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 .

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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 .

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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 .

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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 .

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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.


A . 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 .

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A. 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 .

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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.


A . 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 .

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A. 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 .

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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 .

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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 .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

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 .

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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 .

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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 O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O

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.


A . 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 .

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A. 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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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 .

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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.


A . 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 .

A. 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 .

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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.


A . 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 .

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A. 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 .

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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 .

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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 .

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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.


A . 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 .

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A. 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 .

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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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


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 . 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 .

O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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. 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 .

O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

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.


Human 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 O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Human 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.


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 . A Quantification of cellular reactive oxygen species ( ROS ) using CellROX dye , indicated by mean fluorescence intensity ( MFI ) FITC in human pan / CD4 + / CD8 + T cells , n = 8 / 6 / 6 biological replicates . B Mitochondrial superoxide production quantified through MitoSOX staining , depicted as MFI Phycoerythrin ( PE ) in human pan / CD4 + / CD8 + T cells , n = 8 / 14 / 14 biological replicates . Data information : Data depicted as box plots with median , twenty - fifth and seventy - fifth percentiles and range . Dots indicating individual values . * p < 0 . 05 , * * p < 0 . 01 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate .

O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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. A Quantification of cellular reactive oxygen species (ROS) using CellROX dye, indicated by mean fluorescence intensity (MFI) FITC in human pan/CD4+/CD8+ T cells, n=8/6/6 biological replicates. B Mitochondrial superoxide production quantified through MitoSOX staining, depicted as MFI Phycoerythrin (PE) in human pan/CD4+/CD8+ T cells, n=8/14/14 biological replicates. Data information: Data depicted as box plots with median, twenty-fifth and seventy-fifth percentiles and range. Dots indicating individual values. *p<0.05, **p<0.01, 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 . C Confocal microscopy images of human CD4 + / CD8 + T cells , stained with MitoTracker green , representative of two individual experiments .

O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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. C Confocal microscopy images of human CD4+/CD8+ T cells, stained with MitoTracker green, representative of two individual experiments.


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 . Flow cytometry analyses were performed using the indicated dyes and antibodies identifying T cells and CD4 + / CD8 + T cell subsets . K Flow cytometric quantification of memory T cells : CD4 + / CD8 + T cells were stained for CCR7 ( PE + / - ) and subsequently defined memory phenotype for CD45RO + ( Pacific Blue + ) and CD45RA - ( PerCP - ) staining . Histogram plots depicting the exemplary change of CD45RO and CD45RA distribution for CD8 + T cells following memory cell differentiation ( left side ; NC = black , BHB = red ) . Fractions of CCR7 + CD45RA - CD45RO + central memory ( CM ) and CCR7 - CD45RA - CD45RO + effector memory ( EM ) CD4 + / 8 + T cells ( right side ) , n = 12 / 11 / 8 / 7 biological replicates . Data information : Data depicted as box plots with median , twenty - fifth and seventy - fifth percentiles and range . Dots indicating individual values . * p < 0 . 05 , * * p < 0 . 01 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate .

O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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. Flow cytometry analyses were performed using the indicated dyes and antibodies identifying T cells and CD4+/CD8+ T cell subsets. K Flow cytometric quantification of memory T cells: CD4+/CD8+ T cells were stained for CCR7 (PE+/-) and subsequently defined memory phenotype for CD45RO+ (Pacific Blue+) and CD45RA- (PerCP-) staining. Histogram plots depicting the exemplary change of CD45RO and CD45RA distribution for CD8+ T cells following memory cell differentiation (left side; NC=black, BHB=red). Fractions of CCR7+CD45RA-CD45RO+ central memory (CM) and CCR7-CD45RA-CD45RO+ effector memory (EM) CD4+/8+ T cells (right side), n=12/11/8/7 biological replicates. Data information: Data depicted as box plots with median, twenty-fifth and seventy-fifth percentiles and range. Dots indicating individual values. *p<0.05, **p<0.01, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.


Healthy volunteers conducted a three week KD with a limited carbohydrate consumption of < 30g per day . Blood was taken and analyzed prior to ( T0 ) and post KD ( T1 ) . PBMCs were isolated . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . CD4 + / CD8 + T cells were separated via magnetic cell labeling . A IL2 , IL4 , and CTLA4 mRNA expression in stimulated CD4 + T cells , n = 17 / 18 / 17 individual human subjects . Data information : Data depicted as box plots with median , twenty - fifth and seventy - fifth percentiles and range . Dots indicating individual values . * p < 0 . 05 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood was taken and analyzed prior to (T0) and post KD (T1). PBMCs were isolated. T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. CD4+/CD8+ T cells were separated via magnetic cell labeling. A IL2, IL4, and CTLA4 mRNA expression in stimulated CD4+ T cells, n=17/18/17 individual human subjects. Data information: Data depicted as box plots with median, twenty-fifth and seventy-fifth percentiles and range. Dots indicating individual values. *p<0.05, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.


Healthy volunteers conducted a three week KD with a limited carbohydrate consumption of < 30g per day . Blood was taken and analyzed prior to ( T0 ) and post KD ( T1 ) . PBMCs were isolated . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . CD4 + / CD8 + T cells were separated via magnetic cell labeling . B Tbet and GATA3 mRNA expression in CD4 + T cells , n = 7 individual human subjects . Data information : Data depicted as box plots with median , twenty - fifth and seventy - fifth percentiles and range . Dots indicating individual values . * p < 0 . 05 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood was taken and analyzed prior to (T0) and post KD (T1). PBMCs were isolated. T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. CD4+/CD8+ T cells were separated via magnetic cell labeling. B Tbet and GATA3 mRNA expression in CD4+ T cells, n=7 individual human subjects. Data information: Data depicted as box plots with median, twenty-fifth and seventy-fifth percentiles and range. Dots indicating individual values. *p<0.05, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.


Healthy volunteers conducted a three week KD with a limited carbohydrate consumption of < 30g per day . Blood was taken and analyzed prior to ( T0 ) and post KD ( T1 ) . PBMCs were isolated . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . CD4 + / CD8 + T cells were separated via magnetic cell labeling . C Flow cytometric quantification of Th1 / Th2 cells and respective ratio of Th1 / Th2 cells , n = 7 individual human subjects . Data information : Data depicted as box plots with median , twenty - fifth and seventy - fifth percentiles and range . Dots indicating individual values . * p < 0 . 05 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood was taken and analyzed prior to (T0) and post KD (T1). PBMCs were isolated. T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. CD4+/CD8+ T cells were separated via magnetic cell labeling. C Flow cytometric quantification of Th1/Th2 cells and respective ratio of Th1/Th2 cells, n=7 individual human subjects. Data information: Data depicted as box plots with median, twenty-fifth and seventy-fifth percentiles and range. Dots indicating individual values. *p<0.05, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.


Healthy volunteers conducted a three week KD with a limited carbohydrate consumption of < 30g per day . Blood was taken and analyzed prior to ( T0 ) and post KD ( T1 ) . PBMCs were isolated . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . CD4 + / CD8 + T cells were separated via magnetic cell labeling . D IL10 mRNA expression and flow cytometric quantification of CD4 + CD25 + Foxp3 + regulatory T cells ( Treg ) , n = 19 / 9individual human subjects . Data information : Data depicted as box plots with median , twenty - fifth and seventy - fifth percentiles and range . Dots indicating individual values . * p < 0 . 05 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood was taken and analyzed prior to (T0) and post KD (T1). PBMCs were isolated. T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. CD4+/CD8+ T cells were separated via magnetic cell labeling. D IL10 mRNA expression and flow cytometric quantification of CD4+CD25+Foxp3+ regulatory T cells (Treg), n=19/9individual human subjects. Data information: Data depicted as box plots with median, twenty-fifth and seventy-fifth percentiles and range. Dots indicating individual values. *p<0.05, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.


Healthy volunteers conducted a three week KD with a limited carbohydrate consumption of < 30g per day . Blood was taken and analyzed prior to ( T0 ) and post KD ( T1 ) . PBMCs were isolated . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . CD4 + / CD8 + T cells were separated via magnetic cell labeling . E IFNγ GZMB , PRF1 and CTLA4 mRNA expression in CD8 + T cells , n = 16 / 18 / 17 / 17 individual human subjects . Data information : Data depicted as box plots with median , twenty - fifth and seventy - fifth percentiles and range . Dots indicating individual values . * p < 0 . 05 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood was taken and analyzed prior to (T0) and post KD (T1). PBMCs were isolated. T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. CD4+/CD8+ T cells were separated via magnetic cell labeling. E IFNγ GZMB, PRF1 and CTLA4 mRNA expression in CD8+ T cells, n=16/18/17/17 individual human subjects. Data information: Data depicted as box plots with median, twenty-fifth and seventy-fifth percentiles and range. Dots indicating individual values. *p<0.05, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.


Healthy volunteers conducted a three week KD with a limited carbohydrate consumption of < 30g per day . Blood was taken and analyzed prior to ( T0 ) and post KD ( T1 ) . PBMCs were isolated . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . CD4 + / CD8 + T cells were separated via magnetic cell labeling . F relative CD8 + cell lysis activity as measured by calcein - fluorescence of isolated T cells , n = 4 individual experiments . Data information : Data depicted as box plots with median , twenty - fifth and seventy - fifth percentiles and range . Dots indicating individual values . * p < 0 . 05 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood was taken and analyzed prior to (T0) and post KD (T1). PBMCs were isolated. T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. CD4+/CD8+ T cells were separated via magnetic cell labeling. F relative CD8+ cell lysis activity as measured by calcein-fluorescence of isolated T cells, n=4 individual experiments. Data information: Data depicted as box plots with median, twenty-fifth and seventy-fifth percentiles and range. Dots indicating individual values. *p<0.05, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.


44 healthy volunteers conducted a three week KD with a limited carbohydrate consumption of < 30g per day . Blood was taken and analyzed prior to starting the diet ( T0 ) and again after three weeks of strict adherence to the diet ( T1 ) . PBMCs were isolated . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . Pan / CD4 + / CD8 + T cells were separated via magnetic cell labeling . Mitochondrial metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer . ( A , B ) OCR , maximum respiration , spare respiratory capacity and basal respiration were measured in A CD4 + and B CD8 + T cells , n = 14 individual experiments , each performed in three technical replicates . Data information : Data depicted as mean ± SEM ( OCR ) or as box plots with median , twenty - fifth and seventy - fifth percentiles and range , crosses indicating mean Dots indicating individual values . * p < 0 . 05 , * * p < 0 . 01 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

44 healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood was taken and analyzed prior to starting the diet (T0) and again after three weeks of strict adherence to the diet (T1). PBMCs were isolated. T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Pan/CD4+/CD8+ T cells were separated via magnetic cell labeling. Mitochondrial metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer. (A, B) OCR, maximum respiration, spare respiratory capacity and basal respiration were measured in A CD4+ and B CD8+ T cells, n=14 individual experiments, each performed in three technical replicates. Data information: Data depicted as mean ± SEM (OCR) or as box plots with median, twenty-fifth and seventy-fifth percentiles and range , crosses indicating mean Dots indicating individual values. *p<0.05, **p<0.01, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.


44 healthy volunteers conducted a three week KD with a limited carbohydrate consumption of < 30g per day . Blood was taken and analyzed prior to starting the diet ( T0 ) and again after three weeks of strict adherence to the diet ( T1 ) . PBMCs were isolated . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . Pan / CD4 + / CD8 + T cells were separated via magnetic cell labeling . Mitochondrial metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer . E Western Blot of mitochondrial oxidative phosphorylation proteins in CD4 + / CD8 + T cells as indicated , representative of five individual experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

44 healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood was taken and analyzed prior to starting the diet (T0) and again after three weeks of strict adherence to the diet (T1). PBMCs were isolated. T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Pan/CD4+/CD8+ T cells were separated via magnetic cell labeling. Mitochondrial metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer. E Western Blot of mitochondrial oxidative phosphorylation proteins in CD4+/CD8+ T cells as indicated, representative of five individual experiments.


44 healthy volunteers conducted a three week KD with a limited carbohydrate consumption of < 30g per day . Blood was taken and analyzed prior to starting the diet ( T0 ) and again after three weeks of strict adherence to the diet ( T1 ) . PBMCs were isolated . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . Pan / CD4 + / CD8 + T cells were separated via magnetic cell labeling . Mitochondrial metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer . F Confocal microscopy images of human CD4 + / CD8 + T cells , stained with MitoTracker green , representative of two individual experiments .

44 healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood was taken and analyzed prior to starting the diet (T0) and again after three weeks of strict adherence to the diet (T1). PBMCs were isolated. T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Pan/CD4+/CD8+ T cells were separated via magnetic cell labeling. Mitochondrial metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer. F Confocal microscopy images of human CD4+/CD8+ T cells, stained with MitoTracker green, representative of two individual experiments.


44 healthy volunteers conducted a three week KD with a limited carbohydrate consumption of < 30g per day . Blood was taken and analyzed prior to starting the diet ( T0 ) and again after three weeks of strict adherence to the diet ( T1 ) . PBMCs were isolated . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . Pan / CD4 + / CD8 + T cells were separated via magnetic cell labeling . Mitochondrial metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer . H TruCulture IFNγ , IL4 , IL6 , IL8 , IL12 subunit p40 , IL23 and TNFα protein quantification of LPS - stimulated whole blood samples , n = 11 / 12 / 11 / 10 / 11 / 10 / 11 individual human subjects . Data information : Data depicted as mean ± SEM ( OCR ) or as box plots with median , twenty - fifth and seventy - fifth percentiles and range Dots indicating individual values . * p < 0 . 05 , * * p < 0 . 01 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate .

44 healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood was taken and analyzed prior to starting the diet (T0) and again after three weeks of strict adherence to the diet (T1). PBMCs were isolated. T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Pan/CD4+/CD8+ T cells were separated via magnetic cell labeling. Mitochondrial metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer. H TruCulture IFNγ, IL4, IL6, IL8, IL12 subunit p40, IL23 and TNFα protein quantification of LPS-stimulated whole blood samples, n=11/12/11/10/11/10/11 individual human subjects. Data information: Data depicted as mean ± SEM (OCR) or as box plots with median, twenty-fifth and seventy-fifth percentiles and range Dots indicating individual values. *p<0.05, **p<0.01, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.


( D ) HaloTag pulse - chase analysis in WT MEF monitoring fluorescent Halo - AATNNN during up to 6 h of chase in the presence of BafA1 . The fluorescent JF646 - Halo ligand and the polymer - specific 2C1 antibody stains are shown . ( E ) Same as ( D ) for fluorescent Halo - ATZNNN . ( F ) Quantification of accumulation of fluorescent Halo - AATNNN and Halo - ATZNNN within LAMP1 - positive endolysosomes . Mean ± SEM , n = 20 , 11 , 10 , 13 for Halo - AATNNN , n = 18 , 12 , 12 , 13 for Halo - ATZNNN . Two - way ANOVA and Sidak ' s multiple comparison test , ns P > 0 . 05 , * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) HaloTag pulse-chase analysis in WT MEF monitoring fluorescent Halo-AATNNN during up to 6 h of chase in the presence of BafA1. The fluorescent JF646-Halo ligand and the polymer-specific 2C1 antibody stains are shown. (E) Same as (D) for fluorescent Halo-ATZNNN. (F) Quantification of accumulation of fluorescent Halo-AATNNN and Halo-ATZNNN within LAMP1-positive endolysosomes. Mean ± SEM, n=20, 11, 10, 13 for Halo-AATNNN, n= 18, 12, 12, 13 for Halo-ATZNNN. Two-way ANOVA and Sidak's multiple comparison test, ns P>0.05, *** P<0.001.


A Confocal laser scanning microscopy analyses in cells treated with BafA1 . Total HA - ATZNNN ( red ) or HA - ATZNNN polymers ( magenta ) in LAMP1 - positive endolysosome ( green ) in WT MEF . ( C ) Same as ( A ) in Cnx - KO MEF . ( D ) Same as ( A ) in WT MEF exposed to kifunensine ( KIF ) . ( E ) Same as ( A ) in WT MEF exposed to Castanospermine ( CST ) . ( F ) Same as ( A ) in sCNX MEF . ( G ) Same as ( A ) in Uggt1 - KO MEF .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Confocal laser scanning microscopy analyses in cells treated with BafA1. Total HA-ATZNNN (red) or HA-ATZNNN polymers (magenta) in LAMP1-positive endolysosome (green) in WT MEF. (C) Same as (A) in Cnx-KO MEF. (D) Same as (A) in WT MEF exposed to kifunensine (KIF). (E) Same as (A) in WT MEF exposed to Castanospermine (CST). (F) Same as (A) in sCNX MEF. (G) Same as (A) in Uggt1-KO MEF.


( J ) Halo - ATZNNN in Uggt1 - KO MEF mock - transfected ( empty pcDNA3 plasmid ) . ( K , L ) ( K ) Same as ( J ) , with a plasmid for expression of active or ( L ) inactive UGGT1 . ( M ) Quantification of Halo - ATZNNN - positive endolysosomes ( mean , n = 22 , 10 , 15 cells for panels J - L , respectively ) . One - way ANOVA and Dunnett ' s multiple comparison test , ns P > 0 . 05 , * * * * P < 0 . 0001 ) .

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(J) Halo-ATZNNN in Uggt1-KO MEF mock-transfected (empty pcDNA3 plasmid). (K, L) (K) Same as (J), with a plasmid for expression of active or (L) inactive UGGT1. (M) Quantification of Halo-ATZNNN-positive endolysosomes (mean, n=22, 10, 15 cells for panels J-L, respectively). One-way ANOVA and Dunnett's multiple comparison test, ns P>0.05, **** P<0.0001).


( B - I ) Confocal laser scanning microscopy analyses ( as in Fig . 2 ) in WT MEF treated with BafA1 . ( J , K ) ( J ) Quantification of ATZxxx - positive endolysosomes and ( K ) of ATZxxx polymers - positive endolysosomes ( mean , n = 18 , 30 , 24 , 14 , 27 , 22 , 27 , 18 cells for HA stain and n = 12 , 14 , 14 , 11 , 14 , 14 , 17 , 11 cells for 2C1 stain of panels B - I , respectively ) . One - way ANOVA and Dunnett ' s multiple comparison test , ns P > 0 . 05 , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-I) Confocal laser scanning microscopy analyses (as in Fig. 2) in WT MEF treated with BafA1. (J, K) (J) Quantification of ATZxxx-positive endolysosomes and (K) of ATZxxx polymers-positive endolysosomes (mean, n= 18, 30, 24, 14, 27, 22, 27, 18 cells for HA stain and n= 12, 14, 14, 11, 14, 14, 17, 11 cells for 2C1 stain of panels B-I, respectively). One-way ANOVA and Dunnett's multiple comparison test, ns P>0.05, **** P<0.0001.


( C ) Co - immunoprecipitation of ATZXXX ( Bait , lower panel ) with CNX ( upper panel ) and BiP ( middle panel ) . Protein content in the total cell extracts ( TCE ) is shown . ( D ) Quantification of ( C ) for CNX association ( n = 3 ) . Unpaired two - tailed t - test , * * P < 0 . 01 * * * P < 0 . 001 , * * * * P < 0 . 0001 . ( E ) Quantification of ( C ) for BiP association ( n = 3 for ATZNNN and ATZQQN ; n = 4 for ATZQNQ and ATZQQQ ) . Unpaired two - tailed t - test , * P < 0 . 005 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Co-immunoprecipitation of ATZXXX (Bait, lower panel) with CNX (upper panel) and BiP (middle panel). Protein content in the total cell extracts (TCE) is shown. (D) Quantification of (C) for CNX association (n=3). Unpaired two-tailed t-test, ** P<0.01*** P<0.001, **** P<0.0001. (E) Quantification of (C) for BiP association (n=3 for ATZNNN and ATZQQN; n=4 for ATZQNQ and ATZQQQ). Unpaired two-tailed t-test, * P<0.005, ** P<0.01, *** P<0.001, **** P<0.0001.


( G ) Confocal laser scanning microscopy in Flp - In 3T3 cells stably expressing ATZNNN in the absence of BafA1 ( to allow clearance of misfolded model proteins delivered within endolysosomes ) . Co - localization with Calnexin ( CNX ) . ( H ) Same as ( G ) for BiP co - localization . ( I ) Same as ( G ) for ATZQQQ . ( J ) Same as ( H ) for ATZQQQ .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Confocal laser scanning microscopy in Flp-In 3T3 cells stably expressing ATZNNN in the absence of BafA1 (to allow clearance of misfolded model proteins delivered within endolysosomes). Co-localization with Calnexin (CNX). (H) Same as (G) for BiP co-localization. (I) Same as (G) for ATZQQQ. (J) Same as (H) for ATZQQQ.


( K ) HaloTag pulse - chase analysis in MEF monitoring fluorescent Halo - ATZNNN during up to 24 h . ( L ) Same as ( K ) for fluorescent Halo - ATZQQQ . Arrowheads show select clusters of JF646 - labeled Halo - ATZQQQ . Arrowheads show co - localization Halo - ATZQQQ : BiP .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K) HaloTag pulse-chase analysis in MEF monitoring fluorescent Halo-ATZNNN during up to 24 h. (L) Same as (K) for fluorescent Halo-ATZQQQ. Arrowheads show select clusters of JF646-labeled Halo-ATZQQQ. Arrowheads show co-localization Halo-ATZQQQ:BiP.


( A ) Quantification of AURKB transcripts in non - IPF lung derived resident - fibroblasts treated with indicated mitogens for 16hrs . * P < 0 . 05 , * * * P < 0 . 0005 , and * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Quantification of AURKB transcripts in non-IPF lung derived resident-fibroblasts treated with indicated mitogens for 16hrs. *P < 0.05, ***P < 0.0005, and ****P < 0.00005, 1-way ANOVA, (n=4).


( B ) Quantification of AURKB gene transcripts in isolated fibroblasts from non - IPF and IPF lung stromal cell cultures . * * P < 0 . 005 , unpaired t - test , ( n = 6 ) .

O O O O O O O O O O O O O O O O O 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) Quantification of AURKB gene transcripts in isolated fibroblasts from non-IPF and IPF lung stromal cell cultures. **P < 0.005, unpaired t-test, (n=6).


( C ) IPF and non - IPF lung sections were immunostained using AURKB antibody ( n = 4 ) . Representative images were obtained at 40X magnification . Scale bar : 40µm . Dotted area : black ; myofibroblastic core , blue ; active fibrotic front . Arrows indicate AURKB positive cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) IPF and non-IPF lung sections were immunostained using AURKB antibody (n=4). Representative images were obtained at 40X magnification. Scale bar: 40µm. Dotted area: black; myofibroblastic core, blue; active fibrotic front. Arrows indicate AURKB positive cells.


( D ) Immunoblots of Aurkb and Aurka in total lung lysates of normal ( CCSP / - ) and fibrotic ( CCSP / TGFα ) mice fed with Dox for 6wks . Quantification was performed using phosphor imager software and normalization was done using loading control GAPDH . * P < 0 . 05 , unpaired t - test , ( n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Immunoblots of Aurkb and Aurka in total lung lysates of normal (CCSP/-) and fibrotic (CCSP/TGFα) mice fed with Dox for 6wks. Quantification was performed using phosphor imager software and normalization was done using loading control GAPDH. *P < 0.05, unpaired t-test, (n=4).


( E ) Immunostaining was performed using AURKB antibody in lung sections of control ( CCSP / - ) and TGFα ( CCSP / TGFα ) mice on Dox for 6wks ( n = 6 ) . Representative images were obtained at 20X ( low ; Scale bar : 100µm ) and 63X ( high ; Scale bar : 30µm ) magnification . Dashed box indicates area of the section showing in high magnification .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Immunostaining was performed using AURKB antibody in lung sections of control (CCSP/-) and TGFα (CCSP/TGFα) mice on Dox for 6wks (n=6). Representative images were obtained at 20X (low; Scale bar: 100µm) and 63X (high; Scale bar: 30µm) magnification. Dashed box indicates area of the section showing in high magnification.


( F ) Western blot analysis of Aurkb in total lung lysates from saline and bleomycin treated mice . Quantification was performed using phosphor imager software and normalization was done using loading control Gapdh . * * P < 0 . 005 , unpaired t - test , ( n = 4 - 5 ) .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Western blot analysis of Aurkb in total lung lysates from saline and bleomycin treated mice. Quantification was performed using phosphor imager software and normalization was done using loading control Gapdh. **P < 0.005, unpaired t-test, (n=4-5).


( A ) Human IPF lung fibroblasts were transiently transfected with control or WT1 siRNA for 72hrs and AURKB transcripts were quantified . * * * * P < 0 . 00005 , unpaired t - test , ( n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Human IPF lung fibroblasts were transiently transfected with control or WT1 siRNA for 72hrs and AURKB transcripts were quantified. ****P < 0.00005, unpaired t-test, (n=4).


( B ) Lung - resident fibroblasts from TGFα mice on Dox for 4 wks were transiently transfected with control or WT1 siRNA for 72hrs and AURKB transcripts were quantified . * * * * P < 0 . 00005 , unpaired t - test , ( n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Lung-resident fibroblasts from TGFα mice on Dox for 4 wks were transiently transfected with control or WT1 siRNA for 72hrs and AURKB transcripts were quantified. ****P < 0.00005, unpaired t-test, (n=4).


( C ) Immunoblot analysis of AURKB and WT1 in the lysates of non - IPF fibroblasts transduced with control or WT1 - adenoviral particles for 72hrs . * * P < 0 . 005 , unpaired t - test , ( n = 3 ) .

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(C) Immunoblot analysis of AURKB and WT1 in the lysates of non-IPF fibroblasts transduced with control or WT1-adenoviral particles for 72hrs. **P < 0.005, unpaired t-test, (n=3).


( D ) Quantification of AURKB transcripts in primary fibroblasts from IPF lung treated with either control or WT1 siRNA and stimulated with media or TGFα ( 100ng / mL ) for 16hrs . * * * * P < 0 . 00005 , unpaired t - test , ( n = 4 ) .

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(D) Quantification of AURKB transcripts in primary fibroblasts from IPF lung treated with either control or WT1 siRNA and stimulated with media or TGFα (100ng/mL) for 16hrs. ****P < 0.00005, unpaired t-test, (n=4).


( E ) Primary lung - resident fibroblasts were isolated from stromal cultures of TGFα mice placed on Dox for 8 wks . Cell lysates were prepared , and the ChIP assay was performed with anti - WT1 antibody or normal rabbit IgG as a negative control using AURKB gene promoter - specific PCR primers . Non - immunoprecipitated DNA is represented as input DNA ( product size , 104 bp ) . * * * P < 0 . 0005 , unpaired t - test , ( n = 2 ) .

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(E) Primary lung-resident fibroblasts were isolated from stromal cultures of TGFα mice placed on Dox for 8 wks. Cell lysates were prepared, and the ChIP assay was performed with anti-WT1 antibody or normal rabbit IgG as a negative control using AURKB gene promoter-specific PCR primers. Non-immunoprecipitated DNA is represented as input DNA (product size, 104 bp). ***P < 0.0005, unpaired t-test, (n=2).


( F ) AURKB - promoter luciferase activity was measured in HEK293 cells transfected with control or WT1 overexpressing ( OE ) vector . * * * * P < 0 . 00005 , one - way - ANOVA , ( n = 6 ) .

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(F) AURKB-promoter luciferase activity was measured in HEK293 cells transfected with control or WT1 overexpressing (OE) vector. ****P < 0.00005, one-way-ANOVA, (n=6).


( C ) Lung sections from control and TGFα mice fed with Dox food for 4 wks were stained for AURKB ( green ) and Ki - 67 ( red ) . Merged image shows cells that co - express AURKB and Ki - 67 ( yellow ) .

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(C) Lung sections from control and TGFα mice fed with Dox food for 4 wks were stained for AURKB (green) and Ki-67(red). Merged image shows cells that co-express AURKB and Ki-67 (yellow).


( D ) Quantification of Ki - 67 + and AURKB + double positive cells was performed using five confocal images per mice in control and TGFα mice . Images were obtained at 40X magnification . Scale bar : 50µm . * * * * P < 0 . 00005 , unpaired t - test , ( n = 4 ) .

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(D) Quantification of Ki-67+ and AURKB+ double positive cells was performed using five confocal images per mice in control and TGFα mice. Images were obtained at 40X magnification. Scale bar: 50µm. ****P < 0.00005, unpaired t-test, (n=4).


( E ) Proliferation was measured in primary lung resident fibroblast isolated from either IPF lung or TGFα mice and treated with either control or AURKB siRNA . * * * * P < 0 . 00005 , unpaired t - test , ( n = 9 - 11 ) .

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(E) Proliferation was measured in primary lung resident fibroblast isolated from either IPF lung or TGFα mice and treated with either control or AURKB siRNA. ****P < 0.00005, unpaired t-test, (n=9-11).


( F ) Proliferation was measured in primary lung - resident fibroblasts isolated from stromal cultures of TGFα mice on Dox for 2 wks and transiently transfected with control or AURKB siRNA and stimulated with TGFα ( 20ng / mL ) for 24hrs . * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 9 - 11 ) .

(F) Proliferation was measured in primary lung-resident fibroblasts isolated from stromal cultures of TGFα mice on Dox for 2 wks and transiently transfected with control or AURKB siRNA and stimulated with TGFα (20ng/mL) for 24hrs. ****P < 0.00005, 1-way ANOVA, (n=9-11).


( G ) Quantification of CCNA2 and PLK1 transcripts in human IPF fibroblasts transiently transfected with control or AURKB siRNA for 72hrs . * * P < 0 . 005 , * * * P < 0 . 0005 , unpaired t - test , ( n = 3 ) .

(G) Quantification of CCNA2 and PLK1 transcripts in human IPF fibroblasts transiently transfected with control or AURKB siRNA for 72hrs. **P < 0.005, ***P < 0.0005, unpaired t-test, (n=3).


( H ) Lung sections from control and TGFα mice fed with Dox food for 6 wks were stained for AURKB ( green ) and αSMA ( red ) . Images were obtained at 40X magnification . Scale bar : 50µm . ( n = 4 ) .

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(H) Lung sections from control and TGFα mice fed with Dox food for 6 wks were stained for AURKB (green) and αSMA (red). Images were obtained at 40X magnification. Scale bar: 50µm. (n=4).


( I ) Quantification of apoptotic cells using Incucyte ZOOM ( Caspase - 3 / 7 - positive cells ) in lung - resident fibroblasts isolated from IPF and TGFα mice on Dox for 6 wks and treated with control or AURKB siRNA for 72h . * * P < 0 . 005 , * * * * P < 0 . 00005 , 2 - way ANOVA , ( n = 4 ) .

(I) Quantification of apoptotic cells using Incucyte ZOOM (Caspase-3/7- positive cells) in lung-resident fibroblasts isolated from IPF and TGFα mice on Dox for 6 wks and treated with control or AURKB siRNA for 72h. **P < 0.005, ****P < 0.00005, 2-way ANOVA, (n=4).


( J ) Quantification of BAK , BAX and FAS gene transcripts in human IPF lung resident fibroblasts treated with either control or AURKB siRNA for 72hrs . * * P < 0 . 005 , * * * P < 0 . 0005 , unpaired t - test , ( n = 3 ) .

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(J) Quantification of BAK, BAX and FAS gene transcripts in human IPF lung resident fibroblasts treated with either control or AURKB siRNA for 72hrs. **P < 0.005, ***P < 0.0005, unpaired t-test, (n=3).


( K ) Quantification of Bak , Bax and Fas gene transcripts in lung resident fibroblasts isolated from TGFα mice and treated with either control or AURKB siRNA for 72hrs . * P < 0 . 005 , * * P < 0 . 005 , unpaired t - test , ( n = 3 ) .

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(K) Quantification of Bak, Bax and Fas gene transcripts in lung resident fibroblasts isolated from TGFα mice and treated with either control or AURKB siRNA for 72hrs. *P < 0.005, **P < 0.005, unpaired t-test, (n=3).


( A ) Proliferation was assessed using BrdU incorporation assay in human IPF fibroblasts treated with indicated doses of barasertib for total of 48hrs . * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 9 - 11 ) .

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(A) Proliferation was assessed using BrdU incorporation assay in human IPF fibroblasts treated with indicated doses of barasertib for total of 48hrs. ****P < 0.00005, 1-way ANOVA, (n=9-11).


( B ) Proliferation was assessed using BrdU incorporation assay in fibroblasts isolated from TGFα mice lung and treated with indicated doses of barasertib for total of 48hrs . * * * P < 0 . 0005 , 1 - way ANOVA , ( n = 9 - 11 ) .

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(B) Proliferation was assessed using BrdU incorporation assay in fibroblasts isolated from TGFα mice lung and treated with indicated doses of barasertib for total of 48hrs. ***P < 0.0005, 1-way ANOVA, (n=9-11).


( C ) Primary lung resident fibroblasts isolated from TGFα mice on Dox for 4 wks were treated with vehicle or 5µM barasertib for 48hrs and immunostained using PCNA antibody . Images were obtained at 40X magnification . Scale bar : 50µm . The number of PCNA - positive cells and total DAPI - positive cells were quantified using MetaMorph image analysis software . Proliferation is indicated as the percentage of proliferating cells in total DAPI - positive cells . * * * * P < 0 . 00005 , unpaired t - test , ( n = 3 - 4 ) .

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(C) Primary lung resident fibroblasts isolated from TGFα mice on Dox for 4 wks were treated with vehicle or 5µM barasertib for 48hrs and immunostained using PCNA antibody. Images were obtained at 40X magnification. Scale bar: 50µm. The number of PCNA-positive cells and total DAPI-positive cells were quantified using MetaMorph image analysis software. Proliferation is indicated as the percentage of proliferating cells in total DAPI-positive cells. ****P < 0.00005, unpaired t-test, (n=3-4).


( D ) Proliferation was measured in primary fibroblasts treated with TGFα ( 20ng / mL ) and indicated doses of barasertib for 48hrs . . * * P < 0 . 005 , 1 - way ANOVA , ( n = 9 - 11 ) .

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(D) Proliferation was measured in primary fibroblasts treated with TGFα (20ng/mL) and indicated doses of barasertib for 48hrs.. **P<0.005, 1-way ANOVA, (n=9-11).


( E ) Quantification of apoptotic cells using Incucyte ZOOM ( Caspase - 3 / 7 - positive cells ) in resident fibroblasts isolated from IPF lung stromal cultures and treated with vehicle or 5µM Barasertib . * * * * P < 0 . 00005 , 2 - way ANOVA , ( n = 4 ) .

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(E) Quantification of apoptotic cells using Incucyte ZOOM (Caspase-3/7- positive cells) in resident fibroblasts isolated from IPF lung stromal cultures and treated with vehicle or 5µM Barasertib. ****P < 0.00005, 2-way ANOVA, (n=4).


( B ) Representative images of Masson ' s trichrome - stained lung sections from the vehicle and barasertib treated mice . Images were obtained at 10X magnification . Scale bar : 200μm .

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(B) Representative images of Masson's trichrome-stained lung sections from the vehicle and barasertib treated mice. Images were obtained at 10X magnification. Scale bar: 200μm.


( C ) Quantification of right lung weight of mice treated with vehicle or barasertib . * * * P < 0 . 0005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 8 - 10 mice / group ) .

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(C) Quantification of right lung weight of mice treated with vehicle or barasertib. ***P< 0.0005, ****P < 0.00005, 1-way ANOVA, (n=8-10 mice/group).


( D ) Quantification of total lung hydroxyproline levels in mice treated with vehicle or barasertib . * P < 0 . 05 , * * * P < 0 . 0005 , 1 - way ANOVA , ( n = 8 - 10 mice / group ) .

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(D) Quantification of total lung hydroxyproline levels in mice treated with vehicle or barasertib. *P< 0.05, ***P < 0.0005, 1-way ANOVA, (n=8-10 mice/group).


( E ) Quantification of Col1α , Col5α and Fn1 gene transcripts in total lung of mice treated with vehicle or barasertib . * * * P < 0 . 0005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 8 mice / group ) .

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(E) Quantification of Col1α, Col5α and Fn1 gene transcripts in total lung of mice treated with vehicle or barasertib.***P< 0.0005, ****P < 0.00005, 1-way ANOVA, (n=8 mice/group).


( A ) Lung sections from vehicle and barasertib treated mice were immunostained using Ki - 67 antibody . Representative images were obtained at 20X magnification . Scale bar : 50μm .

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(A) Lung sections from vehicle and barasertib treated mice were immunostained using Ki-67 antibody. Representative images were obtained at 20X magnification. Scale bar: 50μm.


( B ) Immunoblot analysis in lung lysates from vehicle and barasertib treated mice using PCNA antibody . Gapdh is used as loading control . * * P < 0 . 005 , 1 - way ANOVA , ( n = 4 - 5 mice / group ) .

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(B) Immunoblot analysis in lung lysates from vehicle and barasertib treated mice using PCNA antibody. Gapdh is used as loading control. **P< 0.005, 1-way ANOVA, (n=4-5 mice/group).


( C ) Quantification of Aurkb , Plk1 and CcnA2 gene transcripts in total lungs of mice treated with vehicle or barasertib . * * P < 0 . 005 , * * * P < 0 . 0005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 8 mice / group ) .

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(C) Quantification of Aurkb, Plk1 and CcnA2 gene transcripts in total lungs of mice treated with vehicle or barasertib. **P< 0.005, ***P<0.0005, ****P < 0.00005, 1-way ANOVA, (n=8 mice/group).


( B ) Quantification of right lung weight of mice treated with vehicle and barasertib . * * P < 0 . 005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 9 - 10 mice / group ) .

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(B) Quantification of right lung weight of mice treated with vehicle and barasertib. **P< 0.005, ****P < 0.00005, 1-way ANOVA, (n=9-10 mice/group).


( C ) Western blot analysis in lung lysates from vehicle and barasertib treated mice using Col1α and Fn1 antibodies . Gapdh is used as loading control . * P < 0 . 05 , * * P < 0 . 005 , * * * P < 0 . 0005 , 1 - way ANOVA , ( n = 4 - 5 mice / group ) .

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(C) Western blot analysis in lung lysates from vehicle and barasertib treated mice using Col1α and Fn1 antibodies. Gapdh is used as loading control. *P< 0.05, **P<0.005, ***P < 0.0005, 1-way ANOVA, (n=4-5 mice/group).


( D ) Representative images of Masson ' s trichrome stained lung section from mice treated with vehicle and barasertib . Images were obtained at 10X magnification . Scale bar : 200μm .

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(D) Representative images of Masson's trichrome stained lung section from mice treated with vehicle and barasertib. Images were obtained at 10X magnification. Scale bar: 200μm.


( E ) Quantification of lung mechanics in mice treated with vehicle and barasertib . * P < 0 . 05 , * * P < 0 . 005 , * * * P < 0 . 0005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 9 - 10 mice / group ) .

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(E) Quantification of lung mechanics in mice treated with vehicle and barasertib. *P<0.05, **P< 0.005, ***P<0.0005, ****P < 0.00005, 1-way ANOVA, (n=9-10 mice/group).


( B ) Representative images of Masson ' s trichrome stained lung section from mice treated with vehicle and barasertib . Images were obtained at 10X magnification . Scale bar : 200μm .

(B) Representative images of Masson's trichrome stained lung section from mice treated with vehicle and barasertib. Images were obtained at 10X magnification. Scale bar: 200μm.


( C ) Quantification of total lung hydroxyproline in mice treated with vehicle and barasertib . * P < 0 . 05 , * * * P < 0 . 0005 , 1 - way ANOVA , ( n = 7 - 8 mice / group ) .

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(C) Quantification of total lung hydroxyproline in mice treated with vehicle and barasertib. *P< 0.05, ***P < 0.0005, 1-way ANOVA, (n=7-8 mice/group).


( D ) Quantification of Col1α1 , Col3α , Col5α , Col14 , Col15 , αSma , Ccna2 , and Fas gene transcripts in total lungs of mice treated with vehicle or barasertib . * P < 0 . 05 , * * P < 0 . 005 * * * P < 0 . 0005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 6 mice / group ) .

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(D) Quantification of Col1α1, Col3α, Col5α, Col14, Col15, αSma, Ccna2, and Fas gene transcripts in total lungs of mice treated with vehicle or barasertib. *P< 0.05, **P<0.005 ***P<0.0005, ****P < 0.00005, 1-way ANOVA, (n=6 mice/group).


( E ) Quantification of αSma protein levels in lung lysates from vehicle and barasertib treated mice . Gapdh is used as loading control . Data are presented as mean ± SEM . * * P < 0 . 005 , unpaired t - test ( n = 5mice / group ) .

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(E) Quantification of αSma protein levels in lung lysates from vehicle and barasertib treated mice. Gapdh is used as loading control. Data are presented as mean ± SEM. **P<0.005, unpaired t-test (n=5mice /group).


( D ) Western blot of whole zebrafish larvae , Smyhc1 stained with the F59 antibody . smyhc1 + / + larvae express Smyhc1 at 24hpf , but not at 48hpf . smyhc1 - / - larvae do not express Smyhc1 .

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(D) Western blot of whole zebrafish larvae, Smyhc1 stained with the F59 antibody. smyhc1+/+ larvae express Smyhc1 at 24hpf, but not at 48hpf. smyhc1-/- larvae do not express Smyhc1.