Association between SINE Alu RNA ratio ( upper panel ) and gene expression levels ( lower panel ) of genes up - regulated in AD ( Dataset EV3 , | log2FoldChange | > 0 . 5 ) . Every column in both panels corresponds to the same MAP patient of either the no AD or AD group . Patients in each group are sorted from left to right in an ascending order with regard to Alu RNA processing ratio . Every row in the heatmap corresponds to one gene , with color density representing normalized FPKM values from RNA - seq for this gene for each patient ( columns ) ( red represents high and blue low expression levels ) . Association between full length Alu RNA levels ( upper panel ) and gene expression levels ( lower panel ) of genes down - regulated in AD ( Dataset EV4 , | log2FoldChange | > 0 . 5 ) . Every column in both panels corresponds to the same MAP patient of either the no AD or AD group . Patients in each group are sorted from left to right in an ascending order with regard to full length Alu RNA levels . Every row in the heatmap corresponds to one gene , with color density representing normalized FPKM values from RNA - seq for this gene for each patient ( columns ) ( red represents high and blue low expression levels ) .

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

Association between SINE Alu RNA ratio (upper panel) and gene expression levels (lower panel) of genes up-regulated in AD (Dataset EV3, |log2FoldChange|> 0.5). Every column in both panels corresponds to the same MAP patient of either the no AD or AD group. Patients in each group are sorted from left to right in an ascending order with regard to Alu RNA processing ratio. Every row in the heatmap corresponds to one gene, with color density representing normalized FPKM values from RNA-seq for this gene for each patient (columns) (red represents high and blue low expression levels). Association between full length Alu RNA levels (upper panel) and gene expression levels (lower panel) of genes down-regulated in AD (Dataset EV4, |log2FoldChange|> 0.5). Every column in both panels corresponds to the same MAP patient of either the no AD or AD group. Patients in each group are sorted from left to right in an ascending order with regard to full length Alu RNA levels. Every row in the heatmap corresponds to one gene, with color density representing normalized FPKM values from RNA-seq for this gene for each patient (columns) (red represents high and blue low expression levels).


Scatterplot depicting the positive correlation between average gene expression values of upregulated genes in Figure 6B and Alu RNA processing ratio in AD patients ( r = 0 . 88 , p < 0 . 001 ) . Statistical test is based on Pearson ' s product moment correlation coefficient and follows a t distribution using the cor . test function in R package stats . Scatterplot depicting the negative correlation between average gene expression values of down - regulated genes in Figure 6C and full length Alu RNA levels in AD patients ( r = - 0 . 42 , p < 0 . 001 ) . Statistical test as in subfigure 6D .

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

Scatterplot depicting the positive correlation between average gene expression values of upregulated genes in Figure 6B and Alu RNA processing ratio in AD patients (r = 0.88, p<0.001). Statistical test is based on Pearson's product moment correlation coefficient and follows a t distribution using the cor.test function in R package stats. Scatterplot depicting the negative correlation between average gene expression values of down-regulated genes in Figure 6C and full length Alu RNA levels in AD patients (r = -0.42, p<0.001). Statistical test as in subfigure 6D.


Correlation between gene expression and Alu RNA processing ratio for AD upregulated genes ( Dataset EV3 ) ( upper panel ) and a random set of non - differentially expressed genes ( Dataset EV5 ) ( lower panel ) . For every gene a correlation coefficient was calculated ( Pearson correlation ) together with the respective p - value . Statistical test is based on Pearson ' s product moment correlation coefficient and follows a t distribution using the cor . test function in R package stats . Based on the r and p value genes were classified into four categories : i ) NS , non - significant for p value > 0 . 05 , ii ) no correlation or weak correlation ( r ≤ 0 . 05 ) , iii ) significant ( p < 0 . 05 ) and strong correlation ( 0 .

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

Correlation between gene expression and Alu RNA processing ratio for AD upregulated genes (Dataset EV3) (upper panel) and a random set of non-differentially expressed genes (Dataset EV5) (lower panel). For every gene a correlation coefficient was calculated (Pearson correlation) together with the respective p-value. Statistical test is based on Pearson's product moment correlation coefficient and follows a t distribution using the cor.test function in R package stats. Based on the r and p value genes were classified into four categories: i)NS, non-significant for p value > 0.05, ii) no correlation or weak correlation (r≤0.05), iii) significant (p < 0.05) and strong correlation (0.5


Expression levels of full length Alu RNA ( RT - qPCR ) in the Alu RNA KD experiment . Statistical significance ( p value threshold 0 . 05 ) for the comparison between anti - Alu LNA treated samples ( anti - Alu ) and samples treated with a scramble control LNA ( ctrl ) with p = 0 . 049 and n = 3 , unpaired non - directional t - test . Error bars represent standard deviation from the mean . Expression levels of P53 ( RT - qPCR ) in the Alu RNA KD experiment . Statistical significance ( p value threshold 0 . 05 ) for levels in anti - Alu LNA treated samples ( anti - Alu ) greater than samples treated with a scramble control LNA ( ctrl ) with p = 0 . 046 and n = 3 , unpaired directional t - test . Error bars represent standard deviation from the mean .

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

Expression levels of full length Alu RNA (RT-qPCR) in the Alu RNA KD experiment. Statistical significance (p value threshold 0.05) for the comparison between anti-Alu LNA treated samples (anti-Alu) and samples treated with a scramble control LNA (ctrl) with p=0.049 and n=3, unpaired non-directional t-test. Error bars represent standard deviation from the mean. Expression levels of P53 (RT-qPCR) in the Alu RNA KD experiment. Statistical significance (p value threshold 0.05) for levels in anti-Alu LNA treated samples (anti-Alu) greater than samples treated with a scramble control LNA (ctrl) with p=0.046 and n=3, unpaired directional t-test. Error bars represent standard deviation from the mean.


Expression levels ( RT - qPCR ) _ of selected genes from panel A that are either strongly correlated with Alu RNA processing ( left , r > 0 . 5 ) or only weakly / not correlated ( right , r < 0 . 5 ) . Statistical significance ( p value threshold 0 . 05 ) for anti - Alu greater than control ( depicted as asterisk , n = 3 / group , unpaired directional t - test , error bars represent standard deviation from the mean ) . Pearson correlation co - efficient for each gene is depicted below the name of each gene .

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

Expression levels (RT-qPCR)_of selected genes from panel A that are either strongly correlated with Alu RNA processing (left, r> 0.5) or only weakly/not correlated (right, r <0.5). Statistical significance (p value threshold 0.05) for anti-Alu greater than control (depicted as asterisk, n=3/group, unpaired directional t-test, error bars represent standard deviation from the mean). Pearson correlation co-efficient for each gene is depicted below the name of each gene.


In vitro incubation of one of the Alu RNA consensus sequences for different incubation periods . In vitro transcribed Alu RNA ( 67nM ) incubated at 37oC with 250nM HSF1 in the course of 6 hours with time intervals of 1 hour .

O O O O O 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

In vitro incubation of one of the Alu RNA consensus sequences for different incubation periods. In vitro transcribed Alu RNA (67nM) incubated at 37oC with 250nM HSF1 in the course of 6 hours with time intervals of 1 hour.


Comparison among Hsf1 ( ~ 60KDa ) , denatured HSF1 , Poly A polymerase ( ~ 56KDa ) and no protein ( just TAP buffer ) with regard to Alu RNA processing ( estimating relative full length RNA remaining ) ( two replicates ) . The full gels are available as Source Data For Figure 8 . Relative full length RNA remaining was calculated using ImageJ area under the curve software over time . Error bars represent standard deviation from the mean .

O O B-CONTROLLED_VAR 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

Comparison among Hsf1 (~60KDa), denatured HSF1, Poly A polymerase (~56KDa) and no protein (just TAP buffer) with regard to Alu RNA processing (estimating relative full length RNA remaining) (two replicates). The full gels are available as Source Data For Figure 8. Relative full length RNA remaining was calculated using ImageJ area under the curve software over time. Error bars represent standard deviation from the mean.


Comparison among Alu RNA ( three replicates ) , and two control RNAs ( two replicates ) regarding the full - length RNA levels remaining after in vitro incubation for 90 minutes at 37C with HSF1 . Sizes of control RNAs are control for RNA # 1 , 143nt and for control RNA # 2 , 432nt . Incubation in the absence of HSF1 but presence of the same buffer ( TAP ) was used as control to take into account any non HSF1 specific RNA destabilization due to non - specific degradation . Threshold for statistical significance was a p value of 0 . 05 with p = 0 . 03 ( unpaired , non - directional t - test ) for the comparison between HSF1 and no protein incubation ( n = 3 , for Alu RNA ) . The full gels are available as Source Data For Figure 8 . Error bars represent standard deviation from the mean .

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

Comparison among Alu RNA (three replicates), and two control RNAs (two replicates) regarding the full-length RNA levels remaining after in vitro incubation for 90 minutes at 37C with HSF1. Sizes of control RNAs are control for RNA #1, 143nt and for control RNA #2, 432nt. Incubation in the absence of HSF1 but presence of the same buffer (TAP) was used as control to take into account any non HSF1 specific RNA destabilization due to non-specific degradation. Threshold for statistical significance was a p value of 0.05 with p=0.03 (unpaired, non-directional t-test) for the comparison between HSF1 and no protein incubation (n=3, for Alu RNA). The full gels are available as Source Data For Figure 8. Error bars represent standard deviation from the mean.


Plotting of the position of the first base ( 5 ' end ) of Alu RNA fragments across the Alu consensus sequence produced by Alu RNA that has been processed in vitro for 90 minutes at 37C in the presence of HSF1 ( upper panel ) and compared with one of the in vivo samples of Figure 1 ( lower panel ) . The x axis represents an Alu RNA metagene aligned at the start site of the Alu consensus sequence and the y axis shows the 5 ' end count for Alu RNA fragments aligning to any position downstream of position + 1 . The in vivo sample x axis depicts a 11nt sift compared to the in vitro one .

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

Plotting of the position of the first base (5' end) of Alu RNA fragments across the Alu consensus sequence produced by Alu RNA that has been processed in vitro for 90 minutes at 37C in the presence of HSF1 (upper panel) and compared with one of the in vivo samples of Figure 1 (lower panel). The x axis represents an Alu RNA metagene aligned at the start site of the Alu consensus sequence and the y axis shows the 5' end count for Alu RNA fragments aligning to any position downstream of position +1. The in vivo sample x axis depicts a 11nt sift compared to the in vitro one.


Scatterplot depicting the positive correlation between HSF1 mRNA expression values and Alu RNA processing ratio in MAP AD patients ( r = 0 . 72 , p < 0 . 001 ) . Statistical test is based on Pearson ' s product moment correlation coefficient and follows a t distribution using the cor . test function in R package stats . Scatterplot depicting lack of correlation between HSF1 mRNA expression values and full length Alu RNA levels in MAP AD patients ( ns = non - significant , with p value 0 . 05 as the significance threshold ) . Statistical test as in subfigure 8E .

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

Scatterplot depicting the positive correlation between HSF1 mRNA expression values and Alu RNA processing ratio in MAP AD patients (r=0.72, p<0.001). Statistical test is based on Pearson's product moment correlation coefficient and follows a t distribution using the cor.test function in R package stats. Scatterplot depicting lack of correlation between HSF1 mRNA expression values and full length Alu RNA levels in MAP AD patients (ns=non-significant, with p value 0.05 as the significance threshold). Statistical test as in subfigure 8E.


Human Gut microbial species explored for the existence of subspecies show wide phylogenetic spread according to NCBI taxonomy ( A ) and include Methanobrevibacter smithii , the main archaeal member of the human gut microbiome , as well as representatives of all abundant phyla . Species names are according to NCBI taxonomy , with species cluster ( specI ) identifiers according to Mende et al . , which splits some named species into multiple specI clusters ( Mende et al , 2013 ) . Of 71 investigated species , 44 stratify into subspecies ( highlighted in blue ) . Each species ' average abundance across 2 , 144 human gut metagenomes is proportional to the size of the circles on the cladogram . Bars represent the number of subspecies identified in each , with ' 1 ' indicating no subdivision . The black portion of the bar corresponds to subspecies for which no representative genome sequence is available from NCBI . Geographic enrichments of subspecies are displayed as a heat map ( showing only significant enrichment , FDR corrected Fisher test p - value < 0 . 05 , per country as maximum log - odds ratio across con - specific subspecies ) . Subspecies with a restricted geographic range are predominantl y found in the Chinese and Kazakh population . The 71 investigated species captured an average of 95 . 5 % of sequencing reads that were assigned to any reference genome . The subset of 44 species with identified subspecies accounted for the majority of this abundance ( B ) .

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

Human Gut microbial species explored for the existence of subspecies show wide phylogenetic spread according to NCBI taxonomy (A) and include Methanobrevibacter smithii, the main archaeal member of the human gut microbiome, as well as representatives of all abundant phyla. Species names are according to NCBI taxonomy, with species cluster (specI) identifiers according to Mende et al., which splits some named species into multiple specI clusters (Mende et al, 2013). Of 71 investigated species, 44 stratify into subspecies (highlighted in blue). Each species' average abundance across 2,144 human gut metagenomes is proportional to the size of the circles on the cladogram. Bars represent the number of subspecies identified in each, with '1' indicating no subdivision. The black portion of the bar corresponds to subspecies for which no representative genome sequence is available from NCBI. Geographic enrichments of subspecies are displayed as a heat map (showing only significant enrichment, FDR corrected Fisher test p-value < 0.05, per country as maximum log-odds ratio across con-specific subspecies). Subspecies with a restricted geographic range are predominantly found in the Chinese and Kazakh population. The 71 investigated species captured an average of 95.5% of sequencing reads that were assigned to any reference genome. The subset of 44 species with identified subspecies accounted for the majority of this abundance (B).


Subspecies identified for M . smithii and B . vulgatus / dorei are shown in principle coordinate ( PCoA ) projections of the between - sample distances based on single nucleotide variations ( see Methods ) . The first principal coordinate ( PC ) explains over 70 % of the variation in both cases ( panels A and C ) . Reference genomes have been projected into the same PCoA plots ( marked with ' x ' in A and C , see Methods ) . The numbers adjacent to the placed genomes correspond to those shown in parentheses next to NCBI taxonomy identifiers ( leaves ) on the phylogenetic trees in B and D , respectively . The sample density for each subspecies is highlighted by the histogram above and the total number of samples in which the species could be quantified is indicated in headers . Quantification of the frequency of each subspecies ( bottom plot in A and C ) reveals that for M . smithii only one sample has two subspecies co - occurring in one individual , while all the others have a single dominating one . In contrast , for B . vulgatus / dorei , co - occurrence is more commonly observed . Phylogenies reconstructed from the reference genomes ( NCBI taxonomic identifiers , see Methods ) are fully consistent with the SNP - based clustering . The representative genome for each species , relative to which genomic variants were called , is highlighted with a box .

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

Subspecies identified for M. smithii and B. vulgatus/dorei are shown in principle coordinate (PCoA) projections of the between-sample distances based on single nucleotide variations (see Methods). The first principal coordinate (PC) explains over 70% of the variation in both cases (panels A and C). Reference genomes have been projected into the same PCoA plots (marked with 'x' in A and C, see Methods). The numbers adjacent to the placed genomes correspond to those shown in parentheses next to NCBI taxonomy identifiers (leaves) on the phylogenetic trees in B and D, respectively. The sample density for each subspecies is highlighted by the histogram above and the total number of samples in which the species could be quantified is indicated in headers. Quantification of the frequency of each subspecies (bottom plot in A and C) reveals that for M. smithii only one sample has two subspecies co-occurring in one individual, while all the others have a single dominating one. In contrast, for B. vulgatus/dorei, co-occurrence is more commonly observed. Phylogenies reconstructed from the reference genomes (NCBI taxonomic identifiers, see Methods) are fully consistent with the SNP-based clustering. The representative genome for each species, relative to which genomic variants were called, is highlighted with a box.


( E ) In B . vulgatus / dorei , subspecies composition within each individual is generally stable over time , with a change of the dominant subspecies being rare even over a period of up to 1 , 000 days . An exception ( highlighted by purple line ) is seen for an individual , in which one dominant subspecies is replaced by another one after antibiotic treatment . The right hand panel summarizes subspecies frequency changes , underlining remarkable stability over time .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) In B. vulgatus/dorei, subspecies composition within each individual is generally stable over time, with a change of the dominant subspecies being rare even over a period of up to 1,000 days. An exception (highlighted by purple line) is seen for an individual, in which one dominant subspecies is replaced by another one after antibiotic treatment. The right hand panel summarizes subspecies frequency changes, underlining remarkable stability over time.


Three subspecies of Eubacterium rectale ( A ) are color - coded in PCoA plots . In most individuals we observe dominance of one subspecies at a time ( see frequency plots below PCoAs ) .

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

Three subspecies of Eubacterium rectale (A) are color-coded in PCoA plots. In most individuals we observe dominance of one subspecies at a time (see frequency plots below PCoAs).


four of Eubacterium eligens ( B ) are color - coded in PCoA plots . In most individuals we observe dominance of one subspecies at a time ( see frequency plots below PCoAs ) .

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

four of Eubacterium eligens (B) are color-coded in PCoA plots. In most individuals we observe dominance of one subspecies at a time (see frequency plots below PCoAs).


( D ) Grouping E . rectale individuals from the Danish ( DK ) and Swedish ( SE ) studies , based on this deletion shows a significant ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) increase in BMI and blood fasting insulin levels as well as a decrease in overall community diversity in individuals who are predominantly colonised by the flagellum carrying subspecies ( see Methods ) . The same trend is observed for E . eligens subspecies in the same studies , though not statistically significant ( n . s . ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Grouping E. rectale individuals from the Danish (DK) and Swedish (SE) studies, based on this deletion shows a significant (*p < 0.05, **p < 0.01, ***p < 0.001) increase in BMI and blood fasting insulin levels as well as a decrease in overall community diversity in individuals who are predominantly colonised by the flagellum carrying subspecies (see Methods). The same trend is observed for E. eligens subspecies in the same studies, though not statistically significant (n.s.).


Confocal laser scanning fluorescence microscopy ( CLSM ) images showing DNA staining ( blue in the merged images ) , rDNA FISH ( green ) and NCL immunofluorescence ( red ) in untreated HeLa cells or in cells treated with α ‐ amanitin ( 50 μg / ml ) , AMD ( 50 ng / ml ) or the Pol III inhibitor ML ‐ 60218 ( 200 μM ) for 5 h .

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

Confocal laser scanning fluorescence microscopy (CLSM) images showing DNA staining (blue in the merged images), rDNA FISH (green) and NCL immunofluorescence (red) in untreated HeLa cells or in cells treated with α‐amanitin (50 μg/ml), AMD (50 ng/ml) or the Pol III inhibitor ML‐60218 (200 μM) for 5 h.


CLSM images of propidium iodide ‐ stained RNA after microinjection of buffer or nucleolar RNA into α ‐ amanitin ‐ treated HeLa cells . Nucleoli were visualized by immunofluorescence of nucleophosmin ( NPM ) . The graph represents the average number of nucleolar domains ( ± 95 % CI ) based on the analysis of 90 , 87 , 80 and 86 cells , respectively . * * P ‐ value 0 . 01 , 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 B-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

CLSM images of propidium iodide‐stained RNA after microinjection of buffer or nucleolar RNA into α‐amanitin‐treated HeLa cells. Nucleoli were visualized by immunofluorescence of nucleophosmin (NPM). The graph represents the average number of nucleolar domains (± 95% CI) based on the analysis of 90, 87, 80 and 86 cells, respectively. **P‐value 0.01, t‐test.


CLSM images showing nucleolar co ‐ localization of NCL ( immunofluorescence ) with aluRNA ( RNA FISH ) but not with L1 ‐ repeat RNA . Cells were pre ‐ treated in situ with RNase A or an RNase inhibitor . Nuclei were counterstained with DAPI . The signal intensity of nucleolar aluRNA was two ‐ fold higher compared to nucleoplasmic signal ( n = 92 , P ‐ value 0 . 05 , t ‐ test ) .

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

CLSM images showing nucleolar co‐localization of NCL (immunofluorescence) with aluRNA (RNA FISH) but not with L1‐repeat RNA. Cells were pre‐treated in situ with RNase A or an RNase inhibitor. Nuclei were counterstained with DAPI. The signal intensity of nucleolar aluRNA was two‐fold higher compared to nucleoplasmic signal (n = 92, P‐value 0.05, t‐test).


Graphs representing the average number of nucleolar bodies after microinjection of in vitro transcribed RNA into HeLa cells that were pre ‐ treated with α ‐ amanitin ( 50 μg / ml ) for 5 h or left untreated ( control ) ( ± 95 % CI . * * P ‐ value 0 . 01 , n = 90 , 87 , 86 , 83 or 86 cells , respectively ) . Representative CLSM images of propidium iodide ‐ stained RNA are shown on the right side .

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

Graphs representing the average number of nucleolar bodies after microinjection of in vitro transcribed RNA into HeLa cells that were pre‐treated with α‐amanitin (50 μg/ml) for 5 h or left untreated (control) (± 95% CI. **P‐value 0.01, n = 90, 87, 86, 83 or 86 cells, respectively). Representative CLSM images of propidium iodide‐stained RNA are shown on the right side.


CLSMimages showing NPM ( immunofluorescence , red ) in HeLacells transfected with control ASO , aluRNAASO , or treated with transfection reagent only ( mock ) . Nuclei were counterstained with DAPI ( blue ) . A fraction of 80 ± 8 % of the cells treated with aluRNAASO ( n = 143 ) showed irregular nucleoli or dispersed nucleoli ( 53 % of the cells ) . Only 26 ± 9 % of the cells treated with control ASO presented abnormal nucleoli , with none of them showing a dispersion of nucleolar domains ( n = 123 ) , P ‐ value 0 . 01 , t ‐ test .

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CLSMimages showing NPM (immunofluorescence, red) in HeLacells transfected with control ASO, aluRNAASO, or treated with transfection reagent only (mock). Nuclei were counterstained with DAPI (blue). A fraction of 80 ± 8% of the cells treated with aluRNAASO (n = 143) showed irregular nucleoli or dispersed nucleoli (53% of the cells). Only 26 ± 9% of the cells treated with control ASO presented abnormal nucleoli, with none of them showing a dispersion of nucleolar domains (n = 123), P‐value 0.01, t‐test.


CLSM images showing NCL localization ( immunofluorescence ) , aluRNA ( RNA FISH ) and DNA ( DAPI ) . Transfection of HeLa cells was done as in ( A ) .

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CLSM images showing NCL localization (immunofluorescence), aluRNA (RNA FISH) and DNA (DAPI). Transfection of HeLa cells was done as in (A).


HeLa cells were transfected as in ( A ) , and nascent RNAs were visualized by pulse labeling with ethynyl uridine ( EU ) for 30 min . Nuclei were counterstained with DAPI ( DNA ) .

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HeLa cells were transfected as in (A), and nascent RNAs were visualized by pulse labeling with ethynyl uridine (EU) for 30 min. Nuclei were counterstained with DAPI (DNA).


Levels of 47Spre ‐ rRNA in mock ‐ , control ASO ‐ and aluRNAASO ‐ treated HeLacells . Top : Northern blot . Center : Agarose gel electrophoretic analysis of RNA . Bottom panel : Quantification from RT - qPCR levels of 47Spre ‐ rRNA normalized to 18SrRNA . Error bars represent the standard deviation ( n = 6 ) . * * P ‐ value 0 . 01 , t ‐ test .

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Levels of 47Spre‐rRNA in mock‐, control ASO‐ and aluRNAASO‐treated HeLacells. Top: Northern blot. Center: Agarose gel electrophoretic analysis of RNA. Bottom panel: Quantification from RT-qPCR levels of 47Spre‐rRNA normalized to 18SrRNA. Error bars represent the standard deviation (n = 6). **P‐value 0.01, t‐test.


HeLa cells were transfected with an empty vector or plasmids expressing aluRNA or L1 ‐ repeat RNA and GFP from a separate promoter . Transfected cells were identified by GFP fluorescence , and nucleoli volumes were evaluated based on the immunofluorescence signal of the nucleolar marker NPM . The graphs show that the volume of nucleoli is increased by 30 % in the presence of ectopic aluRNA ( ± 95 % CI , n = 20 ) . * * P ‐ value 0 . 01 , t ‐ test . Scale bar , 10 μm .

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HeLa cells were transfected with an empty vector or plasmids expressing aluRNA or L1‐repeat RNA and GFP from a separate promoter. Transfected cells were identified by GFP fluorescence, and nucleoli volumes were evaluated based on the immunofluorescence signal of the nucleolar marker NPM. The graphs show that the volume of nucleoli is increased by 30% in the presence of ectopic aluRNA (± 95% CI, n = 20). **P‐value 0.01, t‐test. Scale bar, 10 μm.


CLSM live ‐ cell imaging of RFP ‐ NCL in cells transfected with a plasmid expressing aluRNA and imaged for 10 h . The arrows indicate nucleolar domains displaying time ‐ dependent increase in the nucleolar size or fusion of two domains into one . Scale bars , 10 μm .

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CLSM live‐cell imaging of RFP‐NCL in cells transfected with a plasmid expressing aluRNA and imaged for 10 h. The arrows indicate nucleolar domains displaying time‐dependent increase in the nucleolar size or fusion of two domains into one. Scale bars, 10 μm.


HeLa cells were transfected as in ( A ) and incubated for 24 h . Nascent RNA was pulse ‐ labeled for 30 min , and nucleoli were visualized by immunofluorescence of NPM . Scale bars , 10 μm .

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HeLa cells were transfected as in (A) and incubated for 24 h. Nascent RNA was pulse‐labeled for 30 min, and nucleoli were visualized by immunofluorescence of NPM. Scale bars, 10 μm.


Graphs represent the intensity of nucleolar fluorescence signals as percentage of the intensity of nuclear signals and reveal a 27 % increase in the presence of ectopic aluRNA ( ± 95 % CI , n = 30 ) . * * P ‐ value 0 . 01 , * P ‐ value = 0 . 01 , t ‐ test .

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Graphs represent the intensity of nucleolar fluorescence signals as percentage of the intensity of nuclear signals and reveal a 27% increase in the presence of ectopic aluRNA (± 95% CI, n = 30). **P‐value 0.01, *P‐value = 0.01, t‐test.


CLSMimages of UBFimmunofluorescence in mouseNIH 3T3cells treated for 5 h with α ‐ amanitin to inhibit Pol II transcription show the dispersion of nucleoli , as previously also imaged via RNAstaining ( Caudron ‐ Herger et al , ) .

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CLSMimages of UBFimmunofluorescence in mouseNIH 3T3cells treated for 5 h with α‐amanitin to inhibit Pol II transcription show the dispersion of nucleoli, as previously also imaged via RNAstaining (Caudron‐Herger et al, ).


CLSMimages showing the localization of NPM ( immunofluorescence , red ) in mouse NIH 3T3 cells treated with ASO as indicated and counterstained with DAPI ( blue ) .

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CLSMimages showing the localization of NPM (immunofluorescence, red) in mouse NIH 3T3 cells treated with ASO as indicated and counterstained with DAPI (blue).


CLSM images of MS2 ‐ tagged aluRNA or MS2 ‐ tagged control RNA ( a fragment from the transcript of the CDV3 gene ) . MS2 ‐ loop ‐ containing RNAs were visualized by RNA FISH using MS2 ‐ loop ‐ specific FISH probes ( green ) ; nucleoli were visualized by immunofluorescence of NCL ( red ) .

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CLSM images of MS2‐tagged aluRNA or MS2‐tagged control RNA (a fragment from the transcript of the CDV3 gene). MS2‐loop‐containing RNAs were visualized by RNA FISH using MS2‐loop‐specific FISH probes (green); nucleoli were visualized by immunofluorescence of NCL (red).


The top panel depicts the experimental approach used to tether MS2 ‐ loop ‐ containing RNAs to lacO arrays via a GFP ‐ tagged LacI ‐ MS2 coat fusion protein . The lacO arrays were stably integrated in the genome of U2OS cells . In the bottom panel , CLSM images of U2OS cells are shown that were transfected with MS2 ‐ aluRNAR . They reveal the localization of LacI ‐ GFP ‐ MS2 and NCL ( immunofluorescence , red ) . The arrow indicates the lacO array , which is associated with a nucleolar domain .

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The top panel depicts the experimental approach used to tether MS2‐loop‐containing RNAs to lacO arrays via a GFP‐tagged LacI‐MS2 coat fusion protein. The lacO arrays were stably integrated in the genome of U2OS cells. In the bottom panel, CLSM images of U2OS cells are shown that were transfected with MS2‐aluRNAR. They reveal the localization of LacI‐GFP‐MS2 and NCL (immunofluorescence, red). The arrow indicates the lacO array, which is associated with a nucleolar domain.


MS2 ‐ loop ‐ containing forward aluRNA or aluRNAL or aluRNAR were recruited to the single stably integrated lacO array in the U2OS F4IIB8 cell line . The propensity for nucleolar localization was evaluated as the average number of lacO arrays with tethered RNA detected in nucleoli ( ± 95 % CI ) and is plotted in the bar chart . Calculations are based on analysis of more than 100 cells . * * P ‐ value 0 . 01 , t ‐ test , from the analysis of two independent biological replicates . As controls , transcripts of the MS2 loops only , the MS2 ‐ CDV3 , MS2 ‐ RepA , MS2 ‐ STARD7 and MS2 ‐ CORO1C RNAs were used . The inset shows a CLSM image with MS2 ‐ GFP ‐ LacI ( green ) and NPM ( red , immunofluorescence ) after recruitment of MS2 ‐ aluRNA .

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MS2‐loop‐containing forward aluRNA or aluRNAL or aluRNAR were recruited to the single stably integrated lacO array in the U2OS F4IIB8 cell line. The propensity for nucleolar localization was evaluated as the average number of lacO arrays with tethered RNA detected in nucleoli (± 95% CI) and is plotted in the bar chart. Calculations are based on analysis of more than 100 cells. **P‐value 0.01, t‐test, from the analysis of two independent biological replicates. As controls, transcripts of the MS2 loops only, the MS2‐CDV3, MS2‐RepA, MS2‐STARD7 and MS2‐CORO1C RNAs were used. The inset shows a CLSM image with MS2‐GFP‐LacI (green) and NPM (red, immunofluorescence) after recruitment of MS2‐aluRNA.


Top : Scheme illustrating the experimental approach used to tether GFP ‐ tagged LacI fusion proteins via a GFP ‐ binding protein ( GBP ) to lacO arrays . The lacO arrays are stably integrated in the genome of U2OS cells . Bottom : CLSM images showing the localization of GBP ‐ LacI ‐ RFP , GFP ‐ NCL , GFP ‐ NPM or GFP ‐ TIP5 ‐ RBD fusion proteins relative to aluRNA that was visualized by RNA FISH . The insets contain an enlarged image of one of the lacO loci .

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Top: Scheme illustrating the experimental approach used to tether GFP‐tagged LacI fusion proteins via a GFP‐binding protein (GBP) to lacO arrays. The lacO arrays are stably integrated in the genome of U2OS cells. Bottom: CLSM images showing the localization of GBP‐LacI‐RFP, GFP‐NCL, GFP‐NPM or GFP‐TIP5‐RBD fusion proteins relative to aluRNA that was visualized by RNA FISH. The insets contain an enlarged image of one of the lacO loci.


CLSM images showing GBP ‐ LacI ‐ RFP and GFP ‐ NCL ( green ) recruited to lacO arrays as indicated by arrows . Immunofluorescence of endogenous NPM ( red ) marks nucleoli . The inset shows decondensation of lacO arrays in the nucleolus . The graph at the bottom depicts the average number of lacO loci tethered to nucleoli ( ± 95 % CI ) by the indicated proteins ( n = 72 ) . NCL ‐ ∆ RNA is an NCL deletion mutant lacking the RBD and GAR domains required for RNA binding . * * P ‐ value 0 . 01 , t ‐ test .

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CLSM images showing GBP‐LacI‐RFP and GFP‐NCL (green) recruited to lacO arrays as indicated by arrows. Immunofluorescence of endogenous NPM (red) marks nucleoli. The inset shows decondensation of lacO arrays in the nucleolus. The graph at the bottom depicts the average number of lacO loci tethered to nucleoli (± 95% CI) by the indicated proteins (n = 72). NCL‐∆RNA is an NCL deletion mutant lacking the RBD and GAR domains required for RNA binding. **P‐value 0.01, t‐test.


CLSM images of UBF ( immunofluorescence , green ) or rDNA ( DNA FISH , green ) and DNA ( DAPI , blue ) after knockdown of NCL and NPM by siRNA in HeLa cells .

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CLSM images of UBF (immunofluorescence, green) or rDNA (DNA FISH, green) and DNA (DAPI, blue) after knockdown of NCL and NPM by siRNA in HeLa cells.


Western blots for detection of TCF7 , LEF1 , TCF7L1 , TCF7L2 and β - catenin ( B " ) proteins in wild - type ( WT ) and knockout ( K . O . )

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Western blots for detection of TCF7, LEF1, TCF7L1, TCF7L2 and β-catenin (B") proteins in wild-type (WT) and knockout (K.O.)


TCF - reporter TOP - flash assay performed on the indicated cell lines treated with 10 μM CHIR , for 24 hrs . The control FOP - flash reporter is not activated upon CHIR administration . The data represent the mean ± s . e . m . of averages of nine independent experiments ( N = 9 ) . dBcat , β - catenin knockout cells ; d4TCF , TCF / LEF quadruple knockout cells Transfection of individual TCF - expressing plasmids could restore the ability of d4TCF cells to respond to Wnt3 as measured in a TOP - flash

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TCF-reporter TOP-flash assay performed on the indicated cell lines treated with 10 μM CHIR, for 24 hrs. The control FOP-flash reporter is not activated upon CHIR administration. The data represent the mean ± s.e.m. of averages of nine independent experiments (N = 9). dBcat, β-catenin knockout cells; d4TCF, TCF/LEF quadruple knockout cells Transfection of individual TCF-expressing plasmids could restore the ability of d4TCF cells to respond to Wnt3 as measured in a TOP-flash assa


Quantitative RT - PCR analyses of Axin2 transcripts , performed on the different cell lines treated with 10 μM CHIR or DMSO ( control ) for 24 hrs . Error bars show that standard deviation obtained from 3 independent experiments Transfection of individual TCF - expressing plasmids could restore the ability of d4TCF cells to respond t CHI as measured by Axin2 mRNA

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Quantitative RT-PCR analyses of Axin2 transcripts, performed on the different cell lines treated with 10 μM CHIR or DMSO (control) for 24 hrs. Error bars show that standard deviation obtained from 3 independent experiments Transfection of individual TCF-expressing plasmids could restore the ability of d4TCF cells to respond t CHI as measured by Axin2 mRNA abundance


a Heat map showing the upregulated genes in wild - type ( WT ) parental HEK 293T cells upon CHIR treatment , ranked based on the log2 fold change . Increased and decreased transcription is depicted in red and blue , respectively . Gene names are indicated along the y - axis . Canonical Wnt targets are among the most upregulated genes in WT cells ( red boxes ) . The genotypes of the cell clones used is indicated on the bottom of the heat map . Triplicates of each cell line are included . b Hierarchical clustering of differentially expressed genes with at least a 2 - fold change in any comparison . Dendrogram clustering indicates the relationships between the different experimental conditions . c Volcano plot displays differentially regulated genes in d4TCF ( C ) and dBcat ( C " ) compared to WT parental cells . Red dots indicate significantly regulated genes based on adjusted p - value and log - fold - change ( logFC ) . d Smear plots for every cell line indicating the behaviour of every gene after CHIR treatment . Differential gene expression is indicated by the log2 fold change ( logFC ) , and the average logCPM ( logarithm of counts per million reads ) was used to estimate the accuracy of the

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a Heat map showing the upregulated genes in wild-type (WT) parental HEK 293T cells upon CHIR treatment, ranked based on the log2 fold change. Increased and decreased transcription is depicted in red and blue, respectively. Gene names are indicated along the y-axis. Canonical Wnt targets are among the most upregulated genes in WT cells (red boxes). The genotypes of the cell clones used is indicated on the bottom of the heat map. Triplicates of each cell line are included. b Hierarchical clustering of differentially expressed genes with at least a 2-fold change in any comparison. Dendrogram clustering indicates the relationships between the different experimental conditions. c Volcano plot displays differentially regulated genes in d4TCF (C) and dBcat (C") compared to WT parental cells. Red dots indicate significantly regulated genes based on adjusted p-value and log-fold-change (logFC). d Smear plots for every cell line indicating the behaviour of every gene after CHIR treatment. Differential gene expression is indicated by the log2 fold change (logFC), and the average logCPM (logarithm of counts per million reads) was used to estimate the accuracy of the expression


e RT - qPCR - based validation of three differentially expressed genes , upon CHIR stimulation in WT , dBcat or d4TCF cell lines . The data represent the mean ± s . e . m . of averages of three independent experiments ( N = 3 ) . Samples were compared using Student " s t - test . Asterisks ( * ) indicate a p - value < 0 . 05 , and ( * * ) a p - value <

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e RT-qPCR-based validation of three differentially expressed genes, upon CHIR stimulation in WT, dBcat or d4TCF cell lines. The data represent the mean ± s.e.m. of averages of three independent experiments (N = 3). Samples were compared using Student"s t-test. Asterisks (*) indicate a p-value<0.05, and (**) a p-value<


b , c RT - qPCR - based validation of selected TCF - independent β - catenin - dependent target genes ( B ) upregulated or ( C ) downregulated in d4TCF cells ( TCF - independent ) treated with 10μM CHIR or DMSO for 24hr . Data represent the mean ± s . e . m . of averages of three independent experiments ( N = 3 ) . Samples were compared using Student " s t - test . Asterisks ( * ) indicate a p - value <

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b, c RT-qPCR-based validation of selected TCF-independent β-catenin-dependent target genes (B) upregulated or (C) downregulated in d4TCF cells (TCF-independent) treated with 10μM CHIR or DMSO for 24hr. Data represent the mean ± s.e.m. of averages of three independent experiments (N = 3). Samples were compared using Student"s t-test. Asterisks (*) indicate a p-value<


( A , B ) β - catenin occupancy on regulatory regions of the prototypical Wnt target genes , AXIN2 and LEF1 ( A ) , and at the promoter regions of HOXC4 and ZNF503 ( B )

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(A, B) β-catenin occupancy on regulatory regions of the prototypical Wnt target genes, AXIN2 and LEF1 (A), and at the promoter regions of HOXC4 and ZNF503 (B)


( C ) β - catenin ChIP - seq enrichment over ENCODE TCF7L2 ( also known as TCF4 ) ChIP peaks in HEK293T cells . The data represent the mean ± 95 % C . I . obtained from three independent experiments ( N = 3 )

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(C) β-catenin ChIP-seq enrichment over ENCODE TCF7L2 (also known as TCF4) ChIP peaks in HEK293T cells. The data represent the mean ± 95% C.I. obtained from three independent experiments (N = 3)


b RT - qPCR validation of the β - catenin - GHOST response . A selection of 7 genes ( colour coded ) that are activated upon CHIR treatment in d4TCF , or in wild - type ( WT ) in combination with ICAT overexpression , but not in pentaKO cells - indicating that their regulation is dependent on β - catenin but can only occur in the absence of TCF / LEF - β - catenin interaction . The genotype and the treatment for each condition is indicated in the x - axis . Error bars show that standard deviation obtained from 3 independent

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b RT-qPCR validation of the β-catenin-GHOST response. A selection of 7 genes (colour coded) that are activated upon CHIR treatment in d4TCF, or in wild-type (WT) in combination with ICAT overexpression, but not in pentaKO cells - indicating that their regulation is dependent on β-catenin but can only occur in the absence of TCF/LEF-β-catenin interaction. The genotype and the treatment for each condition is indicated in the x-axis. Error bars show that standard deviation obtained from 3 independent experiments


c FOXO4 but not FOXO3 overexpression upregulates the β - catenin - GHOST target GADD45 both in CHIR - treated WT ( black bars ) or d4TCF ( grey bars ) cells . Samples were compared using Student " s t - test . Asterisks ( * ) indicate a p - value <

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c FOXO4 but not FOXO3 overexpression upregulates the β-catenin-GHOST target GADD45 both in CHIR-treated WT (black bars) or d4TCF (grey bars) cells. Samples were compared using Student"s t-test. Asterisks (*) indicate a p-value<


d FOXO4 overexpression induced GADD45 transcription in WT ( black bars ) but not in dBcat ( grey bars ) cells . Samples were compared using Student " s t - test . Asterisks ( * ) indicate a p - value <

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d FOXO4 overexpression induced GADD45 transcription in WT (black bars) but not in dBcat (grey bars) cells. Samples were compared using Student"s t-test. Asterisks (*) indicate a p-value<


e Endogenous FOXO4 , when immunoprecipitated ( IP ) pulls down β - catenin . Overexpressed FOXO4 increases the amount of β - catenin detected in the immunoprecipitated , indicating physical

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e Endogenous FOXO4, when immunoprecipitated (IP) pulls down β-catenin. Overexpressed FOXO4 increases the amount of β-catenin detected in the immunoprecipitated, indicating physical association


f GADD45 is upregulated by CHIR in d4TCF cell ; this positive transcriptional regulation is blocked by FOXO4 - specific siRNA but not control scrambled siRNA . All experiments were done at least three times ( N = 3 ) . Samples were compared using Student " s t - test . Asterisks ( * * ) indicate a p - value < 0 . 02 ; ns , non - statistically significant change was

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f GADD45 is upregulated by CHIR in d4TCF cell; this positive transcriptional regulation is blocked by FOXO4-specific siRNA but not control scrambled siRNA. All experiments were done at least three times (N=3). Samples were compared using Student"s t-test. Asterisks (**) indicate a p-value<0.02; ns, non-statistically significant change was obs


A ) U2OS cells were harvested at the indicated time points after treatment with ionizing radiation ( 15 Gy ) followed by analysis by SDS - PAGE and immunoblotting with antibodies recognizing PALB2 and Vinculin . ( NT , no treatment ) . The lysate from the 24 hours time point was treated with phosphatase ( Ppase ) .

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A) U2OS cells were harvested at the indicated time points after treatment with ionizing radiation (15 Gy) followed by analysis by SDS-PAGE and immunoblotting with antibodies recognizing PALB2 and Vinculin. (NT, no treatment). The lysate from the 24 hours time point was treated with phosphatase (Ppase).


B ) U2OS cells were left untreated or treated with ATM ( KU 55933 , 10 μM ) , ATR ( ETP - 464 , 1 μM ) , or DNA - PK ( NU7441 , 1 μM ) inhibitors 30 minutes prior to exposure to IR ( 15 Gy , 2h recovery ) . Immunoblots were performed with indicated antibodies .

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B) U2OS cells were left untreated or treated with ATM (KU 55933, 10 μM), ATR (ETP-464, 1 μM), or DNA-PK (NU7441, 1 μM) inhibitors 30 minutes prior to exposure to IR (15 Gy, 2h recovery). Immunoblots were performed with indicated antibodies.


C ) U2OS cells were left untreated ( NT ) or exposed to HU ( 2 mM ) for the indicated times . The lysate from the 24 hour time point was treated with phosphatase ( Ppase ) . Immunoblots were performed with indicated antibodies .

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C) U2OS cells were left untreated (NT) or exposed to HU (2 mM) for the indicated times. The lysate from the 24 hour time point was treated with phosphatase (Ppase). Immunoblots were performed with indicated antibodies.


D ) Untreated U2OS cells or cells exposed to either HU ( 2 mM , 24h ) or IR ( 15 Gy , 2h recovery ) alone or together with ATM inhibitor ( KU55933 , 10 μM ) or inhibitors to ATR ( ATR # 1 : ETP - 464 , 1 μM ; ATR # 2 : AZ - 20 , 3 μM ) were harvested and lysates were prepared . The inhibitors were added 30 minutes prior to HU or IR treatment . Immunoblots were performed with indicated antibodies .

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D) Untreated U2OS cells or cells exposed to either HU (2 mM, 24h) or IR (15 Gy, 2h recovery) alone or together with ATM inhibitor (KU55933, 10 μM) or inhibitors to ATR (ATR#1: ETP-464, 1 μM; ATR#2: AZ-20, 3 μM) were harvested and lysates were prepared. The inhibitors were added 30 minutes prior to HU or IR treatment. Immunoblots were performed with indicated antibodies.


E ) ATR kinase assay was performed with N - terminal PALB2 ( aa 1 - 560 ; GST - PALB2 - N ) as a substrate and HA - tagged ATR as kinase and visualized by Fujifilm Phosphoimager . 293T cells transfected with empty vector ( EV ) , GST , and ATR inhibition ( AZ - 20 , 30 μM ) are controls . HA - ATR and GST - PALB2 - N was controlled by immunoblotting with HA and GST antibodies .

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E) ATR kinase assay was performed with N-terminal PALB2 (aa 1-560; GST-PALB2-N) as a substrate and HA-tagged ATR as kinase and visualized by Fujifilm Phosphoimager. 293T cells transfected with empty vector (EV), GST, and ATR inhibition (AZ-20, 30 μM) are controls. HA-ATR and GST-PALB2-N was controlled by immunoblotting with HA and GST antibodies.


B ) Characterization of the Doxycycline - inducible cell lines expressing siRNA resistant wild type ( WT ) or mutant ( TMA or TMD ) FLAG / HA - tagged PALB2 . The cell lines were transfected with UNC ( Negative Control ) or PALB2 siRNA for 24 hours prior to induction with Doxycycline . Immunoblots were performed with indicated antibodies .

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B) Characterization of the Doxycycline-inducible cell lines expressing siRNA resistant wild type (WT) or mutant (TMA or TMD) FLAG/HA-tagged PALB2. The cell lines were transfected with UNC (Negative Control) or PALB2 siRNA for 24 hours prior to induction with Doxycycline. Immunoblots were performed with indicated antibodies.


C ) Cell line characterization of FLAG / HA - tagged PALB2 localization 2 hours following exposure to 15 Gy of IR . Z - stack max intensity image projection and background subtraction was done using ImageJ / Fiji . Scale bar = 5μm .

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C) Cell line characterization of FLAG/HA-tagged PALB2 localization 2 hours following exposure to 15 Gy of IR. Z-stack max intensity image projection and background subtraction was done using ImageJ/Fiji. Scale bar = 5μm.


D ) Kinase assay was performed as described in Figure 1E except that the SDS - PAGE gel was Coomassie stained following electrophoresis . WT was used at two different concentrations ( 1x and 0 . 75x ) .

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D) Kinase assay was performed as described in Figure 1E except that the SDS-PAGE gel was Coomassie stained following electrophoresis. WT was used at two different concentrations (1x and 0.75x).


E ) Doxycycline - inducible stable U2OS cell lines were left untreated or treated with Doxycycline and / or IR ( 15 Gy , 2h recovery ) . Following protein extraction the lysates were immunoprecipitated ( IP ) with IgG or antibody recognizing phosphorylated S / Q sites ( pS / Q ) and analyzed by SDS - PAGE . Immunoblots were performed with indicated antibodies .

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E) Doxycycline-inducible stable U2OS cell lines were left untreated or treated with Doxycycline and/or IR (15 Gy, 2h recovery). Following protein extraction the lysates were immunoprecipitated (IP) with IgG or antibody recognizing phosphorylated S/Q sites (pS/Q) and analyzed by SDS-PAGE. Immunoblots were performed with indicated antibodies.


A ) PALB2 cell lines transfected with PALB2 siRNA followed by inducible expression of siRNA resistant PALB2 versions were exposed to 15 Gy of IR and fixed for IF 2 hours later . Z - stack images were acquired with a 60X oil objective ( Deltavision ) . Z stack max intensity projection , background subtraction , and foci count was done using ImageJ / Fiji . ( * p < 0 . 0001 , unpaired Student ' s t - test ) . Scale bar = 5μm .

O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) PALB2 cell lines transfected with PALB2 siRNA followed by inducible expression of siRNA resistant PALB2 versions were exposed to 15 Gy of IR and fixed for IF 2 hours later. Z-stack images were acquired with a 60X oil objective (Deltavision). Z stack max intensity projection, background subtraction, and foci count was done using ImageJ/Fiji. (* p<0.0001, unpaired Student's t-test). Scale bar = 5μm.


B ) PALB2 cell lines were transfected and the exogenous PALB2 induced as in Figure 3A before they were pulsed with 10 μM EdU for 20 minutes prior to addition of 2 mM HU . 24 hours later the cells were fixed and processed for IF as in Figure 3A . Cells in S - phase ( EdU + ) at the time of HU treatment were Click - IT labeled with an Alexa Fluor 647 azide and RAD51 foci in EdU positive cells were enumerated using ImageJ / Fiji . ( * p < 0 . 0001 , unpaired Student ' s t - test ) . Scale bar = 5μm .

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B) PALB2 cell lines were transfected and the exogenous PALB2 induced as in Figure 3A before they were pulsed with 10 μM EdU for 20 minutes prior to addition of 2 mM HU. 24 hours later the cells were fixed and processed for IF as in Figure 3A. Cells in S-phase (EdU+) at the time of HU treatment were Click-IT labeled with an Alexa Fluor 647 azide and RAD51 foci in EdU positive cells were enumerated using ImageJ/Fiji. (* p<0.0001, unpaired Student's t-test). Scale bar=5μm.


C ) HeLa DR - GFP cell line was used to analyze homologous recombination activity . Cells were transfected with PALB2 siRNA followed by transfection of I - SceI together with empty vector ( EV ) , WT - , TMA - , or TMD - PALB2 the next day . 72 hours later cells were fixed and analyzed by FACS . The percentage of cells with GFP signal from the empty vector sample was set to one ( dotted line ) . The graph shows a representative image from three biological repeats ( error bars = SEM ) .

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C) HeLa DR-GFP cell line was used to analyze homologous recombination activity. Cells were transfected with PALB2 siRNA followed by transfection of I-SceI together with empty vector (EV), WT-, TMA-, or TMD-PALB2 the next day. 72 hours later cells were fixed and analyzed by FACS. The percentage of cells with GFP signal from the empty vector sample was set to one (dotted line). The graph shows a representative image from three biological repeats (error bars=SEM).


D ) Phosphorylation of PALB2 supports RAD51 foci . Cells were transfected with siPALB2 overnight followed by forward transfection of siFBH1 / siBLM for 6 hours then induced for expression of PALB2 ( WT and TMA ) . 24 hours following induction cells were pulsed with EdU and exposed to HU as in Figure 3B . Cells in S - phase ( EdU + ) at the time of HU treatment were Click - IT labeled with an Alexa Fluor 647 azide and RAD51 foci in EdU positive cells were enumerated using ImageJ / Fiji ( * p < 0 . 0001 , unpaired Student ' s t - test ) . Representative images of displaying RAD51 ( green ) and HA - PALB2 ( red ) localization in HU treated EdU positive ( not shown ) cells . Scale bar = 5μm .

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D) Phosphorylation of PALB2 supports RAD51 foci. Cells were transfected with siPALB2 overnight followed by forward transfection of siFBH1/siBLM for 6 hours then induced for expression of PALB2 (WT and TMA). 24 hours following induction cells were pulsed with EdU and exposed to HU as in Figure 3B. Cells in S-phase (EdU+) at the time of HU treatment were Click-IT labeled with an Alexa Fluor 647 azide and RAD51 foci in EdU positive cells were enumerated using ImageJ/Fiji (* p<0.0001, unpaired Student's t-test). Representative images of displaying RAD51 (green) and HA-PALB2 (red) localization in HU treated EdU positive (not shown) cells. Scale bar=5μm.


A ) U2OS and PALB2 cell lines were transfected with UNC ( Negative Control ) or PALB2 siRNA and 24 hours later Doxycycline was added to the PALB2 cell lines . Left panel , cells were treated the following day with IR ( 5 Gy ) and 2 hours later Nocodazole was added for 6 hours , or treated with only Nocodazole for 6 hours . Right panel , cells were treated with IR ( 5 Gy ) and left to recover for 16 hours . Cells were fixed and stained with the pMPM2 antibody to detect the mitotic cells by flow cytometry . The percentages of mitotic cells in the IR + Nocodazole samples were normalized to the Nocodazole samples ( left panel , n = 3 , error bars = SEM ) . For the samples with the 16 hour recovery after IR the percentage of mitotic cells in the WT - PALB2 expressing cells was set to one ( right panel ) . ( n = 3 , * * p < 0 . 005 , * p < 0 . 05 , unpaired Student ' s t - test , error bars = SEM ) .

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A) U2OS and PALB2 cell lines were transfected with UNC (Negative Control) or PALB2 siRNA and 24 hours later Doxycycline was added to the PALB2 cell lines. Left panel, cells were treated the following day with IR (5 Gy) and 2 hours later Nocodazole was added for 6 hours, or treated with only Nocodazole for 6 hours. Right panel, cells were treated with IR (5 Gy) and left to recover for 16 hours. Cells were fixed and stained with the pMPM2 antibody to detect the mitotic cells by flow cytometry. The percentages of mitotic cells in the IR+Nocodazole samples were normalized to the Nocodazole samples (left panel, n=3, error bars=SEM). For the samples with the 16 hour recovery after IR the percentage of mitotic cells in the WT-PALB2 expressing cells was set to one (right panel). (n=3, **p<0.005, *p<0.05, unpaired Student's t-test, error bars=SEM).


B ) The relative intensity of phosphorylated H2AX ( pS139 ) was examined in the total population of WT - and TMA - PALB2 expressing cell lines 18 hours post exposure to IR ( 5 Gy ) . Cell lines were prepared as in above , fixed at the indicated time point , and stained for pH2AX . Cells were imaged with a 20X air objective on a Scan ^ R workstation ( Olympus ) , mean relative pH2AX intensity was calculated from background subtracted images using the Scan ^ R analysis software . ( * p < 0 . 0001 , unpaired Student ' s t - test ) .

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B) The relative intensity of phosphorylated H2AX (pS139) was examined in the total population of WT- and TMA-PALB2 expressing cell lines 18 hours post exposure to IR (5 Gy). Cell lines were prepared as in above, fixed at the indicated time point, and stained for pH2AX. Cells were imaged with a 20X air objective on a Scan^R workstation (Olympus), mean relative pH2AX intensity was calculated from background subtracted images using the Scan^R analysis software. (* p<0.0001, unpaired Student's t-test).


C ) Number of 53BP1 nuclear in TMA - PALB2 expressing G1 daughter cells . Cell lines were prepared as above and left untreated ( NT ) or exposed to IR ( 3 Gy ) , and 16 hours later Cytochalasin B was added for 8 hours to block cytokinesis . The irradiated bi - nucleated cells were scored for 53BP1 nuclear bodies . Scale bar = 10μm .

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C) Number of 53BP1 nuclear in TMA-PALB2 expressing G1 daughter cells. Cell lines were prepared as above and left untreated (NT) or exposed to IR (3 Gy), and 16 hours later Cytochalasin B was added for 8 hours to block cytokinesis. The irradiated bi-nucleated cells were scored for 53BP1 nuclear bodies. Scale bar = 10μm.


Nurr1 ( N ) and Foxa2 ( F ) synergistically downregulate SGK1 expression in cultured glia . ( A ) Up - and downregulated genes in the microarray data for the cultured glia transduced with N + F ( vs . mock - transduced control ) . Sgk1 is marked with an arrow in the top downregulated genes .

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Nurr1 (N) and Foxa2 (F) synergistically downregulate SGK1 expression in cultured glia. (A) Up- and downregulated genes in the microarray data for the cultured glia transduced with N+F (vs. mock-transduced control). Sgk1 is marked with an arrow in the top downregulated genes.


Effect of N and F on SGK1 expression in glial cells shown in the microarray ( B ) , RNA - seq ( C ) , Data in ( B and C ) represent values of fold change ( FC ) or read count ( RC ) with color intensities . n = 3 . One - way ANOVA . Data are represented as mean ± SEM . Significantly different at p = 0 . 0493 * , 0 . 0092 * * , 0 . 0009 * * * .

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Effect of N and F on SGK1 expression in glial cells shown in the microarray (B), RNA-seq (C), Data in (B and C) represent values of fold change (FC) or read count (RC) with color intensities. n=3. One-way ANOVA. Data are represented as mean ± SEM. Significantly different at p= 0.0493*, 0.0092**, 0.0009***.


Effect of N and F on SGK1 expression in glial cells shown in the RT - PCR analyses . One - way ANOVA . Data are represented as mean ± SEM . Significantly different at p = 0 . 0493 * , 0 . 0092 * * , 0 . 0009 * * * .

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Effect of N and F on SGK1 expression in glial cells shown in the RT-PCR analyses. One-way ANOVA. Data are represented as mean ± SEM. Significantly different at p= 0.0493*, 0.0092**, 0.0009***.


Downregulation of SGK1 is responsible for N + F function to inhibit NFκB - mediated pro - inflammatory cytokine expression . ( E ) WB analysis exhibiting N + F - induced inhibition of NFκB intracellular signaling in cultured VM - glia . The N + F - mediated downregulation of NFκB signaling is attained by inhibiting IκB phosphorylation

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Downregulation of SGK1 is responsible for N+F function to inhibit NFκB-mediated pro-inflammatory cytokine expression. (E) WB analysis exhibiting N+F-induced inhibition of NFκB intracellular signaling in cultured VM-glia. The N+F-mediated downregulation of NFκB signaling is attained by inhibiting IκB phosphorylation


Downregulation of SGK1 is responsible for N + F function to inhibit NFκB - mediated pro - inflammatory cytokine expression . ( E ) WB analysis exhibiting N + F - induced inhibition of NFκB intracellular signaling in cultured VM - glia . The N + F - mediated downregulation of NFκB signaling is attained by inhibiting IκB

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WB analysis exhibiting N + F - induced inhibition of NFκB intracellular signaling in cultured VM - glia . The N + F - mediated downregulation of NFκB signaling is attained by inhibiting IκB phosphorylation ( E ) and thus blocking the release of NFκB from the NFκB - IκB inhibitory complex ( F ) .

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WB analysis exhibiting N+F-induced inhibition of NFκB intracellular signaling in cultured VM-glia. The N+F-mediated downregulation of NFκB signaling is attained by inhibiting IκB phosphorylation (E) and thus blocking the release of NFκB from the NFκB-IκB inhibitory complex (F).


N + F - mediated downregulation of NFκB signaling ( phosphorylation of p65 , p - p65 , G ) were alleviated by forced SGK1 expression in the VM - glial cultures .

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N+F-mediated downregulation of NFκB signaling (phosphorylation of p65, p-p65, G) were alleviated by forced SGK1 expression in the VM-glial cultures.


H ) N + F - mediated downregulation of pro - inflammatory cytokine expression ( IL - 1β , TNFα , H ) were alleviated by forced SGK1 expression in the VM - glial cultures .

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H) N+F-mediated downregulation of pro-inflammatory cytokine expression (IL-1β, TNFα, H) were alleviated by forced SGK1 expression in the VM-glial cultures.


( I ) SGK1 inhibition blocks glial NFκB signaling . Data are represented as mean ± SEM . n = 4 - 6 . One - way ANOVA . Significantly different at p = 0 . 002 * , 0 . 00004 * * , 0 . 00001 # , 3 . 01E - 10 # # , 3 . 78E - 06 + , 1 . 59E - 07 + + in graph I .

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(I) SGK1 inhibition blocks glial NFκB signaling. Data are represented as mean ± SEM. n=4-6. One-way ANOVA. Significantly different at p=0.002*, 0.00004**, 0.00001#, 3.01E-10##, 3.78E-06+, 1.59E-07++ in graph I.


Glial pro - inflammatory cytokine expression regulated by SGK1 inhibition

Glial pro-inflammatory cytokine expression regulated by SGK1 inhibition


Glial pro - inflammatory cytokine expression regulated by SGK1


Glial pro - inflammatory cytokine expression regulated by SGK1 inhibition ( J - M ) and overexpression ( N ) .

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Glial pro-inflammatory cytokine expression regulated by SGK1 inhibition (J-M) and overexpression (N).


In the RNA - seq data ( GSE145490 , normalized read count ( RC ) > 1 , > 2 . 6 - fold , 3 , 000 genes ) , genes downregulated in the VM - glia treated with the SGK1 inhibitor GSK - 650394 vs the vehicle ( DMSO ) - treated control were analyzed . A and B , Top gene ontologies ( GOs ) and KEGG pathways of the genes downregulated in VM - glia by the SGK1 inhibitor treatment . The purple bar indicates the number of genes under the designated GO term / KEGG pathway . The red bar indicates the p - value , and the negative log of the p - value ( bottom ) is plotted on the X - axis .

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In the RNA-seq data (GSE145490, normalized read count (RC)>1, >2.6-fold, 3,000 genes), genes downregulated in the VM-glia treated with the SGK1 inhibitor GSK-650394 vs the vehicle (DMSO)-treated control were analyzed. A and B, Top gene ontologies (GOs) and KEGG pathways of the genes downregulated in VM-glia by the SGK1 inhibitor treatment. The purple bar indicates the number of genes under the designated GO term/KEGG pathway. The red bar indicates the p-value, and the negative log of the p-value (bottom) is plotted on the X-axis.


In the RNA - seq data ( GSE145490 , normalized read count ( RC ) > 1 , > 2 . 6 - fold , 3 , 000 genes ) , genes downregulated in the VM - glia treated with the SGK1 inhibitor GSK - 650394 vs the vehicle ( DMSO ) - treated control were analyzed .

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In the RNA-seq data (GSE145490, normalized read count (RC)>1, >2.6-fold, 3,000 genes), genes downregulated in the VM-glia treated with the SGK1 inhibitor GSK-650394 vs the vehicle (DMSO)-treated control were analyzed.


C , Gene set enrichment analysis ( GESA ) for inflammatory and immune responses .

C, Gene set enrichment analysis (GESA) for inflammatory and immune responses.


D , Scatterplots for the differentially expressed genes ( DEGs ) highlighting the inflammatory / immune genes that are most significantly downregulated in the inhibiter - treated cultures .

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D, Scatterplots for the differentially expressed genes (DEGs) highlighting the inflammatory/immune genes that are most significantly downregulated in the inhibiter-treated cultures.


E , Heatmap for selected inflammatory genes in the SGK1 inhibitor - treated and - untreated glial cultures . Data represent the RC values ( inside box ) and log2 [ SGK1 inhibitor - treated / control ] ( color intensity ) .

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E, Heatmap for selected inflammatory genes in the SGK1 inhibitor-treated and -untreated glial cultures. Data represent the RC values (inside box) and log2[SGK1 inhibitor-treated/control] (color intensity).


F , Microglia and astrocyte reactivation - specific marker expressions in the RNA - seq data .

F, Microglia and astrocyte reactivation-specific marker expressions in the RNA-seq data.


mRNA expression of the NLRP3 - inflammasome components Nlrp3 , Pycard ( Asc ) , and Caspase1 . Decreased expression of the components shown in the RNA - seq Data are expressed as mean ± SEM . Significantly different at p = 0 . 0018 * , 0 . 0405 * * , 0 . 0039 * * * , 0 . 0002 # , 0 . 0033 # # , 0 . 0099 # # # , 0 . 0001 # # # # , 0 . 0056 + , 0 . 0398 + + , 0 . 0067 + + +

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mRNA expression of the NLRP3-inflammasome components Nlrp3, Pycard (Asc), and Caspase1. Decreased expression of the components shown in the RNA-seq Data are expressed as mean ± SEM. Significantly different at p=0.0018*, 0.0405**, 0.0039***, 0.0002#, 0.0033##, 0.0099###, 0.0001####, 0.0056+, 0.0398++, 0.0067+++


B , mRNA expression of the NLRP3 - inflammasome components Nlrp3 , Pycard ( Asc ) , and Caspase1 . Decreased expression of the components shown in the q - PCR analyses in other independent VM - glial cultures treated with the inhibitor ( GSK - 650394 ) and sh - Sgk1 and si - Sgk1 ( vs . DMSO , sh - control , and si - control , respectively ) ( B ) . n = 3 ; Student ' s t - test . Data are expressed as mean ± SEM . Significantly different at p = 0 . 0018 * , 0 . 0405 * * , 0 . 0039 * * * , 0 . 0002 # , 0 . 0033 # # , 0 . 0099 # # # , 0 . 0001 # # # # , 0 . 0056 + , 0 . 0398 + + , 0 . 0067 + + + in graph B .

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B, mRNA expression of the NLRP3-inflammasome components Nlrp3, Pycard (Asc), and Caspase1. Decreased expression of the components shown in the q-PCR analyses in other independent VM-glial cultures treated with the inhibitor (GSK-650394) and sh-Sgk1 and si-Sgk1 (vs. DMSO, sh-control, and si-control, respectively) (B). n=3; Student's t-test. Data are expressed as mean ± SEM. Significantly different at p=0.0018*, 0.0405**, 0.0039***, 0.0002#, 0.0033##, 0.0099###, 0.0001####, 0.0056+, 0.0398++, 0.0067+++ in graph B.


C , Immunoblot analysis to assess NLRP3 inflammasome activation . To activate the inflammasome pathway , VM - derived glial cells were treated with LPS ( 0 . 25 µg / ml , 3h ) and then ATP ( 2 . 5 mM , 30 min ) in the presence or absence of the SGK1 inhibitor GSK - 650394 . Two days later , intracellular and released levels of pro - and activated caspase - 1 and IL - 1β proteins were determined in the culture media and cell lysates , respectively .

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C, Immunoblot analysis to assess NLRP3 inflammasome activation. To activate the inflammasome pathway, VM-derived glial cells were treated with LPS (0.25 µg/ml, 3h) and then ATP (2.5 mM, 30 min) in the presence or absence of the SGK1 inhibitor GSK-650394. Two days later, intracellular and released levels of pro- and activated caspase-1 and IL-1β proteins were determined in the culture media and cell lysates, respectively.


D , Released level of IL - 1β ( activated ) was further quantified using ELISA . n = 3 ; ANOVA . Significantly different at p = 0 . 0002 # , 0 . 0012 * , 0 . 0004 * * in graph D .

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D, Released level of IL-1β (activated) was further quantified using ELISA. n=3; ANOVA. Significantly different at p=0.0002#, 0.0012*, 0.0004** in graph D.


Expression of CGAS - STING pathway genes in the RNA - seq data . The graph represents log2 RC ratios of [ GSK - 650394 - treated / DMSO - treated control ] .

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Expression of CGAS-STING pathway genes in the RNA-seq data. The graph represents log2 RC ratios of [GSK-650394-treated/DMSO-treated control].


G , WB analysis demonstrating suppression of CGAS - STING signaling by the SGK1 inhibitor in VM - glial cultures . Representative blots are shown on the left . The levels of the active forms of CGAS - STING signaling molecules are quantified from 5 - 10 independent blots ( right ) . Data are represented as mean ± SEM . One - way ANOVA . Significantly different at p = 0 . 001 # , 0 . 014 * ( CGAS ) , 0 . 00004 # , 0 . 0002 * ( STING ) , 0 . 017 # , 0 . 037 * ( pTBK1 ) , 0 . 009 # , 0 . 045 * ( pIRF3 ) , 0 . 012 # , 0 . 036 * ( pP65 ) in graph G .

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G, WB analysis demonstrating suppression of CGAS-STING signaling by the SGK1 inhibitor in VM-glial cultures. Representative blots are shown on the left. The levels of the active forms of CGAS-STING signaling molecules are quantified from 5-10 independent blots (right). Data are represented as mean ± SEM. One-way ANOVA. Significantly different at p=0.001#, 0.014* (CGAS), 0.00004#, 0.0002* (STING), 0.017#, 0.037* (pTBK1), 0.009#, 0.045* (pIRF3), 0.012#, 0.036* (pP65) in graph G.


H , Expression of Ifnb mRNA , a final product of the CGAS - STING pathway , determined by real - time PCR analyses . Data are represented as mean ± SEM . n = 3 ; student ' s t - test . Significantly different at p = 0 . 0459 # , 0 . 0305 * in graph H .

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H, Expression of Ifnb mRNA, a final product of the CGAS-STING pathway, determined by real-time PCR analyses. Data are represented as mean ± SEM. n=3; student's t-test. Significantly different at p=0.0459#, 0.0305* in graph H.


Ppargc1a ( PGC1α ) mRNA expression upregulated in VM - glia treated with the SGK1 inhibitors in the RNA - seq data Data are represented as mean ± SEM . n = 3 ; Student ' s t - test . Significantly different at p = 0 . 0011 # , 0 . 0499 * in graph B .

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Ppargc1a (PGC1α) mRNA expression upregulated in VM-glia treated with the SGK1 inhibitors in the RNA-seq data Data are represented as mean ± SEM. n=3; Student's t-test. Significantly different at p=0.0011#, 0.0499* in graph B.


B , Ppargc1a ( PGC1α ) mRNA expression upregulated in VM - glia treated with the SGK1 inhibitors in the q - PCR analysis Data are represented as mean ± SEM . n = 3 ; Student ' s t - test . Significantly different at p = 0 . 0011 # , 0 . 0499 * in graph B .

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B, Ppargc1a (PGC1α) mRNA expression upregulated in VM-glia treated with the SGK1 inhibitors in the q-PCR analysis Data are represented as mean ± SEM. n=3; Student's t-test. Significantly different at p=0.0011#, 0.0499* in graph B.


C , Immunoblot for PGC1α protein expression . Data are represented as mean ± SEM . n = 3 . Student ' s t - test . Significantly different at p = 0 . 0077 * in graph C .

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C, Immunoblot for PGC1α protein expression. Data are represented as mean ± SEM. n=3. Student's t-test. Significantly different at p=0.0077* in graph C.


D , Mitochondrial biogenesis estimated by Mitotimer reporter gene expression in cultured VM - glia . Green fluorescence indicates newly synthesized mitochondria , and red fluorescence indicates old or damaged mitochondria . MFIs were estimated in 3 independent cultures ( > 5 randomly selected microscopic fields / culture ) . Data are represented as mean ± SEM . One - way ANOVA . Significantly different at p = 3 . 78E - 08 * , 0 . 00003 * * in graph D .

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D, Mitochondrial biogenesis estimated by Mitotimer reporter gene expression in cultured VM-glia. Green fluorescence indicates newly synthesized mitochondria, and red fluorescence indicates old or damaged mitochondria. MFIs were estimated in 3 independent cultures (>5 randomly selected microscopic fields/culture). Data are represented as mean ± SEM. One-way ANOVA. Significantly different at p=3.78E-08*, 0.00003** in graph D.


E , Mitochondrial ROS levels estimated using the MitoSox red probe . Fluorescence intensity was measured using ImageJ , and ROS levels are presented as mean fluorescent intensity ( MFI ) . Mitochondrial damage was induced by treatment with the mitochondrial toxins CCCP ( 2µM ) + H2O2 ( 500 µM ) for 4hr . MFIs were estimated in 3 independent cultures ( > 5 randomly selected microscopic fields / culture ) . Data are represented as mean ± SEM . One - way ANOVA . Significantly different at p = 0 . 0013 * , 0 . 0273 # in graph E .

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E, Mitochondrial ROS levels estimated using the MitoSox red probe. Fluorescence intensity was measured using ImageJ, and ROS levels are presented as mean fluorescent intensity (MFI). Mitochondrial damage was induced by treatment with the mitochondrial toxins CCCP (2µM)+H2O2(500 µM) for 4hr. MFIs were estimated in 3 independent cultures (>5 randomly selected microscopic fields/culture). Data are represented as mean ± SEM. One-way ANOVA. Significantly different at p=0.0013*, 0.0273# in graph E.


F , Mitochondrial membrane potential ( JC - 1 ) . The red fluorescence ( JC - 1 Aggregate ) indicates disrupted mitochondrial membrane potential , and green fluorescence ( JC - 1 Monomer , insets ) indicates intact potential . The inset images were taken from the same microscopic fields of the respective JC - 1 aggregate images . Mitochondrial damage was induced by treatment with the mitochondrial toxins CCCP ( 2µM ) + H2O2 ( 500 µM ) for 4hr . MFIs were estimated in 3 independent cultures ( > 5 randomly selected microscopic fields / culture ) . Data are represented as mean ± SEM . One - way ANOVA . Significantly different at p = 0 . 0006 * , 0 . 0093 # in graph F .

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F, Mitochondrial membrane potential (JC-1). The red fluorescence (JC-1 Aggregate) indicates disrupted mitochondrial membrane potential, and green fluorescence (JC-1 Monomer, insets) indicates intact potential. The inset images were taken from the same microscopic fields of the respective JC-1 aggregate images. Mitochondrial damage was induced by treatment with the mitochondrial toxins CCCP (2µM)+H2O2(500 µM) for 4hr. MFIs were estimated in 3 independent cultures (>5 randomly selected microscopic fields/culture). Data are represented as mean ± SEM. One-way ANOVA. Significantly different at p=0.0006*, 0.0093# in graph F.


G , Intracellular levels of ATP , an indicator of mitochondrial oxidative phosphorylation . Data are expressed as mean ± SEM . n = 3 cultures ; one - way ANOVA . Significantly different at p = 0 . 0009 * , 0 . 00004 * * , 0 . 04913 # , 0 . 0024 + in graph G .

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G, Intracellular levels of ATP, an indicator of mitochondrial oxidative phosphorylation. Data are expressed as mean ± SEM. n=3 cultures; one-way ANOVA. Significantly different at p= 0.0009*, 0.00004**, 0.04913#, 0.0024+in graph G.


Glial cell senescence rescued by SGK1 inhibition . ( H ) Expression of genes associated with cell senescence in the RNA - seq data ( VM - glia with vs . without GSK - 650394 treatment ) .

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Glial cell senescence rescued by SGK1 inhibition. (H) Expression of genes associated with cell senescence in the RNA-seq data (VM-glia with vs. without GSK-650394 treatment).


( I ) Intracellular ROS levels estimated by DCF - DA . Data are represented as mean ± SEM . n = 3 ; one - way ANOVA . Significantly different at p = 0 . 0074 * , 0 . 012 # in graph I .

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(I) Intracellular ROS levels estimated by DCF-DA. Data are represented as mean ± SEM. n=3; one-way ANOVA. Significantly different at p=0.0074*, 0.012# in graph I.


( J ) Senescence - associated β - galactosidase ( SA - β - gal ) activity . Data are represented as mean ± SEM . n = 3 ; one - way ANOVA . Significantly different at p = 2 . 05E - 06 * , 0 . 0042 # , 0 . 0006 + in graph J .

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(J) Senescence-associated β-galactosidase (SA-β-gal) activity. Data are represented as mean ± SEM. n=3; one-way ANOVA. Significantly different at p=2.05E-06*, 0.0042#, 0.0006+ in graph J.


( K ) Immunoblots for proteins associated with cell senescence . Significantly different at p = 0 . 008 * , 0 . 001 # ( TNFA ) , 0 . 01 * , 0 . 009 # ( HMGB1 ) , 7 . 32E - 10 * , 1 . 23E - 8 # ( IL6 ) , 9 . 62E - 11 * , 1 . 01E - 5 # ( MMP3 ) , 0 . 001 * , 5 . 72E - 4 # ( LAMIN B ) in graph K .

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(K) Immunoblots for proteins associated with cell senescence. Significantly different at p=0.008*, 0.001# (TNFA), 0.01*, 0.009# (HMGB1), 7.32E-10*, 1.23E-8# (IL6), 9.62E-11*, 1.01E-5# (MMP3), 0.001*, 5.72E-4# (LAMIN B) in graph K.


( L ) IL - 6 levels secreted from cultured VM - glia . The cytokine levels were estimated using ELISA of the media from the VM - glia with and without the SGK1 inhibitor . Data are represented as mean ± SEM . n = 3 cultures . One - way ANOVA . Significantly different at p = 0 . 0028 * , 0 . 0062 # in graph L .

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(L) IL-6 levels secreted from cultured VM-glia. The cytokine levels were estimated using ELISA of the media from the VM-glia with and without the SGK1 inhibitor. Data are represented as mean ± SEM. n=3 cultures. One-way ANOVA. Significantly different at p=0.0028*, 0.0062# in graph L.


( M ) Glial cell viability following treatment with senolytic compounds ( azithromycin , fisetin ) . Data are represented as mean ± SEM . n = 3 ; Student ' s t - test . Significantly different at p = 0 . 0073 * , 0 . 0018 * * , 0 . 0286 # , 0 . 0269 # # , 0 . 0045 # # # in graph M .

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(M) Glial cell viability following treatment with senolytic compounds (azithromycin, fisetin). Data are represented as mean ± SEM. n=3; Student's t-test. Significantly different at p=0.0073*,0.0018**, 0.0286#, 0.0269##, 0.0045### in graph M.


B , Western blot analysis for detecting SNCA oligomers . Data are represented as mean ± SEM . n = 4 ; one - way ANOVA . Significantly different at p = 2 . 5E - 07 * , 1 . 65E - 07 * * , 0 . 022 # , 0 . 029 # # , 0 . 046 + , 0 . 015 + + , 0 . 0136 & , 0 . 0016 & & in graph B .

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B, Western blot analysis for detecting SNCA oligomers. Data are represented as mean ± SEM. n=4; one-way ANOVA. Significantly different at p=2.5E-07*, 1.65E-07**, 0.022#, 0.029##, 0.046+, 0.015++, 0.0136&, 0.0016&& in graph B.


C , Immunocytochemical detection of p - 129 - SNCA , associated with SNCA aggregation . Data are represented as mean ± SEM . n = 6 . Significantly different at p = 0 . 0363 * in graph C .

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C, Immunocytochemical detection of p-129-SNCA, associated with SNCA aggregation. Data are represented as mean ± SEM. n=6. Significantly different at p=0.0363* in graph C.


E , Bi - FC - based assessment of the inter - neuronal propagation and aggregation of SNCA . Bi - FC + inclusions ( puncta ) were counted in E . Data are represented as mean ± SEM . n = 5 . Significantly different at p = 0 . 0446 * in graph E .

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E, Bi-FC-based assessment of the inter-neuronal propagation and aggregation of SNCA. Bi-FC+ inclusions (puncta) were counted in E. Data are represented as mean ± SEM. n=5. Significantly different at p=0.0446* in graph E.


VM - glial cultures were transduced with sh - Sgk1 ( or sh - cont ) lentivirus . Medium was condition in sh - Sgk1 - glia ( or sh - cont - glia ) , and the conditioned medium ( CM ) treatment effects on neuronal SNCA aggregation were assessed by WB

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VM-glial cultures were transduced with sh-Sgk1 (or sh-cont) lentivirus. Medium was condition in sh-Sgk1-glia (or sh-cont-glia), and the conditioned medium (CM) treatment effects on neuronal SNCA aggregation were assessed by WB


VM - glial cultures were transduced with sh - Sgk1 ( or sh - cont ) lentivirus . Medium was condition in sh - Sgk1 - glia ( or sh - cont - glia ) , and the conditioned medium ( CM ) treatment effects on neuronal SNCA aggregation were assessed by

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