( I ) Immunoblots depicting levels of PSD 95 , GFAP , Synapsin 1 and tubulin in the whole cortical lysate and the synaptoneurosome preparations . Quantification representing the average fold enrichments of PSD 95 , Synapsin 1 and GFAP in the synaptoneurosome preparations compared to the whole cortical lysate . Data presented as mean ± SEM with scattered data points . Data points for all the proteins were normalized to their respective whole lysate values . * * p < 0 . 05 , * * p < 0 . 01 , n ≥ 7 animals for each group . One - sample t - test .

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

(I) Immunoblots depicting levels of PSD 95, GFAP, Synapsin 1 and tubulin in the whole cortical lysate and the synaptoneurosome preparations. Quantification representing the average fold enrichments of PSD 95, Synapsin 1 and GFAP in the synaptoneurosome preparations compared to the whole cortical lysate. Data presented as mean ± SEM with scattered data points. Data points for all the proteins were normalized to their respective whole lysate values. **p<0.05, **p<0.01, n ≥ 7 animals for each group. One-sample t-test.


( J ) Bar graph representing the normalized average value of synaptic ATP level on the basal condition , on DHPG treatment for 5 minutes , on DHPG treatment with MPEP , on DHPG treatment with anisomycin and on DHPG treatment with cycloheximide ( 350μM ) . Data presented as mean ± SEM with scattered data points . Values in all other groups were normalized to the basal group for the corresponding experiment . * p < 0 . 05 , n = 8 animals .

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(J) Bar graph representing the normalized average value of synaptic ATP level on the basal condition, on DHPG treatment for 5 minutes, on DHPG treatment with MPEP, on DHPG treatment with anisomycin and on DHPG treatment with cycloheximide (350μM). Data presented as mean ± SEM with scattered data points. Values in all other groups were normalized to the basal group for the corresponding experiment. *p<0.05, n=8 animals.


( K ) Bar graph representing the normalized average value of the synaptic ATP level on the basal condition , on NMDA treatment for 5 minutes and on NMDA treatment with D - AP5 . Data presented as mean + / - SEM with scattered data points . Values in all other groups were normalized to the basal group for the corresponding experiment . n = 8 independent platings .

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

(K) Bar graph representing the normalized average value of the synaptic ATP level on the basal condition, on NMDA treatment for 5 minutes and on NMDA treatment with D-AP5. Data presented as mean +/- SEM with scattered data points. Values in all other groups were normalized to the basal group for the corresponding experiment. n=8 independent platings.


( B ) Representative neurons showing changes in the PercevalHR fluorescence ratio ( ~ ATP / ADP ratio ) in somatic compartments on the bath application of DHPG and NMDA . Scale bar 10μm . ( C ) Representative neurons showing changes in the PercevalHR fluorescence ratio ( ~ ATP / ADP ratio ) in dendritic compartments on the bath application of DHPG and NMDA . Scale bar 10μm .

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(B) Representative neurons showing changes in the PercevalHR fluorescence ratio (~ATP/ADP ratio) in somatic compartments on the bath application of DHPG and NMDA. Scale bar 10μm. (C) Representative neurons showing changes in the PercevalHR fluorescence ratio (~ATP/ADP ratio) in dendritic compartments on the bath application of DHPG and NMDA. Scale bar 10μm.


( D ) Average traces depicting the time course of the normalized somatic ATP / ADP ratio on the basal conditions , on DHPG treatment , and on NMDA treatment . The curved arrow on the top indicates the time when the agonists were added and kept in the imaging media . Data presented as mean + / - SEM . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . n = 5 - 6 cells from ≥ 3 independent platings .

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

(D) Average traces depicting the time course of the normalized somatic ATP/ADP ratio on the basal conditions, on DHPG treatment, and on NMDA treatment. The curved arrow on the top indicates the time when the agonists were added and kept in the imaging media. Data presented as mean +/- SEM. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. n= 5-6 cells from ≥3 independent platings.


( E ) Average traces depicting the time course of normalized dendritic ATP / ADP ratio on the basal condition , on the basal condition in the presence of anisomycin , on DHPG treatment , on NMDA treatment , on DHPG treatment in the presence of anisomycin and on NMDA treatment in the presence of anisomycin . The curved arrow on the top indicates the time when the agonists were added and kept in the imaging media . Data presented as mean + / - SEM . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . n = 6 - 9 cells from ≥ 3 independent platings .

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

(E) Average traces depicting the time course of normalized dendritic ATP/ADP ratio on the basal condition, on the basal condition in the presence of anisomycin, on DHPG treatment, on NMDA treatment, on DHPG treatment in the presence of anisomycin and on NMDA treatment in the presence of anisomycin. The curved arrow on the top indicates the time when the agonists were added and kept in the imaging media. Data presented as mean +/- SEM. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. n= 6-9 cells from ≥3 independent platings.


( G ) Line graph showing the average normalized synaptic ATP level at various time points after DHPG treatment until 5 minutes of recovery in the presence or absence of anisomycin . Data presented as mean + / - SEM , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 for comparison with 0 minute . # # p < 0 . 01 and # # # # p < 0 . 0001 for comparison between DHPG + anisomycin . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . n ≥ 5 animals per group .

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

(G) Line graph showing the average normalized synaptic ATP level at various time points after DHPG treatment until 5 minutes of recovery in the presence or absence of anisomycin. Data presented as mean +/- SEM, *p<0.05, **p<0.01, ****p<0.0001 for comparison with 0 minute. ##p<0.01 and ####p<0.0001 for comparison between DHPG + anisomycin. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. n≥ 5 animals per group.


( H ) Line graph showing the average normalized synaptic ATP level at various time points after NMDA treatment until 5 minutes after recovery in the presence or absence of anisomycin . Data presented as mean + / - SEM , * * p < 0 . 01 for comparison with 0 minutes . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . n ≥ 4 animals per group .

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(H) Line graph showing the average normalized synaptic ATP level at various time points after NMDA treatment until 5 minutes after recovery in the presence or absence of anisomycin. Data presented as mean +/- SEM, **p<0.01 for comparison with 0 minutes. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. n≥ 4 animals per group.


( B ) Representative images showing newly synthesized proteins on the basal condition visualized through FUNCAT metabolic labeling ( pseudo - colored ) in cortical neurons . MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization . Scale bar 10μm .

O O O O O O O O O O O O O O O O O O O O O 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) Representative images showing newly synthesized proteins on the basal condition visualized through FUNCAT metabolic labeling (pseudo-colored) in cortical neurons. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.


( C ) Representative images showing newly synthesized proteins visualized through FUNCAT metabolic labeling ( pseudo - colored ) in cortical neurons at various time points following DHPG and NMDA treatment . MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization . Scale bar 10μm .

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

(C) Representative images showing newly synthesized proteins visualized through FUNCAT metabolic labeling (pseudo-colored) in cortical neurons at various time points following DHPG and NMDA treatment. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.


( D ) Line graph showing the change in the average normalized FUNCAT intensity representing the quantity of newly synthesized proteins at various time points following DHPG and NMDA treatments . Data presented as mean + / - SEM . Data points in all groups were normalized to the average of the basal group . * * * p < 0 . 001 n = 21 - 54 neurons per group from 3 independent platings .

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(D) Line graph showing the change in the average normalized FUNCAT intensity representing the quantity of newly synthesized proteins at various time points following DHPG and NMDA treatments. Data presented as mean +/- SEM. Data points in all groups were normalized to the average of the basal group. ***p<0.001 n= 21-54 neurons per group from 3 independent platings.


( E ) Representative images showing phospho - eEF2 immunolabeling ( Pseudo - colored ) in low - density cortical neurons on the basal condition , on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes . MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization . Scale bar 10μm .

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(E) Representative images showing phospho-eEF2 immunolabeling (Pseudo-colored) in low-density cortical neurons on the basal condition, on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.


( F ) Box plot showing phospho - eEF2 normalized intensity distribution across multiple neurons on the basal condition , on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes . Data points in all groups were normalized to the average of the basal group . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * p < 0 . 05 , * * p < 0 . 01 , n = 31 - 49 cells per group from 3 independent platings .

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

(F) Box plot showing phospho-eEF2 normalized intensity distribution across multiple neurons on the basal condition, on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, **p<0.01, n=31-49 cells per group from 3 independent platings.


( G ) Representative immunoblots describing changes in the phospho - eEF2 and total - eEF2 levels at various time points after DHPG and NMDA treatment in cortical synaptoneurosomes . Note in each case phospho and total eEF2 levels were normalized individually to their respective tubulin levels for calculating the phospho / total undefined ratio .

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(G) Representative immunoblots describing changes in the phospho-eEF2 and total-eEF2 levels at various time points after DHPG and NMDA treatment in cortical synaptoneurosomes. Note in each case phospho and total eEF2 levels were normalized individually to their respective tubulin levels for calculating the phospho/total undefined ratio.


( H ) Line graph showing the average value of the synaptic phospho / total ratio of eEF2 at various time points after DHPG and NMDA treatment until 5 minutes of recovery . Data presented as mean + / - SEM , * * p < 0 . 01 . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . * * * p < 0 . 001 , * * * * p < 0 . 0001 , n ≥ 5 animals per group .

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

(H) Line graph showing the average value of the synaptic phospho/total ratio of eEF2 at various time points after DHPG and NMDA treatment until 5 minutes of recovery. Data presented as mean +/- SEM, **p<0.01. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. ***p<0.001, ****p<0.0001, n≥ 5 animals per group.


( A ) Representative images showing phospho - AMPK immunolabeling ( Pseudo - colored ) in low - density cortical neurons on the basal condition , on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes . MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization . Scale bar 10μm .

(A) Representative images showing phospho-AMPK immunolabeling (Pseudo-colored) in low-density cortical neurons on the basal condition, on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.


( B ) Box plot showing the normalized phospho - AMPK intensity distribution across multiple neurons on the basal condition , on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes . Data points in all groups were normalized to the average of the basal group . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * p < 0 . 05 , * * p < 0 . 01 , n = 30 - 46 cells per group from 3 independent platings .

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

(B) Box plot showing the normalized phospho-AMPK intensity distribution across multiple neurons on the basal condition, on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, **p<0.01, n=30-46 cells per group from 3 independent platings.


( C ) Representative immunoblots describing changes in the phospho - AMPK and total - AMPK levels at various time points after DHPG treatment in cortical synaptoneurosomes in the presence or absence of anisomycin . Note in each case phospho and total AMPK levels were normalized individually to their respective tubulin levels before calculating the phospho / total ratio of AMPK .

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

(C) Representative immunoblots describing changes in the phospho-AMPK and total-AMPK levels at various time points after DHPG treatment in cortical synaptoneurosomes in the presence or absence of anisomycin. Note in each case phospho and total AMPK levels were normalized individually to their respective tubulin levels before calculating the phospho/total ratio of AMPK.


( D ) Line graph showing the normalized average value of the synaptic phospho / total ratio of AMPK at various time points after DHPG treatment in the presence or absence of anisomycin . Data presented as mean + / - SEM . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , n = 5 animals per group .

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

(D) Line graph showing the normalized average value of the synaptic phospho/total ratio of AMPK at various time points after DHPG treatment in the presence or absence of anisomycin. Data presented as mean +/- SEM. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. *p<0.05, **p<0.01, ****p<0.0001, n= 5 animals per group.


( E ) Representative immunoblots depicting the changes in the α - SNAP protein levels at various time points after DHPG treatment in cortical synaptoneurosomes in the presence or absence of anisomycin .

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(E) Representative immunoblots depicting the changes in the α-SNAP protein levels at various time points after DHPG treatment in cortical synaptoneurosomes in the presence or absence of anisomycin.


( F ) Line graph showing the normalized average value of the synaptic α - SNAP protein levels at various time points after DHPG treatment in the presence or absence of anisomycin . Data presented as mean + / - SEM . * p < 0 . 05 . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . * * p < 0 . 01 , n ≥ 4 animals per group .

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(F) Line graph showing the normalized average value of the synaptic α-SNAP protein levels at various time points after DHPG treatment in the presence or absence of anisomycin. Data presented as mean +/- SEM. *p<0.05. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. **p<0.01, n≥ 4 animals per group.


( G ) Representative images showing the α - SNAP ( cyan ) and AMPK ( magenta ) immunolabeling in low - density cortical neurons . Merge shows the colocalization of the two channels both on the basal condition and on DHPG treatment for 5 minutes . Scale bar 10μm . Zoomed in representative images of dendrites showing the merge of both channels on the basal and DHPG treated conditions . Scale bar 5μm .

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

(G) Representative images showing the α-SNAP (cyan) and AMPK (magenta) immunolabeling in low-density cortical neurons. Merge shows the colocalization of the two channels both on the basal condition and on DHPG treatment for 5 minutes. Scale bar 10μm. Zoomed in representative images of dendrites showing the merge of both channels on the basal and DHPG treated conditions. Scale bar 5μm.


( H ) Box plot depicting the quantification of co - localization through Pearson ' s correlation coefficient between α - SNAP and AMPK in cortical neurons on the basal condition and on DHPG treatment for 5 minutes . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * p < 0 . 01 , n ≥ 35 cells per group from 4 independent platings . Unpaired - sample t - test .

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(H) Box plot depicting the quantification of co-localization through Pearson's correlation coefficient between α-SNAP and AMPK in cortical neurons on the basal condition and on DHPG treatment for 5 minutes. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.01, n≥35 cells per group from 4 independent platings. Unpaired-sample t-test.


( I ) Representative images showing the phospho - AMPK immunolabeling ( Pseudo - colored ) in low - density cortical neurons on the basal condition and on DHPG treatment for 5 minutes in the presence of scrambled siRNA and in the presence of α - SNAP siRNA . MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization . Scale bar 10μm .

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

(I) Representative images showing the phospho-AMPK immunolabeling (Pseudo-colored) in low-density cortical neurons on the basal condition and on DHPG treatment for 5 minutes in the presence of scrambled siRNA and in the presence of α-SNAP siRNA. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.


( J ) Box plot showing the normalized phospho - AMPK intensity distribution across multiple neurons on the basal condition in the presence of scrambled siRNA , on DHPG treatment for 5 minutes in the presence of scrambled siRNA , on the basal condition in the presence of α - SNAP siRNA , on DHPG treatment for 5 minutes in the presence of α - SNAP siRNA . Data points in all groups were normalized to the average of the scrambled siRNA basal group . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * p < 0 . 05 , n = 29 - 35 cells per group from 3 independent platings .

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

(J) Box plot showing the normalized phospho-AMPK intensity distribution across multiple neurons on the basal condition in the presence of scrambled siRNA, on DHPG treatment for 5 minutes in the presence of scrambled siRNA, on the basal condition in the presence of α-SNAP siRNA, on DHPG treatment for 5 minutes in the presence of α-SNAP siRNA. Data points in all groups were normalized to the average of the scrambled siRNA basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, n=29-35 cells per group from 3 independent platings.


( A ) Representative immunoblots describing changes in the phospho - AMPK and total - AMPK levels at various time points after NMDA treatment in cortical synaptoneurosomes in the presence or absence of extracellular Ca2 + . Note in each case phospho and total AMPK levels were normalized individually to their respective tubulin levels before calculating the phospho / total AMPK ratio .

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

(A) Representative immunoblots describing changes in the phospho-AMPK and total-AMPK levels at various time points after NMDA treatment in cortical synaptoneurosomes in the presence or absence of extracellular Ca2+. Note in each case phospho and total AMPK levels were normalized individually to their respective tubulin levels before calculating the phospho/total AMPK ratio.


( B ) Line graph showing the normalized average value for the synaptic phospho / total ratio of AMPK at various time points after NMDA treatment until 5 minutes of recovery in the presence or absence of extracellular Ca2 + . Data presented as mean + / - SEM . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . * * p < 0 . 01 , * * * * p < 0 . 0001 , n = 6 animals per group .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-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 O O O O O O O O O O O O O O O O O O O O O O O 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) Line graph showing the normalized average value for the synaptic phospho/total ratio of AMPK at various time points after NMDA treatment until 5 minutes of recovery in the presence or absence of extracellular Ca2+. Data presented as mean +/- SEM. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. **p<0.01, ****p<0.0001, n= 6 animals per group.


( C ) Representative images depicting intracellular Ca2 + levels through Fluo8 fluorescence ( Pseudo - colored ) of a cortical neuron plated at low - density before stimulation , 15 sec ( immediate ) after NMDA treatment , 300 sec ( delayed ) after NMDA treatment and after ionomycin treatment in the presence of 10mM Ca2 + . Scale bar 20μm .

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

(C) Representative images depicting intracellular Ca2+ levels through Fluo8 fluorescence (Pseudo-colored) of a cortical neuron plated at low-density before stimulation, 15 sec (immediate) after NMDA treatment, 300 sec (delayed) after NMDA treatment and after ionomycin treatment in the presence of 10mM Ca2+. Scale bar 20μm.


( D ) Representative time trace showing the normalized change in Fluo8 fluorescence on NMDA treatment in the presence or absence of extracellular Ca2 + and D - AP5 . Values in all other groups were normalized to the unsimulated condition for the corresponding experiment and all the data points are represented as a fraction of the initial time point . Ionomycin treatment in the presence of 10mM Ca2 + was used to calculate fluorescence maximum for a particular cell .

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

(D) Representative time trace showing the normalized change in Fluo8 fluorescence on NMDA treatment in the presence or absence of extracellular Ca2+ and D-AP5. Values in all other groups were normalized to the unsimulated condition for the corresponding experiment and all the data points are represented as a fraction of the initial time point. Ionomycin treatment in the presence of 10mM Ca2+ was used to calculate fluorescence maximum for a particular cell.


( E ) Box plot showing the distribution of ΔF / F0 across multiple neurons on the basal condition , on NMDA treatment in the presence Ca2 + , on NMDA treatment in the absence of Ca2 + , on NMDA treatment with D - AP5 and on NMDA treatment along with MK - 801 . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * * * * p < 0 . 0001 , n ≥ 30 neurons per group from 3 independent platings .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O 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 O O O O O O 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) Box plot showing the distribution of ΔF/F0 across multiple neurons on the basal condition, on NMDA treatment in the presence Ca2+, on NMDA treatment in the absence of Ca2+, on NMDA treatment with D-AP5 and on NMDA treatment along with MK-801. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. ****p<0.0001, n≥30 neurons per group from 3 independent platings.


( B ) Representative images showing the phospho - eEF2 immunolabeling ( Pseudo - colored ) and newly synthesized proteins as FUNCAT signal ( pseudo - colored ) in cortical neurons on the basal condition and on DHPG ( 50μM ) treatment for 5 minutes in the presence or absence of AICAR ( 1mM ) . MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization . Scale bar 10μm .

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

(B) Representative images showing the phospho-eEF2 immunolabeling (Pseudo-colored) and newly synthesized proteins as FUNCAT signal (pseudo-colored) in cortical neurons on the basal condition and on DHPG (50μM) treatment for 5 minutes in the presence or absence of AICAR (1mM). MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.


( C ) Box plot showing the normalized phospho - eEF2 intensity distribution across multiple neurons on the basal condition , on DHPG treatment for 5 minutes , on AICAR treatment and on DHPG treatment with AICAR . Data points in all groups were normalized to the average of the basal group . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , n = 17 - 46 cells per group from 3 independent platings .

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

(C) Box plot showing the normalized phospho-eEF2 intensity distribution across multiple neurons on the basal condition, on DHPG treatment for 5 minutes, on AICAR treatment and on DHPG treatment with AICAR. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, **p<0.01, ****p<0.0001, n=17-46 cells per group from 3 independent platings.


( D ) Box plot showing the FUNCAT intensity distribution across multiple neurons on the basal condition , on DHPG treatment for 5 minutes , on AICAR treatment and on DHPG treatment with AICAR . Data points in all groups were normalized to the average of the basal group . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * * * * p < 0 . 0001 , n = 18 - 57 cells per group from 3 independent platings .

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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR 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

(D) Box plot showing the FUNCAT intensity distribution across multiple neurons on the basal condition, on DHPG treatment for 5 minutes, on AICAR treatment and on DHPG treatment with AICAR. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. ****p<0.0001, n=18-57 cells per group from 3 independent platings.


( E ) Representative images showing the phospho - eEF2 immunolabeling ( Pseudo - colored ) and newly synthesized proteins as FUNCAT signal ( Pseudo - colored ) in cortical neurons on the basal condition and on NMDA treatment for 5 minutes in the presence or absence of Compound C . MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization . Scale bar 10μm .

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

(E) Representative images showing the phospho-eEF2 immunolabeling (Pseudo-colored) and newly synthesized proteins as FUNCAT signal (Pseudo-colored) in cortical neurons on the basal condition and on NMDA treatment for 5 minutes in the presence or absence of Compound C. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.


( F ) Box plot showing phospho - eEF2 intensity distribution across multiple neurons on basal condition ( Vehicle control ) , on NMDA treatment for 5 minutes , on Compound C treatment and on NMDA treatment with Compound C . Data points in all groups were normalized to the average of the basal group . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * p < 0 . 05 , * * p < 0 . 01 , n = 21 - 36 cells per group from 3 independent platings .

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

(F) Box plot showing phospho-eEF2 intensity distribution across multiple neurons on basal condition (Vehicle control), on NMDA treatment for 5 minutes, on Compound C treatment and on NMDA treatment with Compound C. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, **p<0.01, n=21-36 cells per group from 3 independent platings.


( G ) Box plot showing the FUNCAT intensity distribution across multiple neurons on the basal condition , on NMDA treatment for 5 minutes , on Compound C treatment and on NMDA treatment with Compound C . Data points in all groups were normalized to the average of the basal group . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , n = 24 - 52 cells per group from 3 independent platings .

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

(G) Box plot showing the FUNCAT intensity distribution across multiple neurons on the basal condition, on NMDA treatment for 5 minutes, on Compound C treatment and on NMDA treatment with Compound C. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, **p<0.01, ****p<0.0001, n=24-52 cells per group from 3 independent platings.


( G ) U2OS ( left panel , n = 6 biological replicates ) or HeLa cells ( right panel , n = 3 biological replicates ) were transfected with the indicated siRNA ; 48 hours post - transfection , they were treated with the radiomimetic antibiotic , phleomycin ( 50 μg / mL ) , and collected at the indicated time points . Flow cytometry analysis of phosphorylated - H2AX signal was used to measure γ - H2AX endogenous signal . Data are represented as a bar graph showing the mean ± SEM , each replicate being represented by a round symbol . Significance was determined by two - way ANOVA followed by a Holm - Sidak ' s test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 0005 .

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

(G) U2OS (left panel, n=6 biological replicates) or HeLa cells (right panel, n = 3 biological replicates) were transfected with the indicated siRNA; 48 hours post-transfection, they were treated with the radiomimetic antibiotic, phleomycin (50 μg/mL), and collected at the indicated time points. Flow cytometry analysis of phosphorylated-H2AX signal was used to measure γ-H2AX endogenous signal. Data are represented as a bar graph showing the mean ± SEM, each replicate being represented by a round symbol. Significance was determined by two-way ANOVA followed by a Holm-Sidak's test. *P<0.05, **P<0.01, ***P<0.0005.


( H ) U2OS ( n = 8 biological replicates ) were transfected with the indicated siRNA ; 48 hours post - transfection , they were treated with the radiomimetic antibiotic , phleomycin ( 50 μg / mL ) , and collected at the indicated time points . Data are represented as a bar graph showing the mean ± SEM , each replicate being representing as a round symbol . Significance was determined by two - way ANOVA followed by a Sidak ' s test . * P < 0 . 05 .

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

(H) U2OS (n=8 biological replicates) were transfected with the indicated siRNA; 48 hours post-transfection, they were treated with the radiomimetic antibiotic, phleomycin (50 μg/mL), and collected at the indicated time points. Data are represented as a bar graph showing the mean ± SEM, each replicate being representing as a round symbol. Significance was determined by two-way ANOVA followed by a Sidak's test. *P<0.05.


( A ) U2OS ( left panel ) , RPE1 - hTERT ( middle panel ) , and HeLa cells ( right panel ) were monitored for their sensitivity to the radiomimetic drug NCS using the SRB assay . For each cell line , the following conditions were used : U2OS cells were transfected with a nontargeting siRNA ( siCtrl ) or an siRNA targeting human POGZ ( siPOGZ - 1 or - 2 ) ; RPE1 - hTERT cells were transduced a control shRNA ( shCtrl ) or a shRNA directed against human POGZ ( shPOGZ - 1 or - 2 ) ; HeLa cells expressing a non - targeting sgRNA ( sgCtrl ) or a sgRNA targeting human POGZ and sub - cloned ( POGZ ∆ - 1 or - 2 ) . Cells were pulsed with NCS at the indicated concentrations for 1 hour , replenished with fresh medium and incubated for 4 days before being processed for SRB assays . Data are represented as a bar graph showing the relative mean ± SEM , each replicate being representing as a round symbol ( 3 biological replicates ) . Significance was determined by two - way ANOVA followed by a Bonferroni ' s test . * P < 0 . 01 .

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

(A) U2OS (left panel), RPE1-hTERT (middle panel), and HeLa cells (right panel) were monitored for their sensitivity to the radiomimetic drug NCS using the SRB assay. For each cell line, the following conditions were used: U2OS cells were transfected with a nontargeting siRNA (siCtrl) or an siRNA targeting human POGZ (siPOGZ-1 or -2); RPE1-hTERT cells were transduced a control shRNA (shCtrl) or a shRNA directed against human POGZ (shPOGZ-1 or -2); HeLa cells expressing a non-targeting sgRNA (sgCtrl) or a sgRNA targeting human POGZ and sub-cloned (POGZ∆-1 or -2). Cells were pulsed with NCS at the indicated concentrations for 1 hour, replenished with fresh medium and incubated for 4 days before being processed for SRB assays. Data are represented as a bar graph showing the relative mean ± SEM, each replicate being representing as a round symbol (3 biological replicates). Significance was determined by two-way ANOVA followed by a Bonferroni's test. *P<0.01.


( D ) U2OS ( left panel ) and HeLa ( right panel ) cells containing the DR - GFP reporter construct were transfected with the indicated siRNA . 24h post - transfection . Cells were transfected with the I - SceI expression plasmid or an empty vector ( EV ) , and the GFP + population was analyzed 48h post - plasmid transfection . The percentage of GFP + cells was determined for each individual condition and subsequently normalized to the non - targeting condition provided with I - SceI ( siCtrl , I - SceI ) . Data are represented as the mean ± SEM , each replicate being representing as a round symbol ( n = 3 biological replicates ) . Significance was determined by one - way ANOVA followed by a Dunnett ' s test . * P ≤ 0 . 0001 .

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

(D) U2OS (left panel) and HeLa (right panel) cells containing the DR-GFP reporter construct were transfected with the indicated siRNA. 24h post-transfection. Cells were transfected with the I-SceI expression plasmid or an empty vector (EV), and the GFP+ population was analyzed 48h post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition provided with I-SceI (siCtrl, I-SceI). Data are represented as the mean ± SEM, each replicate being representing as a round symbol (n=3 biological replicates). Significance was determined by one-way ANOVA followed by a Dunnett's test. *P≤0.0001.


( F ) Quantification of γ - H2AX foci in HeLa cells where POGZ has been targeted by CRISPR technology ( POGZ ∆ - 1 or - 2 ) and in control HeLa cells ( sgCtrl ) . Cells were exposed to 1 Gy before being pulsed with Edu for 1hr and were recovered at the indicated time points . Cells were fixed , stained , and imaged via confocal microscopy . Data are the total number of γ - H2AX foci in EdU + cells and represented as a bar graph showing the mean ± SD ( n = 3 biological replicates , with at least 100 cells analyzed for each time point ) . Significance was determined by two - way ANOVA followed by a Dunnett ' s test . * P < 0 . 05 , * * P < 0 . 0005 . ( G ) Representative images used for quantification in ( F ) . Scale bar = 5 μm .

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

(F) Quantification of γ-H2AX foci in HeLa cells where POGZ has been targeted by CRISPR technology (POGZ∆-1 or -2) and in control HeLa cells (sgCtrl). Cells were exposed to 1 Gy before being pulsed with Edu for 1hr and were recovered at the indicated time points. Cells were fixed, stained, and imaged via confocal microscopy. Data are the total number of γ-H2AX foci in EdU+ cells and represented as a bar graph showing the mean ± SD (n = 3 biological replicates, with at least 100 cells analyzed for each time point). Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.05, **P<0.0005. (G) Representative images used for quantification in (F). Scale bar = 5 μm.


( A ) U2OS stably expressing mCherry - LacR - FokI were transfected with the indicated siRNA . 24h post - transfection , DNA damage was induced using Shield1 and 4 - OHT . Immunofluorescence against the indicated DNA repair proteins was subsequently performed to monitor their accumulation at sites of DNA damage by confocal microscopy . Data are represented as a box - and - whisker ( 10 - 90 percentile ) where the fluorescent signal at the mCherry focus was normalized to nuclear background . At least 100 cells per condition were counted ( n = 3 biological replicates ) . Significance was determined by two - way ANOVA followed by a Dunnett ' s test . * P < 0 . 05 , * * P < 0 . 005 , * * P ≤ 0 . 0005 .

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

(A) U2OS stably expressing mCherry-LacR-FokI were transfected with the indicated siRNA. 24h post-transfection, DNA damage was induced using Shield1 and 4-OHT. Immunofluorescence against the indicated DNA repair proteins was subsequently performed to monitor their accumulation at sites of DNA damage by confocal microscopy. Data are represented as a box-and-whisker (10-90 percentile) where the fluorescent signal at the mCherry focus was normalized to nuclear background. At least 100 cells per condition were counted (n=3 biological replicates). Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.05, **P<0.005, **P≤0.0005.


( B ) U2OS ( left panel ) , RPE1 - hTERT ( middle panel ) , and HeLa cells ( right panel ) were monitored for their capacity to form IR - induced BRCA1 foci . For each cell line , the following conditions were used : U2OS cells were transfected with a nontargeting siRNA ( siCtrl ) or an siRNA targeting human POGZ ( siPOGZ - 1 or - 2 ) ; RPE1 - hTERT cells were transduced a control shRNA ( shCtrl ) or a shRNA directed against human POGZ ( shPOGZ - 1 or - 2 ) ; HeLa cells were expressing a non - targeting sgRNA ( sgCtrl ) or a sgRNA targeting human POGZ and sub - cloned ( POGZ ∆ - 1 or - 2 ) . Cells were exposed to 1 Gy before being pulsed with Edu for 1hr and were recovered 1h post - exposure to IR . Cells were fixed , stained , and imaged via confocal microscopy . Data are the total number of ΒRCA1 foci in EdU + cells and represented as a bar graph showing the mean ± SD ( n = 3 biological replicates , with at least 100 cells analyzed for each time point ) . Significance was determined by two - way ANOVA followed by a Dunnett ' s test . * P < 0 . 005 , * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) U2OS (left panel), RPE1-hTERT (middle panel), and HeLa cells (right panel) were monitored for their capacity to form IR-induced BRCA1 foci. For each cell line, the following conditions were used: U2OS cells were transfected with a nontargeting siRNA (siCtrl) or an siRNA targeting human POGZ (siPOGZ-1 or -2); RPE1-hTERT cells were transduced a control shRNA (shCtrl) or a shRNA directed against human POGZ (shPOGZ-1 or -2); HeLa cells were expressing a non-targeting sgRNA (sgCtrl) or a sgRNA targeting human POGZ and sub-cloned (POGZ∆-1 or -2). Cells were exposed to 1 Gy before being pulsed with Edu for 1hr and were recovered 1h post-exposure to IR. Cells were fixed, stained, and imaged via confocal microscopy. Data are the total number of ΒRCA1 foci in EdU+ cells and represented as a bar graph showing the mean ± SD (n = 3 biological replicates, with at least 100 cells analyzed for each time point). Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.005, **P<0.001.


( E ) U2OS stably expressing mCherry - LacR - FokI were transduced with the indicated shRNA and subsequently transfected with the indicated siRNA . 24h post - transfection , DNA damage was induced using Shield - 1 and 4 - OHT . Immunofluorescence against BRCA1 was subsequently performed to monitor its accumulation at sites of DNA damage by confocal microscopy . Data are represented as a box - and - whisker ( 10 - 90 percentile ) where the fluorescent signal at the mCherry focus was normalized to nuclear background . At least 50 cells per condition were counted . Significance was determined by two - way ANOVA followed by a Dunnett ' s test . * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) U2OS stably expressing mCherry-LacR-FokI were transduced with the indicated shRNA and subsequently transfected with the indicated siRNA. 24h post-transfection, DNA damage was induced using Shield-1 and 4-OHT. Immunofluorescence against BRCA1 was subsequently performed to monitor its accumulation at sites of DNA damage by confocal microscopy. Data are represented as a box-and-whisker (10-90 percentile) where the fluorescent signal at the mCherry focus was normalized to nuclear background. At least 50 cells per condition were counted. Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.0001.


( G ) U2OS stably expressing mCherry - LacR - FokI were transduced with the indicated siRNA . 24h post - transfection , DNA damage was induced using Shield - 1 and 4 - OHT . Immunofluorescence against the indicated HP1 isoform was subsequently performed to monitor its respective accumulation at sites of DNA damage by confocal microscopy . Data are represented as a box - and - whisker ( 10 - 90 percentile ) where the fluorescent signal at the mCherry focus was normalized to nuclear background . At least 75 cells per condition were counted . Significance was determined by two - way ANOVA followed by a Dunnett ' s test . * P < 0 . 005 , * * P < 0 . 0001 .

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

(G) U2OS stably expressing mCherry-LacR-FokI were transduced with the indicated siRNA. 24h post-transfection, DNA damage was induced using Shield-1 and 4-OHT. Immunofluorescence against the indicated HP1 isoform was subsequently performed to monitor its respective accumulation at sites of DNA damage by confocal microscopy. Data are represented as a box-and-whisker (10-90 percentile) where the fluorescent signal at the mCherry focus was normalized to nuclear background. At least 75 cells per condition were counted. Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.005, **P<0.0001.


( C ) HeLa cells transfected with the indicated construct were monitored for their capacity to form IR - induced BRCA1 foci . HeLa cells where POGZ has been targeted by CRISPR technology ( POGZ ∆ - 1 ) and in control HeLa cells ( sgCtrl ) were transfected by an empty Flag vector ( EV ) or a sgRNA - resistant FLAG - tagged POGZ cDNA construct corresponding to indicated rescue mutant ( full - length , FL ; POGZ801 - 848 , HPZ ) . Cells were exposed to 1 Gy before being pulsed with Edu for 1hr and were recovered 1h post - exposure to IR . Cells were fixed , stained , and imaged via confocal microscopy . Data are the total number of ΒRCA1 foci in EdU + cells and represented as a bar graph showing the mean ± SD ( n = 3 biological replicates , with at least 100 cells analyzed for each time point ) . Significance was determined by two - way ANOVA followed by a Dunnett ' s test . * P < 0 . 05 , * * P < 0 . 0001 . ( D ) Representative images used for quantification in ( C ) . Scale bar = 5 μm .

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

(C) HeLa cells transfected with the indicated construct were monitored for their capacity to form IR-induced BRCA1 foci. HeLa cells where POGZ has been targeted by CRISPR technology (POGZ∆-1) and in control HeLa cells (sgCtrl) were transfected by an empty Flag vector (EV) or a sgRNA-resistant FLAG-tagged POGZ cDNA construct corresponding to indicated rescue mutant (full-length, FL; POGZ801-848, HPZ). Cells were exposed to 1 Gy before being pulsed with Edu for 1hr and were recovered 1h post-exposure to IR. Cells were fixed, stained, and imaged via confocal microscopy. Data are the total number of ΒRCA1 foci in EdU+ cells and represented as a bar graph showing the mean ± SD (n = 3 biological replicates, with at least 100 cells analyzed for each time point). Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.05, **P<0.0001. (D) Representative images used for quantification in (C). Scale bar = 5 μm.


( E ) Similar as in ( C ) except that γ - H2AX foci were monitored at the indicated time points by confocal microscopy . Data are the total number of γ - H2AX foci in EdU + cells and represented as a bar graph showing the mean ± SD ( n = 3 biological replicates , with at least 100 cells analyzed for each time point ) . Significance was determined by two - way ANOVA followed by a Dunnett ' s test . * P < 0 . 05 . ( F ) Representative images used for quantification in ( E ) . Scale bar = 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Similar as in (C) except that γ-H2AX foci were monitored at the indicated time points by confocal microscopy. Data are the total number of γ-H2AX foci in EdU+ cells and represented as a bar graph showing the mean ± SD (n = 3 biological replicates, with at least 100 cells analyzed for each time point). Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.05. (F) Representative images used for quantification in (E). Scale bar = 5 μm.


Observed genotypic distribution of offspring of heterozygous Pogz + / ∆ crosses at specified embryonic day . Each litter is considered a biological replicate . Data are represented as a bar graph showing the mean ± SEM ( n for each embryonic day is specified ) . ( D )

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

Observed genotypic distribution of offspring of heterozygous Pogz+/∆ crosses at specified embryonic day. Each litter is considered a biological replicate. Data are represented as a bar graph showing the mean ± SEM (n for each embryonic day is specified). (D)


( E ) The body mass of male wild - type ( WT ) or Pogz + / ∆ mice was monitored weekly for 4 weeks post - weaning . Data are represented as a bar graph showing the mean ± SEM and each mouse is represented by a round dot ( WT ) or a square ( Pogz + / ∆ ) . At least 4 mice per genotype was monitored . Significance was determined by two - way ANOVA followed by a Sidak ' s test . * P < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The body mass of male wild-type (WT) or Pogz+/∆ mice was monitored weekly for 4 weeks post-weaning. Data are represented as a bar graph showing the mean ± SEM and each mouse is represented by a round dot (WT) or a square (Pogz+/∆ ). At least 4 mice per genotype was monitored. Significance was determined by two-way ANOVA followed by a Sidak's test. *P<0.05.


( H ) Quantification of the distance travelled ( left panel ) and the average speed ( right panel ) of each mouse ( n = 6 mice per genotype ) in the open field . Data are represented as a bar graph showing the mean ± SEM and each mouse is represented by a a round dot ( WT ) or a square ( Pogz + / ∆ ) . Significance was determined by unpaired two - tailed t - test . * P < 0 . 005 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of the distance travelled (left panel) and the average speed (right panel) of each mouse (n=6 mice per genotype) in the open field. Data are represented as a bar graph showing the mean ± SEM and each mouse is represented by a a round dot (WT) or a square (Pogz+/∆ ). Significance was determined by unpaired two-tailed t-test. *P<0.005.


( L ) Plasma was isolated from cardiac punctures of wild - type ( WT ) or Pogz + / ∆ mice ( 8 weeks ) and assessed for circulating levels of specified immunoglobulin isotypes . Each mouse is represented by a round dot ( WT ) or a square ( Pogz + / ∆ ) ( n = 6 mice per genotype ) . Significance was determined by two - way ANOVA followed by a Bonferroni ' s test . * P < 0 . 005 .

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

(L) Plasma was isolated from cardiac punctures of wild-type (WT) or Pogz+/∆ mice (8 weeks) and assessed for circulating levels of specified immunoglobulin isotypes. Each mouse is represented by a round dot (WT) or a square (Pogz+/∆ ) (n=6 mice per genotype). Significance was determined by two-way ANOVA followed by a Bonferroni's test. *P<0.005.


( A ) Wild - type ( WT ) and Pogz + / ∆ mice were subjected to a lethal dose of ionizing radiation ( 8 . 5 Gy ) before recovering in sterile conditions and being assessed for their sensitivity to IR . Data are represented as a Kaplan - Meier survival curve of each genotype ( ( n = 6 mice per genotype ) . Significance was determined by log - rank ( Mantel - Cox ) test . * P < 0 . 005 .

(A) Wild-type (WT) and Pogz+/∆ mice were subjected to a lethal dose of ionizing radiation (8.5 Gy) before recovering in sterile conditions and being assessed for their sensitivity to IR. Data are represented as a Kaplan-Meier survival curve of each genotype ((n=6 mice per genotype). Significance was determined by log-rank (Mantel-Cox) test. *P<0.005.


( D ) MEFs were monitored for their sensitivity to the radiomimetic drug phleomycin using the SRB assay . Immortalized MEFS obtained from the indicated genotype were treated with increasing concentrations of phleomycin for 1hr , replenished with fresh medium and incubated for 4 days before being processed for SRB assays . Data are represented as a bar graph showing the relative mean ± SEM , each replicate being representing as a round dot ( WT ) or a square ( Pogz + / ∆ ) . Significance was determined by two - way ANOVA followed by a Bonferroni ' s test . * P < 0 . 005 , * * P < 0 . 0005 .

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 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

(D) MEFs were monitored for their sensitivity to the radiomimetic drug phleomycin using the SRB assay. Immortalized MEFS obtained from the indicated genotype were treated with increasing concentrations of phleomycin for 1hr, replenished with fresh medium and incubated for 4 days before being processed for SRB assays. Data are represented as a bar graph showing the relative mean ± SEM, each replicate being representing as a round dot (WT) or a square (Pogz+/∆ ). Significance was determined by two-way ANOVA followed by a Bonferroni's test. *P<0.005, **P<0.0005.


( H ) Stimulated CD43 - negative splenocytes were loaded on slides via Cytospin and processed for γ - H2AX immunofluorescence . Cells were fixed , stained , and imaged via confocal microscopy . Data are the percentage of cells in a field of view with indicated γ - H2AX foci and are represented as a bar graph showing the mean ± SEM . At least 100 cells per genotype were counted . Significance was determined by two - way ANOVA followed by a Sidak ' s test . * P < 0 . 05 , * * P < 0 . 005 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Stimulated CD43-negative splenocytes were loaded on slides via Cytospin and processed for γ-H2AX immunofluorescence. Cells were fixed, stained, and imaged via confocal microscopy. Data are the percentage of cells in a field of view with indicated γ-H2AX foci and are represented as a bar graph showing the mean ± SEM. At least 100 cells per genotype were counted. Significance was determined by two-way ANOVA followed by a Sidak's test. *P<0.05, **P<0.005.


B , Representative images ( left panel ) and quantifications ( right panel ) of NNMT immunostaining in tissue sections from primary tumors ( T ) and metastases ( M ) in different breast cancer models , scale bar : 100 µm . n = 5 tissue sections per model . * * P < 0 . 01 ; n . s . , not significant , n . a . , not available ; Mann - Whitney U - test . All data are means ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Representative images (left panel) and quantifications (right panel) of NNMT immunostaining in tissue sections from primary tumors (T) and metastases (M) in different breast cancer models, scale bar: 100 µm. n = 5 tissue sections per model. **P < 0.01; n.s., not significant, n.a., not available; Mann-Whitney U-test. All data are means ± SD.


C , Kaplan - Meier overall survival analysis of breast cancer patients stratified according to NNMT protein levels ( negative or weak , n = 541 ; intermediate , n = 108 ; strong , n = 44 ) in primary ( n = 517 ) and metastatic ( n = 176 ) tumor tissue . Estimated 5 - year overall survival rates for subjects with strong , intermediate , and negative / weak NNMT levels were 43 . 0 ± 10 . 0 % , 69 . 0 ± 6 . 0 % and 73 . 0 ± 2 . 0 % , respectively . * * P < 0 . 01 ; Log - rank test .

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

C, Kaplan-Meier overall survival analysis of breast cancer patients stratified according to NNMT protein levels (negative or weak, n = 541; intermediate, n = 108; strong, n = 44) in primary (n = 517) and metastatic (n =176) tumor tissue. Estimated 5-year overall survival rates for subjects with strong, intermediate, and negative/weak NNMT levels were 43.0 ± 10.0%, 69.0 ± 6.0% and 73.0 ± 2.0%, respectively. **P < 0.01; Log-rank test.


D , Graphic quantification ( pie charts ) and representative images of immunostaining from 625 primary breast cancer specimens classified according to NNMT protein abundance [ negative ( neg ) / weak , intermediate or strong ] . NNMT levels are significantly higher ( P = 0 , Student ' s T - test ) in ERα negative ( neg ) than in ERα positive ( pos ) breast cancers ( side table ) . Scale bar : 100 µm .

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

D, Graphic quantification (pie charts) and representative images of immunostaining from 625 primary breast cancer specimens classified according to NNMT protein abundance [negative (neg)/weak, intermediate or strong]. NNMT levels are significantly higher (P = 0, Student's T-test) in ERα negative (neg) than in ERα positive (pos) breast cancers (side table). Scale bar: 100 µm.


E , Kaplan - Meier plot depicting metastasis onset after tumor removal in mice injected orthotopically with SUM159PT WT ( n = 10 ) , KO ( KOd : n = 5 ; KOs : n = 5 ) or KO - NNMT ( KOd - NNMT : n = 5 ; KOs - NNMT : n = 5 ) cells . * P < 0 . 05 ; Log - rank test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Kaplan-Meier plot depicting metastasis onset after tumor removal in mice injected orthotopically with SUM159PT WT (n = 10), KO (KOd: n = 5; KOs: n = 5) or KO-NNMT (KOd-NNMT: n= 5; KOs-NNMT: n= 5) cells. *P < 0.05; Log-rank test.


F , Bar graph quantification ( left panel ) and representative bioluminescence images ( right panel ) of metastases at day 75 after cancer cell injection of MDA - MB - 231 KO1 - RFP or KO1 - NNMT cells and post - primary tumor removal . n = 7 to 10 animals per group . * P < 0 . 05 ; Mann - Whitney U - test . All data are means ± SD . G , Bar graph quantification ( left panel ) and representative bioluminescence images ( right panel ) of metastases at day 34 after injection of MDA - MB - 231 KO1 - RFP or KO1 - NNMT cells into the tail veins of mice . n = 10 animals per group . * * * P < 0 . 001 ; Mann - Whitney U - test . All data are means ± SD .

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

F, Bar graph quantification (left panel) and representative bioluminescence images (right panel) of metastases at day 75 after cancer cell injection of MDA-MB-231 KO1- RFP or KO1- NNMT cells and post-primary tumor removal. n = 7 to 10 animals per group. *P < 0.05; Mann-Whitney U-test. All data are means ± SD. G, Bar graph quantification (left panel) and representative bioluminescence images (right panel) of metastases at day 34 after injection of MDA-MB-231 KO1- RFP or KO1- NNMT cells into the tail veins of mice. n = 10 animals per group. ***P < 0.001; Mann-Whitney U-test. All data are means ± SD.


C , Representative histological images of SUM159 PT - WT , - KOd , and KOs tumors immunostained with an anti - human vimentin antibody . n = 5 tumors , 2 sections each . Scale bar : 100 µm .

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

C, Representative histological images of SUM159 PT-WT, -KOd, and KOs tumors immunostained with an anti-human vimentin antibody. n = 5 tumors, 2 sections each. Scale bar: 100 µm.


G , Representative images of human COL1A1 immunostaining ( left panel ) and bar graph quantification of COL1A1 positive area ( right panel ) in tissue sections of SUM159PT WT , KOd , and KOs tumors . n = 10 to 15 tumors per model with 2 - 4 tissue sections per tumor . * * P < 0 . 01 ; Kruskal - Wallis test . All data are means ± SD . Scale bar : 200 µm .

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

G, Representative images of human COL1A1 immunostaining (left panel) and bar graph quantification of COL1A1 positive area (right panel) in tissue sections of SUM159PT WT, KOd, and KOs tumors. n = 10 to 15 tumors per model with 2 - 4 tissue sections per tumor. **P < 0.01; Kruskal-Wallis test. All data are means ± SD. Scale bar: 200 µm.


A , Bar graph depicting increased ratio of S - adenosylmethionine ( SAM ) : S - adenosylhomocysteine ( SAH ) in SUM159PT KOd and KOs compared to WT cells . n = 3 technical replicates . * P < 0 . 05 ; * * P < 0 . 01 ; Mann - Whitney U - test . All data are means ± SD .

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

A, Bar graph depicting increased ratio of S-adenosylmethionine (SAM): S-adenosylhomocysteine (SAH) in SUM159PT KOd and KOs compared to WT cells. n = 3 technical replicates. *P < 0.05; **P < 0.01; Mann-Whitney U-test. All data are means ± SD.


D , Dot plots depicting the overlap of H3K9me3 ChIP - sequencing and mRNA - sequencing data . In the y axis : differential H3K9me3 enrichment at the gene promoter in NNMT KO compared to WT cells . In the x axis : differential mRNA expression of NNMT KO compared to WT cells . n = 3 experimental replicates . Cut - off : adjusted P < 0 . 05 . Bold dots highlight collagen genes and PRDM5 .

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

D, Dot plots depicting the overlap of H3K9me3 ChIP-sequencing and mRNA-sequencing data. In the y axis: differential H3K9me3 enrichment at the gene promoter in NNMT KO compared to WT cells. In the x axis: differential mRNA expression of NNMT KO compared to WT cells. n = 3 experimental replicates. Cut-off: adjusted P < 0.05. Bold dots highlight collagen genes and PRDM5.


F , Bar graph representing average PRDM5 mRNA expression in SUM159PT KOs , KOd , and WT cells . n = 3 experimental replicates with 2 technical replicates each . * * * * P < 0 . 0001 ; Mann - Whitney U - test . All data are means ± SD .

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

F, Bar graph representing average PRDM5 mRNA expression in SUM159PT KOs, KOd, and WT cells. n = 3 experimental replicates with 2 technical replicates each. ****P < 0.0001; Mann-Whitney U-test. All data are means ± SD.


G , H , Bar graphs representing average collagen gene mRNA expression upon overexpression ( OE ) of PRDM5 in SUM159PT KOd and KOs cells . n = 3 to 5 experimental replicates with 2 technical replicates each . * P < 0 . 05 , * * P < 0 . 01 , n . s . , not significant ; Kruskal - Wallis test . All data are means ± SD .

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

G, H, Bar graphs representing average collagen gene mRNA expression upon overexpression (OE) of PRDM5 in SUM159PT KOd and KOs cells. n = 3 to 5 experimental replicates with 2 technical replicates each. *P < 0.05, **P < 0.01, n.s., not significant; Kruskal-Wallis test. All data are means ± SD.


A , Representative images and bar graph quantification depicting 5 - methylcytosine ( 5 - mC ) signal by immunofluorescence in SUM159PT WT , KOs , and KOd cells . n = 2 experimental replicates with 5 to 6 technical replicates each . * * * P < 0 . 001 ; Kruskal - Wallis test . Data are means ± SD . Scale bar , 50 µm .

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

A, Representative images and bar graph quantification depicting 5-methylcytosine (5-mC) signal by immunofluorescence in SUM159PT WT, KOs, and KOd cells. n = 2 experimental replicates with 5 to 6 technical replicates each. ***P < 0.001; Kruskal-Wallis test. Data are means ± SD. Scale bar, 50 µm.


D , Bar graphs depicting 5 - mC abundance at promoters of the indicated genes shown as fold enrichment of methylated DNA immunoprecipitate ( MedIP ) over IgG control . n = 3 experimental replicates with 2 to 3 technical replicates each . * P < 0 . 05 , * * P < 0 . 01 , n . s . , not significant ; Kruskal - Wallis test . Data are means ± SEM .

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

D, Bar graphs depicting 5-mC abundance at promoters of the indicated genes shown as fold enrichment of methylated DNA immunoprecipitate (MedIP) over IgG control. n = 3 experimental replicates with 2 to 3 technical replicates each. *P < 0.05, ** P < 0.01, n.s., not significant; Kruskal-Wallis test. Data are means ± SEM.


E , Bar graphs representing average PRDM5 and collagen gene mRNA expression upon 5 - aza treatment in SUM159PT WT , Kos , and KOd cells . n = 3 experimental replicates with 2 technical replicates each . * P < 0 . 05 , n . s . , not significant ; Kruskal - Wallis test . Data are means ± SEM .

O O O O O O O O O O O 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 O O O O O O O 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, Bar graphs representing average PRDM5 and collagen gene mRNA expression upon 5- aza treatment in SUM159PT WT, Kos, and KOd cells. n = 3 experimental replicates with 2 technical replicates each. *P < 0.05, n.s., not significant; Kruskal-Wallis test. Data are means ± SEM.


A , Bar graph quantification ( left panel ) and representative bioluminescence images ( right panel ) of metastases at day 27 after injection of MDA - MB - 231 cells into the tail veins of mice bearing short hairpin sh NT ( non - targeting ) , sh1 or sh2 targeting PRDM5 . Mice were fed with doxycycline food to sustain shRNA expression during the whole experiment . n = 7 to 8 animals per group . * * P < 0 . 01 ; One - way ANOVA . All data are means ± SD .

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

A, Bar graph quantification (left panel) and representative bioluminescence images (right panel) of metastases at day 27 after injection of MDA-MB-231 cells into the tail veins of mice bearing short hairpin sh NT (non-targeting), sh1 or sh2 targeting PRDM5. Mice were fed with doxycycline food to sustain shRNA expression during the whole experiment. n = 7 to 8 animals per group. **P < 0.01; One-way ANOVA. All data are means ± SD.


B , Box plots depicting bioluminescence quantification ( left panel ) and representative images ( right panel ) of metastases at day 42 and day 62 after intravenous injection of MDA - MB - 231 KO1 _ RFP , KO1 _ NNMT1 and KO1 _ COL1A1 ( COL1A1 over - expression ) cells . n = 4 to 8 animals per group . * * P < 0 . 01 , * * * P < 0 . 001 ; Day 42 : One - way ANOVA , Day 62 : Student t - test . Boxes define the upper and lower quartiles ; central band indicate the median ; whiskers define max to min values .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Box plots depicting bioluminescence quantification (left panel) and representative images (right panel) of metastases at day 42 and day 62 after intravenous injection of MDA-MB-231 KO1_RFP, KO1_NNMT1 and KO1_COL1A1 (COL1A1 over-expression) cells. n = 4 to 8 animals per group. **P < 0.01, ***P < 0.001; Day 42: One-way ANOVA, Day 62: Student t-test. Boxes define the upper and lower quartiles; central band indicate the median; whiskers define max to min values.


E , Box plots showing COL1A1 and PRDM5 expression level ( H - Score ) according to the NNMT status ( NNMT negative , n = 17 ; NNMT positive , n = 47 ) within the hormone receptor - negative ( HR - ) tumor samples . * P < 0 . 05 ; Student t - test . Boxes define the upper and lower quartiles ; central band indicate the median ; whiskers define max to min values .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Box plots showing COL1A1 and PRDM5 expression level (H-Score) according to the NNMT status (NNMT negative, n = 17; NNMT positive, n = 47) within the hormone receptor-negative (HR-) tumor samples. *P < 0.05; Student t-test. Boxes define the upper and lower quartiles; central band indicate the median; whiskers define max to min values.


Western blot of pre - mRNA 3 ' - end processing factors in response to UV treatment ( 40 J / m2 ) of A549 cells ( n = 3 ) , followed by16 hours of recovery . GAPDH was used as a loading control .

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Western blot of pre-mRNA 3'-end processing factors in response to UV treatment (40 J/m2) of A549 cells (n=3), followed by16 hours of recovery. GAPDH was used as a loading control.


RT - qPCR assay on nuclear RNA for assessing the uncleaved / total ratio of p53 and TATA - binding protein ( TBP ) pre - mRNAs in A549 cells ( n = 3 ) transfected for 48 hours with two different siRNA targeting PCF11 prior to exposure ( UV + ) or not ( UV - ) to UV irradiation ( 40 J / m2 ) . " n " indicates the number of biological replicates for each experiment . All data are presented as the mean ± s . e . m . P values were calculated using two - sided unpaired t - test .

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RT-qPCR assay on nuclear RNA for assessing the uncleaved/total ratio of p53 and TATA-binding protein (TBP) pre-mRNAs in A549 cells (n=3) transfected for 48 hours with two different siRNA targeting PCF11 prior to exposure (UV+) or not (UV-) to UV irradiation (40 J/m2). "n" indicates the number of biological replicates for each experiment. All data are presented as the mean ± s.e.m. P values were calculated using two-sided unpaired t-test.


RT - qPCR analysis on RNA extracted from nucleoplasm and chromatin fractions . The ratio of uncleaved pre - mRNA ( nucleoplasm / chromatin ) was calculated to quantify the level of unprocessed p53 , TBP , WDR13 and GAPDH pre - mRNA ( n = 3 biological replicates ) released in the nucleoplasm compared to the chromatin - bound unprocessed pre - mRNA . WDR13 and GAPDH were included as controls as they have been previously reported to be processed post - and co - transcriptionally , respectively . Data are presented as the mean ± s . e . m .

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RT-qPCR analysis on RNA extracted from nucleoplasm and chromatin fractions. The ratio of uncleaved pre-mRNA (nucleoplasm/chromatin) was calculated to quantify the level of unprocessed p53, TBP, WDR13 and GAPDH pre-mRNA (n=3 biological replicates) released in the nucleoplasm compared to the chromatin-bound unprocessed pre-mRNA. WDR13 and GAPDH were included as controls as they have been previously reported to be processed post- and co-transcriptionally, respectively. Data are presented as the mean ± s.e.m.


RT - PCR analysis of p53 3 ' flanking region using the same primers employed in the data panel above ( n = 3 ) . Lane 1 is a control PCR amplification of cDNA derived from the reverse transcription of a control mRNA using oligo ( dT ) . Lanes 2 - 7 are PCR amplification of reverse transcribed p53 chromatin - associated pre - mRNA using oligo oligo ( dT ) .

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RT-PCR analysis of p53 3' flanking region using the same primers employed in the data panel above (n=3). Lane 1 is a control PCR amplification of cDNA derived from the reverse transcription of a control mRNA using oligo (dT). Lanes 2-7 are PCR amplification of reverse transcribed p53 chromatin-associated pre-mRNA using oligo oligo (dT).


Representative images of smFISH in untreated ( - UV ) or UV - treated ( + UV ) A549 cells with the indicated probes ( A , B , C and D ) .

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Representative images of smFISH in untreated (-UV) or UV-treated (+UV) A549 cells with the indicated probes (A, B, C and D).


RT - qPCR assay on nuclear RNA for assessing the uncleaved / total ratio of p53 pre - mRNA in wild type ( WT ) and CoTC deleted ( ΔCoTC ) A549 cells ( n = 3 ) treated with or without UV irradiation ( 40 J / m2 ) . " n " indicates the number of biological replicates for each experiment . All data are presented as the mean ± s . e . m . P values were calculated using two - sided unpaired t - test .

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RT-qPCR assay on nuclear RNA for assessing the uncleaved/total ratio of p53 pre-mRNA in wild type (WT) and CoTC deleted (ΔCoTC) A549 cells (n=3) treated with or without UV irradiation (40 J/m2). "n" indicates the number of biological replicates for each experiment. All data are presented as the mean ± s.e.m. P values were calculated using two-sided unpaired t-test.


RT - qPCR measuring relative p53 and p21 mRNA levels in wild type ( WT ) and CoTC - deleted ( ΔCoTC ) cells ( n = 3 ) in response to UV treatment ( 40 J / m2 ) . The expression was normalized to HPRT . " n " indicates the number of biological replicates for each experiment . All data are presented as the mean ± s . e . m . P values were calculated using two - sided unpaired t - test .

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RT-qPCR measuring relative p53 and p21 mRNA levels in wild type (WT) and CoTC-deleted (ΔCoTC) cells (n=3) in response to UV treatment (40 J/m2). The expression was normalized to HPRT. "n" indicates the number of biological replicates for each experiment. All data are presented as the mean ± s.e.m. P values were calculated using two-sided unpaired t-test.


Western blot analysis of p53 and p21 expression in wild type ( WT ) and CoTC deleted ( ΔCoTC ) A549 cells ( n = 3 ) treated with or without UV irradiation ( 40 J / m2 ) .

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Western blot analysis of p53 and p21 expression in wild type (WT) and CoTC deleted (ΔCoTC) A549 cells (n=3) treated with or without UV irradiation (40 J/m2).


RT - qPCR ( uncleaved / total RNA ) on nuclear RNA extracted from UV - treated or untreated A549 cells ( n = 3 ) , to assess the regulation of 3 ' end processing of 20 pre - mRNAs randomly selected from the previous RNA - sequencing data . " n " indicates the number of biological replicates for each experiment . All data are presented as the mean ± s . e . m .

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RT-qPCR (uncleaved/total RNA) on nuclear RNA extracted from UV-treated or untreated A549 cells (n=3), to assess the regulation of 3'end processing of 20 pre-mRNAs randomly selected from the previous RNA-sequencing data. "n" indicates the number of biological replicates for each experiment. All data are presented as the mean ± s.e.m.


RT - qPCR ( Released uncleaved pre - mRNA nucleoplasm / chromatin ) on RNA extracted from the nucleoplasm and the chromatin fractions of A549 UV - treated cells ( n = 3 ) . " n " indicates the number of biological replicates for each experiment . All data are presented as the mean ± s . e . m .

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RT-qPCR (Released uncleaved pre-mRNA nucleoplasm/chromatin) on RNA extracted from the nucleoplasm and the chromatin fractions of A549 UV-treated cells (n=3). "n" indicates the number of biological replicates for each experiment. All data are presented as the mean ± s.e.m.


A , B . Height ( A ) and body mass index ( B ) of the participants by age in the diet groups compared to 2 SD intervals ( shaded area ) and median in the Finnish reference population .

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A, B. Height (A) and body mass index (B) of the participants by age in the diet groups compared to 2 SD intervals (shaded area) and median in the Finnish reference population.


C . Dietary intake of selected macro - and micronutrients of participants . The shaded areas represent Finnish dietary recommendations for 2 to 5 years olds that covers majority of participants ( THL , 2019 ) . Energy intake recommendations vary by weight and therefore unambiguous limits could not be represented .

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C. Dietary intake of selected macro- and micronutrients of participants. The shaded areas represent Finnish dietary recommendations for 2 to 5 years olds that covers majority of participants (THL, 2019). Energy intake recommendations vary by weight and therefore unambiguous limits could not be represented.


A . Serum , plasma or urine levels of vitamin D , vitamin A , folate , zinc , iron and iodine status biomarkers in diet groups . Solid lines indicate cut - offs for deficiency , and dashed lines indicate cut - offs for insufficiency . The RBP limits for vitamin A insufficiency and deficiency are validated for the method at 1 . 17 µmol / l and 0 . 83 µmol / l , respectively . Vitamin A status was calculated based on RBP , transthyretin , and CRP . ( Talsma et al , 2015 ) .

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A. Serum, plasma or urine levels of vitamin D, vitamin A, folate, zinc, iron and iodine status biomarkers in diet groups. Solid lines indicate cut-offs for deficiency, and dashed lines indicate cut-offs for insufficiency. The RBP limits for vitamin A insufficiency and deficiency are validated for the method at 1.17 µmol/l and 0.83 µmol/l, respectively. Vitamin A status was calculated based on RBP, transthyretin, and CRP.(Talsma et al, 2015).


B . Cholesterol metabolism biomarkers in the diet groups . The subfigure summarizes cholesterol metabolism in humans and highlights cholesterol - related metabolites measured in this study with black font . ( Nelson & Cox , 2012 ; Risley , 2002 ) .

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B. Cholesterol metabolism biomarkers in the diet groups. The subfigure summarizes cholesterol metabolism in humans and highlights cholesterol-related metabolites measured in this study with black font. (Nelson & Cox, 2012; Risley, 2002).


B . Targeted analysis of bile acid concentrations in serum of omnivores , vegetarians and vegans .

B. Targeted analysis of bile acid concentrations in serum of omnivores, vegetarians and vegans.


C . Amino acid concentrations in untargeted metabolomics . Numeric means and standard deviations in this figure can be found in Appendix Table S4 . # Valine and betaine are two major components under the same m / z - peak , causing uncertainty in the interpretation of valine levels .

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C. Amino acid concentrations in untargeted metabolomics. Numeric means and standard deviations in this figure can be found in Appendix Table S4. #Valine and betaine are two major components under the same m/z-peak, causing uncertainty in the interpretation of valine levels.


A . Fatty acid analysis from untargeted MS metabolomics including free alpha - linolenic acid ( ALA ) and docosahexaenoic acid ( DHA ) .

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A. Fatty acid analysis from untargeted MS metabolomics including free alpha-linolenic acid (ALA) and docosahexaenoic acid (DHA).


B . Carnitine - bound fatty acids commonly found in serum that were detected in mass spectrometry .

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B. Carnitine-bound fatty acids commonly found in serum that were detected in mass spectrometry.


C . Lysophosphatidylcholine and lysophosphatidylethanolamine with C16 : 0 ( palmitic acid ) and C18 : 1 ( oleic acid ) .

C. Lysophosphatidylcholine and lysophosphatidylethanolamine with C16:0 (palmitic acid) and C18:1 (oleic acid).


D . Combined analysis of all found triglycerides correlating to medium - chain fatty acid lengths and long or very long chain fatty acid lengths .

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D. Combined analysis of all found triglycerides correlating to medium-chain fatty acid lengths and long or very long chain fatty acid lengths.


( D , E ) HTM - mediated quantification of MPM - 2 ( D ) and H3S10P ( E ) levels per individual nucleus in U2OS cells treated with DMSO as a control or 50 μM P22077 ( USP7i ) for 4 hrs . Nuclei were stained with DAPI ( blue ) . Scale bar , 30 μm . The quantification is shown to the right of representative images for each analysis . ( * * * p < 0 . 05 ; t - test ) . Grey lines indicate mean values .

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(D, E) HTM-mediated quantification of MPM-2 (D) and H3S10P (E) levels per individual nucleus in U2OS cells treated with DMSO as a control or 50 μM P22077 (USP7i) for 4 hrs. Nuclei were stained with DAPI (blue). Scale bar, 30 μm. The quantification is shown to the right of representative images for each analysis. (*** p<0.05; t-test). Grey lines indicate mean values.


( A ) Western Blot showing the levels of USP7 , MPM - 2 , CDK1Y15P and CDK1 in whole cell extracts of RPE cells treated with 25 μM P22077 for 2 - 4 hrs ( USP7i ) , an asterisk indicates a non - specific band .

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(A) Western Blot showing the levels of USP7, MPM-2, CDK1Y15P and CDK1 in whole cell extracts of RPE cells treated with 25 μM P22077 for 2-4 hrs (USP7i), an asterisk indicates a non-specific band.


( B ) Immunofluorescence of CCNB1 ( red ) and CCNB1S126P ( green ) in RPE cells in control conditions ( Control ) and after treatment with 25 μM P22077 for 2 hrs ( USP7i ) . Nuclei were stained with DAPI ( blue ) . Scale bar , 30 μm .

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(B) Immunofluorescence of CCNB1 (red) and CCNB1S126P (green) in RPE cells in control conditions (Control) and after treatment with 25 μM P22077 for 2 hrs (USP7i). Nuclei were stained with DAPI (blue). Scale bar, 30 μm.


( B ) Western blot showing the levels of MPM - 2 , pAKT , AKT , pPRC1 , pKAP , KAP , pMAPKAPK - 2 , pSTAT3 , STAT3 and β - Actin in whole cell extracts of RPE cells treated with DMSO ( Control ) , 0 . 5 μM Okadaic acid for 2 hrs , 25 μM P22077 for 4 hrs or 25 μM BAY 11 - 7082 for 2 hrs . Ponceau staining is shown as loading control .

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(B) Western blot showing the levels of MPM-2, pAKT, AKT, pPRC1, pKAP, KAP, pMAPKAPK-2, pSTAT3, STAT3 and β-Actin in whole cell extracts of RPE cells treated with DMSO (Control), 0.5 μM Okadaic acid for 2 hrs, 25 μM P22077 for 4 hrs or 25 μM BAY 11-7082 for 2 hrs. Ponceau staining is shown as loading control.


( D ) Immunofluorescence of PP2A - A ( green ) in U2OS cells treated with DMSO ( CONTROL ) , 50 μM P22077 ( USP7i ) for 4 hrs or 0 . 5 μM Okadaic acid ( OA ) for 1 hr 30 min . Nuclei were stained with DAPI ( blue ) . Scale bar , 10 μm .

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(D) Immunofluorescence of PP2A-A (green) in U2OS cells treated with DMSO (CONTROL), 50 μM P22077 (USP7i) for 4 hrs or 0.5 μM Okadaic acid (OA) for 1 hr 30 min. Nuclei were stained with DAPI (blue). Scale bar, 10 μm.


( E ) Western blot showing the levels of MPM - 2 , pAKT , AKT , pPRC1 , pMAPKAPK - 2 , p4e - BP1 , 4e - BP1 and β - Actin , in whole cell extracts of RPE cells treated with DMSO ( Control ) , 25 μM P22077 ( USP7i ) and 10 μM of the PP2A activator DT - 061 for 2 - 4 hrs . Ponceau staining is shown as loading control .

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(E) Western blot showing the levels of MPM-2, pAKT, AKT, pPRC1, pMAPKAPK-2, p4e-BP1, 4e-BP1 and β-Actin, in whole cell extracts of RPE cells treated with DMSO (Control), 25 μM P22077 (USP7i) and 10 μM of the PP2A activator DT-061 for 2-4 hrs. Ponceau staining is shown as loading control.