Fig 2: The effect of ARNT on viral genome replication and transcription. HeLa cells treated with ARNT-specific siRNA (siARNT) and non-targeting siRNA (siScr) were infected with GS/65 virus at an MOI of 2. At 8 h p.i., the RNA was extracted, and then the expression levels of vRNA (A), cRNA (B), and mRNA (C) of the NP gene were determined by using qRT-PCR. Nuclear proteins were extracted for Western blot analysis (D). ** p < 0.01.

Fig 3: Inhibition of IDO1 in cervical CSCs reduces Notch1 activation. Tumorspheres from HeLa and SiHa cells were dissociated into single cell suspension. (A,B) The tumorsphere cells were transduced with lentiviruses carrying IDO1 specific shRNAs (IDO1#1 or IDO1#2) or control LacZ specific shRNA (LacZ) followed by selection with puromycin (A) or treatment with 20 µM of INCB-024360 for 48 h. The expression of NICD or IDO1 was determined by Western blotting. The insert numbers indicated the relative expression level in comparison to sh-LacZ transduced cells. (C) The putative binding sites of aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator (AhR/ARNT) (-GCGTG-) in the Notch1 promoter were analyzed by the EPD website and the binding of ARNT to -309 site of Notch1 promoter in HeLa and SiHa tumorsphere cells after the knockdown of IDO1 was analyzed by the ChIP method with an anti-ARNT antibody and quantitated by the qPCR method. ** p < 0.01. All the experiments were repeated three times and data from one experiment were presented.

Fig 4: The effect of ARNT on the distribution of PA protein in 293T cells. (A) Confirmation of the expression of eGFP-PA and DsRed-ARNT in 293T cells by Western blotting. (B) The distribution of eGFP-PA and DsRed-ARNT in cells. 293T cells were transfected with peGFP-PA and pDsRED-ARNT individually or in combination. At 24 h p.t., the cells were fixed, and the nuclei were stained by DAPI. The cells were then scanned using a confocal microscope. Typical images are presented. (C) Statistical analysis of the intracellular localization patterns of PA in 293T cells. Based on the confocal microscopy data in panel A, the localization of PA was categorized into three types: clear nuclear localization (blue), simultaneous localization in the cytoplasm and nucleus (red), and predominantly cytoplasmic localization (green). The localization of PA was analyzed in more than 50 cells. The scale bar indicates 0–7.5 µm in length.

Fig 5: Interaction between subdomains or domains of ARNT and PA in 293T cells. (A) Schematic diagram of the full-length ARNT and ARNT mutants tested in this study. (B) Co-immunoprecipitation analysis of the interaction between PA and ARNT mutants. 293T cells were co-transfected with pCAGGS-PA-Flag and different c-Myc-tagged ARNT mutants. At 48 h p.t., cell lysates were immunoprecipitated with an anti-Flag antibody followed by Western blotting with anti-c-Myc and anti-Flag antibodies. (C) Schematic diagram of the full-length PA and PA mutants. (D) Co-immunoprecipitation analysis of the interaction between PA mutants and ARNT. 293T cells were co-transfected with pCAGGS-ARNT-c-Myc and different Flag-tagged PA mutants. At 48 h p.t., cell lysates were immunoprecipitated with an anti-Flag antibody followed by Western blotting analysis with anti-c-Myc and anti-Flag antibodies. *, the target protein.