Fig 1: HPV16 oncogenes induce TREX1 expression in human keratinocytes and sensitize these cells to TREX1 silencing. TREX1 expression levels were determined by western blot in 30 micrograms of total protein extracts from monolayer (A) and organotypic (B) cultures of keratinocytes transduced with constructs expressing E6 and E7 from HPV16 and HPV11. (C) TREX1 expression was analyzed by immunohistochemistry in sections from organotypic cultures established from low passage number keratinocytes transduced with retroviral vectors expressing HPV16 E6 and/or E7 with. Besides, organotypic cultures established from keratinocytes transfected with HPV16 or HPV18 whole genomes and cultured for different passage number (p18 to p82) were used. Magnification: X400. (D) The effect of TREX1 silencing on cell viability was determined in control or HPV16 E6 and/or E7 transduced PHKs. Gene silencing was performed as described in Fig. 1. Cells were cultured in 96 wells plates (2000 cells/well) and after 72 hours 10 µL of Alamar blue per well were added. Cells were incubated at 37 °C and Alamar Blue’s reduction was monitored every hour in a spectrophotometer through absorbance measurement at 570 e 600 nm. (E) Clonogenic assays with low- (p4) and high-passage (p81) PHK expressing HPV16 E6 and E7 were performed as described in Fig. 1. The results shown are representative of at least three independent experiments performed in triplicate. *p-value = 0.05.
Fig 2: TREX1 is upregulated in CIN 2/3 and invasive carcinoma samples. A representative immunoreactivity of TREX1 in clinical samples is shown. (A) A sample of cervicitis showing a positive reaction for TREX1 predominantly decorating the basal layer. (B) A cervical intraepithelial neoplasia grade 2 (CIN2) sample exhibiting a positive reaction weakly staining the basal and parabasal layers of stratified epithelium. (C) A CIN3 (or in situ carcinoma) sample strongly stained by TREX1 immunoreaction, mainly expressed at nuclei. (D) Highlight of the junction between a normal epithelium (right side) and a CIN3 lesion (left side). (E) Example of an invasive squamous cells carcinoma with cytoplasmic positive reaction for TREX1. (F) Example of an intense positive reaction for TREX1 decorating the cytoplasmic area of an invasive cervical adenocarcinoma. Note the strong TREX1 cytoplasmic staining in CIN3 and carcinoma with sparse nuclear staining (D–F). Magnification: X400.
Fig 3: ANKLE1 deficiency induces the activation of the cGAS-STING pathway. a) Cell extracts of HCT116 wild-type, ANKLE1-/-, TREX1-/- and ANKLE1-/-/TREX1-/- cells were analyzed by western blotting for the indicated proteins. b) Clonogenic survival assay was carried out on cells treated with the indicated concentrations of cisplatin. Graph shows mean ± SD of n = 3 independent experiments. Statistical significance values were determined with two-way ANOVA. c) Cell extracts were analyzed by western blotting for the indicated proteins. d–h) Relative mRNA levels of the indicated ISGs in cells normalized to untreated HCT116 wild-type. Bars represent mean ± SD of n = 5 independent experiments. Statistical significance values were determined with unpaired two-tailed t-tests. i) Cells were untreated or treated with cisplatin (0.5 µg mL-1, 3 days) and fixed for immunofluorescence. dsDNA (green) and DNA (blue) were visualized. Scale bars, 10 µm. j) Quantification of the signal intensities of the cytoplasmic staining of dsDNA. Intensity of cytosolic dsDNA of a cell is calculated as: the total intensity of dsDNA staining of the whole cell minus the intensity of dsDNA staining in the nucleus. n = 100 cells were measured per condition. Statistical significance values were determined with unpaired two-tailed t-tests. Black lines represent the means in arbitrary unit (a.u.). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns = not significant.
Fig 4: TREX1 mRNA levels are up-regulated in CIN and invasive carcinomas. TREX1 mRNA levels were analyzed in CIN and SCC series form GEO expression datasets. (A) The mRNA expression of TREX1 was upregulated in CIN1 and CIN3 when compared with normal cervical tissues in GSE51993 series. (B,C) The mRNA expression of TREX1 was increased in invasive carcinomas and cervical cancer cell lines when compared with normal cervical tissues in two different series GSE7803 and GSE9750. Note the TREX1 mRNA is also upregulated in high-grade squamous intraepithelial lesions (HSIL). (D) The mRNA expression of TREX1 was upregulated in HPV-infected women who developed CIN3+ lesions when compared with HPV- infected women without lesions or HPV-negative women (GSE75132). (E) The levels of TREX1 mRNA exhibit a moderate negative correlation with miR103 expression in CIN3 (GSE51993).
Fig 5: TREX1 silencing causes accumulation of sub-G1 cells and p53 upregulation. TREX1 silencing in SiHa, HeLa, C33A and PHK was performed as described in Fig. 1. (A) For cell cycle study, monolayers cultures of the different cell lines silenced for TREX1 expression were seeded in triplicate in 24 well plates (5000 cells/well). After 5 days cells and supernatants were harvested and analyzed in a FACSCalibur. At least 10,000 events were acquired for each condition. The data obtained were analyzed with the FlowJo software. (B) To analyze the effect of TREX1 silencing on the levels of regulators of the cell cycle 30 µg of total protein extracts from monolayer cultures of each cell type were analyzed using antibodies against cyclin A, PCNA, p53 and TREX1. (C) To determine the effect of TREX1 superexpression on the levels of regulators of the cell cycle in PHK expressing HPV16 E6 and/or E7 the cells were transfected with pcDNA5 expression vector harboring the TREX1 sequence. The analysis of the expression of PCNA, p53 and TREX1 was performed as described in (B). Western blot signals were quantified using ImageJ software using housekeeping genes actin or tubulin as normalizers and presented as expression relative to normal keratinocytes.
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