Figure 7 ( A ) TRPV2 RNA expression levels ( microarray probe : 219282 _ s _ at ) measured in normal skin ( n = 7 biological replicates ) , nevi ( n = 9 biological replicates ) , primary ( n = 31 biological replicates ) and metastatic ( n = 73 biological replicates ) melanomas ( GEO : GSE46517 ) ( Data ref : Kwong LN , 2013 ) . TRPV2 levels are increased in malignant lesions compared to normal skin and nevi . Data is represented as a whisker - box plot with outliers plotted as individual points ( Boxes extend from the 25th to 75th percentiles , whiskers from the 10th to 90th percentiles , the horizontal line in each box is plotted at the median ; ns P = 0 , 1383 , * P = 0 , 0488 and * * P = 0 , 0016 , the Kruskal - Wallis test ) . The horizontal dotted line corresponds to TRPV2 median expression in nevi . ( B ) Representative melanoma tissue lesions from a tissue microarray ( TMA ) comparing TRPV2 staining between a benign nevus and a malignant melanoma ( See also appendix Fig S10 ) ( Scale bar = 200µm ) . Top right insets show the same images stained with an anti - melanoma triple cocktail ( HMB45 + A103 + T311 ) . Regions depicted with a black line represent the interface between the diffusely stained tumor ( T ) and the surrounding normal stroma ( S ) . Right panel shows the scatter plot of the complete quantification of TRPV2 staining in 77 patients with malignant melanoma compared to 16 nevi tissues ( dot , single patient sample ) with lines for mean ± SEM ( * P = 0 . 0473 , the Mann - Whitney test ) . ( F ) Analysis of TRPV2 expression by quantitative RT - PCR ( left panel ) and western - blot ( right panel ) in the WM115 / WM266 . 4 pair of isogenic melanoma cell lines . The WM115 cell line was derived from an in situ tumor while the WM266 . 4 was established from a skin metastasis of the same patient . Relative transcript levels are presented as mean ± SEM from n = 4 - 9 biological replicates ( * * P = 0 . 0028 , the Mann - Whitney test ) . ( G ) Kaplan - Meier plot showing the association of TRPV2 expression ( 10 % highest versus 10 % lowest expressers in the skin cutaneous melanoma cohort TCGA data set ) with melanoma patient survival ( ( * ) P = 0 , 0144 , the Log - rank ( Mantel - Cox ) test ) .

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Figure 7(A) TRPV2 RNA expression levels (microarray probe: 219282_s_at) measured in normal skin (n=7 biological replicates), nevi (n=9 biological replicates) , primary (n=31 biological replicates) and metastatic (n=73 biological replicates) melanomas (GEO: GSE46517)(Data ref: Kwong LN, 2013). TRPV2 levels are increased in malignant lesions compared to normal skin and nevi. Data is represented as a whisker-box plot with outliers plotted as individual points (Boxes extend from the 25th to 75th percentiles, whiskers from the 10th to 90th percentiles, the horizontal line in each box is plotted at the median; ns P = 0,1383, *P = 0,0488 and **P = 0,0016, the Kruskal-Wallis test). The horizontal dotted line corresponds to TRPV2 median expression in nevi.(B) Representative melanoma tissue lesions from a tissue microarray (TMA) comparing TRPV2 staining between a benign nevus and a malignant melanoma (See also appendix Fig S10) (Scale bar = 200µm). Top right insets show the same images stained with an anti-melanoma triple cocktail (HMB45+A103+T311). Regions depicted with a black line represent the interface between the diffusely stained tumor (T) and the surrounding normal stroma (S). Right panel shows the scatter plot of the complete quantification of TRPV2 staining in 77 patients with malignant melanoma compared to 16 nevi tissues (dot, single patient sample) with lines for mean ± SEM (*P = 0.0473, the Mann-Whitney test).(F) Analysis of TRPV2 expression by quantitative RT-PCR (left panel) and western-blot (right panel) in the WM115/WM266.4 pair of isogenic melanoma cell lines. The WM115 cell line was derived from an in situ tumor while the WM266.4 was established from a skin metastasis of the same patient. Relative transcript levels are presented as mean ± SEM from n=4-9 biological replicates (**P = 0.0028, the Mann-Whitney test).(G) Kaplan-Meier plot showing the association of TRPV2 expression (10% highest versus 10% lowest expressers in the skin cutaneous melanoma cohort TCGA data set) with melanoma patient survival ((*)P = 0,0144, the Log-rank (Mantel-Cox) test).


Figure 1 ( A ) ROS burst response in wild - type ( WT ) and map4k4 lines in response to 1 μM flg22 . Values are means ± SEM , n = 16 ( biological replicates ) . Results are representative of three independent experiments . Statistical analysis was performed using a one - way ANOVA with Tukey post - test , p < 0 . 01 . Data information Different letters indicate statistical significant base on one - way ANOVA with Tukey post - test , samples sharing letters are not significantly different . ( B ) qRT - PCR analysis of the expression of PTI marker gene FRK1 in wild - type and map4k4 - 1 seedlings 1 h after treatment with 1 μM flg22 . Bars represent means ± SD based on three biological replicates , p < 0 . 01 . Data information : Different letters indicate statistical significant base on one - way ANOVA with Tukey post - test , samples sharing letters are not significantly different . ( D ) Heat map of gene expression response to flg22 in wild - type and map4k4 - 1 , red and blue indicate high and low expression levels . ( F ) qRT - PCR analysis of 14 days old seedlings treated with H2O or 1 µM flg22 for 1 hour . Values are the means of three biological replicates ± SD , p < 0 . 01 . Data information : Different letters indicate statistical significant base on one - way ANOVA with Tukey post - test , samples sharing letters are not significantly different .

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Figure 1(A) ROS burst response in wild-type (WT) and map4k4 lines in response to 1 μM flg22. Values are means ± SEM, n = 16 (biological replicates). Results are representative of three independent experiments. Statistical analysis was performed using a one-way ANOVA with Tukey post-test, p < 0.01. Data information Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different.(B) qRT-PCR analysis of the expression of PTI marker gene FRK1 in wild-type and map4k4-1 seedlings 1 h after treatment with 1 μM flg22. Bars represent means ± SD based on three biological replicates, p < 0.01. Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different.(D) Heat map of gene expression response to flg22 in wild-type and map4k4-1, red and blue indicate high and low expression levels.(F) qRT-PCR analysis of 14 days old seedlings treated with H2O or 1 µM flg22 for 1 hour. Values are the means of three biological replicates ± SD, p < 0.01. Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different.


Figure 2 ( A ) left panel , map4k4 - 1 shows enhanced resistance to Pseudomonas syringae pv . tomato DC3000 . The growth of Pst DC3000 after inoculation in the indicated genotypes ( Col - 0 , white bars , map4k4 - 1 , green bars ) . Bacterial titers were determined 2 hours ( day 0 ) and 3 days after inoculation . Right panel , disease symptoms of wild - type and map4k4 - 1 at 3 days after inoculation . Values are the means ± SD , n = 6 ( biological replicates ) , p < 0 . 01 . Data information : Statistical analysis were performed using a one - way ANOVA with Newman - Keuls multiple comparison tests or t - tests . n . s . indicate non - significant . ( B ) map4k4 - 1 disease resistance phenotype was recovered to wild - type levels in two independent complementation lines , c867 and c872 . Bacterial titers were determined as described above . Values are the means ± SD , n = 6 ( biological replicates ) , p < 0 . 01 . Data information : Statistical analysis were performed using a one - way ANOVA with Newman - Keuls multiple comparison tests or t - tests . n . s . indicate non - significant . ( C ) qRT - PCR analysis of PR1 expression levels in Col - 0 , map4k4 - 1 and c867 after mock ( MgCl2 ) or Pst DC3000 inoculation , values are the means ± SD , n = 3 ( biological replicates ) , p < 0 . 01 . Data information : Statistical analysis were performed using a one - way ANOVA with Newman - Keuls multiple comparison tests or t - tests . n . s . indicate non - significant . ( D ) Flg22 - protection assay . Col - 0 , map4k4 - 1 and c867 were pretreated with H2O or flg22 1 day before Pst DC3000 inoculation . The bacterial population was measured 3 days after infection . Values are the means ± SD , n = 6 . * * p < 0 . 01 , Results are representative of three independent experiments . Data information : Statistical analysis were performed using a one - way ANOVA with Newman - Keuls multiple comparison tests or t - tests . n . s . indicate non - significant .

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Figure 2(A) left panel, map4k4-1 shows enhanced resistance to Pseudomonas syringae pv. tomato DC3000. The growth of Pst DC3000 after inoculation in the indicated genotypes (Col-0, white bars, map4k4-1, green bars). Bacterial titers were determined 2 hours (day 0) and 3 days after inoculation. Right panel, disease symptoms of wild-type and map4k4-1 at 3 days after inoculation. Values are the means ± SD, n = 6 (biological replicates), p < 0.01. Data information: Statistical analysis were performed using a one-way ANOVA with Newman-Keuls multiple comparison tests or t-tests. n.s. indicate non-significant.(B) map4k4-1 disease resistance phenotype was recovered to wild-type levels in two independent complementation lines, c867 and c872. Bacterial titers were determined as described above. Values are the means ± SD, n = 6 (biological replicates), p < 0.01. Data information: Statistical analysis were performed using a one-way ANOVA with Newman-Keuls multiple comparison tests or t-tests. n.s. indicate non-significant.(C) qRT-PCR analysis of PR1 expression levels in Col-0, map4k4-1 and c867 after mock (MgCl2) or Pst DC3000 inoculation, values are the means ± SD, n = 3 (biological replicates), p < 0.01. Data information: Statistical analysis were performed using a one-way ANOVA with Newman-Keuls multiple comparison tests or t-tests. n.s. indicate non-significant.(D) Flg22-protection assay. Col-0, map4k4-1 and c867 were pretreated with H2O or flg22 1 day before Pst DC3000 inoculation. The bacterial population was measured 3 days after infection. Values are the means ± SD, n = 6. **p < 0.01, Results are representative of three independent experiments. Data information: Statistical analysis were performed using a one-way ANOVA with Newman-Keuls multiple comparison tests or t-tests. n.s. indicate non-significant.


Figure 3 ( A ) Subcellular localization of MAP4K4 - GFP in Arabidopsis mesophyll protoplasts . MAP4K4 - GFP ( green ) partially co - localized with the plasma membrane dye FM4 - 64 ( red ) . 14 hours after transformation , photographs were taken when the plasma membrane of mesophyll protoplasts was stained by FM4 - 64 ( < 3 min ) . Scale bars = 10 µm . ( B ) Auto - phosphorylation of recombinant HisMBP - MAP4K4 wild - type , no auto - phosphorylation in the kinase - inactive HisMBP - MAP4K4 - D139A protein . The phosphorylation level was determined by Pro - Q Diamond phosphoprotein gel stain ( upper panel ) , protein loading amounts were visualized by Coomassie G - 250 staining ( lower panel ) . The predicted molecular weight of HisMBP - MAP4K4 is 118 . 4 kDa . ( C ) BiFC visualization of interaction between MAP4K4 and BIK1 in Arabidopsis mesophyll protoplasts . BIK1 - nYFP and MAP4K4 - cYFP were co - expressed in protoplasts to show fluorescence of complemented YFP signal ( green ) , 14 h after transformation , the results were photographed by confocal laser scanning microscopy , scale bars = 10 µm . Chl indicates Chloroplast , BF indicate Bright field . nYFP and cYFP constructs were used as empty controls . MAP3K5 - K375M and MKK4 were used as control . ( D ) MAP4K4 associates with BIK1 in vivo by Co - IP . BIK1 - HA and MAP4K4 - GFP were co - expressed in plants by crossing of 35S : : BIK1 - HA and 35S : : MAP4K4 - GFP transgenic lines . 14 days old plants were treated ( + ) with 1 µM flg22 or H2O ( - ) for 10 min . GFP trap beads were used for immunoprecipitation , and target proteins were detected by western blots using anti - GFP or anti - HA antibodies . The predicted molecular weight of BIK1 - HA , MAP4K4 - GFP is 49 . 3 kDa and 103 . 8 kDa respectively .

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Figure 3(A) Subcellular localization of MAP4K4-GFP in Arabidopsis mesophyll protoplasts. MAP4K4-GFP (green) partially co-localized with the plasma membrane dye FM4-64 (red). 14 hours after transformation, photographs were taken when the plasma membrane of mesophyll protoplasts was stained by FM4-64 (< 3 min). Scale bars = 10 µm.(B) Auto-phosphorylation of recombinant HisMBP-MAP4K4 wild-type, no auto-phosphorylation in the kinase-inactive HisMBP-MAP4K4-D139A protein. The phosphorylation level was determined by Pro-Q Diamond phosphoprotein gel stain (upper panel), protein loading amounts were visualized by Coomassie G-250 staining (lower panel). The predicted molecular weight of HisMBP-MAP4K4 is 118.4 kDa.(C) BiFC visualization of interaction between MAP4K4 and BIK1 in Arabidopsis mesophyll protoplasts. BIK1-nYFP and MAP4K4-cYFP were co-expressed in protoplasts to show fluorescence of complemented YFP signal (green), 14 h after transformation, the results were photographed by confocal laser scanning microscopy, scale bars = 10 µm. Chl indicates Chloroplast, BF indicate Bright field. nYFP and cYFP constructs were used as empty controls. MAP3K5-K375M and MKK4 were used as control.(D) MAP4K4 associates with BIK1 in vivo by Co-IP. BIK1-HA and MAP4K4-GFP were co-expressed in plants by crossing of 35S::BIK1-HA and 35S::MAP4K4-GFP transgenic lines. 14 days old plants were treated (+) with 1 µM flg22 or H2O (-) for 10 min. GFP trap beads were used for immunoprecipitation, and target proteins were detected by western blots using anti-GFP or anti-HA antibodies. The predicted molecular weight of BIK1-HA, MAP4K4-GFP is 49.3 kDa and 103.8 kDa respectively.


Figure 4 ( A ) Decreased BIK1 abundance in map4k4 - 1 . 35S : : BIK1 - HA was introduced into map4k4 - 1 by crossing , sibling lines carrying either map4k4 - 1 or MAP4K4 genotype were isolated from F3 generation . BIK1 levels were detected by western blot analysis using anti - HA antibodies . ( B ) MAP4K4 regulates BIK1 accumulation through the proteasome pathway . 10 days old 35S : : BIK1 - HA seedlings of WT or map4k4 - 1 were treated with DMSO ( - ) or the proteasome inhibitor MG132 ( 50 µM ) for 5h , then the plants were collected for immunoblot analysis . ( C ) Flg22 - induced BIK1 phosphorylation assay . 10 days old 35S : : BIK1 - HA seedlings of WT or map4k4 - 1 were treated with H2O ( - ) or 1 µM flg22 for 15 min , BIK1 phosphorylation levels were detected by mobility - shift assay using anti - HA immunoblot . ( D , E and F ) Identification of phosphorylation sites by LC - MS / MS analysis . Five residues in GST - BIK1 - K105E were phosphorylated in vitro by HisMBP - MAP4K4 . The peptide sequences are shown above the spectrum with the PH designating phosphorylated residue . In the spectrum , the matched fragment ions shows in red line for terminal series ions and in orange for immonium ions and modified ions . ( G ) S233 and T242 were required for BIK1 hyper - phosphorylation . BIK1 - HA , BIK1S233 - HA and BIK1S242 - HA mutants were expressed in Col - 0 protoplasts and treated with 100 nM flg22 for 15 min , BIK1 phosphorylation levels were detected by mobility - shift assay using anti - HA immunoblot . Each experiment was performed at least three times with similar results .

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Figure 4(A) Decreased BIK1 abundance in map4k4-1. 35S::BIK1-HA was introduced into map4k4-1 by crossing, sibling lines carrying either map4k4-1 or MAP4K4 genotype were isolated from F3 generation. BIK1 levels were detected by western blot analysis using anti-HA antibodies.(B) MAP4K4 regulates BIK1 accumulation through the proteasome pathway. 10 days old 35S::BIK1-HA seedlings of WT or map4k4-1 were treated with DMSO (-) or the proteasome inhibitor MG132 (50 µM) for 5h, then the plants were collected for immunoblot analysis.(C) Flg22-induced BIK1 phosphorylation assay. 10 days old 35S::BIK1-HA seedlings of WT or map4k4-1 were treated with H2O (-) or 1 µM flg22 for 15 min, BIK1 phosphorylation levels were detected by mobility-shift assay using anti-HA immunoblot.(D, E and F) Identification of phosphorylation sites by LC-MS/MS analysis. Five residues in GST-BIK1-K105E were phosphorylated in vitro by HisMBP-MAP4K4. The peptide sequences are shown above the spectrum with the PH designating phosphorylated residue. In the spectrum, the matched fragment ions shows in red line for terminal series ions and in orange for immonium ions and modified ions. (G) S233 and T242 were required for BIK1 hyper-phosphorylation. BIK1-HA, BIK1S233-HA and BIK1S242-HA mutants were expressed in Col-0 protoplasts and treated with 100 nM flg22 for 15 min, BIK1 phosphorylation levels were detected by mobility-shift assay using anti-HA immunoblot. Each experiment was performed at least three times with similar results.


Figure 5 ( A ) MAP4K4 associates with PP2C38 in vivo by Co - IP . MAP4K4 - GFP and PP2C38 - HA were co - expressed in protoplasts . GFP trap beads were used for immunoprecipitation , and target proteins were detected by western blots using anti - GFP or anti - HA antibodies . The predicted molecular weight of MAP4K4 - GFP and PP2C38 - HA is 103 . 8kDa and 48 . 1 kDa respectively . ( B ) Identification of phosphorylation sites by LC - MS / MS analysis . In vitro phosphorylation site of HisMBP - PP2C38 by HisMBP - MAP4K4 . The peptide sequence is shown above the spectrum with the PH designating phosphorylated residue . In the spectrum , the matched fragment ions shows in red line for terminal series ions and in orange for immonium ions and modified ions . ( C ) S77 is required for flg22 - induced PP2C38 band shift . PP2C38 - HA and PP2C38 - S77A - HA were expressed in Col - 0 and map4k4 protoplasts and treated with 1µM flg22 for 30 min . The phosphorylated PP2C38 was detected by western blot mobility shift assay . ( D ) flg22 - induced PP2C38 band shift was compromised in map4k4 mutants . PP2C38 - HA were expressed in Col - 0 and map4k4 protoplasts and treated with 1µM flg22 for 30 min . ( E ) PP2C38 dephosphorylates MAP4K4 by its phosphatase activity . HisMBP - MAP4K4 was incubated with HisMBP - PP2C38 or HisMBP - PP2C38D88N D289N ( phosphatase - inactive ) or alone , and the phosphorylation level of MAP4K4 was determined by Pro - Q Diamond phosphoprotein gel stain . All the experiments were performed three times with similar results .

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Figure 5(A) MAP4K4 associates with PP2C38 in vivo by Co-IP. MAP4K4-GFP and PP2C38-HA were co-expressed in protoplasts. GFP trap beads were used for immunoprecipitation, and target proteins were detected by western blots using anti-GFP or anti-HA antibodies. The predicted molecular weight of MAP4K4-GFP and PP2C38-HA is 103.8kDa and 48.1 kDa respectively.(B) Identification of phosphorylation sites by LC-MS/MS analysis. In vitro phosphorylation site of HisMBP-PP2C38 by HisMBP-MAP4K4. The peptide sequence is shown above the spectrum with the PH designating phosphorylated residue. In the spectrum, the matched fragment ions shows in red line for terminal series ions and in orange for immonium ions and modified ions.(C) S77 is required for flg22-induced PP2C38 band shift. PP2C38-HA and PP2C38-S77A-HA were expressed in Col-0 and map4k4 protoplasts and treated with 1µM flg22 for 30 min. The phosphorylated PP2C38 was detected by western blot mobility shift assay.(D) flg22-induced PP2C38 band shift was compromised in map4k4 mutants. PP2C38-HA were expressed in Col-0 and map4k4 protoplasts and treated with 1µM flg22 for 30 min.(E) PP2C38 dephosphorylates MAP4K4 by its phosphatase activity. HisMBP-MAP4K4 was incubated with HisMBP-PP2C38 or HisMBP-PP2C38D88N D289N (phosphatase-inactive) or alone, and the phosphorylation level of MAP4K4 was determined by Pro-Q Diamond phosphoprotein gel stain. All the experiments were performed three times with similar results.


Figure 6 ( A ) BIK1 transgene restores flg22 - induced ROS burst in map4k4 - 1 . 35S : : BIK1 - HA WT or 35S : : BIK1 - HA map4k4 - 1 were generated by crossing as described before . ROS burst response in the indicated genotypes in response to 1 μM flg22 . Values are means ± SEM , n = 16 . Results are representative of three independent experiments . Data information : Different letters indicate statistical significant base on one - way ANOVA with Tukey post - test , samples sharing letters are not significantly different , p < 0 . 05 . ( B ) Morphological phenotype of 6 - week - old of Col - 0 , map4k4 - 1 , bik1 and map4k4 - 1 bik1 mutants . ( C ) ROS burst response in Col - 0 , map4k4 - 1 , bik1 and map4k4 - 1 bik1 mutants . Values are means ± SEM , n = 16 . Results are representative of three independent experiments . Data information : Different letters indicate statistical significant base on one - way ANOVA with Tukey post - test , samples sharing letters are not significantly different , p < 0 . 05 . ( D ) Relative expression level of PR1 in Col - 0 , map4k4 - 1 , bik1 and map4k4 - 1 bik1mutants by qRT - PCR analysis . Values are means ± SD , n = 3 ( biological replicates ) . Data information : Different letters indicate statistical significant base on one - way ANOVA with Tukey post - test , samples sharing letters are not significantly different , p < 0 . 05 . ( E ) Morphological phenotype of 6 - week - old of Col - 0 , map4k4 - 1 , map4k3 and map4k4 - 1 map4k3 mutants . ( F ) Relative expression level of PR1 in Col - 0 , map4k4 - 1 , map4k3 and map4k4 - 1 map4k3 mutants by qRT - PCR analysis . Values are means ± SD , n = 3 ( biological replicates ) . Data information : Different letters indicate statistical significant base on one - way ANOVA with Tukey post - test , samples sharing letters are not significantly different , p < 0 . 05 . ( G ) ROS burst response in Col - 0 , map4k4 - 1 , map4k3 and map4k4 - 1 map4k3 mutants . Values are means ± SEM , n = 16 ( biological replicates ) . Data information : Different letters indicate statistical significant base on one - way ANOVA with Tukey post - test , samples sharing letters are not significantly different , p < 0 . 05 .

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

Figure 6(A) BIK1 transgene restores flg22-induced ROS burst in map4k4-1. 35S::BIK1-HA WT or 35S::BIK1-HA map4k4-1 were generated by crossing as described before. ROS burst response in the indicated genotypes in response to 1 μM flg22. Values are means ± SEM, n = 16. Results are representative of three independent experiments. Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different, p < 0.05.(B) Morphological phenotype of 6-week-old of Col-0, map4k4-1, bik1 and map4k4-1 bik1 mutants.(C) ROS burst response in Col-0, map4k4-1, bik1 and map4k4-1 bik1 mutants. Values are means ± SEM, n = 16. Results are representative of three independent experiments. Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different, p < 0.05.(D) Relative expression level of PR1 in Col-0, map4k4-1, bik1 and map4k4-1 bik1mutants by qRT-PCR analysis. Values are means ± SD, n = 3 (biological replicates). Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different, p < 0.05.(E) Morphological phenotype of 6-week-old of Col-0, map4k4-1, map4k3 and map4k4-1 map4k3 mutants.(F) Relative expression level of PR1 in Col-0, map4k4-1, map4k3 and map4k4-1 map4k3 mutants by qRT-PCR analysis. Values are means ± SD, n = 3 (biological replicates). Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different, p < 0.05.(G) ROS burst response in Col-0, map4k4-1, map4k3 and map4k4-1 map4k3 mutants. Values are means ± SEM, n = 16 (biological replicates). Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different, p < 0.05.


Figure 1B ) Western blots showing absence of mitochondrial proteins from the different mitochondrial complexes : NDUFB8 ( complex I ) , SDHA ( complex II ) , UQCRC2 ( complex III ) and MTCO - 1 ( complex IV ) , in senescent ( 10 days after 20Gy X - ray ) Control and Parkin - expressing MRC5 fibroblasts . Data is representative of 3 independent experiments ; C ) Cellular oxygen consumption rates ( OCR ) in senescent ( 10 days after 20Gy X - ray ) Control and Parkin - expressing MRC5 fibroblasts . Data were obtained using Seahorse XF24 analyzer and shows mean ± S . D . n = 4 technical repeats ( representative of 2 independent experiments ) ; D ) Representative 3D EM pictures of senescent ( 20 days after 20Gy X - ray ) Parkin - expressing MRC5 fibroblasts with or without CCCP . Mitochondria are in red , the nucleus in blue ; E ) Representative images of cell size , Sen - β - Gal activity ( blue cytoplasmic staining ) , macro - H2A foci and SDHA immunofluorescence in proliferating and senescent ( 10 days after 20Gy X - ray ) Parkin - expressing MRC5 fibroblasts ; F ) Quantification of ROS levels ( DHE fluorescence ) , Sen - β - Gal positive cells and Senescence - associated heterochromatin foci ( SAHF ) observed by DAPI in proliferating and senescent ( 10 days after 20Gy X - ray ) Control and Parkin - expressing MRC5 fibroblasts . Data are mean ± S . E . M of n = 3 independent experiments ; Asterisks denote statistical significant P < 0 . 05 One - way ANOVA . G ) Representative western blots showing p21 and p16 expression in proliferating and senescent ( 10 days after 20Gy X - ray ) Control and Parkin - expressing MRC5 fibroblasts . Data are representative of 3 independent experiments ; H ) Secreted protein measured in a cytokine array ( RayBiotech ) of a variety of inflammatory proteins in proliferating and senescent ( 10 days after 20Gy X - ray ) Parkin - expressing MRC5 fibroblasts . Data are mean ± S . E . M of n = 3 independent experiments . Asterisks denote statistical significant P < 0 . 05 One - way ANOVA .

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

Figure 1B) Western blots showing absence of mitochondrial proteins from the different mitochondrial complexes: NDUFB8 (complex I), SDHA (complex II), UQCRC2 (complex III) and MTCO-1 (complex IV), in senescent (10 days after 20Gy X-ray) Control and Parkin-expressing MRC5 fibroblasts. Data is representative of 3 independent experiments;C) Cellular oxygen consumption rates (OCR) in senescent (10 days after 20Gy X-ray) Control and Parkin-expressing MRC5 fibroblasts. Data were obtained using Seahorse XF24 analyzer and shows mean±S.D. n=4 technical repeats (representative of 2 independent experiments);D) Representative 3D EM pictures of senescent (20 days after 20Gy X-ray) Parkin-expressing MRC5 fibroblasts with or without CCCP. Mitochondria are in red, the nucleus in blue;E) Representative images of cell size, Sen-β-Gal activity (blue cytoplasmic staining), macro-H2A foci and SDHA immunofluorescence in proliferating and senescent (10 days after 20Gy X-ray) Parkin- expressing MRC5 fibroblasts;F) Quantification of ROS levels (DHE fluorescence), Sen-β-Gal positive cells and Senescence-associated heterochromatin foci (SAHF) observed by DAPI in proliferating and senescent (10 days after 20Gy X-ray) Control and Parkin-expressing MRC5 fibroblasts. Data are mean±S.E.M of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.G) Representative western blots showing p21 and p16 expression in proliferating and senescent (10 days after 20Gy X-ray) Control and Parkin-expressing MRC5 fibroblasts. Data are representative of 3 independent experiments;H) Secreted protein measured in a cytokine array (RayBiotech) of a variety of inflammatory proteins in proliferating and senescent (10 days after 20Gy X-ray) Parkin-expressing MRC5 fibroblasts. Data are mean±S.E.M of n=3 independent experiments. Asterisks denote statistical significant P<0.05 One-way ANOVA.


Figure 3A ) Representative images of colony assays of wild - type and PGC - 1β - / - MEFs grown at 3 % or 21 % O2 ( 10 days after seeding 5 , 000 cells / well ) . Data are representative of 3 independent experiments ; B ) Effect of 3 % or 21 % O2 and X - ray irradiation ( at 3 % O2 ) on the percentage of Ki67 ( at day 6 ) and Sen - β - Gal positive cells ( at day 10 ) and number ( N ) of 53BP1 foci ( at day 6 ) in wild - type and PGC - 1β - / - MEFs . Data are mean ± S . E . M of n = 3 independent experiments ; Asterisks denote statistical significant P < 0 . 05 One - way ANOVA . C ) Representative images of Sen - β - Gal activity ( blue cytoplasmatic staining ) , Ki - 67 and 53BP1 foci in proliferating and senescent wild - type and PGC - 1β - / - MEFs ( scale bar = 10µm ) ; D ) mRNA expression of PGC - 1β CXCL - 1 and p16 in proliferating and senescent ( 10 days after 10Gy X - ray ) wild type and PGC - 1β - / - MEFs . Data are mean ± S . E . M . of n = 3 independent experiments ; Asterisks denote statistical significant P < 0 . 05 One - way ANOVA . E ) Effects of overexpression of PGC - 1β on the percentage of Ki67 and Sen - β - Gal positive cells , number ( N ) of 53BP1 foci and percentage change of mitochondrial mass ( NAO intensity ) in proliferating and senescent ( 2 days after 10Gy X - ray ) MEFs cultured at 3 % O2 . Data are mean ± S . E . M of n = 3 independent experiments . Asterisks denote statistical significant P < 0 . 05 One - way ANOVA .

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

Figure 3A) Representative images of colony assays of wild-type and PGC-1β -/- MEFs grown at 3% or 21% O2 (10 days after seeding 5,000 cells/well). Data are representative of 3 independent experiments;B) Effect of 3% or 21% O2 and X-ray irradiation (at 3% O2) on the percentage of Ki67 (at day 6) and Sen-β-Gal positive cells (at day 10) and number (N) of 53BP1 foci (at day 6) in wild-type and PGC-1β -/- MEFs. Data are mean±S.E.M of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.C) Representative images of Sen-β-Gal activity (blue cytoplasmatic staining), Ki-67 and 53BP1 foci in proliferating and senescent wild-type and PGC-1β-/- MEFs (scale bar=10µm);D) mRNA expression of PGC-1β CXCL-1 and p16 in proliferating and senescent (10 days after 10Gy X-ray) wild type and PGC-1β-/- MEFs. Data are mean±S.E.M. of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.E) Effects of overexpression of PGC-1β on the percentage of Ki67 and Sen-β-Gal positive cells, number (N) of 53BP1 foci and percentage change of mitochondrial mass (NAO intensity) in proliferating and senescent (2 days after 10Gy X-ray) MEFs cultured at 3% O2. Data are mean±S.E.M of n=3 independent experiments. Asterisks denote statistical significant P<0.05 One-way ANOVA.


Figure 4A ) Representative western blot of mTORC1 activity measured by phosphorylated p70S6K ( T389 ) from 6 to 72 hours after 20Gy in MRC5 fibroblasts . Data are representative of 3 independent experiments ; B ) Representative western blots of the mitochondrialproteins TOMM20 , NDUFB8 ( complex I ) , SDHA ( complex II ) , UQCRC2 ( complex III ) and MT - CO1 ( complex IV ) following 20Gy irradiation with or without 100nM Rapamycin treatment in MRC5fibroblasts . Data are representative of 4 independent experiments ; C ) Effect of 100nM rapamycin on mitochondrial mass ( measured by NAO fluorescence ) 2 - 4 days following replication exhaustion ( RS ) or genotoxic stress ( generated by X - ray irradiation , Etoposide , Neocarcinostatin ( NCS ) , H2O2 or telomere dysfunction ( TRF2ΔBΔM ) ) in a variety of cell lines . Data are mean from 3 independent experiments per cell line or treatment ; D ) ( top ) Representative transmission electron microscopy ( T . E . M . ) micrographs of proliferating and senescent ( 3 days after 20Gy X - ray ) MRC5 fibroblasts treated with or without 100nM rapamycin . Mitochondria are labeled in pink . Scale bar = 2µm ; ( bottom left and middle ) Quantification of mitochondrial volume fraction ( % Vv ) and mitochondrial number per cross section in proliferating and senescent ( 3 days after 20Gy X - ray ) MRC5 fibroblasts treated with or without 100nM rapamycin . T . E . M . mitochondrial analysis are mean ± S . E . M of 24 electron micrographs per condition ; ( bottom right ) mtDNA copy number analysis by q - PCR in proliferating and senescent ( 3 days after 20Gy X - ray ) MRC5 fibroblasts treated with or without 100nM Rapamycin . Data are mean ± S . E . M of n = 3 independent experiments ; Asterisks denote statistical significant P < 0 . 05 One - way ANOVA . E ) Representative western blots showing expression of phosphorylated p70S6K ( T389 ) and AKT ( S473 ) , the mitochondrial protein NDUFB8 and the DDR downstream target p21 in MRC5 fibroblasts treated with or without 10µM of the ATM inhibitor KU55933 and in fibroblasts from a patient with Ataxia Telangiectasis ( AT ) at different time points after 20Gy X - ray . Data are representative of 3 independent experiments ( ATM inhibitor ) and 1 experiment ( AT patient ) ; F ) Western blots showing the effect of the ATM inhibitor KU55933 on γH2A . X , AKT phosphorylation and p21 expression in MRC5fibroblasts after 20Gy X - ray . Data is from one experiment ; G ) Effect of rapamycin and / or ATM inhibitor ( KU55933 ) on mitochondrial mass ( NAO intensity ) in proliferating and senescent ( 3 days after 20Gy X - ray ) MRC5 fibroblasts . Data are mean ± S . E . M of n = 3 independent experiments . Asterisks denote statistical significant P < 0 . 05 One - way ANOVA .

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

Figure 4A) Representative western blot of mTORC1 activity measured by phosphorylated p70S6K (T389) from 6 to 72 hours after 20Gy in MRC5 fibroblasts. Data are representative of 3 independent experiments;B) Representative western blots of the mitochondrialproteins TOMM20, NDUFB8 (complex I), SDHA (complex II), UQCRC2 (complex III) and MT-CO1 (complex IV) following 20Gy irradiation with or without 100nM Rapamycin treatment in MRC5fibroblasts. Data are representative of 4 independent experiments;C) Effect of 100nM rapamycin on mitochondrial mass (measured by NAO fluorescence) 2-4 days following replication exhaustion (RS) or genotoxic stress (generated by X-ray irradiation, Etoposide, Neocarcinostatin (NCS), H2O2 or telomere dysfunction (TRF2ΔBΔM)) in a variety of cell lines. Data are mean from 3 independent experiments per cell line or treatment;D) (top) Representative transmission electron microscopy (T.E.M.) micrographs of proliferating and senescent (3 days after 20Gy X-ray) MRC5 fibroblasts treated with or without 100nM rapamycin. Mitochondria are labeled in pink. Scale bar=2µm; (bottom left and middle) Quantification of mitochondrial volume fraction (%Vv) and mitochondrial number per cross section in proliferating and senescent (3 days after 20Gy X-ray) MRC5 fibroblasts treated with or without 100nM rapamycin. T.E.M. mitochondrial analysis are mean±S.E.M of 24 electron micrographs per condition; (bottom right) mtDNA copy number analysis by q-PCR in proliferating and senescent (3 days after 20Gy X-ray) MRC5 fibroblasts treated with or without 100nM Rapamycin. Data are mean±S.E.M of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.E) Representative western blots showing expression of phosphorylated p70S6K (T389) and AKT (S473), the mitochondrial protein NDUFB8 and the DDR downstream target p21 in MRC5 fibroblasts treated with or without 10µM of the ATM inhibitor KU55933 and in fibroblasts from a patient with Ataxia Telangiectasis (AT) at different time points after 20Gy X-ray. Data are representative of 3 independent experiments (ATM inhibitor) and 1 experiment (AT patient);F) Western blots showing the effect of the ATM inhibitor KU55933 on γH2A.X, AKT phosphorylation and p21 expression in MRC5fibroblasts after 20Gy X-ray. Data is from one experiment;G) Effect of rapamycin and/or ATM inhibitor (KU55933) on mitochondrial mass (NAO intensity) in proliferating and senescent (3 days after 20Gy X-ray) MRC5 fibroblasts. Data are mean±S.E.M of n=3 independent experiments. Asterisks denote statistical significant P<0.05 One-way ANOVA.


Figure 5A ) ROS levels ( DHE intensity ) and mean number ( N ) of yH2A . X foci after mTOR knockdown ( 72 hours ) in proliferating and senescent ( 2 days after 20Gy X - ray ) MRC5 fibroblasts . Data are mean ± S . E . M of n = 3 independent experiments ; Asterisks denote statistical significant P < 0 . 05 One - way ANOVA . B ) ROS levels ( DHE intensity ) and mean number ( N ) of yH2A . X foci in proliferating and senescent ( 3 days after 20Gy X - ray ) MRC5 fibroblasts treated with or without 100nM Rapamycin and / or 2 . 5mM of the antioxidant NAC . Data are mean ± S . E . M of n = 3 independent experiments ; Asterisks denote statistical significant P < 0 . 05 One - way ANOVA . C ) ( top ) Scheme illustrating the experimental design : human MRC5 fibroblasts were irradiated with 20Gy X - ray and treated at day 3 after IR with 10µM of the ATM inhibitor ( ATMi ) KU55933 and / or 100nM Rapamycin ( Rap ) ; ( bottom ) Effect of single or combined inhibition of ATM and mTORC1 on the mean number ( N ) of γH2A . X foci , Sen - β - Gal activity and p21 expression in proliferating and senescent ( 10 days after 20Gy X - ray ) MRC5fibroblasts . Data are mean ± S . E . M of n = 3 independent experiments . Western blots are representative of 3 independent experiments ; Asterisks denote statistical significant P < 0 . 05 One - way ANOVA . D ) ROS levels ( DHE intensity ) in proliferating and senescent ( 3 days after 10Gy X - ray ) wild - type and PGC - 1β - / - MEFs ( top ) and PGC - 1β overexpressing MEFs ( bottom ) . Data are mean ± S . E . M . of n = 3 independent experiments ; Asterisks denote statistical significant P < 0 . 05 One - way ANOVA . E ) ( top ) Representative images and quantification of immunofluorescence staining against FLAG - PGC - 1β ( red ) and 53BP1 ( green ) in proliferating and senescent ( 2 days after 10Gy X - ray ) PGC - 1β overexpressing MEFs cultured at 3 % O2 , with or without 250µM of the antioxidant Trolox ( scale bar = 10µm ) . Data are mean ± S . E . M , n = 3 independent experiments ; Asterisks denote statistical significant P < 0 . 05 One - way ANOVA . F ) ( top ) Effect of overexpression of mutated Rheb ( N153T ) and ( bottom ) effect of 100nM rapamycin on the mean number ( N ) of 53BP1 foci in proliferating and senescent ( 3 days after 10Gy X - ray ) wild - type and PGC - 1β - / - MEFs . Data are mean ± S . E . M of n = 3 independent experiments ( at least 125 cells were analyzed per condition ) . Asterisks denote statistical significant P < 0 . 05 One - way ANOVA .

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

Figure 5A) ROS levels (DHE intensity) and mean number (N) of yH2A.X foci after mTOR knockdown (72 hours) in proliferating and senescent (2 days after 20Gy X-ray) MRC5 fibroblasts. Data are mean±S.E.M of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.B) ROS levels (DHE intensity) and mean number (N) of yH2A.X foci in proliferating and senescent (3 days after 20Gy X-ray) MRC5 fibroblasts treated with or without 100nM Rapamycin and/or 2.5mM of the antioxidant NAC. Data are mean±S.E.M of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.C) (top) Scheme illustrating the experimental design: human MRC5 fibroblasts were irradiated with 20Gy X-ray and treated at day 3 after IR with 10µM of the ATM inhibitor (ATMi) KU55933 and/or 100nM Rapamycin (Rap); (bottom) Effect of single or combined inhibition of ATM and mTORC1 on the mean number (N) of γH2A.X foci, Sen-β-Gal activity and p21 expression in proliferating and senescent (10 days after 20Gy X-ray) MRC5fibroblasts. Data are mean±S.E.M of n=3 independent experiments. Western blots are representative of 3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.D) ROS levels (DHE intensity) in proliferating and senescent (3 days after 10Gy X-ray) wild-type and PGC-1β-/- MEFs (top) and PGC-1β overexpressing MEFs (bottom). Data are mean±S.E.M. of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.E) (top) Representative images and quantification of immunofluorescence staining against FLAG-PGC-1β (red) and 53BP1 (green) in proliferating and senescent (2 days after 10Gy X-ray) PGC-1β overexpressing MEFs cultured at 3% O2, with or without 250µM of the antioxidant Trolox (scale bar=10µm). Data are mean±S.E.M, n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.F) (top) Effect of overexpression of mutated Rheb (N153T) and (bottom) effect of 100nM rapamycin on the mean number (N) of 53BP1 foci in proliferating and senescent (3 days after 10Gy X-ray) wild-type and PGC-1β -/- MEFs. Data are mean±S.E.M of n=3 independent experiments (at least 125 cells were analyzed per condition). Asterisks denote statistical significant P<0.05 One-way ANOVA.


Figure 6A ) ( top ) Representative image of immunofluorescence double staining for the mitochondrial protein MT - CO1 and the DDR marker yH2A . X ( scale bar = 5µm ) and ( bottom ) quantification of MT - CO1 intensity vs number of yH2A . X foci in hepatocytes from 12 months old mice . Data are mean ± S . E . M of n = 3 mice ( at least 30 cells were analyzed per mouse ) ; Asterisks denote statistical significant P < 0 . 05 using Two - tailed t - test . B ) Representative western blots of pS6 , S6 , p21 , PGC - 1β , MT - CO1 and NDUFB8 protein expression in mouse liver tissue at 3 and 12 months of age . Data are from n = 3 mice per group ; C ) Effect of 4 months of rapamycin supplemented diet on PGC - 1β expression in liver tissue of 16 months old mice . Data are from n = 3 mice per group ; D ) ( top ) Quantification of mitochondrial number per cross section and mitochondrial volume fraction ( % Vv ) in hepatocytes from 16 months old mice with or without 4 months rapamycin diet . Data are mean ± S . E . M of n = 3 mice per group ( at least 20 cells were analyzed per mouse ) ; ( bottom ) Representative electron micrographs of hepatocytes from 16 months old mice with or without 4 months rapamycin diet , ( mitochondria are labelled in pink ) . Scale bar = 5µm ; E ) mtDNA copy number ( measured by q - PCR ) in mice liver tissue at 3 , 12 , 16 months and at 16 months after 4 months rapamycin diet . Data are mean ± S . E . M of n = 3 - 4 mice per group ; Asterisks denote statistical significant P < 0 . 05 using One - way ANOVA . F ) Oxygen consumption rates ( OCR ) in isolated liver mitochondria from 16 months old mice fed with or without rapamycin for 4 months , in the presence of pyruvate / malate . State III was induced by injection of ADP . State IV was induced by inhibition of the ATP synthase with oligomycin and uncoupled respiration rates were determined by injection of FCCP . Antimycin A ( AA ) was used to determine background , non - mitochondrial OXPHOS , OCR . Data are mean ± S . E . M of n = 5 mice per group ; G ) ( top ) Representative immuno - TeloFISH images of hepatocytes from 3 and 15 months old mice with or without rapamycin ( 12 months diet ) . Co - localizing foci are amplified in the right panel ( amplified images are from single Z - planes where co - localization was found ) ; ( bottom ) Dot plot graph of Telomere - associated foci ( TAF ) in 3 , 15 and 16 months old mice ( 15 and 16 months old mice were fed with rapamycin for 12 and 4 months respectively ) . Data are from n = 3 - 9 mice per group ( at least 50 cells were analyzed per mice ) ; H ) ( top ) 4 months old and 15 months old micelivers [ control ( - ) or rapamycin ( + ) ] stained with Sen - - β - Gal solution ( Sen - - β - Gal activity is evidenced by blue staining ) . Data are from n = 3 mice per group ; ( bottom ) Representative image showing Sen - - β - Gal staining ( Sen - - β - Gal activity is evidenced by blue staining ) in hepatocytes and corresponding immuno - TeloFISH ( arrows represent co - localizing foci ) ; Asterisks denote statistical significant P < 0 . 05 using One - way ANOVA . I ) Representative western blot showing the effect of 4 months rapamycin feeding on p21 expression in 16 months old mice . Data are from n = 3 mice per group ; j ) Effect of 4 months rapamycin feeding on mRNA expression of the SASP components CXCL1 , CXCL5 and Inhibin A in liver tissue of 16 months old mice with or without 4 months rapamycin treatment . Data are from n = 5 mice per condition ; Asterisks denote statistical significant P < 0 . 05 using Two - tailed t - test . K ) Mean number of TAF in hepatocytes of wild type and PGC - 1 - β - / - mice with 18 months of age . Data are mean ± S . E . M of n = 4 mice per group ; Asterisks denote statistical significant P < 0 . 05 using Two - tailed t - test .

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

Figure 6A) (top) Representative image of immunofluorescence double staining for the mitochondrial protein MT-CO1 and the DDR marker yH2A.X (scale bar=5µm) and (bottom) quantification of MT-CO1 intensity vs number of yH2A.X foci in hepatocytes from 12 months old mice. Data are mean±S.E.M of n=3 mice (at least 30 cells were analyzed per mouse); Asterisks denote statistical significant P<0.05 using Two-tailed t-test.B) Representative western blots of pS6, S6, p21, PGC-1β, MT-CO1 and NDUFB8 protein expression in mouse liver tissue at 3 and 12 months of age. Data are from n=3 mice per group;C) Effect of 4 months of rapamycin supplemented diet on PGC-1β expression in liver tissue of 16 months old mice. Data are from n=3 mice per group;D) (top) Quantification of mitochondrial number per cross section and mitochondrial volume fraction (%Vv) in hepatocytes from 16 months old mice with or without 4 months rapamycin diet. Data are mean±S.E.M of n=3 mice per group (at least 20 cells were analyzed per mouse); (bottom) Representative electron micrographs of hepatocytes from 16 months old mice with or without 4 months rapamycin diet, (mitochondria are labelled in pink). Scale bar=5µm;E) mtDNA copy number (measured by q-PCR) in mice liver tissue at 3, 12, 16 months and at 16 months after 4 months rapamycin diet. Data are mean±S.E.M of n=3-4 mice per group; Asterisks denote statistical significant P<0.05 using One-way ANOVA.F) Oxygen consumption rates (OCR) in isolated liver mitochondria from 16 months old mice fed with or without rapamycin for 4 months, in the presence of pyruvate/malate. State III was induced by injection of ADP. State IV was induced by inhibition of the ATP synthase with oligomycin and uncoupled respiration rates were determined by injection of FCCP. Antimycin A (AA) was used to determine background, non-mitochondrial OXPHOS, OCR. Data are mean±S.E.M of n=5 mice per group;G) (top) Representative immuno-TeloFISH images of hepatocytes from 3 and 15 months old mice with or without rapamycin (12 months diet). Co-localizing foci are amplified in the right panel (amplified images are from single Z-planes where co-localization was found); (bottom) Dot plot graph of Telomere-associated foci (TAF) in 3, 15 and 16 months old mice (15 and 16 months old mice were fed with rapamycin for 12 and 4 months respectively). Data are from n=3-9 mice per group (at least 50 cells were analyzed per mice);H) (top) 4 months old and 15 months old micelivers [control (-) or rapamycin (+)] stained with Sen--β-Gal solution (Sen--β-Gal activity is evidenced by blue staining). Data are from n=3 mice per group; (bottom) Representative image showing Sen--β-Gal staining (Sen--β-Gal activity is evidenced by blue staining) in hepatocytes and corresponding immuno-TeloFISH (arrows represent co-localizing foci); Asterisks denote statistical significant P<0.05 using One-way ANOVA.I) Representative western blot showing the effect of 4 months rapamycin feeding on p21 expression in 16 months old mice. Data are from n=3 mice per group;j) Effect of 4 months rapamycin feeding on mRNA expression of the SASP components CXCL1, CXCL5 and Inhibin A in liver tissue of 16 months old mice with or without 4 months rapamycin treatment. Data are from n=5 mice per condition; Asterisks denote statistical significant P<0.05 using Two-tailed t-test.K) Mean number of TAF in hepatocytes of wild type and PGC-1-β-/- mice with 18 months of age. Data are mean±S.E.M of n=4 mice per group; Asterisks denote statistical significant P<0.05 using Two-tailed t-test.


Figure 1 ( C ) Quantification of AQR position in adult WT , cst - 1 and / or cst - 2 mutant animals . The full length between the URXs and the end of the nematode tail is divided into 10 blocks , and the percent of AQR that stopped within each block is indicated . * * * , P < 0 . 001 by Fisher ' s exact test . N = 50 - 109 . ( E - F ) Fluorescence time - lapse images of F - actin , plasma membrane and histone during AQR / QR . ap migration in WT ( E ) or cst - 1 ; cst - 2 double mutant ( F ) animals . Merged images , left ; inverted fluorescence images of F - actin , right ; white arrows , migration direction ; asterisks , nucleus ; double headed arrows , migration distance . F - actin is labeled with GFP - tagged with the actin - binding domain of Moesin ( GFP : : Moesin ABD ) , and the plasma membrane and Histone are labeled with mCherry - tagged with a myristoylation signal and a histone . The dotted lines indicate the cell periphery . Time is presented in minutes . Scale bars , 5 μm . ( G - J ) Quantification of the QR . ap migration angle ( G H ) , distance ( I ) and speed ( J ) . Each line in ( G I ) represents the measurement from one time - lapse movie . Error bars indicate SD . N = 10 - 17 . Statistical significance is based on Student ' s t - tests , * * , P 0 . 01 ; * * * , P 0 . 001 .

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

Figure 1(C) Quantification of AQR position in adult WT, cst-1 and/or cst-2 mutant animals. The full length between the URXs and the end of the nematode tail is divided into 10 blocks, and the percent of AQR that stopped within each block is indicated. ***, P < 0.001 by Fisher's exact test. N = 50 - 109.(E - F) Fluorescence time-lapse images of F-actin, plasma membrane and histone during AQR/QR.ap migration in WT (E) or cst-1; cst-2 double mutant (F) animals. Merged images, left; inverted fluorescence images of F-actin, right; white arrows, migration direction; asterisks, nucleus; double headed arrows, migration distance. F-actin is labeled with GFP-tagged with the actin-binding domain of Moesin (GFP::Moesin ABD), and the plasma membrane and Histone are labeled with mCherry-tagged with a myristoylation signal and a histone. The dotted lines indicate the cell periphery. Time is presented in minutes. Scale bars, 5 μm. (G - J) Quantification of the QR.ap migration angle (G H), distance (I) and speed (J). Each line in (G I) represents the measurement from one time-lapse movie. Error bars indicate SD. N = 10 - 17. Statistical significance is based on Student's t-tests, **, P 0.01; ***, P  0.001.


Figure 2 ( A ) Fluorescence time - lapse images of F - actin , plasma membrane and histone during QR . ap migration in mig - 2 ( rh17 ) gain - of - function mutant animals . Merged images , left ; inverted fluorescence images of F - actin , right ; white arrows , migration direction ; asterisks , nucleus ; double headed arrow , migration distance . The dotted lines indicate the cell periphery . Time is presented in minutes . Scale bar , 5 μm . ( B - C ) Quantification of the QR . ap migration angle ( B ) and distance ( C ) . N = 10 . ( D ) Mass spectrum of a MIG - 2 peptide that was phosphorylated at Serine 139 in C . elegans . The peptide was identified from the GFP : : MIG - 2 immunoprecipitate . ( E ) In vitro phosphorylation assay of CST - 1 / 2 to MIG - 2 . Bacterially expressed His - tagged MIG - 2 or His - tagged MIG - 2S139A were incubated with the GST - tagged kinase domain of GST - CST - 1 / 2 ( 1 - 318 aa ) . The kinase may use ATP - γ - S as a phosphodonor to thiophosphorylate its substrate ; thus , the phosphorylation reaction was performed with ATP - γ - S , and anti - thiophosphate ester antibody was used to detect substrate phosphorylation ( Allen et al , 2005 ; Allen et al , 2007 ) . The final reaction was probed via immunoblotting with antibodies as indicated . Thio , thiophosphate ester . ( G H ) Three - dimensional ribbon ( PDB 3TH5 ) representations of the human Rac1 polypeptide which indicate the position of the T135 ( S139 in MIG - 2 ) located between the last two α - helixes ( Krauthammer et al , 2012 ) .

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

Figure 2(A) Fluorescence time-lapse images of F-actin, plasma membrane and histone during QR.ap migration in mig-2(rh17) gain-of-function mutant animals. Merged images, left; inverted fluorescence images of F-actin, right; white arrows, migration direction; asterisks, nucleus; double headed arrow, migration distance. The dotted lines indicate the cell periphery. Time is presented in minutes. Scale bar, 5 μm. (B - C) Quantification of the QR.ap migration angle (B) and distance (C). N = 10.(D) Mass spectrum of a MIG-2 peptide that was phosphorylated at Serine 139 in C. elegans. The peptide was identified from the GFP::MIG-2 immunoprecipitate.(E) In vitro phosphorylation assay of CST-1/2 to MIG-2. Bacterially expressed His-tagged MIG-2 or His-tagged MIG-2S139A were incubated with the GST-tagged kinase domain of GST-CST-1/2 (1-318 aa). The kinase may use ATP-γ-S as a phosphodonor to thiophosphorylate its substrate; thus, the phosphorylation reaction was performed with ATP-γ-S, and anti-thiophosphate ester antibody was used to detect substrate phosphorylation (Allen et al, 2005; Allen et al, 2007). The final reaction was probed via immunoblotting with antibodies as indicated. Thio, thiophosphate ester.(G H) Three-dimensional ribbon (PDB 3TH5) representations of the human Rac1 polypeptide which indicate the position of the T135 (S139 in MIG-2) located between the last two α-helixes (Krauthammer et al, 2012).


Figure 3 ( A ) Quantification of AQR position in mig - 2 ( mu28 ) null mutant animals with different rescue transgenes . N = 108 - 114 . * , P < 0 . 05 ; # # , P < 0 . 01 , phenotype enhanced . P values are based on Fisher ' s exact tests . ( B ) Quantification of AQR position in WT and knock - in animals that harbor mig - 2S139 point mutations . N = 100 - 111 . * * * , P < 0 . 001 by Fisher ' s exact test . ( C - F ) Quantification of the QR . ap migration angle ( C D ) , distance ( E ) and speed ( F ) in mig - 2S139A / E mutant animals . Each line in ( C E ) represents the measurement from one movie of QR . ap migration . Error bars indicate SD . N = 12 - 17 . * * , P 0 . 01 ; * * * , P 0 . 001 by Student ' s t tests . ( G ) Inverted fluorescence images ( up ) and schematics ( down ) of QR . ap in WT , cst - 1 ; cst - 2 double or mig - 2 mutant animals . The plasma membrane and histones were labeled with mCherry . Scale bar , 5 μm . ( H ) Area ratio between the cell body and the nucleus in WT and mutant animals . Error bars indicate SD . N = 17 - 22 . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 by Student ' s t tests . ( I - K ) Representative frames ( upper ) and quantification ( lower ) of F - actin enrichment at the QR . ap periphery in WT ( I ) and mig - 2S139A / E mutant animals ( J K ) . The trace starts from the rear of the QR . ap and moves along the cell periphery to the leading edge ( L ) and back to the rear ( R ) . F - actin was labeled with GFP : : Moesin ABD ; the plasma membrane and histone were marked with mCherry fluorescence . Green lines , F - actin fluorescence intensity ; red lines , plasma membrane fluorescence intensity ; dotted lines , cell periphery . Double - headed arrows indicate the F - actin enrichment region , in which the fluorescence intensity is 2 - fold higher than the background . Scale bars , 5 μm . ( L ) Quantification of F - actin enrichment ratio in WT and mig - 2 mutants . Error bars indicate SD . N = 28 - 68 . * * , P 0 . 01 ; * * * , P 0 . 001 by Student ' s t - tests .

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

Figure 3(A) Quantification of AQR position in mig-2(mu28) null mutant animals with different rescue transgenes. N = 108 - 114. *, P < 0.05; ##, P < 0.01, phenotype enhanced. P values are based on Fisher's exact tests.(B) Quantification of AQR position in WT and knock-in animals that harbor mig-2S139 point mutations. N = 100 - 111. ***, P < 0.001 by Fisher's exact test.(C - F) Quantification of the QR.ap migration angle (C D), distance (E) and speed (F) in mig-2S139A/E mutant animals. Each line in (C  E) represents the measurement from one movie of QR.ap migration. Error bars indicate SD. N = 12 -17. **, P 0.01; ***, P  0.001 by Student's t tests.(G) Inverted fluorescence images (up) and schematics (down) of QR.ap in WT, cst-1; cst-2 double or mig-2 mutant animals. The plasma membrane and histones were labeled with mCherry. Scale bar, 5 μm. (H) Area ratio between the cell body and the nucleus in WT and mutant animals. Error bars indicate SD. N = 17 - 22. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Student's t tests.(I - K) Representative frames (upper) and quantification (lower) of F-actin enrichment at the QR.ap periphery in WT (I) and mig-2S139A/E mutant animals (J  K). The trace starts from the rear of the QR.ap and moves along the cell periphery to the leading edge (L) and back to the rear (R). F-actin was labeled with GFP::Moesin ABD; the plasma membrane and histone were marked with mCherry fluorescence. Green lines, F-actin fluorescence intensity; red lines, plasma membrane fluorescence intensity; dotted lines, cell periphery. Double-headed arrows indicate the F-actin enrichment region, in which the fluorescence intensity is 2-fold higher than the background. Scale bars, 5 μm. (L) Quantification of F-actin enrichment ratio in WT and mig-2 mutants. Error bars indicate SD. N = 28 - 68. **, P  0.01; ***, P  0.001 by Student's t-tests.


Figure 4 ( A - C ) Fluorescence images ( upper ) and quantifications ( lower ) of GFP - tagged CST - 1 ( A B ) or GFP - labeled F - actin ( C ) and mCherry tagged plasma membrane and histone in WT and mig - 2 null mutant animals . The membrane protrusions in the white boxes are enlarged 3 times at the bottom . Yellow dashed lines indicate the cell periphery . White lines indicate the line to measure the fluorescence intensity . Green lines denote the CST - 1 fluorescence intensity ; red lines denote the plasma membrane fluorescence intensity ; double - headed arrows denote the distance between the GFP signal and cell periphery . Scale bars , 5 μm . ( D ) Distance between GFP : : CST - 1 or GFP : : Moesin ABD and the plasma membrane at the leading edge in WT and mig - 2 null mutants . Error bars indicate SD . N = 9 - 14 . * * * , P 0 . 001 by Student ' s t test . ( E - F ) Fluorescence images ( left ) and quantifications ( right ) of GFP - tagged CST - 1 and mCherry tagged plasma membrane and histone in WT and mig - 2 null mutant animals . Blue lines , sites of measurements ; dotted lines , cell periphery ; Green lines ; CST - 1 fluorescence intensity ; red lines , mCherry fluorescence intensity . Scale bars , 5 μm . ( G ) Quantification of the fluorescence intensity ratio of GFP : : CST - 1 and mCherry - membrane between the leading edge and the lagging edge of AQR in WT and mig - 2 null mutant animals . Error bars indicate SD . N = 20 . * * * , P < 0 . 001 by Student ' s t test .

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

Figure 4(A - C) Fluorescence images (upper) and quantifications (lower) of GFP-tagged CST-1 (A  B) or GFP-labeled F-actin (C) and mCherry tagged plasma membrane and histone in WT and mig-2 null mutant animals. The membrane protrusions in the white boxes are enlarged 3 times at the bottom. Yellow dashed lines indicate the cell periphery. White lines indicate the line to measure the fluorescence intensity. Green lines denote the CST-1 fluorescence intensity; red lines denote the plasma membrane fluorescence intensity; double-headed arrows denote the distance between the GFP signal and cell periphery. Scale bars, 5 μm. (D) Distance between GFP::CST-1 or GFP::Moesin ABD and the plasma membrane at the leading edge in WT and mig-2 null mutants. Error bars indicate SD. N = 9 - 14. ***, P  0.001 by Student's t test.(E - F) Fluorescence images (left) and quantifications (right) of GFP-tagged CST-1 and mCherry tagged plasma membrane and histone in WT and mig-2 null mutant animals. Blue lines, sites of measurements; dotted lines, cell periphery; Green lines; CST-1 fluorescence intensity; red lines, mCherry fluorescence intensity. Scale bars, 5 μm. (G) Quantification of the fluorescence intensity ratio of GFP::CST-1 and mCherry-membrane between the leading edge and the lagging edge of AQR in WT and mig-2 null mutant animals. Error bars indicate SD. N = 20. ***, P < 0.001 by Student's t test.


Figure 1 ( A ) Primary hippocampal neurons ( DIV6 + 3 ) were transfected with shRNA targeting TDP - 43 ( shTDP ) or control ( shCtrl ) together with GFP - RAB11 to visualize trafficking of recycling endosomes ( RE ) . At least four dendrite segments per neuron were live imaged at 5 Hz for 1 minute to analyze recycling endosome transport . Representative dendrite segments and kymographs of GFP - RAB11 vesicle movement . Quantitative analysis of vesicle motility ( B ) and total number ( C ) from kymographs . ( D ) iPSC - derived human neurons were transduced with shRNA targeting human TDP - 43 ( shTDP ) or control ( shCtrl ) seven days after thawing for three days . TDP - 43 levels were analyzed by quantitative RT - PCR . Expression was normalized to the housekeeping gene YWHAZ and PGK1 . ( E ) iPSC - derived human neurons ( DIV7 + 3 ) were transduced with shTDP or shCtrl and GFP - RAB11 and at least three dendrite segments per neuron analyzed as in ( a ) . Quantitative analysis of vesicle motility ( F ) and number ( G ) from kymographs . Mean + / - s . e . m . , n = 3 , unpaired two - tailed t - test : * denotes p < 0 . 05 , * * denotes p < 0 . 01 , * * * denotes p < 0 . 001 .

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

Figure 1(A) Primary hippocampal neurons (DIV6+3) were transfected with shRNA targeting TDP-43 (shTDP) or control (shCtrl) together with GFP-RAB11 to visualize trafficking of recycling endosomes (RE). At least four dendrite segments per neuron were live imaged at 5 Hz for 1 minute to analyze recycling endosome transport. Representative dendrite segments and kymographs of GFP-RAB11 vesicle movement. Quantitative analysis of vesicle motility (B) and total number (C) from kymographs.(D) iPSC-derived human neurons were transduced with shRNA targeting human TDP-43 (shTDP) or control (shCtrl) seven days after thawing for three days. TDP-43 levels were analyzed by quantitative RT-PCR. Expression was normalized to the housekeeping gene YWHAZ and PGK1.(E) iPSC-derived human neurons (DIV7+3) were transduced with shTDP or shCtrl and GFP-RAB11 and at least three dendrite segments per neuron analyzed as in (a).Quantitative analysis of vesicle motility (F) and number (G) from kymographs. Mean +/- s.e.m., n = 3, unpaired two-tailed t-test: * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.001.


Figure 2 ( A ) Primary hippocampal neurons ( DIV6 + 4 ) were transduced with shRNA targeting TDP - 43 or a control together with tagRFP ( not depicted ) . Representative images of neurons subjected to Alexa - transferrin ( Alexa - Tf ) for 20 min ( pulse ) and chased for 0 , 20 or 60 min in complete media . Scale bar represents 100 µm . ( B ) Quantification of labeled cellular Alexa - Tf normalized to cell area ( using tagRFP ) after chase period . At least ten images per condition were analyzed per experiment in three independent experiments . ( C ) ELISA measurement of β2 - transferrin in CSF of control subjects ( n = 65 ) and sporadic ALS patients ( n = 55 ) , FTD - ALS patients ( n = 12 ) and behavioral variant FTD patients ( bvFTD , n = 16 ) . ( D ) Primary hippocampal neurons ( DIV6 + 3 ) were transfected shTDP or shCtrl and GFP as transfection marker . After 3 days neurons were subjected to Alexa - Tf for 20 min and intracellular Alexa - Tf movement was imaged as in Figure 1 afterwards . Quantitative analysis of Alexa - Tf movement ( E ) and vesicle number ( F ) from kymographs .

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

Figure 2(A) Primary hippocampal neurons (DIV6+4) were transduced with shRNA targeting TDP-43 or a control together with tagRFP (not depicted). Representative images of neurons subjected to Alexa-transferrin (Alexa-Tf) for 20 min (pulse) and chased for 0, 20 or 60 min in complete media. Scale bar represents 100 µm. (B) Quantification of labeled cellular Alexa-Tf normalized to cell area (using tagRFP) after chase period. At least ten images per condition were analyzed per experiment in three independent experiments.(C) ELISA measurement of β2-transferrin in CSF of control subjects (n=65) and sporadic ALS patients (n=55), FTD-ALS patients (n=12) and behavioral variant FTD patients (bvFTD, n=16).(D) Primary hippocampal neurons (DIV6+3) were transfected shTDP or shCtrl and GFP as transfection marker. After 3 days neurons were subjected to Alexa-Tf for 20 min and intracellular Alexa-Tf movement was imaged as in Figure 1 afterwards. Quantitative analysis of Alexa-Tf movement (E) and vesicle number (F) from kymographs.


Figure 3 ( A ) Primary hippocampal neurons ( DIV6 + 4 ) were transfected with myc - tagged TDP - 43 wild - type or a mutant lacking the nuclear localization signal ( ΔNLS ) or an empty vector control . Immunostaining with the indicated antibodies and DAPI to label nuclei . Scale bar represents 50 µm . ( B ) Primary hippocampal neurons ( DIV6 + 3 ) were transfected with either TDP - 43 wild - type , TDP - 43ΔNLS or an empty vector control together with GFP - RAB11 to visualize recycling endosomes and analyzed as in Figure 1 . Quantitative analysis of recycling endosome movement ( C ) and vesicle number ( D ) .

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

Figure 3(A) Primary hippocampal neurons (DIV6+4) were transfected with myc-tagged TDP-43 wild-type or a mutant lacking the nuclear localization signal (ΔNLS) or an empty vector control. Immunostaining with the indicated antibodies and DAPI to label nuclei. Scale bar represents 50 µm.(B) Primary hippocampal neurons (DIV6+3) were transfected with either TDP-43 wild-type, TDP-43ΔNLS or an empty vector control together with GFP-RAB11 to visualize recycling endosomes and analyzed as in Figure 1. Quantitative analysis of recycling endosome movement (C) and vesicle number (D).


Figure 4 ( A ) Primary cortical neurons were transduced with shTDP or control ( DIV2 + 7 ) and subjected to quantitative proteomic analysis . Volcano plot shows up and down regulated proteins from three biological replicates . Significant changes are colored . Compare Table EV1 . ( C - E ) Primary hippocampal neurons ( DIV6 + 4 ) were transduced with lentivirus expressing shRNA targeting TDP - 43 or a control . Immunoblots with the indicated antibodies ( C ) . Quantification of VPS4B and TDP - 43 protein level using densitometry ( n = 4 ) . Expression was normalized to the housekeeping gene β - actin ( D ) . ( C - E ) Primary hippocampal neurons ( DIV6 + 4 ) were transduced with lentivirus expressing shRNA targeting TDP - 43 or a control . VPS4B mRNA level were analyzed by quantitative RT - PCR . Expression was normalized to the housekeeping genes YWHAZ and PGK1 ( E ) . ( F ) Primary hippocampal neurons ( DIV6 + 3 ) were transfected with either shTDP or shCtrl and iRFP670 . Immunostaining with the indicated antibodies . Scale bar represents 50 µm . ( G ) iPSC - derived human neurons were transduced with shRNA targeting human TDP - 43 ( shTDP ) or control ( shCtrl ) seven days after thawing for three days . VPS4B levels were analyzed by quantitative RT - PCR . Expression was normalized to the housekeeping gene YWHAZ and PGK1 ( n = 3 ) .

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

Figure 4(A) Primary cortical neurons were transduced with shTDP or control (DIV2+7) and subjected to quantitative proteomic analysis. Volcano plot shows up and down regulated proteins from three biological replicates. Significant changes are colored. Compare Table EV1.(C-E) Primary hippocampal neurons (DIV6+4) were transduced with lentivirus expressing shRNA targeting TDP-43 or a control. Immunoblots with the indicated antibodies (C). Quantification of VPS4B and TDP-43 protein level using densitometry (n = 4). Expression was normalized to the housekeeping gene β-actin (D).(C-E) Primary hippocampal neurons (DIV6+4) were transduced with lentivirus expressing shRNA targeting TDP-43 or a control. VPS4B mRNA level were analyzed by quantitative RT-PCR. Expression was normalized to the housekeeping genes YWHAZ and PGK1 (E).(F) Primary hippocampal neurons (DIV6+3) were transfected with either shTDP or shCtrl and iRFP670. Immunostaining with the indicated antibodies. Scale bar represents 50 µm.(G) iPSC-derived human neurons were transduced with shRNA targeting human TDP-43 (shTDP) or control (shCtrl) seven days after thawing for three days. VPS4B levels were analyzed by quantitative RT-PCR. Expression was normalized to the housekeeping gene YWHAZ and PGK1 (n = 3).


Figure 5 ( A - B ) Luciferase assay to analyze transcriptional regulation of VPS4B by TDP - 43 . ( B ) HEK293 cells were transfected with shRNAs targeting human TDP - 43 , a control shRNA , TDP - 43 wild - type or empty vector control together with the luciferase reporter containing the rat VPS4B promoter . VPS4B promoter - driven renilla luciferase activity was normalized to TK promoter - driven firefly luciferase . Quantification from six independent experiments . ( C - D ) ChIP assay to analyze binding of TDP - 43 to VPS4B promoter region in rat cortical neurons , HEK293 cells and human brain tissue . PCR reaction from input , negative control and TDP - 43 immunoprecipitates . Signal intensities from at least three independent experiments for each condition were quantified by densitometry and depicted as percent of input . ( E - H ) Analysis of VPS4B in tissue of seven patients with the neuropathological diagnosis of FTLD / ALS - TDP and five healthy controls . ( E , G ) Immunofluorescence with the indicated antibodies in the superior frontal gyrus or spinal cord . Scale bar represents 50 µm . ( F , H ) Quantification of cellular VPS4B levels . At least 50 cells per case were analyzed .

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

Figure 5(A-B) Luciferase assay to analyze transcriptional regulation of VPS4B by TDP-43. (B) HEK293 cells were transfected with shRNAs targeting human TDP-43, a control shRNA, TDP-43 wild-type or empty vector control together with the luciferase reporter containing the rat VPS4B promoter. VPS4B promoter-driven renilla luciferase activity was normalized to TK promoter-driven firefly luciferase. Quantification from six independent experiments.(C-D) ChIP assay to analyze binding of TDP-43 to VPS4B promoter region in rat cortical neurons, HEK293 cells and human brain tissue. PCR reaction from input, negative control and TDP-43 immunoprecipitates. Signal intensities from at least three independent experiments for each condition were quantified by densitometry and depicted as percent of input.(E-H) Analysis of VPS4B in tissue of seven patients with the neuropathological diagnosis of FTLD/ALS-TDP and five healthy controls. (E, G) Immunofluorescence with the indicated antibodies in the superior frontal gyrus or spinal cord. Scale bar represents 50 µm. (F, H) Quantification of cellular VPS4B levels. At least 50 cells per case were analyzed.


Figure 6 ( A ) Primary hippocampal neurons ( DIV6 + 3 ) were transfected with either VPS4B or an empty vector control together with GFP - RAB11 to visualize recycling endosomes . Neurons were imaged as in Figure 1 to analyze recycling endosome transport . Quantitative analysis of vesicle motility ( B ) and number ( C ) from kymographs ( n = 4 ) . ( D ) Primary hippocampal neurons ( DIV6 + 3 ) were transfected with the indicated combinations of shRNA targeting TDP - 43 , VPS4B and control together with GFP - RAB11 and imaged as in Figure 1A . Quantitative analysis of vesicle motility ( E ) and number ( F ) from kymographs ( n = 3 ) .

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

Figure 6(A) Primary hippocampal neurons (DIV6+3) were transfected with either VPS4B or an empty vector control together with GFP-RAB11 to visualize recycling endosomes. Neurons were imaged as in Figure 1 to analyze recycling endosome transport. Quantitative analysis of vesicle motility (B) and number (C) from kymographs (n = 4).(D) Primary hippocampal neurons (DIV6+3) were transfected with the indicated combinations of shRNA targeting TDP-43, VPS4B and control together with GFP-RAB11 and imaged as in Figure 1A. Quantitative analysis of vesicle motility (E) and number (F) from kymographs (n=3).


Figure 7 ( A ) Primary hippocampal neurons ( DIV6 + 4 ) were transduced with shRNA targeting TDP - 43 ( shTDP ) or control ( shCtrl ) and surface expression levels of glycoproteins was analyzed by metabolic labeling followed by surface biotinylation and subsequent streptavidin purification . Volcano plot depicts proteins that are significantly ( p < 0 . 01 , t - test , Benjamini - Hochberg , FDR : 0 . 05 ) decreased or increased on the cell surface in three independent experiments with two technical replicates . For full dataset see Table EV2 . ( B - C ) Primary cortical neurons ( DIV6 + 5 ) were transduced with shCtrl or shTDP . Neurons were treated with the ErbB4 ligand NRG1 ( 10 nM ) for 10 min right before cell lysis . Immunoblots with the indicated antibodies ( B ) . Quantification of total and phosho - ERBB4 ( Y1056 ) using densitometry 3 - 7 biological replicates ( C ) . ( D - E ) Primary cortical neurons ( DIV6 + 5 ) were transduced with shCtrl or shTDP . Neurons were treated with the FGFR1 ligand FGF1 ( 10 ng / ml ) for 10 min right before cell lysis . Immunoblots with the indicated antibodies ( D ) . Quantification of total and phosho - FGFR1 ( Y654 ) using densitometry 3 - 7 biological replicates ( E ) . ( F ) Primary hippocampal neurons ( DIV6 + 4 ) were transduced with shTDP or shCtrl . Gene expression was analyzed by quantitative RT - PCR normalized to the housekeeping genes YWHAZ and GAPDH ( n = 3 ) ( G ) Hippocampal neurons were transfected with ErbB4 containing a luminal HA - tag ( HA / T - ErbB4 ) and shCtrl or shTDP ( DIV9 + 3 ) . Surface HA / T - ErbB4 was stained in living cells and intracellular HA / T - ErbB4 was stained after fixation and permeabilization with HA - antibodies . ( H ) Quantification of HA / T - ErbB4 surface levels . At least 10 images per condition per experiment were analyzed in three independent experiments . Scale bar represents 50 µm .

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

Figure 7(A) Primary hippocampal neurons (DIV6+4) were transduced with shRNA targeting TDP-43 (shTDP) or control (shCtrl) and surface expression levels of glycoproteins was analyzed by metabolic labeling followed by surface biotinylation and subsequent streptavidin purification. Volcano plot depicts proteins that are significantly (p < 0.01, t-test, Benjamini-Hochberg, FDR: 0.05) decreased or increased on the cell surface in three independent experiments with two technical replicates. For full dataset see Table EV2.(B-C) Primary cortical neurons (DIV6+5) were transduced with shCtrl or shTDP. Neurons were treated with the ErbB4 ligand NRG1 (10 nM) for 10 min right before cell lysis. Immunoblots with the indicated antibodies (B). Quantification of total and phosho-ERBB4 (Y1056) using densitometry 3-7 biological replicates (C).(D-E) Primary cortical neurons (DIV6+5) were transduced with shCtrl or shTDP. Neurons were treated with the FGFR1 ligand FGF1 (10 ng/ml) for 10 min right before cell lysis. Immunoblots with the indicated antibodies (D). Quantification of total and phosho-FGFR1 (Y654) using densitometry 3-7 biological replicates (E).(F) Primary hippocampal neurons (DIV6+4) were transduced with shTDP or shCtrl. Gene expression was analyzed by quantitative RT-PCR normalized to the housekeeping genes YWHAZ and GAPDH (n=3)(G) Hippocampal neurons were transfected with ErbB4 containing a luminal HA-tag (HA/T-ErbB4) and shCtrl or shTDP (DIV9+3). Surface HA/T-ErbB4 was stained in living cells and intracellular HA/T-ErbB4 was stained after fixation and permeabilization with HA-antibodies. (H) Quantification of HA/T-ErbB4 surface levels. At least 10 images per condition per experiment were analyzed in three independent experiments. Scale bar represents 50 µm.


Figure 8 ( A , B ) Primary hippocampal neurons ( DIV6 + 3 ) were transfected with shCtrl or shTDP and ErbB4 containing an N - terminal HA epitope tag and followed by thrombin cleavage site ( HA / T - ErbB4 ) . Surface HA / T - ErbB4 was stained in living cells and intracellular HA / T - ErbB4 was stained after fixation and permeabilization with two different HA - antibodies . After removing the HA - signal of surface ErbB4 ( 0 min ) using thrombin cleavage ( 5min , 1U / ml ) membrane insertion of HA / T - ErbB4 was measured at the indicated time points . At least 10 images per condition per experiment were analyzed in three independent experiments . Scale bar represents 50 µm .

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Figure 8(A,B) Primary hippocampal neurons (DIV6+3) were transfected with shCtrl or shTDP and ErbB4 containing an N-terminal HA epitope tag and followed by thrombin cleavage site (HA/T-ErbB4). Surface HA/T-ErbB4 was stained in living cells and intracellular HA/T-ErbB4 was stained after fixation and permeabilization with two different HA-antibodies. After removing the HA-signal of surface ErbB4 (0 min) using thrombin cleavage (5min, 1U/ml) membrane insertion of HA/T-ErbB4 was measured at the indicated time points. At least 10 images per condition per experiment were analyzed in three independent experiments. Scale bar represents 50 µm.