respectively. (E) Quantitation of nuclear ?Np63 protein expression in KPNA1 knockdown Ca9-22 cells. The results are shown as relative ?Np63 protein expression (?Np63/Lamin B1). * p < 0.05 vs. control siRNA.