Fig 2: Stabilization of DNA Secondary Structures Leads to Aberrant Accumulation of Telomerase at Telomeres, Related to Figure 4(A) Analysis of telomerase activity determined by TRAP assay in the different indicated genotypes. Error bars indicate ± SD from two independent experiments.(B and C) Quantification of the interaction between TERT and TRF2 as determined by in situ PLA assay in the cells indicated. Data represents quantification from at least 150 nuclei from a representative experiment (****p < 0.0001; two-way ANOVA). Dashed lines indicate nucleus (as determined by DAPI in blue).(D and E) Representative images and quantification of the localisation of Terc RNA (red) at telomeres (TelC-Green) as determined by in situ RNAscope assay coupled to telomere FISH in cells of the indicated genotype. Arrows indicate colocalization between Terc RNA and TelC (telomere). Data represents quantification from at least 150 nuclei from a representative experiment (****p < 0.0001; two-way ANOVA).

Fig 3: Stabilization of DNA Secondary Structures Leads to Aberrant Accumulation of Telomerase at Telomeres, Related to Figure 4(A) Quantification of telomere length heterogeneity per metaphase 96 hours after Ad-GFP or Ad-Cre infection. Boxplots represent the quantification from at least 30 metaphases from a representative experiment (****p < 0.0001; two-way ANOVA).(B) qRT-PCR analysis of the indicated genes. Data are means ± SD normalized to the expression ß–Actin and relative to control. (**p < 0.01***p < 0.001; two-way ANOVA).(C) Western Blot analysis of the different genotypes to monitor loss of endogenous TPP1 96 hours after Cre infection.(D) Images show a representative metaphase telomere FISH of the indicated drug treatments. Telomere loss, indicated with yellow arrows; telomere fragility, indicated with red arrows; telomere length heterogeneity, indicated with white arrows.(E and F) Quantification of telomere length heterogeneity per metaphase 96 hours after Ad-GFP or Ad-Cre infection in cells treated with GRN163L (E) or BIBR1532 (F). Boxplots represent the quantification from at least 30 metaphases from a representative experiment (****p < 0.0001; two-way ANOVA).(G) Western Blot analysis of the different genotypes to monitor expression of TERT.(H) Images show a representative metaphase telomere FISH of the indicated genotypes. Telomere loss, indicated with yellow arrows; telomere fragility, indicated with red arrows; telomere length heterogeneity, indicated with white arrows.(I) Quantification of telomere length heterogeneity per metaphase 96 hours after Ad-GFP or Ad-Cre infection. Boxplots represent the quantification from at least 30 metaphases from a representative experiment (****p < 0.0001; two-way ANOVA).

Fig 4: A schematic model of the molecular mechanisms by which TERT acts in a feedback loop with NF-?B pathway to regulate macrophage polarization in a mouse model of chronic binge alcohol-induced ALD.On one hand, Up-regulation of TERT upon the EtOH treatment can induce the expression of M1 macrophage markers including TNF-a, IL-1ß, IL-6, NOS2, and CCL2 by interacting with p65, which suggests TERT promotes macrophage polarize towards M1 phenotype via NF-?B signaling. On the other hand, as a stimulus of NF-?B signaling, LPS, can bind LPS-binding protein and then deliver the LPS ligand to CD14 receptor. The LPS-CD14 complex interplays with TLR4 in conjunction with the small extracellular protein MD2 to activate intracellular signaling via NF-?B pathway. Upon activation of TLR4, signaling intermediates IRAK1/4 are recruited to the TLR4 complex via interplay with MyD88 leading to IKK kinase activation.TLR4-induced MyD88- independent (TRIF) signalling results in activation of IKKe and NF-?B as well. Afterwards, NF-?B forms p65/p50 heterodimers in macrophages and binds to the promoter region of TERT and various pro-inflammatory genes to result in gene transcription. In short, this crosstalk mechanism may provide a possible explanation for the ethanol-mediated hepatic proinflammatory response and M1 macrophage polarization in vitro.

Fig 5: Rtel1 Telomeric Dysfunction Is Rescued by Blocking Replication Fork Reversal(A) Schematic of the process of replication fork reversal and the different genes involved in fork reversal and fork restart.(B and C) Representative images (B) and quantification of the frequency (C) of PARP1 foci (red) coincident with telomeres (TelC, green) in cells of the indicated genotypes. Arrows indicate TelC-PARP1 colocalization events. Boxplots represent the quantification from at least 150 nuclei from a representative experiment (****p < 0.0001; two-way ANOVA).(D and E) Quantification of telomere loss (D) and telomere fragility (E) per metaphase in cells of the indicated genotype 96 hr after Ad-GFP or Ad-Cre infection. Boxplots represent the quantification from at least 30 metaphases from a representative experiment (****p < 0.0001; two-way ANOVA).(F) Western blot analysis showing PARylated proteins in cells subject to the indicated PARP inhibitor treatments (5 µM Olaparib and 10 µM NU1025).(G and H) Quantification of telomere loss (G) and telomere fragility (H) per metaphase in cells of the indicated genotype 96 hr after Ad-GFP or Ad-Cre infection. Representative images of telomere FISH on metaphases are shown in Figure S7A. Boxplots represent the quantification from at least 30 metaphases from a representative experiment (****p < 0.0001; two-way ANOVA).(I) Representative images and quantification of the frequency of interaction between TERT and TRF1 as determined by in situ PLA assay in Rtel1-deficient cells subject to the indicated siRNA treatment. Data represent quantification from at least 150 nuclei from a representative experiment (****p < 0.0001; two-way ANOVA). Dashed lines indicate nucleus (as determined by DAPI in blue).See also Figures S3.