Figure 2A - C Reporter cells were treated with and without 1 μM A - 769662 for 3 h followed by heat shock at 43 ° C for 30 min and recovery at 37 ° C for 5 h . Hspa1a and Hspb1 mRNA levels were quantitated by qRT - PCR ( mean ± SD , n = 3 , Student ' s t - test , * * * P < 0 . 001 ) . ACC phosphorylation was detected by immunoblotting ( C ) . D - F Primary MEFs were derived from Ampkα1fl / fl ; Ampkα2fl / fl embryos and transduced with either adenoviral GFP or Cre to delete Ampkα1 / 2 in vitro . AMPKα levels were detected 4 days after transductions ( D ) Following viral transductions , Hsp mRNA levels were quantitated by qRT - PCR in MEFs with and without 10 mM metformin treatment overnight followed by 43 ° C heat shock for 30 min and recovery for 4 h ( mean ± SD , n = 3 , one - way ANOVA , n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . G Transcriptional activities of HSF1 were measured by a dual reporter system consisting of two plasmids : the HSF1 - dependent reporter pHSE - SEAP , in which ideal HSEs drive the expression of secreted alkaline phosphatase ( SEAP ) , and the control reporter pCMV - Gaussia Luc , in which a CMV promoter drives the expression of secreted Gaussia luciferase . In HEK293T cells , either LacZ or GST - AMPKα1CA plasmid was co - transfected with the two reporter plasmids . After 24 h , the transfected cells were heat - shocked at 44 ° C for 45 min . The culture supernatants were collected to measure SEAP and luciferase activities 24 h after heat shock . SEAP signals were normalized to Gaussia luciferase signals ( mean ± SD , n = 5 , Student ' s t - test , * * P < 0 . 01 , * * * P < 0 . 001 ) . H , I Following treatment with 10 mM metformin overnight , endogenous AMPKα and HSF1 proteins were co - precipitated using anti - AMPKα agarose conjugates from lysates of immortalized Hsf1 + / + MEFs ( H ) . HC : heavy chain . Endogenous HSF1 - AMPKα interactions in immortalized Hsf1 + / + and Hsf1 − / − MEFs were visualized in situ by PLA ( I ) . Experimental details are described in Materials and Methods . Scale bars : 50 μm .

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Figure 2A-C Reporter cells were treated with and without 1 μM A-769662 for 3 h followed by heat shock at 43°C for 30 min and recovery at 37°C for 5 h. Hspa1a and Hspb1 mRNA levels were quantitated by qRT-PCR (mean ± SD, n = 3, Student's t-test, ***P < 0.001). ACC phosphorylation was detected by immunoblotting (C).D-F Primary MEFs were derived from Ampkα1fl/fl; Ampkα2fl/fl embryos and transduced with either adenoviral GFP or Cre to delete Ampkα1/2 in vitro. AMPKα levels were detected 4 days after transductions (D)Following viral transductions, Hsp mRNA levels were quantitated by qRT-PCR in MEFs with and without 10 mM metformin treatment overnight followed by 43°C heat shock for 30 min and recovery for 4 h (mean ± SD, n = 3, one-way ANOVA, n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001).G Transcriptional activities of HSF1 were measured by a dual reporter system consisting of two plasmids: the HSF1-dependent reporter pHSE-SEAP, in which ideal HSEs drive the expression of secreted alkaline phosphatase (SEAP), and the control reporter pCMV-Gaussia Luc, in which a CMV promoter drives the expression of secreted Gaussia luciferase. In HEK293T cells, either LacZ or GST-AMPKα1CA plasmid was co-transfected with the two reporter plasmids. After 24 h, the transfected cells were heat-shocked at 44°C for 45 min. The culture supernatants were collected to measure SEAP and luciferase activities 24 h after heat shock. SEAP signals were normalized to Gaussia luciferase signals (mean ± SD, n = 5, Student's t-test, **P < 0.01, ***P < 0.001).H, I Following treatment with 10 mM metformin overnight, endogenous AMPKα and HSF1 proteins were co-precipitated using anti-AMPKα agarose conjugates from lysates of immortalized Hsf1+/+MEFs (H).HC: heavy chain. Endogenous HSF1-AMPKα interactions in immortalized Hsf1+/+ and Hsf1−/− MEFs were visualized in situ by PLA (I). Experimental details are described in Materials and Methods. Scale bars: 50 μm.


Figure 3A In HEK293T cells that stably express an shRNA targeting the 3 ′ UTR of human HSF1 , HSF1WT or HSF1S121A was expressed . Following treatment with 10 mM metformin overnight , the levels of HSF1 phosphorylation at Ser121 were detected by immunoblotting using a specific phospho - HSF1 Ser121 antibody ( A8041 , Assay Biotechnology ) . B Primary Ampkα1fl / fl ; Ampkα2fl / fl MEFs were transduced with adenoviral GFP or Cre . Following treatment with 10 mM metformin overnight or treatment with 10 μM A - 769662 for 3 h , HSF1 Ser121 phosphorylation was examined by immunoblotting . C HSF1WT or HSF1S121A was co - expressed with either GFP or GST - AMPKαCA in HSF1 - deficient HEK293T cells . HSF1 Ser121 phosphorylation was examined by immunoblotting . D AMPK complexes were immunoprecipitated from HEK293T cells treated with and without 10 mM metformin overnight . Aliquots of AMPK complexes were incubated with 400 ng purified recombinant His - tagged HSF1 proteins with and without 100 μM AMP or 20 μM compound C ( CC ) . HSF1 Ser121 phosphorylation was detected by immunoblotting . E , F HSF1 activities were measured by the dual reporter system in HSF1 - deficient HEK293T cells . Either FLAG - HSF1WT or FLAG - HSF1S121A was co - expressed with LacZ orAMPKα1CA ( E ) . E , F HSF1 activities were measured by the dual reporter system in HSF1 - deficient HEK293T cells . Either FLAG - HSF1WT or FLAG - HSF1S121A was co - expressed with LacZ or AMPKα1CA ( E ) . Following expression of HSF1WT or HSF1S121A , cells were treated with and without 10 mM metformin overnight ( F ) ( mean ± SD , n = 5 , one - way ANOVA , n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . G , H FLAG - HSF1WT or FLAG - HSF1S121A plasmids were co - transfected with LacZ or AMPKα1CA plasmids into HSF1 - deficient HEK293T cells for 3 days ( G ) . Following transfection with FLAG - HSF1WT or FLAG - HSF1S121A plasmids for 3 days , HSF1 - deficient HEK293T cells were treated with and without 10 mM metformin overnight ( H ) . Nuclear proteins were extracted to measure HSF1 - DNA binding by the ELISA - based DNA binding assay using anti - FLAG antibodies ( mean ± SD , n = 3 , one - way ANOVA , n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . I , J Following transfections and metformin treatment as described in ( G ) and ( H ) , cytoplasmic and nuclear proteins were extracted for immunoblotting using anti - FLAG antibodies .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A In HEK293T cells that stably express an shRNA targeting the 3′ UTR of human HSF1, HSF1WT or HSF1S121A was expressed. Following treatment with 10 mM metformin overnight, the levels of HSF1 phosphorylation at Ser121 were detected by immunoblotting using a specific phospho-HSF1 Ser121 antibody (A8041, Assay Biotechnology).B Primary Ampkα1fl/fl; Ampkα2fl/fl MEFs were transduced with adenoviral GFP or Cre. Following treatment with 10 mM metformin overnight or treatment with 10 μM A-769662 for 3 h, HSF1 Ser121 phosphorylation was examined by immunoblotting.C HSF1WT or HSF1S121A was co-expressed with either GFP or GST-AMPKαCA in HSF1-deficient HEK293T cells. HSF1 Ser121 phosphorylation was examined by immunoblotting.D AMPK complexes were immunoprecipitated from HEK293T cells treated with and without 10 mM metformin overnight. Aliquots of AMPK complexes were incubated with 400 ng purified recombinant His-tagged HSF1 proteins with and without 100 μM AMP or 20 μM compound C (CC). HSF1 Ser121 phosphorylation was detected by immunoblotting.E, F HSF1 activities were measured by the dual reporter system in HSF1-deficient HEK293T cells. Either FLAG-HSF1WT or FLAG-HSF1S121A was co-expressed with LacZ orAMPKα1CA (E).E, F HSF1 activities were measured by the dual reporter system in HSF1-deficient HEK293T cells. Either FLAG-HSF1WT or FLAG-HSF1S121A was co-expressed with LacZ or AMPKα1CA (E). Following expression of HSF1WT or HSF1S121A, cells were treated with and without 10 mM metformin overnight (F) (mean ± SD, n = 5, one-way ANOVA, n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001).G, H FLAG-HSF1WT or FLAG-HSF1S121A plasmids were co-transfected with LacZ or AMPKα1CA plasmids into HSF1-deficient HEK293T cells for 3 days (G). Following transfection with FLAG-HSF1WT or FLAG-HSF1S121A plasmids for 3 days, HSF1-deficient HEK293T cells were treated with and without 10 mM metformin overnight (H). Nuclear proteins were extracted to measure HSF1-DNA binding by the ELISA-based DNA binding assay using anti-FLAG antibodies (mean ± SD, n = 3, one-way ANOVA, n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001).I, J Following transfections and metformin treatment as described in (G) and (H), cytoplasmic and nuclear proteins were extracted for immunoblotting using anti-FLAG antibodies.


Figure 4A , B NIH3T3 reporter cells were cultured overnight in DMEM with and without leucine ( A ) or 4 . 5 g / l glucose ( B ) . Following heat shock at 43 ° C for 30 min and recovery for 5 h , GFP induction was quantitated by flow cytometry . C , D Nuclear proteins were extracted from reporter cells with and without nutrient deprivations overnight for the ELISA - based DNA binding assay ( mean ± SD , n = 3 , Student ' s t - test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . E , F Following nutrient deprivations overnight , the reporter cells were heat - shocked at 43 ° C for 30 min and recovered overnight . Individual proteins were detected by immunoblotting . G , H Reporter cells were subjected to nutrient starvation for the indicated time . AMPKα Thr172 and HSF1 Ser121 phosphorylation was measured by immunoblotting . G , H Reporter cells were subjected to nutrient starvation for the indicated time . AMPKα Thr172 and HSF1 Ser121 phosphorylation was measured by immunoblotting . I , J HEK293T cells were sequentially transfected with control or AMPKα1 / 2 - targeting siRNAs and dual HSF1 reporter plasmids . 24 h later , transfected cells were starved overnight before reporter activities were measured ( mean ± SD , n = 6 , Student ' s t - test , n . s . , not significant , * * P < 0 . 01 , * * * P < 0 . 001 ) . I , J HEK293T cells were sequentially transfected with control or AMPKα1 / 2 - targeting siRNAs and dual HSF1 reporter plasmids . 24 h later , transfected cells were starved overnight before reporter activities were measured ( mean ± SD , n = 6 , Student ' s t - test , n . s . , not significant , * * P < 0 . 01 , * * * P < 0 . 001 ) . K , L FLAG - HSF1WT or FLAG - HSF1S121A plasmids were co - transfected with the dual HSF1 reporter plasmids into HSF1 - deficient HEK293T cells for 2 days , followed by overnight starvation ( mean ± SD , n = 5 , Student ' s t - test , n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A, B NIH3T3 reporter cells were cultured overnight in DMEM with and without leucine (A) or 4.5 g/l glucose (B). Following heat shock at 43°C for 30 min and recovery for 5 h, GFP induction was quantitated by flow cytometry.C, D Nuclear proteins were extracted from reporter cells with and without nutrient deprivations overnight for the ELISA-based DNA binding assay (mean ± SD, n = 3, Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001).E, F Following nutrient deprivations overnight, the reporter cells were heat-shocked at 43°C for 30 min and recovered overnight. Individual proteins were detected by immunoblotting.G, H Reporter cells were subjected to nutrient starvation for the indicated time. AMPKα Thr172 and HSF1 Ser121 phosphorylation was measured by immunoblotting.G, H Reporter cells were subjected to nutrient starvation for the indicated time. AMPKα Thr172 and HSF1 Ser121 phosphorylation was measured by immunoblotting.I, J HEK293T cells were sequentially transfected with control or AMPKα1/2-targeting siRNAs and dual HSF1 reporter plasmids. 24 h later, transfected cells were starved overnight before reporter activities were measured (mean ± SD, n = 6, Student's t-test, n.s., not significant, **P < 0.01, ***P < 0.001).I, J HEK293T cells were sequentially transfected with control or AMPKα1/2-targeting siRNAs and dual HSF1 reporter plasmids. 24 h later, transfected cells were starved overnight before reporter activities were measured (mean ± SD, n = 6, Student's t-test, n.s., not significant, **P < 0.01, ***P < 0.001).K, L FLAG-HSF1WT or FLAG-HSF1S121A plasmids were co-transfected with the dual HSF1 reporter plasmids into HSF1-deficient HEK293T cells for 2 days, followed by overnight starvation (mean ± SD, n = 5, Student's t-test, n.s., not significant, *P < 0.05, **P < 0.01).


Figure 5A Reporter cells were heat - shocked at 43 ° C for the indicated time . AMPKα Thr172 and HSF1 Ser121 phosphorylation was measured by immunoblotting . B Immediately following heat shock at 43 ° C for 40 min , AMPK complexes were immunoprecipitated from HEK293 cells and in vitro kinase assays were performed as described in Figure 3D using recombinant human ACC1 proteins as substrates . C Following heat shock at 43 ° C for 40 min , both detergent - soluble and detergent - insoluble fractions of cellular proteins were extracted from HEK293 cells as described in Materials and Methods for immunoblotting . D , E Immediately following heat shock at 43 ° C for 40 min , HEK293 cells were subjected to nutrient deprivations for 4 h . AMPKα Thr172 and ACC Ser79 phosphorylation was measured by immunoblotting . F - H HSF1WT or HSF1S121A plasmids were transfected into HSF1 - deficient HEK293T cells . Transfected cells were heat - shocked at 43 ° C for 40 min . HSP mRNAs were quantitated by qRT - PCR following overnight recovery . HSF1 - DNA binding was measured immediately after heat shock ( mean ± SD , n = 3 , Student ' s t - test , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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Figure 5A Reporter cells were heat-shocked at 43°C for the indicated time. AMPKα Thr172 and HSF1 Ser121 phosphorylation was measured by immunoblotting.B Immediately following heat shock at 43°C for 40 min, AMPK complexes were immunoprecipitated from HEK293 cells and in vitro kinase assays were performed as described in Figure 3D using recombinant human ACC1 proteins as substrates.C Following heat shock at 43°C for 40 min, both detergent-soluble and detergent-insoluble fractions of cellular proteins were extracted from HEK293 cells as described in Materials and Methods for immunoblotting.D, E Immediately following heat shock at 43°C for 40 min, HEK293 cells were subjected to nutrient deprivations for 4 h. AMPKα Thr172 and ACC Ser79 phosphorylation was measured by immunoblotting.F-H HSF1WT or HSF1S121A plasmids were transfected into HSF1-deficient HEK293T cells. Transfected cells were heat-shocked at 43°C for 40 min. HSP mRNAs were quantitated by qRT-PCR following overnight recovery. HSF1-DNA binding was measured immediately after heat shock (mean ± SD, n = 3, Student's t-test, **P < 0.01, ***P < 0.001).


Figure 6A , B Following treatment with 10 μM metformin for 3 days , the levels of HSF1 binding to endogenous HSP promoters were quantitated by chromatin IP ( mean ± SD , n = 3 , one - way ANOVA , * * P < 0 . 01 , * * * P < 0 . 001 ) . Normal rabbit IgG served as the negative control ( A ) . Following the same treatment , HSP mRNA levels were quantitated by qRT - PCR ( mean ± SD , n = 3 , Student ' s t - test , * * * P < 0 . 001 ) ( B ) . C , D Following transfection with AMPKα1 / 2 - targeting siRNAs for 2 days , WM115 cells were treated with 10 μM metformin for 3 days . HSP mRNA levels were quantitated by qRT - PCR ( mean ± SD , n = 6 , Student ' s t - test , n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 ) . C , D Following transfection with AMPKα1 / 2 - targeting siRNAs for 2 days , WM115 cells were treated with 10 μM metformin for 3 days . HSP mRNA levels were quantitated by qRT - PCR ( mean ± SD , n = 6 , Student ' s t - test , n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 ) . E Following treatment with 10 μM metformin for 7 days , protein levels in WM115 and S462 cells were measured by immunoblotting . F , G Following treatment with 10 μM metformin for 7 days , levels of polyubiquitinated proteins were detected in both detergent - soluble and detergent - insoluble fractions in WM115 and A2058 cells using a Lys48 - specific polyubiquitin antibody . H A2058 cells were treated with 10 μM metformin alone or co - treated with 2 μM compound C for 7 days . Individual proteins were detected by immunoblotting . I HEK293T cells stably expressing either scramble or HSF1 - targeting shRNAs were treated with 10 μM metformin for 7 days . Individual proteins were detected by immunoblotting . J In HEK293T cells , a plasmid encoding HA - polyQ79 - GFP was co - transfected with the indicated plasmids . Following treatment with and without 10 μM metformin for 5 days , the sizes of aggregates in detergent - insoluble fractions were quantitated by a Multisizer ™ 3 Coulter Counter . Experimental details are described in Materials and Methods . K , L Immortalized and RAS - transformed MEFs were treated with either 10 μM metformin ( K ) or different concentrations of glucose ( L ) for 7 days . Individual proteins were detected by immunoblotting . M Following transfection with LacZ or polyQ79 , HEK293T cells were grown under different concentrations of glucose for 5 days before measuring aggregate size .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A, B Following treatment with 10 μM metformin for 3 days, the levels of HSF1 binding to endogenous HSP promoters were quantitated by chromatin IP (mean ± SD, n = 3, one-way ANOVA, **P < 0.01, ***P < 0.001). Normal rabbit IgG served as the negative control (A). Following the same treatment, HSP mRNA levels were quantitated by qRT-PCR (mean ± SD, n = 3, Student's t-test, ***P < 0.001) (B).C, D Following transfection with AMPKα1/2-targeting siRNAs for 2 days, WM115 cells were treated with 10 μM metformin for 3 days. HSP mRNA levels were quantitated by qRT-PCR (mean ± SD, n = 6, Student's t-test, n.s., not significant, *P < 0.05, **P < 0.01).C, D Following transfection with AMPKα1/2-targeting siRNAs for 2 days, WM115 cells were treated with 10 μM metformin for 3 days. HSP mRNA levels were quantitated by qRT-PCR (mean ± SD, n = 6, Student's t-test, n.s., not significant, *P < 0.05, **P < 0.01).E Following treatment with 10 μM metformin for 7 days, protein levels in WM115 and S462 cells were measured by immunoblotting.F, G Following treatment with 10 μM metformin for 7 days, levels of polyubiquitinated proteins were detected in both detergent-soluble and detergent-insoluble fractions in WM115 and A2058 cells using a Lys48-specific polyubiquitin antibody.H A2058 cells were treated with 10 μM metformin alone or co-treated with 2 μM compound C for 7 days. Individual proteins were detected by immunoblotting.I HEK293T cells stably expressing either scramble or HSF1-targeting shRNAs were treated with 10 μM metformin for 7 days. Individual proteins were detected by immunoblotting.J In HEK293T cells, a plasmid encoding HA-polyQ79-GFP was co-transfected with the indicated plasmids. Following treatment with and without 10 μM metformin for 5 days, the sizes of aggregates in detergent-insoluble fractions were quantitated by a Multisizer™ 3 Coulter Counter. Experimental details are described in Materials and Methods.K, L Immortalized and RAS-transformed MEFs were treated with either 10 μM metformin (K) or different concentrations of glucose (L) for 7 days. Individual proteins were detected by immunoblotting.M Following transfection with LacZ or polyQ79, HEK293T cells were grown under different concentrations of glucose for 5 days before measuring aggregate size.


Figure 7A , B A2058 cells were transduced with lentiviral scramble or HSF1 - targeting ( hA6 and hA9 ) shRNAs . ( A ) Cell numbers were quantitated by Hoechst 33342 DNA staining ( mean ± SD , n = 4 , two - way ANOVA , * * * P < 0 . 001 ) . ( B ) Protein levels were measured by immunoblotting . C A2058 cells stably expressing LacZ or HSF1 were grown in medium containing 4 . 5 , 1 . 0 , or 0 . 45 g / l glucose . Following treatment with 10 μM metformin , cell proliferation was measured by Hoechst 33342 DNA staining ( mean ± SD , n = 5 , two - way ANOVA , * * * P < 0 . 001 ) . D , E 1 × 106 LacZ - or HSF1 - expressing A2058 cells were transplanted into female NOD / SCID mice . One day after transplantation , metformin was administered via drinking water at 1 mg / ml . Tumor incidence ( log - rank test ; D ) and volumes ( E ) were measured ( mean ± SEM , two - way ANOVA n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Tumor growth curves were fitted to exponential growth models to calculate tumor doubling time ( DT ) . F Individual proteins were measured by immunoblotting using lysates of three tumors from each treatment group . G Protein levels were measured by immunoblotting using lysates of mouse liver tissues . H A2058 cells stably expressing FLAG - HSF1WT or FLAG - HSF1S121A were treated with 10 μM metformin for 7 days . Protein levels were measured by immunoblotting . I A2058 cells stably expressing HSF1WT or HSF1S121A were grown in Corning ® ultra - low attachment 96 - well culture plates with 10 μM metformin and different concentrations of glucose for 7 days , 10 , 000 cells per well . Viable cells were counted by Guava flow cytometer using ViaCount ® reagent ( mean ± SEM , n = 8 , Student ' s t - test , n . s . , not significant , * * P < 0 . 01 , * * * P < 0 . 001 ) . J Tukey box plots showing the inverse correlation between AMPKα Thr172 phosphorylation and HSP mRNA expression in ccRCC . Patients were stratified on the median value of AMPKα Thr172 phosphorylation reverse - phase protein array ( RPPA ) scores ( Student ' s t - test ) .

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Figure 7A, B A2058 cells were transduced with lentiviral scramble or HSF1-targeting (hA6 and hA9) shRNAs. (A) Cell numbers were quantitated by Hoechst 33342 DNA staining (mean ± SD, n = 4, two-way ANOVA, ***P < 0.001). (B) Protein levels were measured by immunoblotting.C A2058 cells stably expressing LacZ or HSF1 were grown in medium containing 4.5, 1.0, or 0.45 g/l glucose. Following treatment with 10 μM metformin, cell proliferation was measured by Hoechst 33342 DNA staining (mean ± SD, n = 5, two-way ANOVA, ***P < 0.001).D, E 1 × 106 LacZ- or HSF1-expressing A2058 cells were transplanted into female NOD/SCID mice. One day after transplantation, metformin was administered via drinking water at 1 mg/ml. Tumor incidence (log-rank test; D) and volumes (E) were measured (mean ± SEM, two-way ANOVA n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001). Tumor growth curves were fitted to exponential growth models to calculate tumor doubling time (DT).F Individual proteins were measured by immunoblotting using lysates of three tumors from each treatment group.G Protein levels were measured by immunoblotting using lysates of mouse liver tissues.H A2058 cells stably expressing FLAG-HSF1WT or FLAG-HSF1S121A were treated with 10 μM metformin for 7 days. Protein levels were measured by immunoblotting.I A2058 cells stably expressing HSF1WT or HSF1S121A were grown in Corning®ultra-low attachment 96-well culture plates with 10 μM metformin and different concentrations of glucose for 7 days, 10,000 cells per well. Viable cells were counted by Guava flow cytometer using ViaCount® reagent (mean ± SEM, n = 8, Student's t-test, n.s., not significant, **P < 0.01, ***P < 0.001).J Tukey box plots showing the inverse correlation between AMPKα Thr172 phosphorylation and HSP mRNA expression in ccRCC. Patients were stratified on the median value of AMPKα Thr172 phosphorylation reverse-phase protein array (RPPA) scores (Student's t-test).


Figure 1 ( D ) Violin plots showing the distribution of sheath polymerization speed for both modes of T6SS assemblies . 300 assemblies ( 100 from each biological replicate ; 3 biological replicates ) were analyzed over at least 3 min at 5 s intervals . The central dash line indicates the median while the upper and lower dash lines indicate the quartiles . Unpaired t - test was performed ( ns : not significant ) .

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Figure 1(D) Violin plots showing the distribution of sheath polymerization speed for both modes of T6SS assemblies. 300 assemblies (100 from each biological replicate; 3 biological replicates) were analyzed over at least 3 min at 5 s intervals. The central dash line indicates the median while the upper and lower dash lines indicate the quartiles. Unpaired t-test was performed (ns: not significant).


Figure 3 ( D ) The frequency of contact - dependent assembly ( left panel ) and number of assemblies per cell per min ( right panel ) in cells from different strains as indicated on x - axis . Three biological replicates were performed for each strain . At least 500 cells were analyzed per replicate on 3D - SIM time - lapse imaging of sheath dynamics ( TssB - sfGFP ) in 3 min period . Bar graph ( left panel ) and scatter dot plot ( right panel ) show mean ± SD . Unpaired t - test was performed .

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Figure 3(D) The frequency of contact-dependent assembly (left panel) and number of assemblies per cell per min (right panel) in cells from different strains as indicated on x-axis. Three biological replicates were performed for each strain. At least 500 cells were analyzed per replicate on 3D-SIM time-lapse imaging of sheath dynamics (TssB-sfGFP) in 3 min period. Bar graph (left panel) and scatter dot plot (right panel) show mean ± SD. Unpaired t-test was performed.


Figure 1Kaplan - Meier analysis of the correlation between expression level of LOC113230 and overall survival of colon adenocarcinoma ( COAD ) patients from TCGA database . P < 0 . 001 . P - value for Kaplan - Meier curves was determined using log - rank test . qRT - PCR analysis of LOC113230 expression level in 65 paired CRC samples . Representative RNA - Scope immunostaining images of LOC113230 levels in CRC tissue and corresponding normal tissue from TMA chips . Scale bar represents 100 μM . Kaplan - Meier analysis for overall survival of CRC patients with high and low expression of LOC113230 from TMA chips . * P < 0 . 001 . P - value for Kaplan - Meier curves was determined using log - rank test .

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Figure 1Kaplan-Meier analysis of the correlation between expression level of LOC113230 and overall survival of colon adenocarcinoma (COAD) patients from TCGA database. P<0.001. P-value for Kaplan-Meier curves was determined using log-rank test.qRT-PCR analysis of LOC113230 expression level in 65 paired CRC samples.Representative RNA-Scope immunostaining images of LOC113230 levels in CRC tissue and corresponding normal tissue from TMA chips. Scale bar represents 100 μM.Kaplan-Meier analysis for overall survival of CRC patients with high and low expression of LOC113230 from TMA chips. *P < 0.001. P-value for Kaplan-Meier curves was determined using log-rank test.


Figure 2Overexpression of LOC113230 in SW - 1116 cells inhibited lung metastasis . Left : Exponentially growing SW - 1116 - LOC113230 - Luc and SW - 1116 - Vector - Luc cells were injected into the tail vein of 8 - week - old nude mice and intensities of lung metastatic tumor cells at the 8th week were analyzed by in vivo photon flux imaging equipment . The red circles in images are to help to see the tumors . Right : Box plot ( central band : median ; box limits : first and third quartile ; whiskers : minimum and maximum ) of lung photon flux at the 8th weeks . 5 ng / ml TGF - β and DMSO were applied to stimulate SW - 1116 - Vector or SW - 1116 - LOC113230 cells for 24 and 48 hrs , respectively . Cell migration was detected by transwell assays . Scale bar represents 100 μM .

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Figure 2Overexpression of LOC113230 in SW-1116 cells inhibited lung metastasis. Left: Exponentially growing SW-1116 - LOC113230-Luc and SW-1116-Vector-Luc cells were injected into the tail vein of 8-week-old nude mice and intensities of lung metastatic tumor cells at the 8th week were analyzed by in vivo photon flux imaging equipment. The red circles in images are to help to see the tumors. Right: Box plot (central band: median; box limits: first and third quartile; whiskers: minimum and maximum) of lung photon flux at the 8th weeks.5 ng/ml TGF-β and DMSO were applied to stimulate SW-1116 -Vector or SW-1116 -LOC113230 cells for 24 and 48 hrs, respectively. Cell migration was detected by transwell assays. Scale bar represents 100 μM.


Figure 3I ) Stable expression of LOC113230 300 - 800 inhibited cell migration , while LOC113230 antisense showed no apparent effect on cell migration . Scale bar represents 100 μM .

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Figure 3I)Stable expression of LOC113230 300-800 inhibited cell migration, while LOC113230 antisense showed no apparent effect on cell migration. Scale bar represents 100 μM.


Figure 4 ( A ) Top : Images taken by confocal microscopy showed the localization of LOC113230 detected by RNA scope assays in SW - 1116 . Scale bar represents 50 μM . Bottom : qRT - PCR assays showed LOC113230 expression level in the nuclear and cytoplasmic fractions extracted from SW - 1116 cells . SW - 1116 cells bearing stable expression or knockdown of LOC113230 were treated with MG132 ( 5 μM ) for 12 hrs . Cell lysates were immunoprecipitated ( IP ) assays were performed with α - Flag antibody . The co - eluted proteins were detected by western blot assays with ubiquitin ( Ub ) , ASS1 and α - Flag antibodies , respectively . SW - 1116 cells stably expressing ASS1WT , or ASS1 K234R and LOC113230 or not were treated with MG132 ( 5 μM ) for 12 hrs . Cell lysates were immunoprecipitated ( IP ) assays were performed with α - Flag antibody . The co - eluted proteins were detected by western blot assays with ubiquitin ( Ub ) and α - Flag antibodies , respectively .

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Figure 4(A)Top: Images taken by confocal microscopy showed the localization of LOC113230 detected by RNA scope assays in SW-1116. Scale bar represents 50 μM. Bottom: qRT-PCR assays showed LOC113230 expression level in the nuclear and cytoplasmic fractions extracted from SW-1116 cells.SW-1116 cells bearing stable expression or knockdown of LOC113230 were treated with MG132 (5 μM) for 12 hrs. Cell lysates were immunoprecipitated (IP) assays were performed with α-Flag antibody. The co-eluted proteins were detected by western blot assays with ubiquitin (Ub), ASS1 and α-Flag antibodies, respectively.SW-1116 cells stably expressing ASS1WT, or ASS1 K234R and LOC113230 or not were treated with MG132 (5 μM) for 12 hrs. Cell lysates were immunoprecipitated (IP) assays were performed with α-Flag antibody. The co-eluted proteins were detected by western blot assays with ubiquitin (Ub) and α-Flag antibodies, respectively.


Figure 5Arginine production from each group was determined by a commercially available Arginine assay kit . The cells were stimulated with 5 ng / ml TGF - β or DMSO for 24 hrs . Urea production for each group was determined by a commercially available urea assay kit . The cells were stimulated with 5 ng / ml TGF - β or DMSO for 24 hrs .

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Figure 5Arginine production from each group was determined by a commercially available Arginine assay kit. The cells were stimulated with 5 ng/ml TGF-β or DMSO for 24 hrs. Urea production for each group was determined by a commercially available urea assay kit. The cells were stimulated with 5 ng/ml TGF-β or DMSO for 24 hrs.


Figure 6Depletion of ASS1 decreased cell migration in SW - 1116 cells . Scale bar represents 100 μM . Transwell assays showed the effect of ASS1WT and ASS1K234R on Caco - 2 - Vector or Caco - 2 - LOC113323 cell migration . Scale bar represents 100 μM . . ( N ) Kaplan - Meier analysis for overall survival of patients with high or low expression of ASS1 . P < 0 . 001 . P - value for Kaplan - Meier curves was determined using log - rank test .

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Figure 6Depletion of ASS1 decreased cell migration in SW-1116 cells. Scale bar represents 100 μM.Transwell assays showed the effect of ASS1WT and ASS1K234R on Caco-2-Vector or Caco-2-LOC113323 cell migration. Scale bar represents 100 μM..(N) Kaplan-Meier analysis for overall survival of patients with high or low expression of ASS1. P < 0.001. P-value for Kaplan-Meier curves was determined using log-rank test.


Figure 7The interaction of endogenous TRAF2 , ASS1 and LRPPRC was detected in SW - 1116 cells by co - IP assays . GST - TRAF2 , HA - LRPPRC , His - ASS1 and Biotin - LOC113230 were expressed in E . coli BL21 cells , and were used for in vitro binding assays . SW - 1116 - LOC113230 cells transiently transfected with siRNAs to LRPPRC or TRAF2 or scrambled control ( SiRNA - NC ) were treated with MG132 ( 5 µM ) for 12 hrs . Cell lysates were immunoprecipitated with α - Flag antibody . The precipitate proteins and input proteins were analyzed by immunoblotting . NC : Negative control .

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Figure 7The interaction of endogenous TRAF2, ASS1 and LRPPRC was detected in SW-1116 cells by co-IP assays. GST-TRAF2, HA-LRPPRC, His-ASS1 and Biotin-LOC113230 were expressed in E. coli BL21 cells, and were used for in vitro binding assays.SW-1116-LOC113230 cells transiently transfected with siRNAs to LRPPRC or TRAF2 or scrambled control (SiRNA-NC) were treated with MG132 (5 µM) for 12 hrs. Cell lysates were immunoprecipitated with α-Flag antibody. The precipitate proteins and input proteins were analyzed by immunoblotting. NC: Negative control.


Figure 1A . Left : Telomerase activity measured using ddTRAP assay . The horizontal pink lines represent the threshold used in these samples for background correction . Each dot on the ddPCR output represents a unique droplet that is either positive or negative for fluorescent signal . Right : Quantification of TRAP activity after normalization to SW39 / TEL + cells ( n = 3 ) . Data information : Data are presented as mean ± SD . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution unless specified otherwise . PTM , post - translational modification . B . hTR and hTERT expression , normalized to ACTB mRNA and to SW39 / TEL + . Data information : Data are presented as mean ± SD . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution unless specified otherwise . PTM , post - translational modification . C . RNA Pol II binding at hTR promoter in SI14 / TEL + , SI24 / ALT + and 8G12 / ALT + cell lines , normalized to H3 ChIP . Control loci with , respectively , high or low transcriptional activity : c - fos promoter and subtelomeric 4qHox locus ( n = 3 ) . Statistical analyses were performed using one - way ANOVA with post - hoc Tukey ' s test for multiple comparisons . Data information : Data are presented as mean ± SD . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution unless specified otherwise . PTM , post - translational modification . D . H3K9me3 and H3K27me3 ( left ) or H3K4me3 and H3Ac ( right ) densities at hTR promoter in SI14 / TEL + and SI24 / ALT + , normalized to H3 ( n = 3 ) . Control loci as in C . Data information : Data are presented as mean ± SD . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution unless specified otherwise . PTM , post - translational modification . E . NFYB binding at hTR promoter in SI14 / TEL + , SI24 / ALT + , SW39 / TEL + and IMRB / ALT + cells normalized to H3 . Control loci with , respectively , high and low NFYB binding : hnRNPA1 promoter and subtelomeric 4qHox ( n = 3 ) . Data information : Data are presented as mean ± SD . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution unless specified otherwise . PTM , post - translational modification .

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Figure 1A. Left: Telomerase activity measured using ddTRAP assay. The horizontal pink lines represent the threshold used in these samples for background correction. Each dot on the ddPCR output represents a unique droplet that is either positive or negative for fluorescent signal. Right: Quantification of TRAP activity after normalization to SW39/TEL+ cells (n=3). Data information: Data are presented as mean ± SD. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise. PTM, post-translational modification.B. hTR and hTERT expression, normalized to ACTB mRNA and to SW39/TEL+. Data information: Data are presented as mean ± SD. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise. PTM, post-translational modification.C. RNA Pol II binding at hTR promoter in SI14/TEL+, SI24/ALT+ and 8G12/ALT+ cell lines, normalized to H3 ChIP. Control loci with, respectively, high or low transcriptional activity: c-fos promoter and subtelomeric 4qHox locus (n=3). Statistical analyses were performed using one-way ANOVA with post-hoc Tukey's test for multiple comparisons. Data information: Data are presented as mean ± SD. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise. PTM, post-translational modification.D. H3K9me3 and H3K27me3 (left) or H3K4me3 and H3Ac (right) densities at hTR promoter in SI14/TEL+ and SI24/ALT+, normalized to H3 (n=3). Control loci as in C. Data information: Data are presented as mean ± SD. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise. PTM, post-translational modification.E. NFYB binding at hTR promoter in SI14/TEL+, SI24/ALT+, SW39/TEL+ and IMRB/ALT+ cells normalized to H3. Control loci with, respectively, high and low NFYB binding: hnRNPA1 promoter and subtelomeric 4qHox (n=3). Data information: Data are presented as mean ± SD. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise. PTM, post-translational modification.


Figure 2A . Western blot for hTR chaperone proteins in TEL + and ALT + hybrids . β - Actin is used as loading control . Quantification of western blots is shown on the right , normalized to β - Actin and to SI14 / TEL + cells ( n = 2 - 4 ) . Statistical analyses were performed using one - way ANOVA with post - hoc Tukey ' s test for multiple comparisons . Data information : Data are presented as mean ± SD . B . NHP2 mRNA expression levels , normalized to ACTB mRNA and to SI14 / TEL + cells ( n = 3 ) . Data information : Data are presented as mean ± SD . C . Western blot for NHP2 in SI14 / TEL + , SI24 / ALT + and 8G12 / ALT + cell lines treated with DMSO or 10 μM MG132 for 6 h . β - Actin served as loading control . Quantifications are shown on the right , normalized to β - Actin and to SI14 / TEL + cells ( n = 5 ) . Statistical analyses were performed with Student ' s two - tailed unpaired t - test . Data information : Data are presented as mean ± SD . D . hTR RNA and NHP2 mRNA expression , normalized to ACTB mRNA and to SI14 / TEL + . Data information : Data are presented as mean ± SD . E . Telomerase activity measured with ddTRAP assay in NHP2 - overexpressing 8G12 / ALT + cells , normalized to NHP2 - overexpressing SI14 / TEL + . Data information : Data are presented as mean ± SD . F . ( Top ) Western blot for NHP2 overexpression in 8G12 / ALT + clonal cell lines . β - Actin served as loading control . SI14 / TEL + cells are shown as reference for NHP2 expression in hybrids . ( Bottom ) Western blot for NHP2 overexpression in SI14 / TEL + and 8G12 / ALT + cells compared to empty vector control . β - Actin served as loading control . Data information : Data are presented as mean ± SD . G . hTR RNA , hTERT and NHP2 mRNA expression , normalized to ACTB mRNA and to SI14 / TEL + ( n = 3 ) . Data information : Data are presented as mean ± SD . H . RNA Pol II binding at hTR promoter , normalized to H3 ChIP ( n = 3 ) . See Fig . 1C legend for control loci . Statistical analyses were performed using one - way ANOVA with post - hoc Tukey ' s test for multiple comparisons . Data information : Data are presented as mean ± SD .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Western blot for hTR chaperone proteins in TEL+ and ALT+ hybrids. β-Actin is used as loading control. Quantification of western blots is shown on the right, normalized to β-Actin and to SI14/TEL+ cells (n=2-4). Statistical analyses were performed using one-way ANOVA with post-hoc Tukey's test for multiple comparisons. Data information: Data are presented as mean ± SD.B. NHP2 mRNA expression levels, normalized to ACTB mRNA and to SI14/TEL+ cells (n=3). Data information: Data are presented as mean ± SD.C. Western blot for NHP2 in SI14/TEL+, SI24/ALT+ and 8G12/ALT+ cell lines treated with DMSO or 10 μM MG132 for 6 h. β-Actin served as loading control. Quantifications are shown on the right, normalized to β-Actin and to SI14/TEL+ cells (n=5). Statistical analyses were performed with Student's two-tailed unpaired t-test. Data information: Data are presented as mean ± SD.D. hTR RNA and NHP2 mRNA expression, normalized to ACTB mRNA and to SI14/TEL+. Data information: Data are presented as mean ± SD.E. Telomerase activity measured with ddTRAP assay in NHP2-overexpressing 8G12/ALT+cells, normalized to NHP2-overexpressing SI14/TEL+. Data information: Data are presented as mean ± SD.F. (Top) Western blot for NHP2 overexpression in 8G12/ALT+ clonal cell lines. β-Actin served as loading control. SI14/TEL+ cells are shown as reference for NHP2 expression in hybrids. (Bottom) Western blot for NHP2 overexpression in SI14/TEL+ and 8G12/ALT+ cells compared to empty vector control. β-Actin served as loading control. Data information: Data are presented as mean ± SD.G. hTR RNA, hTERT and NHP2 mRNA expression, normalized to ACTB mRNA and to SI14/TEL+ (n=3). Data information: Data are presented as mean ± SD.H. RNA Pol II binding at hTR promoter, normalized to H3 ChIP (n=3). See Fig. 1C legend for control loci. Statistical analyses were performed using one-way ANOVA with post-hoc Tukey's test for multiple comparisons. Data information: Data are presented as mean ± SD.


Figure 3A . hTR RNA , hTERT and NHP2 mRNA expression , normalized to ACTB mRNA and to 143B / TEL + cells , in a series of TEL + and ALT + human cell lines as indicated ( n = 3 ) . Data information : Data are presented as mean ± SD . Statistical analyses were performed using one - way ANOVA with post - hoc Tukey ' s test for multiple comparisons . B . Western blot for NHP2 expression . β - Actin served as loading control . Quantification is shown on the right after normalization to β - Actin and to 143B / TEL + cells ( n = 3 ) . Data information : Data are presented as mean ± SD . Statistical analyses were performed using one - way ANOVA with post - hoc Tukey ' s test for multiple comparisons .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. hTR RNA, hTERT and NHP2 mRNA expression, normalized to ACTB mRNA and to 143B/TEL+ cells, in a series of TEL+ and ALT+ human cell lines as indicated (n=3). Data information: Data are presented as mean ± SD. Statistical analyses were performed using one-way ANOVA with post-hoc Tukey's test for multiple comparisons.B. Western blot for NHP2 expression. β-Actin served as loading control. Quantification is shown on the right after normalization to β-Actin and to 143B/TEL+ cells (n=3). Data information: Data are presented as mean ± SD. Statistical analyses were performed using one-way ANOVA with post-hoc Tukey's test for multiple comparisons.


Figure 4A . hTR RNA expression , normalized to ACTB mRNA and to U2OS cells ( n = 3 ) . Data information : Data are shown as mean ± SD , with the red line marking the mean value . B . Western blot for NHP2 expression in the early passages of hTR - expressing IMRB or U2OS cells , with β - Actin as loading control . C . Quantification of doubling time for U2OS , U2OS - hTR , IMRB and IMRB - hTR cell lines ( n = 3 ) . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution Data information : Data are shown as mean ± SD , with the red line marking the mean value . D . Western blot in U2OS and U2OS - hTR cells treated with or without Hydroxyurea ( 2 mM for 24 h ) , for the indicated proteins . β - Actin and Vinculin served as loading controls . E . Representative images for co - localization between p - RPA32S33 ( green ) and telomeres ( red FISH probe ) . White arrows indicate co - localization events . Scale bars : 5 μm . Quantification is shown on the right . At least 50 nuclei were scored in each of the three independent experiments performed . Each dot represents the number of p - RPA32S33 - telomere co - localizations in one nucleus . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution . Data information : Data are shown as mean ± SD , with the red line marking the mean value . F . Quantification of live cell numbers with trypan blue staining and manual counting , 72 h post - transfection with siLuc , sihTR # 1 or sihTR # 2 , measured as the percentage of siLuc - treated cells ( n = 3 ) . On the right , representative images of U2OS cells treated with sihTR # 1 for 72 h . Statistical analyses were performed using one - way ANOVA with post - hoc Tukey ' s test for multiple comparisons . Data information : Data are shown as mean ± SD , with the red line marking the mean value . G . Western blot analyses in the indicated cell lines cells transfected for 48 h with siLuc or sihTR , with the indicated antibodies . U2OS cells treated with Hydroxyurea ( 2 mM for 24 h ) were used as positive controls .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. hTR RNA expression, normalized to ACTB mRNA and to U2OS cells (n=3). Data information: Data are shown as mean ± SD, with the red line marking the mean value.B. Western blot for NHP2 expression in the early passages of hTR-expressing IMRB or U2OS cells, with β-Actin as loading control.C. Quantification of doubling time for U2OS, U2OS-hTR, IMRB and IMRB-hTR cell lines (n=3). Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution Data information: Data are shown as mean ± SD, with the red line marking the mean value.D. Western blot in U2OS and U2OS-hTR cells treated with or without Hydroxyurea (2 mM for 24 h), for the indicated proteins. β-Actin and Vinculin served as loading controls.E. Representative images for co-localization between p-RPA32S33 (green) and telomeres (red FISH probe). White arrows indicate co-localization events. Scale bars: 5 μm. Quantification is shown on the right. At least 50 nuclei were scored in each of the three independent experiments performed. Each dot represents the number of p-RPA32S33-telomere co-localizations in one nucleus. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution. Data information: Data are shown as mean ± SD, with the red line marking the mean value.F. Quantification of live cell numbers with trypan blue staining and manual counting, 72 h post-transfection with siLuc, sihTR#1 or sihTR#2, measured as the percentage of siLuc-treated cells (n=3). On the right, representative images of U2OS cells treated with sihTR#1 for 72 h. Statistical analyses were performed using one-way ANOVA with post-hoc Tukey's test for multiple comparisons. Data information: Data are shown as mean ± SD, with the red line marking the mean value.G. Western blot analyses in the indicated cell lines cells transfected for 48 h with siLuc or sihTR, with the indicated antibodies. U2OS cells treated with Hydroxyurea (2 mM for 24 h) were used as positive controls.


Figure 5A . Representative images for fragile telomeres using the CO - FISH assay . Lagging strands ( C - probe ) and leading strands ( G - probe ) were marked with green PNA probes and red LNA probes , respectively . White circles and arrows indicate " smeary " or " doublet " telomeric signals , respectively . Quantification of fragile telomeres is shown on the right as the percentage of total telomeres counted ( n = 932 - 1073 ) . Comparison of fragile to non - fragile telomere percentage was performed using Χ2 - test . Data information : All scale bars : 5 μm . Data are shown as mean ± SD . B . Representative images for co - localization between RAD51 ( green ) and telomeres ( red FISH probe ) . White arrows indicate representative RAD51 - telomere co - localization events . Quantifications are shown on the right . A total of 120 nuclei were scored from two independent experiments . Data information : All scale bars : 5 μm . Data are shown as mean ± SD . B , the red line marks the mean value . Statistical analyses were performed with Student ' s two - tailed unpaired t - tests with Welch ' s correction for normality of distribution , unless specified otherwise . C . Representative images for TIF quantification : co - localization between 53BP1 ( green ) and telomeres ( red FISH probe ) in U2OS or IMRB cells with or without ectopic hTR overexpression . White arrows indicate TIF . Quantification of TIF is on the right . A total of 110 - 170 nuclei were scored from three independent experiments . Each dot represents the number of TIF per nucleus . Data information : All scale bars : 5 μm . Data are shown as mean ± SD . , C , the red line marks the mean value . Statistical analyses were performed with Student ' s two - tailed unpaired t - tests with Welch ' s correction for normality of distribution , unless specified otherwise . D . Representative images for TIF quantification , as in ( A ) , in U2OS or U2OS - hTR cells , treated as indicated with DMSO or 5 μM ATRi or 5 μM ATMi for 20 h . White arrows indicate TIF . Data information : All scale bars : 5 μm . Data are shown as mean ± SD . E . Quantification of TIF from ( D ) . At least 65 nuclei were scored in each of the two independent experiments performed . Each dot represents the number of TIF per nucleus . Statistical analyses were performed using one - way ANOVA with post - hoc Games - Howell ' s test for multiple comparisons . Data information : All scale bars : 5 μm . Data are shown as mean ± SD . the red line marks the mean value . Statistical analyses were performed with Student ' s two - tailed unpaired t - tests with Welch ' s correction for normality of distribution , unless specified otherwise . F . Representative images for co - localization between POLD3 ( red ) and RAP1 ( green ) . White arrows indicate co - localization events . Quantification of co - localization events between POLD3 and RAP1 is shown on the right . A total of 190 - 230 nuclei were scored from three independent experiments . Data information : All scale bars : 5 μm . Data are shown as mean ± SD . F : the red line marks the mean value . Statistical analyses were performed with Student ' s two - tailed unpaired t - tests with Welch ' s correction for normality of distribution , unless specified otherwise . G . C - circle analysis using slot - blot assay hybridized with radioactive C - rich telomeric probe . Quantification is shown below ( n = 7 ) . Statistical analyses were performed using the non - parametrical Mann Whitney test . Data information : All scale bars : 5 μm . Data are shown as mean ± SD . H , I . C - circle analyses in the indicated cell lines 72 h after treatment with siPOLD3 or siLuc used as control for transfection . Quantifications are shown below ( n = 3 ) . siSMARCAL1 was used as positive control for increased C - circle production in cells with defective fork reversal activity ( Zhang et al , 2019b ) . Data information : All scale bars : 5 μm . Data are shown as mean ± SD .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. Representative images for fragile telomeres using the CO-FISH assay. Lagging strands (C-probe) and leading strands (G-probe) were marked with green PNA probes and red LNA probes, respectively. White circles and arrows indicate "smeary" or "doublet" telomeric signals, respectively. Quantification of fragile telomeres is shown on the right as the percentage of total telomeres counted (n=932-1073). Comparison of fragile to non-fragile telomere percentage was performed using Χ2-test. Data information: All scale bars: 5 μm. Data are shown as mean ± SD.B. Representative images for co-localization between RAD51 (green) and telomeres (red FISH probe). White arrows indicate representative RAD51-telomere co-localization events. Quantifications are shown on the right. A total of 120 nuclei were scored from two independent experiments. Data information: All scale bars: 5 μm. Data are shown as mean ± SD. B, the red line marks the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-tests with Welch's correction for normality of distribution, unless specified otherwise.C. Representative images for TIF quantification: co-localization between 53BP1 (green) and telomeres (red FISH probe) in U2OS or IMRB cells with or without ectopic hTR overexpression. White arrows indicate TIF. Quantification of TIF is on the right. A total of 110-170 nuclei were scored from three independent experiments. Each dot represents the number of TIF per nucleus. Data information: All scale bars: 5 μm. Data are shown as mean ± SD. , C, the red line marks the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-tests with Welch's correction for normality of distribution, unless specified otherwise.D. Representative images for TIF quantification, as in (A), in U2OS or U2OS-hTR cells, treated as indicated with DMSO or 5 μM ATRi or 5 μM ATMi for 20 h. White arrows indicate TIF. Data information: All scale bars: 5 μm. Data are shown as mean ± SD.E. Quantification of TIF from (D). At least 65 nuclei were scored in each of the two independent experiments performed. Each dot represents the number of TIF per nucleus. Statistical analyses were performed using one-way ANOVA with post-hoc Games-Howell's test for multiple comparisons. Data information: All scale bars: 5 μm. Data are shown as mean ± SD. the red line marks the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-tests with Welch's correction for normality of distribution, unless specified otherwise.F. Representative images for co-localization between POLD3 (red) and RAP1 (green). White arrows indicate co-localization events. Quantification of co-localization events between POLD3 and RAP1 is shown on the right. A total of 190-230 nuclei were scored from three independent experiments. Data information: All scale bars: 5 μm. Data are shown as mean ± SD. F: the red line marks the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-tests with Welch's correction for normality of distribution, unless specified otherwise.G. C-circle analysis using slot-blot assay hybridized with radioactive C-rich telomeric probe. Quantification is shown below (n=7). Statistical analyses were performed using the non-parametrical Mann Whitney test. Data information: All scale bars: 5 μm. Data are shown as mean ± SD.H,I. C-circle analyses in the indicated cell lines 72 h after treatment with siPOLD3 or siLuc used as control for transfection. Quantifications are shown below (n=3). siSMARCAL1 was used as positive control for increased C-circle production in cells with defective fork reversal activity (Zhang et al, 2019b). Data information: All scale bars: 5 μm. Data are shown as mean ± SD.


Figure 6A . Representative images for co - localization between PML ( green ) and telomeres ( red FISH probe ) . White arrows indicate co - localization events , marking APBs . Quantification of images is shown on the right . A total of 85 - 300 nuclei were scored from four independent experiments . Each dot represents the number of events per nucleus : PML foci or APBs . Data information : All scale bars : 5 μm . Data are shown as mean ± SD , with the red line marking the mean value . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution . C . Representative images for co - localization between EdU ( green ) and telomeres ( red FISH probe ) within PML bodies ( cyan ) in U2OS and U2OS - hTR cells . White arrows indicate co - localization events . D , E . Quantification of images shown in ( C ) . Co - localization events between EdU foci and APBs were analyzed ( D ) and the percentage of APBs that are actively engaged in G2 / M telomeric synthesis was calculated ( E ) . A total of at least 50 nuclei were scored from three independent experiments . Data information : All scale bars : 5 μm . Data are shown as mean ± SD , with the red line marking the mean value . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. Representative images for co-localization between PML (green) and telomeres (red FISH probe). White arrows indicate co-localization events, marking APBs. Quantification of images is shown on the right. A total of 85-300 nuclei were scored from four independent experiments. Each dot represents the number of events per nucleus: PML foci or APBs. Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution.C. Representative images for co-localization between EdU (green) and telomeres (red FISH probe) within PML bodies (cyan) in U2OS and U2OS-hTR cells. White arrows indicate co-localization events. D, E. Quantification of images shown in (C). Co-localization events between EdU foci and APBs were analyzed (D) and the percentage of APBs that are actively engaged in G2/M telomeric synthesis was calculated (E). A total of at least 50 nuclei were scored from three independent experiments. Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution.


Figure 7A . Representative images for co - localization between RPA32 ( green ) and single stranded C - rich telomeric sequences ( ss - Telo - C , red FISH probe ) , in G2 / M - synchronized cells . White arrows indicate co - localization events . B . Quantification of images in ( A ) . A total of at least 55 nuclei were scored from three independent experiments . Each dot represents the number of events per nucleus . C . Quantification of images in ( A ) as the percentage of single stranded C - rich telomeric sequences associated with RPA32 to total single stranded C - rich telomeric sequences A total of at least 55 nuclei were scored from three independent experiments . Data information : All scale bars : 5 μm . Data are shown as mean ± SD , with the red line marking the mean value . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution unless specified otherwise . D . Representative images for co - localization between RPA32 or p - RPA32S33 ( green ) and telomeres ( red FISH probe ) , in the indicated cell lines treated with siLuc or sihnRNPA1 for 48 h . White arrows indicate co - localization events . Data information : All scale bars : 5 μm . Data are shown as mean ± SD , with the red line marking the mean value . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution unless specified otherwise . E . Quantification of images in ( D ) . A total of 85 - 135 nuclei were scored from three independent experiments . Each dot represents the number of events per nucleus . Statistical analyses were performed using one - way ANOVA with post - hoc Games - Howell ' s test for multiple comparisons . Data information : All scale bars : 5 μm . Data are shown as mean ± SD , with the red line marking the mean value . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution unless specified otherwise . F . Representative images for co - localization between RPA32 ( green ) and telomeres ( red FISH probe ) in the indicated cell lines treated with either DMSO or 10 μM NU7026 DNA - PK - inhibitor ( DNA - PK - i ) for 48 h . White arrows indicate co - localization events . Quantifications are shown on the right . A total of 53 - 84 nuclei were scored from three independent experiments . Statistical analyses were performed using one - way ANOVA with post - hoc Games - Howell ' s test for multiple comparisons . Data information : All scale bars : 5 μm . Data are shown as mean ± SD , with the red line marking the mean value . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution unless specified otherwise .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A. Representative images for co-localization between RPA32 (green) and single stranded C-rich telomeric sequences (ss-Telo-C, red FISH probe), in G2/M-synchronized cells. White arrows indicate co-localization events. B. Quantification of images in (A). A total of at least 55 nuclei were scored from three independent experiments. Each dot represents the number of events per nucleus. C. Quantification of images in (A) as the percentage of single stranded C-rich telomeric sequences associated with RPA32 to total single stranded C-rich telomeric sequences A total of at least 55 nuclei were scored from three independent experiments. Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise.D. Representative images for co-localization between RPA32 or p-RPA32S33 (green) and telomeres (red FISH probe), in the indicated cell lines treated with siLuc or sihnRNPA1 for 48 h. White arrows indicate co-localization events. Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise.E. Quantification of images in (D). A total of 85-135 nuclei were scored from three independent experiments. Each dot represents the number of events per nucleus. Statistical analyses were performed using one-way ANOVA with post-hoc Games-Howell's test for multiple comparisons. Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise.F. Representative images for co-localization between RPA32 (green) and telomeres (red FISH probe) in the indicated cell lines treated with either DMSO or 10 μM NU7026 DNA-PK-inhibitor (DNA-PK-i) for 48 h. White arrows indicate co-localization events. Quantifications are shown on the right. A total of 53-84 nuclei were scored from three independent experiments. Statistical analyses were performed using one-way ANOVA with post-hoc Games-Howell's test for multiple comparisons. Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise.


Figure 8A . hTR and NHP2 mRNA expression in U2OS - hTR cells 72 h post - transfection with siLuc or siNHP2 , normalized to ACTB mRNA and to SI14 / TEL + cells ( n = 2 ) . Data information : All scale bars : 5 μm . Data are shown as mean ± SD , with the red line marking the mean value . B . Western blot showing NHP2 protein levels in the indicated cells treated with siLuc or siNHP2 , 72 h post - transfection . β - Actin served as loading control . C . Representative images for co - localization between p - RPA32S33 ( green ) and telomeres ( red FISH probe ) in U2OS - hTR cells treated with control siLuc or siNHP2 , 72 h post - transfection . White arrows indicate co - localization events . Quantification of images is shown on the right . Each dot indicates the number of co - localization events per nucleus ( n = 2 ) . Statistical analyses were performed with Student ' s two - tailed unpaired t - tests with Welch ' s correction for normality of distribution . Data information : All scale bars : 5 μm . Data are shown as mean ± SD , with the red line marking the mean value . D . Representative images for TIF quantification : co - localization between 53BP1 ( green ) and telomeres ( red FISH probe ) , measured 72 h post - transfection with siLuc or siNHP2 in cells further treated with either DMSO or Doxorubicin ( DOXO , 1 . 5 μM ) for 2 h . White arrows indicate TIF . Quantification of TIF is shown on the right . A total of at least 100 nuclei were scored from two independent experiments . Each dot represents the number of TIF per nucleus . Statistical analyses were performed using one - way ANOVA with post - hoc Games - Howell ' s test for multiple comparisons . Data information : All scale bars : 5 μm . Data are shown as mean ± SD , with the red line marking the mean value . E . Western blot analyses in U2OS and U2OS - hTR cells treated with the indicated siRNAs , 72 h post - transfection to detect total protein levels using the indicated antibodies . β - Actin and Vinculin served as loading controls . F . Representative images for 53BP1 foci formation in U2OS and U2OS - hTR cells , 72 h post - transfection with siLuc or siNHP2 , exposed to 1 Gy irradiation and fixed at various time points post - irradiation as indicated . G . Quantification of images shown in ( F ) . Each dot represents the number of 53BP1 foci per nucleus . A total of 114 - 140 nuclei were scored from two independent experiments . Statistical analyses were performed using two - way ANOVA Mixed effect model . Data information : All scale bars : 5 μm . Data are shown as mean ± SD , with the red line marking the mean value .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A. hTR and NHP2 mRNA expression in U2OS-hTR cells 72 h post-transfection with siLuc or siNHP2, normalized to ACTB mRNA and to SI14/TEL+ cells (n=2). Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value.B. Western blot showing NHP2 protein levels in the indicated cells treated with siLuc or siNHP2, 72 h post-transfection. β-Actin served as loading control.C. Representative images for co-localization between p-RPA32S33 (green) and telomeres (red FISH probe) in U2OS-hTR cells treated with control siLuc or siNHP2, 72 h post-transfection. White arrows indicate co-localization events. Quantification of images is shown on the right. Each dot indicates the number of co-localization events per nucleus (n=2). Statistical analyses were performed with Student's two-tailed unpaired t-tests with Welch's correction for normality of distribution. Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value.D. Representative images for TIF quantification: co-localization between 53BP1 (green) and telomeres (red FISH probe), measured 72 h post-transfection with siLuc or siNHP2 in cells further treated with either DMSO or Doxorubicin (DOXO, 1.5 μM) for 2 h. White arrows indicate TIF. Quantification of TIF is shown on the right. A total of at least 100 nuclei were scored from two independent experiments. Each dot represents the number of TIF per nucleus. Statistical analyses were performed using one-way ANOVA with post-hoc Games-Howell's test for multiple comparisons. Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value.E. Western blot analyses in U2OS and U2OS-hTR cells treated with the indicated siRNAs, 72 h post-transfection to detect total protein levels using the indicated antibodies. β-Actin and Vinculin served as loading controls.F. Representative images for 53BP1 foci formation in U2OS and U2OS-hTR cells, 72 h post-transfection with siLuc or siNHP2, exposed to 1 Gy irradiation and fixed at various time points post-irradiation as indicated.G. Quantification of images shown in (F). Each dot represents the number of 53BP1 foci per nucleus. A total of 114-140 nuclei were scored from two independent experiments. Statistical analyses were performed using two-way ANOVA Mixed effect model. Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value.


Figure 1Human peripheral blood mononuclear cells ( PBMCs ) were cultivated for 48h in RPMI containing 80mg / dl glucose ( NC ) and supplemented with 10mM beta - hydroxybutyrate ( BHB ) . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . Human pan T cell RNA was isolated and cell culture supernatant was sampled . A mRNA expression of CD4 + cytokines Il2 , IL4 , IL8 and IL22 relative to endogenous controls , n = 13 / 11 / 10 / 8 biological replicates . Data information : Data depicted as mean ± SEM ( protein data ) and box plots with median , twenty - fifth and seventy - fifth percentiles and range ( all other ) . Dots indicating individual values . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate . Human peripheral blood mononuclear cells ( PBMCs ) were cultivated for 48h in RPMI containing 80mg / dl glucose ( NC ) and supplemented with 10mM beta - hydroxybutyrate ( BHB ) . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . Human pan T cell RNA was isolated and cell culture supernatant was sampled . D mRNA expression of CD8 + cytokines IFNγ , PRF1 , TNFα , GZMB and CTLA4 in stimulated human T cells relative to internal controls , n = 15 / 14 / 10 / 14 / 9 biological replicates . Data information : Data depicted as mean ± SEM ( protein data ) and box plots with median , twenty - fifth and seventy - fifth percentiles and range ( all other ) . Dots indicating individual values . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate . Human peripheral blood mononuclear cells ( PBMCs ) were cultivated for 48h in RPMI containing 80mg / dl glucose ( NC ) and supplemented with 10mM beta - hydroxybutyrate ( BHB ) . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . Human pan T cell RNA was isolated and cell culture supernatant was sampled . F Foxp3 mRNA relative to internal control in human PBMCs and quantification of CD4 + CD25 + Foxp3 + regulatory T cells ( Treg ) following 5 days of Treg differentiation with a representative Foxp3 histogram plot ( right side ) , n = 7 / 11 biological replicates . Data information : Data depicted as mean ± SEM ( protein data ) and box plots with median , twenty - fifth and seventy - fifth percentiles and range ( all other ) . Dots indicating individual values . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate . Human peripheral blood mononuclear cells ( PBMCs ) were cultivated for 48h in RPMI containing 80mg / dl glucose ( NC ) and supplemented with 10mM beta - hydroxybutyrate ( BHB ) . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . Human pan T cell RNA was isolated and cell culture supernatant was sampled . G IL10 and TGFβ1 mRNA and protein expression of human Treg cells , n = 8 / 9 ( IL10 ) , 5 / 9 ( TGFβ1 ) biological replicates . Data information : Data depicted as mean ± SEM ( protein data ) and box plots with median , twenty - fifth and seventy - fifth percentiles and range ( all other ) . Dots indicating individual values . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Human pan T cell RNA was isolated and cell culture supernatant was sampled. A mRNA expression of CD4+ cytokines Il2, IL4, IL8 and IL22 relative to endogenous controls, n=13/11/10/8 biological replicates. Data information: Data depicted as mean ± SEM (protein data) and box plots with median, twenty-fifth and seventy-fifth percentiles and range (all other). Dots indicating individual values. *p<0.05, **p<0.01, ***p<0.001, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Human pan T cell RNA was isolated and cell culture supernatant was sampled. D mRNA expression of CD8+ cytokines IFNγ, PRF1, TNFα, GZMB and CTLA4 in stimulated human T cells relative to internal controls, n=15/14/10/14/9 biological replicates. Data information: Data depicted as mean ± SEM (protein data) and box plots with median, twenty-fifth and seventy-fifth percentiles and range (all other). Dots indicating individual values. *p<0.05, **p<0.01, ***p<0.001, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Human pan T cell RNA was isolated and cell culture supernatant was sampled. F Foxp3 mRNA relative to internal control in human PBMCs and quantification of CD4+CD25+Foxp3+ regulatory T cells (Treg) following 5 days of Treg differentiation with a representative Foxp3 histogram plot (right side), n=7/11 biological replicates. Data information: Data depicted as mean ± SEM (protein data) and box plots with median, twenty-fifth and seventy-fifth percentiles and range (all other). Dots indicating individual values. *p<0.05, **p<0.01, ***p<0.001, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Human pan T cell RNA was isolated and cell culture supernatant was sampled. G IL10 and TGFβ1 mRNA and protein expression of human Treg cells, n=8/9 (IL10), 5/9 (TGFβ1) biological replicates. Data information: Data depicted as mean ± SEM (protein data) and box plots with median, twenty-fifth and seventy-fifth percentiles and range (all other). Dots indicating individual values. *p<0.05, **p<0.01, ***p<0.001, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.


Figure 2Human PBMCs were cultivated for 48h in RPMI containing 80mg / dl glucose ( NC ) and supplemented with 10mM beta - hydroxybutyrate ( BHB ) . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . Pan T cells , CD4 + and CD8 + T cells were isolated through magnetic cell separation . Mitochondrial metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer . ( A - C ) OCR , maximum respiration , spare respiratory capacity and basal respiration were measured in A pan T cells , B CD4 + T cells and C CD8 + T cells , n = 9 ( pan T ) , n = 3 / 4 ( CD4 unstimulated ) , n = 7 / 8 / 9 ( CD4 stimulated ) , n = 5 ( CD8 unstimulated ) and n = 8 / 7 / 7 ( CD8 stimulated ) individual experiments . Data depicted as mean ± SEM ( OCR ) and box plots with median , twenty - fifth and seventy - fifth percentiles and range ( all other ) . Dots indicating individual values . * p < 0 . 05 , * * p < 0 . 01 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Human PBMCs were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Pan T cells, CD4+ and CD8+ T cells were isolated through magnetic cell separation. Mitochondrial metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer. (A-C) OCR, maximum respiration, spare respiratory capacity and basal respiration were measured in A pan T cells, B CD4+ T cells and C CD8+ T cells, n=9 (pan T), n=3/4 (CD4 unstimulated), n=7/8/9 (CD4 stimulated), n=5 (CD8 unstimulated) and n=8/7/7 (CD8 stimulated) individual experiments. Data depicted as mean ± SEM (OCR) and box plots with median, twenty-fifth and seventy-fifth percentiles and range (all other). Dots indicating individual values. *p<0.05, **p<0.01, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.