Fig 2: Indoleamine 2,3-dioxygenase 1 (IDO1) is upregulated in cervical cancer stem cells (CSCs) and irradiated cervical cancer cells. (A) The expression levels of IDO1 mRNA among normal cervical or cervical cancer tissues were analyzed by the Gene Expression Profiling Interactive Analysis (GEPIA) website using the data of the Cancer Genome Atlas (TCGA). * p < 0.01. (B, C) The total cell proteins were harvested from HeLa and SiHa cells under 2-dimensional culture (2D) or tumorsphere culture (S). The IDO1 protein expression was determined by Western blotting (B). The inserted numbers indicated the relative expression level of S in comparison to 2D. The IDO1 activity was determined by the conversion of kynurenine from tryptophan using Ehlrich reagent and measured the absorbance at 492 nm (C). The data were presented as fold change to 2D group. (D) The total cell proteins were harvested from parental (P) or irradiated (R) HeLa and SiHa cells and the IDO1 protein expression was determined by Western blotting. The inserted numbers indicate the relative expression level of R in comparison to P. (E) The culture supernatant of parental (P) or irradiated (R) HeLa and SiHa cells was mixed with RPMI-1640 medium at a ratio of 1:1 and used for examining the T cell suppression effect by measuring the proliferation of Jurkat T cells. The control indicated the mixture of fresh DMEM medium and RPMI-1640 medium.

Fig 3: Tryptophan metabolism by IDO1 positively regulates self-renewal of cervical CSCs. (A) SiHa 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. The surviving cells were collected at 48 h and 2 × 103 cells were used to perform primary tumorsphere cultivation and the tumorsphere numbers were counted at day 7, followed by collecting formed primary tumorspheres with 100 µm cell strainer. The collected primary tumorspheres were dissociated into single cells and 1 × 103 cells were used for secondary tumorsphere formation followed by counting the tumorsphere number at day 7. * p < 0.05; ** p < 0.01 when compared to sh-LacZ group. (B,C) HeLa and SiHa cells were performed tumorsphere cultivation in the supplement of 100 µM kynurenine. The tumorsphere numbers were counted at day 7. The total RNA was collected at 48 h after kynurenine treatment and the expression of BMI1, Oct4 or Sox2 were determined by qRT-PCR method. * p < 0.05; ** p < 0.01. All the experiments were repeated three times and data from one experiment were presented.

Fig 4: Notch1 activation in cervical CSCs participates to the IDO1 induction. (A) Total cell lysates were collected from conventional adherent culture (N) or tumorspheres (S) from HeLa and SiHa cells and the expression of Notch1 and NICD was determined by Western blotting. (B–D) Tumorspheres from HeLa and SiHa cells were dissociated into single cell suspension and were transduced with lentiviruses carrying Notch1 specific shRNAs (sh-Notch1#1 or sh-Notch1#2) followed by puromycin selection. The surviving cells were collected and detected the expression of NICD or IDO1 by Western blotting (B). The insert numbers indicate the relative expression level in comparison to sh-LacZ transduced cells. The kynurenine concentration in the culture supernatant was determined by Ehrlich’s reagent (C). * p < 0.05; ** p < 0.01. Tumorsphere cells were treated with Ro-4929097 as the indicated concentration and detected the expression of NICD or IDO1 by western blot (D). The insert numbers indicate the relative expression level in comparison to 0.1% DMSO control (labeled as 0 µM). (E) The putative binding site of RBPJ/CSL (-TGTGGGAA-) in IDO1 promoter was analyzed by the Eukaryotic Promoter Database (EPD) website and the binding of NICD to the RBPJ/CSL binding site of IDO1 promoter at +140 site in HeLa and SiHa tumorsphere cells was detected by the chromatin immunoprecipitation (ChIP) method with an anti-Notch1 antibody and quantitated by the qPCR method. Data were presented as percentage of input DNA. ** p < 0.01. All the experiments were repeated three times and the data from one experiment were presented.

Fig 5: IDO1-siRNA silences IDO1 expression in vitro. (A) IDO1 mRNA expression. LLC cells were transfected with IDO1-siRNA or GL2-siRNA (negative control). Untransfected cells were used as a blank control. IDO1 mRNA expression level was detected by reverse transcription-quantitative PCR (n=3). (B) IDO1 protein expression. Silencing of IDO1 was confirmed by western blotting (n=3) in LLC cells after transfection with IDO1-siRNA, GL2-siRNA or blank control. *P<0.05 and **P<0.01. GL2, glabra 2; IDO1, indoleamine 2,3-dioxygenase 1; siRNA, small interfering RNA.