( E ) Upper : schematic representation of various Sigmar1 truncations . Bottom : mapping of Sigmar1 domains critical for SERCA2 binding . HEK - 293T cells were transfected with different Sigmar1 truncations , and cell lysates were immunoprecipitated and subjected to western blotting . Data information : All results are representative data generated from at least three independent experiments .

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(E) Upper: schematic representation of various Sigmar1 truncations. Bottom: mapping of Sigmar1 domains critical for SERCA2 binding. HEK-293T cells were transfected with different Sigmar1 truncations, and cell lysates were immunoprecipitated and subjected to western blotting. Data information: All results are representative data generated from at least three independent experiments.


( F ) Upper : schematic representation of various SERCA2 truncations . Bottom : mapping of SERCA2 domains crucial for Sigmar1 binding . Different truncated SERCA2 plasmids were transfected into HEK - 293T cells . After immunoprecipitation , the interaction between truncated SERCA2 and Sigmar1 was detected by western blotting . Red asterisks indicate specific SERCA2 truncation bands . Data information : All results are representative data generated from at least three independent experiments .

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(F) Upper: schematic representation of various SERCA2 truncations. Bottom: mapping of SERCA2 domains crucial for Sigmar1 binding. Different truncated SERCA2 plasmids were transfected into HEK-293T cells. After immunoprecipitation, the interaction between truncated SERCA2 and Sigmar1 was detected by western blotting. Red asterisks indicate specific SERCA2 truncation bands. Data information: All results are representative data generated from at least three independent experiments.


( H ) Western blots showing Q615A mutant truncated SERCA2 expression in HEK - 293T cells transfected with different amounts of Sigmar1 plasmids . The cells were transfected with 1 μg Q615A mutant of truncated SERCA2 plasmid and Sigmar1 plasmid ( 0 . 125 μg , 0 . 25 μg , 0 . 5 μg , 1 μg ) . Data information : All results are representative data generated from at least three independent experiments .

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(H) Western blots showing Q615A mutant truncated SERCA2 expression in HEK-293T cells transfected with different amounts of Sigmar1 plasmids. The cells were transfected with 1 μg Q615A mutant of truncated SERCA2 plasmid and Sigmar1 plasmid (0.125 μg, 0.25 μg, 0.5 μg, 1 μg). Data information: All results are representative data generated from at least three independent experiments.


( I ) Interactions between Sigmar1 and WT or Q615 mutants of truncated SERCA2 were detected by Co - IP assays . Data information : All results are representative data generated from at least three independent experiments .

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(I) Interactions between Sigmar1 and WT or Q615 mutants of truncated SERCA2 were detected by Co-IP assays. Data information: All results are representative data generated from at least three independent experiments.


( J ) TRAP staining to detect osteoclastogenesis of BMMs from WT and gKO mice treated with different siRNAs . Scale bars , 200 μm . Data information : All results are representative data generated from at least three independent experiments .

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(J) TRAP staining to detect osteoclastogenesis of BMMs from WT and gKO mice treated with different siRNAs. Scale bars, 200 μm. Data information: All results are representative data generated from at least three independent experiments.


( A and B ) Western blots showing SERCA2 expression in HEK - 293T cells with or without Sigmar1 cotransfection treated with eeyarestatin I or MG132 at the indicated concentration . All inhibitors were applied to cells 8 hours prior to protein collection . Data information : All results are representative data generated from at least three independent experiments .

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(A and B) Western blots showing SERCA2 expression in HEK-293T cells with or without Sigmar1 cotransfection treated with eeyarestatin I or MG132 at the indicated concentration. All inhibitors were applied to cells 8 hours prior to protein collection. Data information: All results are representative data generated from at least three independent experiments.


( C ) Western blots showing changes in SERCA2 expression in HEK - 293T cells in the presence of different doses of flag - tagged Hrd1 plasmids . Data information : All results are representative data generated from at least three independent experiments .

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(C) Western blots showing changes in SERCA2 expression in HEK-293T cells in the presence of different doses of flag-tagged Hrd1 plasmids. Data information: All results are representative data generated from at least three independent experiments.


( D ) Western blots showing alterations of SERCA2 expression in HEK - 293T cells pretreated with negative control siRNA ( si - NC ) or Sel1L siRNA in the presence or absence of Sigmar1 . Data information : All results are representative data generated from at least three independent experiments .

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(D) Western blots showing alterations of SERCA2 expression in HEK-293T cells pretreated with negative control siRNA (si-NC) or Sel1L siRNA in the presence or absence of Sigmar1. Data information: All results are representative data generated from at least three independent experiments.


( E ) Truncated myc - tagged SERCA2 ubiquitination levels in HEK - 293T cells transfected with empty vector or Sigmar1 were analyzed by immunoprecipitation . Cells were treated with MG132 ( 10 μM ) 8 hours before harvest . Data information : All results are representative data generated from at least three independent experiments .

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(E) Truncated myc-tagged SERCA2 ubiquitination levels in HEK-293T cells transfected with empty vector or Sigmar1 were analyzed by immunoprecipitation. Cells were treated with MG132 (10 μM) 8 hours before harvest. Data information: All results are representative data generated from at least three independent experiments.


( F ) Expression of different truncated SERCA2 lysine mutants in HEK - 293T cells transfected with different amounts of Sigmar1 plasmid . Data information : All results are representative data generated from at least three independent experiments .

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(F) Expression of different truncated SERCA2 lysine mutants in HEK-293T cells transfected with different amounts of Sigmar1 plasmid. Data information: All results are representative data generated from at least three independent experiments.


( G ) Expression of WT or 2KR ( K460A and K541A ) mutants of full - length SERCA2 in HEK - 293T cells transfected with different amounts of Sigmar1 plasmid . Data information : All results are representative data generated from at least three independent experiments .

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(G) Expression of WT or 2KR (K460A and K541A) mutants of full-length SERCA2 in HEK-293T cells transfected with different amounts of Sigmar1 plasmid. Data information: All results are representative data generated from at least three independent experiments.


( B ) Micro - CT images of calvaria from mice that received sham or LPS injection with PBS or dimemorfan treatment . Scale bars , 1 mm . ( C ) Quantification of bone volume / tissue volume ( BV / TV ) of calvaria from different groups ( n = 6 biological replicates ) . Data information : All results are representative data generated from at least three independent experiments . Data are presented as mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test ( C , was used for statistical analysis .

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(B) Micro-CT images of calvaria from mice that received sham or LPS injection with PBS or dimemorfan treatment. Scale bars, 1 mm. (C) Quantification of bone volume/tissue volume (BV/TV) of calvaria from different groups (n = 6 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with Tukey's multiple comparisons test (C, was used for statistical analysis.


( D ) H & E and TRAP staining of calvaria from the three groups . Scale bars , 100 μm . ( E and F ) Quantification of osteoclast number per bone surface ( N . Oc / BS ) and percentage of osteoclast surface per bone surface ( Oc . S / BS ) ( n = 6 biological replicates ) . Data information : All results are representative data generated from at least three independent experiments . Data are presented as mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test E - F , was used for statistical analysis .

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(D) H&E and TRAP staining of calvaria from the three groups. Scale bars, 100 μm. (E and F) Quantification of osteoclast number per bone surface (N. Oc/BS) and percentage of osteoclast surface per bone surface (Oc. S/BS) (n = 6 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with Tukey's multiple comparisons test E-F, was used for statistical analysis.


( H ) Micro - CT images of proximal femurs from sham or ovariectomized mice with different treatments . Scale bars , 1 mm .

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(H) Micro-CT images of proximal femurs from sham or ovariectomized mice with different treatments. Scale bars, 1 mm.


( I ) Quantification of bone volume / tissue volume ( BV / TV ) , trabecular number ( Tb . N ) , trabecular separation ( Tb . Sp ) and trabecular thickness ( Tb . Th ) ( n = 6 biological replicates ) . Data information : All results are representative data generated from at least three independent experiments . Data are presented as mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test , I , was used for statistical analysis .

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(I) Quantification of bone volume/tissue volume (BV/TV), trabecular number (Tb. N), trabecular separation (Tb. Sp) and trabecular thickness (Tb. Th) (n = 6 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with Tukey's multiple comparisons test , I, was used for statistical analysis.


( J ) TRAP staining of femurs from the three groups . Scale bars , 200 μm .

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(J) TRAP staining of femurs from the three groups. Scale bars, 200 μm.


( K and L ) Quantification of osteoclast number per bone surface ( N . Oc / BS ) and percentage of osteoclast surface per bone surface ( Oc . S / BS ) ( n = 6 biological replicates ) . Data information : All results are representative data generated from at least three independent experiments . Data are presented as mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test was used for statistical analysis .

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(K and L) Quantification of osteoclast number per bone surface (N. Oc/BS) and percentage of osteoclast surface per bone surface (Oc. S/BS) (n = 6 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with Tukey's multiple comparisons test was used for statistical analysis.


( A ) TRAP staining of hPBMCs treated with human RANKL ( 50 ng / mL ) or dimemorfan for 12 days . Scale bars , 500 μm .

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(A) TRAP staining of hPBMCs treated with human RANKL (50 ng/mL) or dimemorfan for 12 days. Scale bars, 500 μm.


( B ) Western blot analysis of NFATc1 expression during a 7 - day induction of hPBMCs to osteoclasts .

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(B) Western blot analysis of NFATc1 expression during a 7-day induction of hPBMCs to osteoclasts.


( C ) hPBMCs were induced to differentiate into osteoclasts for 7 days , and the relative mRNA levels of marker genes were evaluated by RT - qPCR ( n = 6 biological replicates ) . Data information : All results are representative data generated from at least three independent experiments . Data are presented as mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test ( C ) was used for statistical analysis .

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(C) hPBMCs were induced to differentiate into osteoclasts for 7 days, and the relative mRNA levels of marker genes were evaluated by RT-qPCR (n=6 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with Tukey's multiple comparisons test (C) was used for statistical analysis.


A , B Detection of γb palmitoylation through the biotin - switch assay in N . benthamiana . The γb - 3xFlag and γb3CS - 3xFlag proteins were treated with ( Hyd + ) or without ( Hyd - ) hydroxylamine , the thioester cleavage reagent . Lanes labelled ' Palmitoylation ' show γb - 3xFlag or γb3CS - 3xFlag amounts recovered from the neutravidin beads . The loading controls indicate sample loading onto the neutravidin beads . Data information : In ( A , representative data are shown and three biological replicates had similar results .

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A, B Detection of γb palmitoylation through the biotin-switch assay in N. benthamiana. The γb-3xFlag and γb3CS-3xFlag proteins were treated with (Hyd+) or without (Hyd-) hydroxylamine, the thioester cleavage reagent. Lanes labelled ' Palmitoylation ' show γb-3xFlag or γb3CS-3xFlag amounts recovered from the neutravidin beads. The loading controls indicate sample loading onto the neutravidin beads. Data information: In (A, representative data are shown and three biological replicates had similar results.


D Western blot detection of viral accumulation in BSMV - and BSMV3CS - infected N . benthamiana plants with anti - TGB1 or anti - γb antibodies . SL8 , eighth systemically infected leaf ; SL9 , ninth systemically infected leaf . Ino , agroinfiltrated leaves . Rubisco large subunit ( RbcL ) was used as a loading control . Data information : In representative data are shown and three biological replicates had similar results .

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D Western blot detection of viral accumulation in BSMV- and BSMV3CS-infected N. benthamiana plants with anti-TGB1 or anti-γb antibodies. SL8, eighth systemically infected leaf; SL9, ninth systemically infected leaf. Ino, agroinfiltrated leaves. Rubisco large subunit (RbcL) was used as a loading control. Data information: In representative data are shown and three biological replicates had similar results.


F Western blot analysis of protein accumulation in BSMV - and BSMV3CS - systemically infected barley leaves with anti - TGB1 or anti - γb antibodies . RbcL was used as a loading control . Data information : In F ) , representative data are shown and three biological replicates had similar results .

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F Western blot analysis of protein accumulation in BSMV- and BSMV3CS- systemically infected barley leaves with anti-TGB1 or anti-γb antibodies. RbcL was used as a loading control. Data information: In F), representative data are shown and three biological replicates had similar results.


A Effects of γb palmitoylation on BSMV replication . Movement - deficient BSMV ( RNAα + RNAγ / RNAγ3CS ) was agroinfiltrated into N . benthamiana leaves . At 3 dpi , total protein and RNA were extracted for Western and Northern blot analyses . Antibodies and probes used for molecular analyses are indicated on the right of each panel . Actin was used to monitor equal protein loading , and methylene blue - stained rRNAs served as RNA loading controls . Data information : representative data are shown and three biological replicates had similar results .

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A Effects of γb palmitoylation on BSMV replication. Movement-deficient BSMV (RNAα + RNAγ/RNAγ3CS) was agroinfiltrated into N. benthamiana leaves. At 3 dpi, total protein and RNA were extracted for Western and Northern blot analyses. Antibodies and probes used for molecular analyses are indicated on the right of each panel. Actin was used to monitor equal protein loading, and methylene blue-stained rRNAs served as RNA loading controls. Data information: representative data are shown and three biological replicates had similar results.


B Western blot analyses of chloroplast localization of γb and γb3CS . Transiently expressed αa - GFP in intact chloroplasts isolated from BSMVγb - 3xFlag - or BSMV3CS - 3xFlag - infected N . benthamiana leaves at 4 dpi . Detection of γb - 3xFlag and γb3CS - 3xFlag with an anti - Flag antibody ; TGB1 analysis with an anti - TGB1 antibody ; αa - GFP detection with an anti - GFP antibody . NTRC was a loading control for intact chloroplasts Phosphoenolpyruvate carboxylase ( PEPC ) was used to assess cytosolic contamination of isolated chloroplasts . Data information : , representative data are shown and three biological replicates had similar results .

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B Western blot analyses of chloroplast localization of γb and γb3CS. Transiently expressed αa-GFP in intact chloroplasts isolated from BSMVγb-3xFlag- or BSMV3CS-3xFlag-infected N. benthamiana leaves at 4 dpi. Detection of γb-3xFlag and γb3CS-3xFlag with an anti-Flag antibody; TGB1 analysis with an anti-TGB1 antibody; αa-GFP detection with an anti-GFP antibody. NTRC was a loading control for intact chloroplasts Phosphoenolpyruvate carboxylase (PEPC) was used to assess cytosolic contamination of isolated chloroplasts. Data information: , representative data are shown and three biological replicates had similar results.


C Subcellular localization of γb - GFP and γb3CS - GFP in N . benthamiana epidermal leaf cells . The first and second rows show subcellular localization of γb - GFP and γb3CS - GFP transient expression in leaves . The third and fourth rows show localization of γb - GFP and γb3CS - GFP in BSMVγb - GFP - and BSMV3CS - GFP - infected leaves at 3 dpi . Figures on the right indicate the normalized fluorescence intensities of the GFP ( green ) or the chloroplast autofluorescence ( red ) channels along the dashed white lines shown in the merged images of the left panel . Scale bars , 20 μm . Data information : representative data are shown and three biological replicates had similar results .

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C Subcellular localization of γb-GFP and γb3CS-GFP in N. benthamiana epidermal leaf cells. The first and second rows show subcellular localization of γb-GFP and γb3CS-GFP transient expression in leaves. The third and fourth rows show localization of γb-GFP and γb3CS-GFP in BSMVγb-GFP- and BSMV3CS-GFP-infected leaves at 3 dpi. Figures on the right indicate the normalized fluorescence intensities of the GFP (green) or the chloroplast autofluorescence (red) channels along the dashed white lines shown in the merged images of the left panel. Scale bars, 20 μm. Data information: representative data are shown and three biological replicates had similar results.


D Subcellular localization of γb - GFP or γb3CS - GFP in isolated N . benthamiana protoplasts harvested at 3 dpi . The fifth and sixth columns show γb localization in BSMVγb - GFP - ( movement - deficient ) N . benthamiana protoplasts after treatment with 200 μM 2 - BP inhibitor . The released protoplasts were collected after 48 h incubation in DMSO or 2 - BP followed by confocal microscopy visualization . Figures at the bottom indicate the normalized fluorescence intensities of the GFP ( green ) and the chloroplast autofluorescence ( red ) channels along the dashed white lines in the merged images above . Scale bars , 20 μm . Data information : representative data are shown and three biological replicates had similar results .

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D Subcellular localization of γb-GFP or γb3CS-GFP in isolated N. benthamiana protoplasts harvested at 3 dpi. The fifth and sixth columns show γb localization in BSMVγb-GFP- (movement-deficient) N. benthamiana protoplasts after treatment with 200 μM 2-BP inhibitor. The released protoplasts were collected after 48 h incubation in DMSO or 2-BP followed by confocal microscopy visualization. Figures at the bottom indicate the normalized fluorescence intensities of the GFP (green) and the chloroplast autofluorescence (red) channels along the dashed white lines in the merged images above. Scale bars, 20 μm. Data information: representative data are shown and three biological replicates had similar results.


A Effects of γb palmitoylation on virus cell - to - cell movement at 3 dpi with the BSMV duplex fluorescence ( dfBSMV ) reporter system The dfBSMVmγb mutant control contains an ATG substitution of the γb initiation codon Red fluorescence produced from the mCherry expression cassette illustrates the primary infection foci , whereas GFP fluorescence outside the red region shows invaded cells surrounding the primary infection foci . Fluorescence was evaluated by confocal microscopy . Scale bars , 100 μm . B Quantification of virus movement in Panel A . To determine the movement capacity of the viruses depicted in each panel , the mCherry and GFP fluorescent intensities were first determined by ImageJ software . Then , the intensities within the secondarily invaded cells were divided by the fluorescent mCherry plus the GFP intensities in the primary infection foci ( n = 10 ) . The Y - axis indicates the relative sizes of the green areas in comparison to that of the red - colored areas . Data information : In ( A , representative data are shown and three biological replicates had similar results . In ( B ) , the box boundaries indicate the upper ( 25th percentile ) and lower ( 75th percentile ) quartiles , whiskers indicate minimum and maximum values , central band indicates the median . Letters in the chart denote statistically significant differences among groups according to the Duncan ' s multiple range test ( p < 0 . 05 ) .

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A Effects of γb palmitoylation on virus cell-to-cell movement at 3 dpi with the BSMV duplex fluorescence (dfBSMV) reporter system The dfBSMVmγb mutant control contains an ATG substitution of the γb initiation codon Red fluorescence produced from the mCherry expression cassette illustrates the primary infection foci, whereas GFP fluorescence outside the red region shows invaded cells surrounding the primary infection foci. Fluorescence was evaluated by confocal microscopy. Scale bars, 100 μm. B Quantification of virus movement in Panel A. To determine the movement capacity of the viruses depicted in each panel, the mCherry and GFP fluorescent intensities were first determined by ImageJ software. Then, the intensities within the secondarily invaded cells were divided by the fluorescent mCherry plus the GFP intensities in the primary infection foci (n = 10). The Y-axis indicates the relative sizes of the green areas in comparison to that of the red-colored areas. Data information: In (A, representative data are shown and three biological replicates had similar results. In (B), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).


D Biotin - switch assay to access palmitoylation of γb in BSMV - infected N . benthamiana leaves at different timepoints . BSMVγb - 3xFlag was treated with the hydroxylamine ( Hyd + ) thioester cleavage reagent . Equal sample amounts used for palmitoylation analyses were monitored by Western blot analysis with an anti - Flag antibody . Data information : In representative data are shown and three biological replicates had similar results .

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D Biotin-switch assay to access palmitoylation of γb in BSMV-infected N. benthamiana leaves at different timepoints. BSMVγb-3xFlag was treated with the hydroxylamine (Hyd+) thioester cleavage reagent. Equal sample amounts used for palmitoylation analyses were monitored by Western blot analysis with an anti-Flag antibody. Data information: In representative data are shown and three biological replicates had similar results.


E Subcellular localization of BSMVγb - GFP and BSMV3CS - GFP in N . benthamiana leaf epidermal cells . The mCherry - HDEL and mTalin - RFP reporters were used as markers to indicate the ER and actin localization Figures at the bottom indicate the normalized fluorescence intensities of the GFP and mCherry channels along the white dashed lines in the merged images above . Scale bars , 10 μm . Data information : In E ) , representative data are shown and three biological replicates had similar results .

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E Subcellular localization of BSMVγb-GFP and BSMV3CS-GFP in N. benthamiana leaf epidermal cells. The mCherry-HDEL and mTalin-RFP reporters were used as markers to indicate the ER and actin localization Figures at the bottom indicate the normalized fluorescence intensities of the GFP and mCherry channels along the white dashed lines in the merged images above. Scale bars, 10 μm. Data information: In E), representative data are shown and three biological replicates had similar results.


F , G Western blot analysis of subcellular fractions extracted from N . benthamiana leaf tissues at 3 dpi . S3 indicates the supernatant separated by centrifugation at 3 , 000 g ; and S30 is the 30 , 000 g supernatant ; P30 is the 30 , 000 g pellet . The association of γb with membranes was determined by 1 % Triton X - 100 extraction .

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F, G Western blot analysis of subcellular fractions extracted from N. benthamiana leaf tissues at 3 dpi. S3 indicates the supernatant separated by centrifugation at 3,000 g; and S30 is the 30,000 g supernatant; P30 is the 30,000 g pellet. The association of γb with membranes was determined by 1% Triton X-100 extraction.


A BiFC co - localization analyses of αa and TGB1 in N . benthamiana leaf cells at 3 dpi . YFPn and YFPc was fused to the TGB1 and the αa proteins , respectively . Combinations of γb , γb3CS and the TBSV P19 control are shown above the micrographs . YFP signals were visualized by confocal microscopy at 3 dpi and depicted as a false - green color . Scale bars , 30 μm . B Quantification of the numbers of YFP foci per visual field ( n = 10 ) . Data information : In ( A , , representative data are shown and three biological replicates had similar results . In ( B , the box boundaries indicate the upper ( 25th percentile ) and lower ( 75th percentile ) quartiles , whiskers indicate minimum and maximum values , central band indicates the median . Letters in the chart denote statistically significant differences among groups according to the Duncan ' s multiple range test ( p < 0 . 05 ) .

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A BiFC co-localization analyses of αa and TGB1 in N. benthamiana leaf cells at 3 dpi. YFPn and YFPc was fused to the TGB1 and the αa proteins, respectively. Combinations of γb, γb3CS and the TBSV P19 control are shown above the micrographs. YFP signals were visualized by confocal microscopy at 3 dpi and depicted as a false-green color. Scale bars, 30 μm. B Quantification of the numbers of YFP foci per visual field (n = 10). Data information: In (A, , representative data are shown and three biological replicates had similar results. In (B, the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).


C Co - IP assays to determine in vivo interactions between TGB1 and γb or γb3CS . N . benthamiana leaf tissues co - agroinfiltrated with RNAα , RNAγγb - GFP or RNAγ3CS - GFP , and TGB1 - 3xFlag plasmids were harvested at 3 dpi . Total proteins were immunoprecipitated with anti - Flag beads . Input and immunoprecipitated proteins ( IP ) were analyzed by Western blotting with anti - GFP or anti - Flag antibodies .

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C Co-IP assays to determine in vivo interactions between TGB1 and γb or γb3CS. N. benthamiana leaf tissues co-agroinfiltrated with RNAα, RNAγγb-GFP or RNAγ3CS-GFP, and TGB1-3xFlag plasmids were harvested at 3 dpi. Total proteins were immunoprecipitated with anti-Flag beads. Input and immunoprecipitated proteins (IP) were analyzed by Western blotting with anti-GFP or anti-Flag antibodies.


D Confocal microscopy analyses showing RFP - TGB1 localization at the chloroplast periphery in N . benthamiana leaves agroinfiltrated with αa - GFP and CFP - γb plasmids . CFP - γb or CFP - γb3CS were co - expressed with RFP - TGB1 and αa - GFP in N . benthamiana leaves . Chloroplast autofluorescence was displayed in false pink color . The third and fourth rows show leaves treated with 400 μM 2 - BP inhibitor or the DMSO control at 1 dpi . Two days later , confocal microscopy observations of RFP - TGB1 , αa - GFP , and CFP - γb were performed . Figures on the right indicate the normalized fluorescence intensity of GFP , RFP , and CFP channels along the white dashed line in the merged confocal images . Scale bars , 10 μm . Data information : In , representative data are shown and three biological replicates had similar results .

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D Confocal microscopy analyses showing RFP-TGB1 localization at the chloroplast periphery in N. benthamiana leaves agroinfiltrated with αa-GFP and CFP-γb plasmids. CFP-γb or CFP-γb3CS were co-expressed with RFP-TGB1 and αa-GFP in N. benthamiana leaves. Chloroplast autofluorescence was displayed in false pink color. The third and fourth rows show leaves treated with 400 μM 2-BP inhibitor or the DMSO control at 1 dpi. Two days later, confocal microscopy observations of RFP-TGB1, αa-GFP, and CFP-γb were performed. Figures on the right indicate the normalized fluorescence intensity of GFP, RFP, and CFP channels along the white dashed line in the merged confocal images. Scale bars, 10 μm. Data information: In , representative data are shown and three biological replicates had similar results.


E Statistical analyses of the GFP and RFP co - localization in panel D . Pearson correlation coefficient ( Pearson ' s r ) of GFP and RFP at the periphery of the chloroplasts was measured with the ImageJ PSC colocalization tool ( n = 8 ) . F Relative percentages of RFP - TGB1 foci that co - localized with αa - GFP and CFP - γb at the periphery of the chloroplast images shown in panel D ( n = 8 ) . Data information : In E and F ) , the box boundaries indicate the upper ( 25th percentile ) and lower ( 75th percentile ) quartiles , whiskers indicate minimum and maximum values , central band indicates the median . Letters in the chart denote statistically significant differences among groups according to the Duncan ' s multiple range test ( p < 0 . 05 ) .

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E Statistical analyses of the GFP and RFP co-localization in panel D. Pearson correlation coefficient (Pearson's r) of GFP and RFP at the periphery of the chloroplasts was measured with the ImageJ PSC colocalization tool (n = 8). F Relative percentages of RFP-TGB1 foci that co-localized with αa-GFP and CFP-γb at the periphery of the chloroplast images shown in panel D (n = 8). Data information: In E and F), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).


G Chloroplast extraction experiment for detection of TGB1 chloroplast localization . Plasmids harboring αa - GFP and RFP - TGB1 were co - infiltrated into N . benthamiana along with CFP - γb or CFP - γb3CS . Intact chloroplast preparations isolated from infiltrated N . benthamiana leaves at 4 dpi were subjected to Western blot analyses . RFP - TGB1 was analyzed with an anti - TGB1 antibody . The αa - GFP , CFP - γb , and CFP - γb3CS proteins were detected with an anti - GFP antibody . NTRC was the loading control for the chloroplast preparations Phosphoenolpyruvate carboxylase ( PEPC ) served as a control to assess cytosolic contamination of isolated chloroplasts . Data information : In G ) , representative data are shown and three biological replicates had similar results .

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G Chloroplast extraction experiment for detection of TGB1 chloroplast localization. Plasmids harboring αa-GFP and RFP-TGB1 were co-infiltrated into N. benthamiana along with CFP-γb or CFP-γb3CS. Intact chloroplast preparations isolated from infiltrated N. benthamiana leaves at 4 dpi were subjected to Western blot analyses. RFP-TGB1 was analyzed with an anti-TGB1 antibody. The αa-GFP, CFP-γb, and CFP-γb3CS proteins were detected with an anti-GFP antibody. NTRC was the loading control for the chloroplast preparations Phosphoenolpyruvate carboxylase (PEPC) served as a control to assess cytosolic contamination of isolated chloroplasts. Data information: In G), representative data are shown and three biological replicates had similar results.


B Analysis of γb palmitoylation in NbPAT15 - or NbPAT21 - silenced N . benthamiana plants . NbPAT15 and NbPAT21 were silenced by intron - spliced hairpin RNA - mediated RNAi approaches The γb - 3xFlag plasmid was agroinfiltrated into the leaves , followed by biotin - switch assays at 3 dpi . Data information : In ( B representative data are shown and three biological replicates had similar results .

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B Analysis of γb palmitoylation in NbPAT15- or NbPAT21-silenced N. benthamiana plants. NbPAT15 and NbPAT21 were silenced by intron-spliced hairpin RNA-mediated RNAi approaches The γb-3xFlag plasmid was agroinfiltrated into the leaves, followed by biotin-switch assays at 3 dpi. Data information: In (B representative data are shown and three biological replicates had similar results.


C Effects of γb palmitoylation by NbPAT15 or NbPAT21 on BSMV cell - to - cell movement . Infiltrated leaves were observed by confocal microscopy at 3 dpi . Red fluorescence produced from the mCherry expression cassette shows the primary infection foci , and GFP fluorescence outside the red regions shows virus spread into secondary infected cells . Scale bars , 100 μm . Data information : In C ) , representative data are shown and three biological replicates had similar results .

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C Effects of γb palmitoylation by NbPAT15 or NbPAT21 on BSMV cell-to-cell movement. Infiltrated leaves were observed by confocal microscopy at 3 dpi. Red fluorescence produced from the mCherry expression cassette shows the primary infection foci, and GFP fluorescence outside the red regions shows virus spread into secondary infected cells. Scale bars, 100 μm. Data information: In C), representative data are shown and three biological replicates had similar results.


D Quantification of BSMV movement . The green and red fluorescent areas were measured by ImageJ software ( n = 10 ) . Data information : In ( D ) , the box boundaries indicate the upper ( 25th percentile ) and lower ( 75th percentile ) quartiles , whiskers indicate minimum and maximum values , central band indicates the median . Letters in the chart denote statistically significant differences among groups according to the Duncan ' s multiple range test ( p < 0 . 05 ) .

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D Quantification of BSMV movement. The green and red fluorescent areas were measured by ImageJ software (n = 10). Data information: In (D), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).


A , B BiFC assays to identify interactions of γb with NbPAT15 and NbPAT21 during BSMV infection in N . benthamiana at 3 dpi . PAT15 - YFPn and PAT21 - YFPn were co - expressed with BSMVγb - YFPc . Chloroplast autofluorescence is depicted as a false red color and mCherry - HDEL provides the ER marker . Figures on the right show the normalized fluorescence intensities of GFP , mCherry and the chloroplast autofluorescence channels along the white dashed lines in the merged images . Scale bars , 20 μm . Data information : In ( A , B , representative data are shown and three biological replicates had similar results .

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A, B BiFC assays to identify interactions of γb with NbPAT15 and NbPAT21 during BSMV infection in N. benthamiana at 3 dpi. PAT15-YFPn and PAT21-YFPn were co-expressed with BSMVγb-YFPc. Chloroplast autofluorescence is depicted as a false red color and mCherry-HDEL provides the ER marker. Figures on the right show the normalized fluorescence intensities of GFP, mCherry and the chloroplast autofluorescence channels along the white dashed lines in the merged images. Scale bars, 20 μm. Data information: In (A, B, representative data are shown and three biological replicates had similar results.


C Co - localization analyses of αa - GFP , RFP - TGB1 , and CFP - γb in N . benthamiana leaves at 3 dpi . Mixtures of A . tumefaciens containing αa - GFP , RFP - TGB1 , and CFP - γb were infiltrated into NbPATs - silenced N . benthamiana leaves or the non - silenced TRV : GUS control . Chloroplast autofluorescence is displayed as a false pink color . Figures on the right show the normalized fluorescence intensities of GFP , RFP , CFP and the chloroplast autofluorescence channels along the white dashed lines in the merged images . Scale bars , 10 μm . Data information : In , representative data are shown and three biological replicates had similar results .

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C Co-localization analyses of αa-GFP, RFP-TGB1, and CFP-γb in N. benthamiana leaves at 3 dpi. Mixtures of A. tumefaciens containing αa-GFP, RFP-TGB1, and CFP-γb were infiltrated into NbPATs-silenced N. benthamiana leaves or the non-silenced TRV:GUS control. Chloroplast autofluorescence is displayed as a false pink color. Figures on the right show the normalized fluorescence intensities of GFP, RFP, CFP and the chloroplast autofluorescence channels along the white dashed lines in the merged images. Scale bars, 10 μm. Data information: In , representative data are shown and three biological replicates had similar results.


D Co - IP assays of interactions between γb and TGB1 in NbPAT15 - and NbPAT21 - silenced N . benthamiana leaves , TRV : GUS and TRV : GUS combined with the 2 - BP inhibitor provided positive and negative controls , respectively . The silenced leaves were inoculated with BSMV3xFlag - TGB1 / γb - GFP mixtures ( RNAα + RNAβ3xFlag - TGB1 + RNAγγb - GFP ) and harvested at 3 dpi . Total proteins were immunoprecipitated with anti - Flag beads . Input and IP proteins were analyzed by Western blotting with anti - GFP or anti - Flag antibodies . E Average amounts of RFP - TGB1 foci co - localizing with αa - GFP and CFP - γb at the chloroplast peripheries shown in panel C ( n = 12 ) . Data information : In E ) , representative data are shown and three biological replicates had similar results . In ( E ) , the box boundaries indicate the upper ( 25th percentile ) and lower ( 75th percentile ) quartiles , whiskers indicate minimum and maximum values , central band indicates the median . Letters in the chart denote statistically significant differences among groups according to the Duncan ' s multiple range test ( p < 0 . 05 ) .

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D Co-IP assays of interactions between γb and TGB1 in NbPAT15- and NbPAT21-silenced N. benthamiana leaves, TRV:GUS and TRV:GUS combined with the 2-BP inhibitor provided positive and negative controls, respectively. The silenced leaves were inoculated with BSMV3xFlag-TGB1/γb-GFP mixtures (RNAα + RNAβ3xFlag-TGB1 + RNAγγb-GFP) and harvested at 3 dpi. Total proteins were immunoprecipitated with anti-Flag beads. Input and IP proteins were analyzed by Western blotting with anti-GFP or anti-Flag antibodies. E Average amounts of RFP-TGB1 foci co-localizing with αa-GFP and CFP-γb at the chloroplast peripheries shown in panel C (n = 12). Data information: In E), representative data are shown and three biological replicates had similar results. In (E), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).


B Fractionation assay to detect γb membrane - associations . Separation of total protein ( T ) extracted from BSMV infected leaves into soluble ( S ) and membrane ( M ) fractions by ultracentrifugation at 100 , 000 g . PEPC and BiP , respectively , provide soluble and membrane fraction markers .

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B Fractionation assay to detect γb membrane-associations. Separation of total protein (T) extracted from BSMV infected leaves into soluble (S) and membrane (M) fractions by ultracentrifugation at 100,000 g. PEPC and BiP, respectively, provide soluble and membrane fraction markers.


C Aqueous two - phase partitioning of γb PM localization . The microsomal pellet from BSMVγb - GFP - infected leaves was obtained by ultracentrifugation at 100 , 000 g , and the two - phase fractionation procedure was used to separate the plasma membrane ( upper phase ) from other cellular membranes ( lower phase ) . H + - ATPase is a PM marker .

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C Aqueous two-phase partitioning of γb PM localization. The microsomal pellet from BSMVγb-GFP-infected leaves was obtained by ultracentrifugation at 100,000 g, and the two-phase fractionation procedure was used to separate the plasma membrane (upper phase) from other cellular membranes (lower phase). H+-ATPase is a PM marker.


D PM associations of γb - GFP and γb3CS - GFP in N . benthamiana protoplasts . The first and second columns show localization of γb at 3 dpi after infiltrations for transient expression of γb - GFP and γb3CS - GFP . The third and fourth columns show γb localization in BSMVγb - GFP and BSMV3CS - GFP - infected leaves . PIP2A provided a PM marker ( Nelson et al . , 2007 ) . Figures below the confocal images show the normalized fluorescence intensities of the GFP and mCherry proteins along the white dashed lines in the merged images . Scale bars , 10 μm . Data information : In D , , representative data are shown and three biological replicates had similar results .

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D PM associations of γb-GFP and γb3CS-GFP in N. benthamiana protoplasts. The first and second columns show localization of γb at 3 dpi after infiltrations for transient expression of γb-GFP and γb3CS-GFP. The third and fourth columns show γb localization in BSMVγb-GFP and BSMV3CS-GFP-infected leaves. PIP2A provided a PM marker (Nelson et al., 2007). Figures below the confocal images show the normalized fluorescence intensities of the GFP and mCherry proteins along the white dashed lines in the merged images. Scale bars, 10 μm. Data information: In D, , representative data are shown and three biological replicates had similar results.


E Western blot analysis of subcellular fractions of BSMV infected N . benthamiana leaves at 3 dpi . The S3 and S30 fractions are the 3000 and 30000 g supernatants , and P30 is the 30000 g pellet . Membrane associated γb was released by 1 % Triton X - 100 treatment .

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E Western blot analysis of subcellular fractions of BSMV infected N. benthamiana leaves at 3 dpi. The S3 and S30 fractions are the 3000 and 30000 g supernatants, and P30 is the 30000 g pellet. Membrane associated γb was released by 1% Triton X-100 treatment.


F Western blot detection of BSMV accumulation in N . benthamiana plants . A . tumefaciens mixtures harboring RNAα and RNAβmTGB2 , along with RNAγ , RNAγγb - ROP , or RNAγγb - mROP , were agroinfiltrated into N . benthamiana leaves . Western blotting to detect virus accumulation at 3 dpi used anti - TGB1 or anti - CP antibodies . Actin provided loading controls .

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F Western blot detection of BSMV accumulation in N. benthamiana plants. A. tumefaciens mixtures harboring RNAα and RNAβmTGB2, along with RNAγ, RNAγγb-ROP, or RNAγγb-mROP, were agroinfiltrated into N. benthamiana leaves. Western blotting to detect virus accumulation at 3 dpi used anti-TGB1 or anti-CP antibodies. Actin provided loading controls.


G Analysis of the effects of enhanced PM association of γb on cell - to - cell virus movement with the BSMV duplex fluorescence ( dfBSMV ) reporter system . Mixtures of A . tumefaciens containing dfBSMV , dfBSMVγb - GFP - ROP , and dfBSMVγb - GFP - mROP were infiltrated into N . benthamiana leaves . Infiltrated leaves were observed by confocal microscope at 3 dpi . Scale bars , 100 μm . ( n = 6 ) . Data information : In G ) , representative data are shown and three biological replicates had similar results . In ( G ) , the box boundaries indicate the upper ( 25th percentile ) and lower ( 75th percentile ) quartiles , whiskers indicate minimum and maximum values , central band indicates the median . Letters in the chart denote statistically significant differences among groups according to the Duncan ' s multiple range test ( p < 0 . 05 ) .

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G Analysis of the effects of enhanced PM association of γb on cell-to-cell virus movement with the BSMV duplex fluorescence (dfBSMV) reporter system. Mixtures of A. tumefaciens containing dfBSMV, dfBSMVγb-GFP-ROP, and dfBSMVγb-GFP-mROP were infiltrated into N. benthamiana leaves. Infiltrated leaves were observed by confocal microscope at 3 dpi. Scale bars, 100 μm. (n = 6). Data information: In G), representative data are shown and three biological replicates had similar results. In (G), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).


B Co - IP analyses to analyze interactions of NbREM1 with γb or γb3CS in N . benthamiana leaf tissues infiltrated with A . tumefaciens mixtures containing movement - deficient BSMV plasmids ( RNAα + RNAγγb - GFP or RNAγ3CS - GFP ) and NbREM1 - 3xFlag . Total proteins were extracted at 3 dpi and immunoprecipitated with anti - Flag beads . Input and immunoprecipitated proteins ( IP ) were analyzed by Western blot analyses with an anti - GFP or anti - Flag antibodies . Data information : In B , , representative data are shown and three biological replicates had similar results .

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B Co-IP analyses to analyze interactions of NbREM1 with γb or γb3CS in N. benthamiana leaf tissues infiltrated with A. tumefaciens mixtures containing movement-deficient BSMV plasmids (RNAα + RNAγγb-GFP or RNAγ3CS-GFP) and NbREM1-3xFlag. Total proteins were extracted at 3 dpi and immunoprecipitated with anti-Flag beads. Input and immunoprecipitated proteins (IP) were analyzed by Western blot analyses with an anti-GFP or anti-Flag antibodies. Data information: In B, , representative data are shown and three biological replicates had similar results.


D Analysis of the cleaved 5 ( 6 ) - carboxyfluorescein ( CF ) movement in N . benthamiana leaves . RNAα + RNAγ / RNAγ3CS were co - infiltrated into transiently overexpressed NbREM1 N . benthamiana leaves , and 1 μL CFDA was loaded onto the adaxial leaf blade surface and incubated for 5 min followed by confocal analysis at 3 dpi . The areas of dye diffusion are marked by dashed white circles . Scale bars , 100 μm . Data information : In D , representative data are shown and three biological replicates had similar results .

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D Analysis of the cleaved 5(6)-carboxyfluorescein (CF) movement in N. benthamiana leaves. RNAα + RNAγ/RNAγ3CS were co-infiltrated into transiently overexpressed NbREM1 N. benthamiana leaves, and 1 μL CFDA was loaded onto the adaxial leaf blade surface and incubated for 5 min followed by confocal analysis at 3 dpi. The areas of dye diffusion are marked by dashed white circles. Scale bars, 100 μm. Data information: In D, representative data are shown and three biological replicates had similar results.


E Quantification of CF movement . Dye diffusion areas identified by the dashed white circles in panel D were measured by image J software ( n = 6 ) . Data information : In ( E , the box boundaries indicate the upper ( 25th percentile ) and lower ( 75th percentile ) quartiles , whiskers indicate minimum and maximum values , central band indicates the median . Letters in the chart denote statistically significant differences among groups according to the Duncan ' s multiple range test ( p < 0 . 05 ) .

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E Quantification of CF movement. Dye diffusion areas identified by the dashed white circles in panel D were measured by image J software (n = 6). Data information: In (E, the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).


F Aniline blue staining of callose in N . benthamiana leaf tissues . Leaves were infiltrated with mixtures of different A . tumefaciens constructs indicated beside the panels . At 3 dpi , the 0 . 1 % aniline blue fluorochrome was infiltrated into N . benthamiana leaves . After incubation at 5 min in the dark , the leaves were observed by confocal microscopy at 405 nm . Scale bars , 20 μm . Data information : representative data are shown and three biological replicates had similar results .

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F Aniline blue staining of callose in N. benthamiana leaf tissues. Leaves were infiltrated with mixtures of different A. tumefaciens constructs indicated beside the panels. At 3 dpi, the 0.1% aniline blue fluorochrome was infiltrated into N. benthamiana leaves. After incubation at 5 min in the dark, the leaves were observed by confocal microscopy at 405 nm. Scale bars, 20 μm. Data information: representative data are shown and three biological replicates had similar results.


G Quantification of callose staining intensity as shown in panel F . The relative callose staining intensity was set to 1 in the REM samples ( n = 10 ) . Data information : In , the box boundaries indicate the upper ( 25th percentile ) and lower ( 75th percentile ) quartiles , whiskers indicate minimum and maximum values , central band indicates the median . Letters in the chart denote statistically significant differences among groups according to the Duncan ' s multiple range test ( p < 0 . 05 ) .

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G Quantification of callose staining intensity as shown in panel F. The relative callose staining intensity was set to 1 in the REM samples (n = 10). Data information: In , the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).


H Aniline blue staining of callose in leaves with different treatments above the panel in NbPAT15 and NbPAT21 RNAi plants . Scale bars , 20 μm . Data information : In H ) , representative data are shown and three biological replicates had similar results .

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H Aniline blue staining of callose in leaves with different treatments above the panel in NbPAT15 and NbPAT21 RNAi plants. Scale bars, 20 μm. Data information: In H), representative data are shown and three biological replicates had similar results.


I Quantification of callose staining intensity as shown in panel H . The relative callose staining intensity was set as 1 in the samples of EV ( n = 8 ) . Data information : In I ) , the box boundaries indicate the upper ( 25th percentile ) and lower ( 75th percentile ) quartiles , whiskers indicate minimum and maximum values , central band indicates the median . Letters in the chart denote statistically significant differences among groups according to the Duncan ' s multiple range test ( p < 0 . 05 ) .

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I Quantification of callose staining intensity as shown in panel H. The relative callose staining intensity was set as 1 in the samples of EV (n = 8). Data information: In I), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).


( A ) HeLa cells were exposed to 5 Gy ionizing radiation ( γIR ) , collected at the indicated time points and stained for RASSF1A and γH2AX . Images for representative intra - nuclear RASSF1A foci distribution are shown . Nucleolar boundaries are marked with dashed lines .

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(A) HeLa cells were exposed to 5 Gy ionizing radiation (γIR), collected at the indicated time points and stained for RASSF1A and γH2AX. Images for representative intra-nuclear RASSF1A foci distribution are shown. Nucleolar boundaries are marked with dashed lines.


( B ) Quantification of the number of cells with RASSF1A foci at the time points presented in ( A ) . Error bars represent standard deviation and derive from 3 independent experiments .

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(B) Quantification of the number of cells with RASSF1A foci at the time points presented in (A). Error bars represent standard deviation and derive from 3 independent experiments.


( C ) HeLa cells were treated with 5 Gy γIR and 2 h post treatment fixed and stained with the indicated antibodies . ( D ) HeLa cells were irradiated with 5 Gy and 2 h post treatment stained for RASSF1A ( R1A ) and NUCLEOLIN ( NCL ) . ( E ) HeLa cells were treated with 50 ng / ml NCS for 30 min , washed and 2 h later fixed and stained with the indicated antibodies . The distance of RASSF1A + / γH2AX + foci or RASSF1A - / γH2AX + foci to the closest nucleolus ( based on Fibrillarin ( Fb ) staining ) was measured . Middle line represents the median and the boxes 25th and 75th percentiles . The whiskers mark the smallest and largest values .

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(C) HeLa cells were treated with 5 Gy γIR and 2 h post treatment fixed and stained with the indicated antibodies. (D) HeLa cells were irradiated with 5 Gy and 2 h post treatment stained for RASSF1A (R1A) and NUCLEOLIN (NCL). (E) HeLa cells were treated with 50 ng/ml NCS for 30 min, washed and 2 h later fixed and stained with the indicated antibodies. The distance of RASSF1A+/γH2AX+ foci or RASSF1A-/γH2AX+ foci to the closest nucleolus (based on Fibrillarin (Fb) staining) was measured. Middle line represents the median and the boxes 25th and 75th percentiles. The whiskers mark the smallest and largest values.


( F ) AsiSI expression in U2OS cells was induced by OHT and cells were stained for RASSF1A and γH2AX . Boxed areas are shown in higher magnification . Nucleolar boundaries are marked with dashed lines . ( G ) Quantification of the number of cells with RASSF1A foci in ( F ) . Error bars represent standard deviation and derive from 3 independent experiments .

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(F) AsiSI expression in U2OS cells was induced by OHT and cells were stained for RASSF1A and γH2AX. Boxed areas are shown in higher magnification. Nucleolar boundaries are marked with dashed lines. (G) Quantification of the number of cells with RASSF1A foci in (F). Error bars represent standard deviation and derive from 3 independent experiments.


( H ) U2OS cells with stable integration of a Lac operator ( LacO ) sequence adjacent to an I - SceI endonuclease site were transfected with Lac Repressor ( LacR ) - mCherry and GFP or GFP - NLS - HA - I - SceI . 24 h post transfection cells were fixed and stained for 53BP1 or RASSF1A . ( I ) Quantification of the LacO arrays enriched for 53BP1 or RASSF1A in ( H ) . Error bars represent standard deviation and derive from 3 independent experiments .

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(H) U2OS cells with stable integration of a Lac operator (LacO) sequence adjacent to an I-SceI endonuclease site were transfected with Lac Repressor (LacR)-mCherry and GFP or GFP-NLS-HA-I-SceI. 24 h post transfection cells were fixed and stained for 53BP1 or RASSF1A. (I) Quantification of the LacO arrays enriched for 53BP1 or RASSF1A in (H). Error bars represent standard deviation and derive from 3 independent experiments.


( J ) HeLa cells were transfected with I - PpoI endonuclease and stained for RASSF1A and γH2AX . Fluorescence intensity profiles of RASSF1A ( green ) and γH2AX ( red ) signals across the HeLa nuclei are shown . Position of line scan indicated by the yellow line .

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(J) HeLa cells were transfected with I-PpoI endonuclease and stained for RASSF1A and γH2AX. Fluorescence intensity profiles of RASSF1A (green) and γH2AX (red) signals across the HeLa nuclei are shown. Position of line scan indicated by the yellow line.


( A ) HeLa cells were transfected with I - PpoI mRNA and collected at the indicated time points . Cells were co - stained for RASSF1A ( R1A ) and NBS1 . Boxed areas are shown in higher magnification . Nucleolar boundaries are marked with dashed lines . ( B ) Quantification of ( A ) . Error bars represent standard deviation and derive from 3 independent experiments .

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(A) HeLa cells were transfected with I-PpoI mRNA and collected at the indicated time points. Cells were co-stained for RASSF1A (R1A) and NBS1. Boxed areas are shown in higher magnification. Nucleolar boundaries are marked with dashed lines. (B) Quantification of (A). Error bars represent standard deviation and derive from 3 independent experiments.


( C ) Assessment of RASSF1A localisation after induction of rDNA DSBs using the I - PpoI endonuclease in methanol fixed HeLa cells . Fluorescence intensity profile of RASSF1A ( green ) and LAMIN A / C ( red ) signals across the HeLa nuclei in the presence and absence of rDNA DSBs in control and I - PpoI treated cells . Position of line scan indicated by the yellow line .

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(C) Assessment of RASSF1A localisation after induction of rDNA DSBs using the I-PpoI endonuclease in methanol fixed HeLa cells. Fluorescence intensity profile of RASSF1A (green) and LAMIN A/C (red) signals across the HeLa nuclei in the presence and absence of rDNA DSBs in control and I-PpoI treated cells. Position of line scan indicated by the yellow line.


( D ) HeLa cells were transfected with the I - PpoI mRNA and 6 h later stained for RASSF1A - pS131 ( R1A - pS131 ) and γH2AX . Fluorescence intensity profile of RASSF1A - pS131 ( green ) and γH2AX ( red ) signals across the HeLa nuclei are shown . Position of line scan indicated by the yellow line .

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(D) HeLa cells were transfected with the I-PpoI mRNA and 6 h later stained for RASSF1A-pS131 (R1A-pS131) and γH2AX. Fluorescence intensity profile of RASSF1A-pS131 (green) and γH2AX (red) signals across the HeLa nuclei are shown. Position of line scan indicated by the yellow line.


( E ) Quantification of cells with RASSF1A - pS131 positive nucleolar caps 6 h post I - PpoI transfection . Error bars represent standard deviation and derive from 3 independent experiments . Representative images are shown in Fig EV3A .

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(E) Quantification of cells with RASSF1A-pS131 positive nucleolar caps 6 h post I-PpoI transfection. Error bars represent standard deviation and derive from 3 independent experiments. Representative images are shown in Fig EV3A.


( F ) HeLa cells were treated with the ATMi or ATRi prior to transfection with I - PpoI mRNA . 6 h post mRNA transfection cells were stained for RASSF1A - pS131 ( R1A - pS131 ) . ( G ) Quantification of ( F ) . Error bars represent standard deviation and derive from 3 independent experiments .

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(F) HeLa cells were treated with the ATMi or ATRi prior to transfection with I-PpoI mRNA. 6 h post mRNA transfection cells were stained for RASSF1A-pS131 (R1A-pS131). (G) Quantification of (F). Error bars represent standard deviation and derive from 3 independent experiments.


( H ) HeLa cells were treated or not with ATM inhibitor ( ATMi ) followed by I - PpoI mRNA transfection and 6 h later cell lysates were analyzed by western blot for the indicated antibodies .

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(H) HeLa cells were treated or not with ATM inhibitor (ATMi) followed by I-PpoI mRNA transfection and 6 h later cell lysates were analyzed by western blot for the indicated antibodies.


( I ) HeLa cells were treated or not with ATMi followed by I - PpoI mRNA transfection and 6 h later cells were fixed and RASSF1A recruitment at rDNA DSBs was assessed . ( J ) Quantification of ( I ) . Error bars represent standard deviation and derive from 3 independent experiments .

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(I) HeLa cells were treated or not with ATMi followed by I-PpoI mRNA transfection and 6 h later cells were fixed and RASSF1A recruitment at rDNA DSBs was assessed. (J) Quantification of (I). Error bars represent standard deviation and derive from 3 independent experiments.


( A ) HeLa cells with I - PpoI induced rDNA breaks were stained with the indicated antibodies . Intra - nucleolar cap localization for RASSF1A and 53BP1 in correlation with other proteins was accessed . Boxed areas are shown in higher magnification .

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(A) HeLa cells with I-PpoI induced rDNA breaks were stained with the indicated antibodies. Intra-nucleolar cap localization for RASSF1A and 53BP1 in correlation with other proteins was accessed. Boxed areas are shown in higher magnification.


( B ) HeLa cells were transfected with I - PpoI mRNA and stained for RASSF1A and 53BP1 . Fluorescence intensity profiles of RASSF1A ( green ) and 53BP1 ( red ) signals across the HeLa nuclei are shown . Position of line scan indicated by the yellow line .

(B) HeLa cells were transfected with I-PpoI mRNA and stained for RASSF1A and 53BP1. Fluorescence intensity profiles of RASSF1A (green) and 53BP1 (red) signals across the HeLa nuclei are shown. Position of line scan indicated by the yellow line.


( C ) HeLa cells were treated with siRNAs against LUCIFERASE ( siLUC ) or 53BP1 ( si53BP1 ) , 48 h later transfected with I - PpoI mRNA , fixed and stained for RASSF1A ( R1A ) and γH2AX . ( D ) Quantification of ( C ) and western blot analysis from siLUCIFERASE ( siLUC ) or si53BP1 treated cells for the indicated antibodies . Error bars represent standard deviation and derive from 3 independent experiments .

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(C) HeLa cells were treated with siRNAs against LUCIFERASE (siLUC) or 53BP1 (si53BP1), 48 h later transfected with I-PpoI mRNA, fixed and stained for RASSF1A (R1A) and γH2AX. (D) Quantification of (C) and western blot analysis from siLUCIFERASE (siLUC) or si53BP1 treated cells for the indicated antibodies. Error bars represent standard deviation and derive from 3 independent experiments.


( E ) HeLa cells were treated with siRNAs against LUSIFERASE ( siLUC ) or RNF8 ( siRNF8 ) , 48 h later transfected for I - PpoI mRNA , fixed and stained for RASSF1A and γH2AX . ( F ) Quantification of ( E ) and western blot analysis from siLUCIFERASE ( siLUC ) or siRNF8 treated cells for the indicated antibodies . Error bars represent standard deviation and derive from 3 independent experiments .

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(E) HeLa cells were treated with siRNAs against LUSIFERASE (siLUC) or RNF8 (siRNF8), 48 h later transfected for I-PpoI mRNA, fixed and stained for RASSF1A and γH2AX. (F) Quantification of (E) and western blot analysis from siLUCIFERASE (siLUC) or siRNF8 treated cells for the indicated antibodies. Error bars represent standard deviation and derive from 3 independent experiments.


( G ) HeLa cells were treated with siRNA against 53BP1 , or an ATM inhibitor ( ATMi ) followed by 5 Gy of ionizing radiation ( γIR ) . Cells were fixed 2 h post treatment and stained for RASSF1A . Nucleolar boundaries are marked with dashed lines . ( H ) Quantification of cells with RASSF1A foci in ( G ) . Error bars represent standard deviation and derive from 3 independent experiments .

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(G) HeLa cells were treated with siRNA against 53BP1, or an ATM inhibitor (ATMi) followed by 5 Gy of ionizing radiation (γIR). Cells were fixed 2 h post treatment and stained for RASSF1A. Nucleolar boundaries are marked with dashed lines. (H) Quantification of cells with RASSF1A foci in (G). Error bars represent standard deviation and derive from 3 independent experiments.


( I ) Western blot analysis from total cell extracts with the indicated treatments and RASSF1A immuneprecipitates ( IPs ) with the indicated antibodies . IgG immunoprecipitation served as a control .

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(I) Western blot analysis from total cell extracts with the indicated treatments and RASSF1A immuneprecipitates (IPs) with the indicated antibodies. IgG immunoprecipitation served as a control.


( J ) Western blot analysis from total cell extracts and RASSF1A immunoprecipitates with the indicated treatments with the indicated antibodies . IgG immunoprecipitation served as a control .

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(J) Western blot analysis from total cell extracts and RASSF1A immunoprecipitates with the indicated treatments with the indicated antibodies. IgG immunoprecipitation served as a control.


( K ) U2OS cells were transfected with FLAG - RASSF1A ( FLAG - R1A ) , FLAG - RASSF1A131A ( FLAG - R1A131A ) or pcDNA3 and 24 h later transfected with the I - PpoI mRNA . Cell lysates were subjected to immunoprecipitation against FLAG . Western blot analysis of total cell extracts and the IPs is shown .

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(K) U2OS cells were transfected with FLAG-RASSF1A (FLAG-R1A), FLAG-RASSF1A131A (FLAG-R1A131A) or pcDNA3 and 24 h later transfected with the I-PpoI mRNA. Cell lysates were subjected to immunoprecipitation against FLAG. Western blot analysis of total cell extracts and the IPs is shown.


( A ) U2OS cells were transfected with either HA - 53BP1WT ( aa1 - 1972 ) , HA - 53BP1ΔBRCT ( aa 1 - 1709 ) , HA - 53BP1ΔΝterminus ( aa 921 - 1972 ) or empty pcDNA3 vector and 24 h later transfected with I - PpoI mRNA . Cell lysates were subjected to immunoprecipitation with an HA tag antibody . Western blot analysis of total cell extracts and the IPs is shown .

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(A) U2OS cells were transfected with either HA-53BP1WT (aa1-1972), HA-53BP1ΔBRCT (aa 1-1709), HA-53BP1ΔΝterminus (aa 921-1972) or empty pcDNA3 vector and 24 h later transfected with I-PpoI mRNA. Cell lysates were subjected to immunoprecipitation with an HA tag antibody. Western blot analysis of total cell extracts and the IPs is shown.


( B ) U2OS cells were transfected with either full length MYC - RASSF1A or with the indicated RASSF1A deletion mutants and 24 h later transfected with I - PpoI mRNA . Cell lysates were subjected to immunoprecipitation with a MYC - tag antibody . Western blot analysis of the total cell extracts and the IPs is shown .

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(B) U2OS cells were transfected with either full length MYC-RASSF1A or with the indicated RASSF1A deletion mutants and 24 h later transfected with I-PpoI mRNA. Cell lysates were subjected to immunoprecipitation with a MYC-tag antibody. Western blot analysis of the total cell extracts and the IPs is shown.


( C ) HeLa cells were treated with siLUCIFERASE ( siLUC ) or si53BP1 and 48 h later transfected with I - PpoI mRNA and stained for ATM - pS1981 . ( D ) Quantification of ( C ) . Error bars represent standard deviation and derive from 3 independent experiments .

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(C) HeLa cells were treated with siLUCIFERASE (siLUC) or si53BP1 and 48 h later transfected with I-PpoI mRNA and stained for ATM-pS1981. (D) Quantification of (C). Error bars represent standard deviation and derive from 3 independent experiments.


( E - H ) HeLa cells were treated with siLUCIFERASE ( siLUC ) or two different siRNAs against RASSF1A ( siR1A _ 1 and siR1A _ 2 ) and 48 h later cells were transfected with I - PpoI mRNA to induce rDNA DSBs . 6 h post mRNA transfection , cells were stained for V5 to identify I - PpoI transfected cells and the other indicated antibodies . Representative images ( E , F ) and quantification of staining ( G , H ) for the indicated antibodies are shown . Error bars represent standard deviation and derive from 3 independent experiments .

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

(E-H) HeLa cells were treated with siLUCIFERASE (siLUC) or two different siRNAs against RASSF1A (siR1A_1 and siR1A_2) and 48 h later cells were transfected with I-PpoI mRNA to induce rDNA DSBs. 6 h post mRNA transfection, cells were stained for V5 to identify I-PpoI transfected cells and the other indicated antibodies. Representative images (E, F) and quantification of staining (G, H) for the indicated antibodies are shown. Error bars represent standard deviation and derive from 3 independent experiments.


( I ) HeLa cells were treated with siLUCIFERASE ( siLUC ) , siRASSF1A or si53BP1 and 48 h later transfected with the I - PpoI mRNA . Cells were fixed and underwent ImmunoFISH against UBF and an rDNA probe . Boxed areas are shown in higher magnification . Nucleolar boundaries are marked with dashed lines . ( J ) HeLa cells were treated with siLUCIFERASE ( siLUC ) , siRASSF1A or si53BP1 and 48 h later transfected with the I - PpoI mRNA . Cells were fixed and underwent ImmunoFISH against ATM - pS1981 and an rDNA probe . Boxed areas are shown in higher magnification . Nucleolar boundaries are marked with dashed lines .

O O O O O O O O O O O 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 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

(I) HeLa cells were treated with siLUCIFERASE (siLUC), siRASSF1A or si53BP1 and 48 h later transfected with the I-PpoI mRNA. Cells were fixed and underwent ImmunoFISH against UBF and an rDNA probe. Boxed areas are shown in higher magnification. Nucleolar boundaries are marked with dashed lines. (J) HeLa cells were treated with siLUCIFERASE (siLUC), siRASSF1A or si53BP1 and 48 h later transfected with the I-PpoI mRNA. Cells were fixed and underwent ImmunoFISH against ATM-pS1981 and an rDNA probe. Boxed areas are shown in higher magnification. Nucleolar boundaries are marked with dashed lines.


( K ) HeLa cells were treated with siLUCIFERASE ( siLUC ) or siRASSF1A followed by I - PpoI mRNA transfection . 6 h later cells were stained for 53BP1 . ( L ) Quantification of ( K ) . Error bars represent standard deviation and derive from 3 independent experiments .

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(K) HeLa cells were treated with siLUCIFERASE (siLUC) or siRASSF1A followed by I-PpoI mRNA transfection. 6 h later cells were stained for 53BP1. (L) Quantification of (K). Error bars represent standard deviation and derive from 3 independent experiments.


( M ) U2OS cells were transfected with MYC - RASSF1A ( 1 - 340 ) or a RASSF1A mutant that lacks the SARAH domain , MYC - RASSF1A ( 1 - 288 ) . Cells were transfected with I - PpoI mRNA and stained with the indicated antibodies .

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

(M) U2OS cells were transfected with MYC-RASSF1A (1-340) or a RASSF1A mutant that lacks the SARAH domain, MYC-RASSF1A (1-288). Cells were transfected with I-PpoI mRNA and stained with the indicated antibodies.


( N ) U2OS cells were transfected with MYC - RASSF1A ( 1 - 340 ) or MYC - RASSF1A ( 1 - 288 ) . 24 h later cells were transfected with I - PpoI mRNA and harvested after 6 h . Cell lysates underwent cell fractionation and the chromatin bound fraction was analyzed by western blot .

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(N) U2OS cells were transfected with MYC-RASSF1A (1-340) or MYC-RASSF1A (1-288). 24 h later cells were transfected with I-PpoI mRNA and harvested after 6 h. Cell lysates underwent cell fractionation and the chromatin bound fraction was analyzed by western blot.


( O ) U2OS cells were treated with siRNA against RASSF1A followed by transfection with MYC - tag , MYC - RASSF1A ( 1 - 340 ) or MYC - RASSF1A ( 1 - 288 ) . 24 h later cells were transfected with I - PpoI mRNA and stained for MYC expression and ATM - pS1981 .

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

(O) U2OS cells were treated with siRNA against RASSF1A followed by transfection with MYC-tag, MYC-RASSF1A (1-340) or MYC-RASSF1A (1-288). 24 h later cells were transfected with I-PpoI mRNA and stained for MYC expression and ATM-pS1981.


( P ) Quantification of cells with ATM - pS1981 positive nucleolar caps in ( O ) . Error bars represent standard deviation and derive from 3 independent experiments .

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

(P) Quantification of cells with ATM-pS1981 positive nucleolar caps in (O). Error bars represent standard deviation and derive from 3 independent experiments.


( A ) HeLa cells were pre - treated with the ATMi or ATRi prior to transfection with I - PpoI mRNA . Cells were stained for RPA and quantified . Error bars represent standard deviation and derive from 3 independent experiments . ( B ) HeLa cells were pre - treated with the ATMi or ATRi prior to transfection with I - PpoI mRNA . Cells were stained for RAD51 and quantified . Error bars represent standard deviation and derive from 3 independent experiments .

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(A) HeLa cells were pre-treated with the ATMi or ATRi prior to transfection with I-PpoI mRNA. Cells were stained for RPA and quantified. Error bars represent standard deviation and derive from 3 independent experiments. (B) HeLa cells were pre-treated with the ATMi or ATRi prior to transfection with I-PpoI mRNA. Cells were stained for RAD51 and quantified. Error bars represent standard deviation and derive from 3 independent experiments.


( C - F ) HeLa cells were treated with siLUCIFERASE ( siLUC ) or two different siRNAs against RASSF1A ( siR1A _ 1 and siR1A _ 2 ) and 48 h later cells were transfected with I - PpoI mRNA to induce rDNA DSBs . 6 h post mRNA transfection , cells were stained for V5 to identify I - PpoI transfected cells and the other indicated antibodies . Representative images ( C , E ) and quantification of staining ( D , F ) for the indicated antibodies are shown . Error bars represent standard deviation and derive from 3 independent experiments .

(C-F) HeLa cells were treated with siLUCIFERASE (siLUC) or two different siRNAs against RASSF1A (siR1A_1 and siR1A_2) and 48 h later cells were transfected with I-PpoI mRNA to induce rDNA DSBs. 6 h post mRNA transfection, cells were stained for V5 to identify I-PpoI transfected cells and the other indicated antibodies. Representative images (C, E) and quantification of staining (D, F) for the indicated antibodies are shown. Error bars represent standard deviation and derive from 3 independent experiments.


( G ) Western blot analysis from HeLa cell extracts treated with the indicated siRNAs for the indicated antibodies .

(G) Western blot analysis from HeLa cell extracts treated with the indicated siRNAs for the indicated antibodies.


( H ) Cells were treated with siLUCIFERASE ( siLUC ) or si53BP1 and 48 h later transfected with I - PpoI mRNA and stained for RPA . ( I ) Quantification of ( H ) . 107 values were analysed in siLUC and 90 values were analysed in si53BP1 .

O O 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

(H) Cells were treated with siLUCIFERASE (siLUC) or si53BP1 and 48 h later transfected with I-PpoI mRNA and stained for RPA. (I) Quantification of (H). 107 values were analysed in siLUC and 90 values were analysed in si53BP1.


( J ) U2OS cells were treated with siLUCIFERASE ( siLUC ) , siRASSF1A ( siR1A ) or si53BP1 , treated with 10 μM BrdU for 24 h prior to I - PpoI mRNA transfection . 6 h post I - PpoI treated cells were pre - extracted , fixed and stained for BrdU under non denaturing conditions to visualize ssDNA . ( K ) Quantification of ( J ) . Fold change of nuclear BrdU signal relative to siLUC is shown . Error bars represent standard deviation and derive from 3 independent 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

(J) U2OS cells were treated with siLUCIFERASE (siLUC), siRASSF1A (siR1A) or si53BP1, treated with 10 μM BrdU for 24 h prior to I-PpoI mRNA transfection. 6 h post I-PpoI treated cells were pre-extracted, fixed and stained for BrdU under non denaturing conditions to visualize ssDNA. (K) Quantification of (J). Fold change of nuclear BrdU signal relative to siLUC is shown. Error bars represent standard deviation and derive from 3 independent experiments.


( L ) HeLa cells were transfected with I - PpoI endonuclease and stained for MST2 and γH2AX . Fluorescence intensity profiles of MST2 ( green ) and γH2AX ( red ) signals across the HeLa nuclei are shown . Position of line scan indicated by the yellow line .

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(L) HeLa cells were transfected with I-PpoI endonuclease and stained for MST2 and γH2AX. Fluorescence intensity profiles of MST2 (green) and γH2AX (red) signals across the HeLa nuclei are shown. Position of line scan indicated by the yellow line.


( M ) Validation of the MST2 siRNA with western blot .

(M) Validation of the MST2 siRNA with western blot.


( N ) HeLa cells treated with siLUCIFERASE ( siLUC ) or siMST2 and 48 h later transfected with I - PpoI mRNA . Cells were treated with 5 - EU for 30 min prior to fixation and assessed for incorporation . ( O ) Quantification of 5 - EU intensity in siLUCIFERASE ( siLUC ) or siMST2 treated cells transfected with I - PpoI mRNA . Middle line represents the median and the boxes 25th and 75th percentiles . The whiskers mark the smallest and largest values . 100 values were analyzed in each condition .

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

(N) HeLa cells treated with siLUCIFERASE (siLUC) or siMST2 and 48 h later transfected with I-PpoI mRNA. Cells were treated with 5-EU for 30 min prior to fixation and assessed for incorporation. (O) Quantification of 5-EU intensity in siLUCIFERASE (siLUC) or siMST2 treated cells transfected with I-PpoI mRNA. Middle line represents the median and the boxes 25th and 75th percentiles. The whiskers mark the smallest and largest values. 100 values were analyzed in each condition.


( P ) HeLa cells were treated with siLUCIFERASE ( siLUC ) or siMST2 and 48 h post transfection cells were transfected with I - PpoI mRNA . 6 h post transfection cells were fixed and stained for RASSF1A ( R1A ) and γH2AX . ( Q ) Quantification of ( P ) . Error bars represent standard deviation and derive from 3 independent 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 B-MEASURED_VAR O B-MEASURED_VAR 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

(P) HeLa cells were treated with siLUCIFERASE (siLUC) or siMST2 and 48 h post transfection cells were transfected with I-PpoI mRNA. 6 h post transfection cells were fixed and stained for RASSF1A (R1A) and γH2AX. (Q) Quantification of (P). Error bars represent standard deviation and derive from 3 independent experiments.


( R - W ) HeLa cells treated with siLUCIFERASE ( siLUC ) or siMST2 and 48 h later transfected with I - PpoI mRNA . Cells were stained for ATM - pS1981 ( R ) , KAP1 - pS824 ( T ) and RPA ( V ) and quantified ( S , U , W ) . 108 values in ( S ) , 95 values in ( U ) and 100 values in ( W ) were analysed .

O O O O O O 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 B-MEASURED_VAR 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

(R-W) HeLa cells treated with siLUCIFERASE (siLUC) or siMST2 and 48 h later transfected with I-PpoI mRNA. Cells were stained for ATM-pS1981 (R), KAP1-pS824 (T) and RPA (V) and quantified (S, U, W). 108 values in (S), 95 values in (U) and 100 values in (W) were analysed.


( Β ) Clonogenic survival analysis of HeLa cells treated with the indicated siRNAs and the indicated doses of Ionizing radiation ( γIR ) . Survival fraction was calculated . Error bars represent SEM and derive from 3 independent 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

(Β) Clonogenic survival analysis of HeLa cells treated with the indicated siRNAs and the indicated doses of Ionizing radiation (γIR). Survival fraction was calculated. Error bars represent SEM and derive from 3 independent experiments.


( C ) Clonogenic survival analysis and representative images of HeLa cells treated with the indicated siRNAs followed by I ‐ PpoI WT or I - PpoI H98A mRNA transfections . Survival ratio I - PpoI WT / I - PpoI H98A in each siRNA condition is presented . Error bars represent standard deviation and derive from 3 independent experiments .

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

(C) Clonogenic survival analysis and representative images of HeLa cells treated with the indicated siRNAs followed by I‐PpoI WT or I-PpoI H98A mRNA transfections. Survival ratio I-PpoI WT/I-PpoI H98A in each siRNA condition is presented. Error bars represent standard deviation and derive from 3 independent experiments.


( E ) 45S rDNA copy number analysis in patients ' tumors compared to adjacent controls in the LUAD cohort . 67 tumor samples and 43 samples from adjacent tissue were analyzed . ( F ) 45S rDNA copy number analysis in the LUAD patient samples based on RASSF1A CpG promoter methylation . 67 samples were analysed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 45S rDNA copy number analysis in patients' tumors compared to adjacent controls in the LUAD cohort. 67 tumor samples and 43 samples from adjacent tissue were analyzed. (F) 45S rDNA copy number analysis in the LUAD patient samples based on RASSF1A CpG promoter methylation. 67 samples were analysed.


F - M ) Total cellularity of F ) HSPCs , G ) LT - HSCs , H ) ST - HSCs , I ) Tom + HSCs , J ) GFP + drHSCs , K ) MPP2 , L ) MPP3 , M ) MPP4 in E16 . 5 fetuses following saline or maternal infection with Pru or RH as shown in Fig . 1A . n = 9 - 15 fetuses from at least 3 litters / condition .

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F-M) Total cellularity of F) HSPCs, G) LT-HSCs, H) ST-HSCs, I) Tom+ HSCs, J) GFP+ drHSCs, K) MPP2, L) MPP3, M) MPP4 in E16.5 fetuses following saline or maternal infection with Pru or RH as shown in Fig. 1A. n = 9-15 fetuses from at least 3 litters/condition.