( D , E ) - Seq . of transcriptome isolated from pax2amKO2 - High pax2amKO2 - Low subpopulations . ( D ) Volcano plot representing differential expression of genes between two thyrocyte subpopulations . Dots for pax2a , tg , mKO2 EGFP are marked . Dots for genes that display significant differential expression ( DESeq2 , adjusted p - value < 0 . 05 ) are coloured . ( E ) displaying expression of selected genes in two subpopulations . Biological replicates are represented by columns .

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

(D , E) RNA-Seq. analysis of transcriptome isolated from pax2amKO2-High and pax2amKO2-Low subpopulations. (D) Volcano plot representing the differential expression of genes between the two thyrocyte subpopulations. Dots for pax2a, tg, mKO2 and EGFP are marked. Dots for genes that display significant differential expression (DESeq2, adjusted p-value < 0.05) are coloured. (E) Heatmap displaying the expression of selected genes in the two subpopulations. Biological replicates are represented by individual columns.


Plotting of position of 5 ' end of Alu fragments across Alu metagene to depict potential processing areas of Alu RNAs in post - mortem hippocampal tissues ( PM hippo ) from from CBB . Each row in depicts distribution of of 5 ' ends of reads mapped across Alu metagene for each . x - axis represents a metagene combining unique Alu sequences ( ALUome ) aligned at start site of their consensus with numbers representing from Transcriptional Start Site ( TSS ) . density corresponds to normalized of 5 ' end of reads with red corresponding to higher density of these 5 ' ends at a specific position . XL1 , XR1 XR2 denote Alu processing areas defined by high - density areas in at specific positions of Alu metagene , with middle letter corresponding to arm of Alu ( see Figure 1C , L = Left , R = Right ) , in which is located .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5'end of Alu RNA fragments across the Alu metagene to depict potential processing areas of Alu RNAs in all post-mortem hippocampal tissues (PM hippo) from patients from the CBB. Each row in the heatmap depicts the distribution of counts of the 5'ends of reads mapped across the Alu metagene for each patient. The x-axis represents a metagene combining all unique Alu RNA sequences (ALUome) aligned at the start site of their consensus sequence with numbers representing the distance from the Transcriptional Start Site (TSS) area. Heatmap density corresponds to normalized counts of the 5'end of the reads with red corresponding to higher density of these 5'ends at a specific position. XL1, XR1 and XR2 denote the Alu processing areas defined by the high-density areas in the heatmap at specific positions of the Alu metagene, with the middle letter corresponding to the arm of the Alu RNA (see Figure 1C, L=Left, R=Right), in which the area is located.


Normalized of processed fragments mapping to right arm of Alu RNAs that are differentially processed ( rows ) between AD no AD for each CBB ( columns ) ( Dataset EV1 ) . Red corresponds to higher normalized of processed Alu fragments .

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

Normalized counts of processed fragments mapping to the right arm of Alu RNAs that are differentially processed (rows) between AD and no AD for each CBB patient (columns) (Dataset EV1). Red corresponds to higher normalized counts of processed Alu RNA fragments.


Boxplots depict differences in hippocampi of AD no AD regarding SINE Alu processing ( left panel ) ( a p value of 0 . 05 was considered as threshold for statistical significance with p = 0 . 01 , n = 24 , unpaired non - directional t - ) in total Alu ( right panel ) ( ns = not significant ) . In boxplots , central band ( that divides box into 2 parts ) represents median of data , ends of box shows upper ( Q3 ) lower ( Q1 ) quartiles , extreme shows Q3 + 1 . 5xIQR to Q1 - 1 . 5xIQR ( highest lowest value excluding potential outliers ) , while dots beyond extreme show potential outliers .

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

Boxplots depict differences in hippocampi of AD and no AD patients regarding SINE Alu RNA processing ratio (left panel) (a p value of 0.05 was considered as threshold for statistical significance with p=0.01 , n=24, unpaired non-directional t-test) and in total Alu RNA levels (right panel) (ns=not significant). In the boxplots, the central band (the line that divides the box into 2 parts) represents the median of the data, the ends of the box shows the upper (Q3) and lower (Q1) quartiles, the extreme line shows Q3+1.5xIQR to Q1-1.5xIQR (the highest and lowest value excluding potential outliers), while dots beyond the extreme line show potential outliers.


Plotting of position of 5 ' end of Alu fragments across Alu metagene to depict potential processing areas of Alu RNAs in post - mortem cortex tissues from from MAP cohort . As in Figure 1B each row represents distribution of normalized of 5 ' ends of reads mapped across Alu metagene for each . As in Figure 1B x - axis numbers represent from Transcriptional Start Site ( TSS ) density corresponds to normalized of 5 ' end of reads with red for higher density of these 5 ' ends at a specific position . XR1 as in Figure 1B .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5'end of Alu RNA fragments across the Alu metagene to depict potential processing areas of Alu RNAs in all post-mortem cortex tissues from patients from the MAP cohort. As in Figure 1B each row represents the distribution of normalized counts of the 5'ends of reads mapped across the Alu metagene for each patient. As in Figure 1B x-axis numbers represent the distance from the Transcriptional Start Site (TSS) area and heatmap density corresponds to normalized counts of the 5'end of the reads with red for higher density of these 5'ends at a specific position. XR1 as in Figure 1B.


As in Figure 2B , normalized of processed fragments mapping to right arm of Alu RNAs that are differentially processed ( rows ) between AD no AD for each MAP ( columns ) ( Dataset EV2 ) . Red corresponds to higher normalized of processed Alu fragments .

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

As in Figure 2B, normalized counts of processed fragments mapping to the right arm of Alu RNAs that are differentially processed (rows) between AD and no AD for each MAP patient (columns) (Dataset EV2). Red corresponds to higher normalized counts of processed Alu RNA fragments.


Boxplots depict differences in cortex of AD no AD regarding SINE Alu processing ( left panel ) ( a p value of 0 . 05 was considered as threshold for statistical significance with p < 0 . 001 , n = 118 , unpaired non - directional t - ) in full Alu RNAs ( p = 0 . 005 ) . Boxplot representation as in subfigure 2D .

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

Boxplots depict differences in cortex of AD and no AD patients regarding SINE Alu RNA processing ratio (left panel) (a p value of 0.05 was considered as threshold for statistical significance with p<0.001, n=118, unpaired non-directional t-test) and in full length Alu RNAs (p=0.005). Boxplot representation as in subfigure 2D.


Boxplots depict full Alu in cortex of AD no AD separated into three groups . No significant difference observed between different groups of either AD or no AD or for comparisons between no AD AD of each group ( unpaired non - directional t - , with p < 0 . 05 considered threshold for statistical significance , n for no AD = 19 ( below 85 ) , 19 ( between 85 to 90 ) , 13 ( above 90 ) ; n for AD = 8 ( below 85 ) , 21 ( between 85 to 90 ) , 38 ( above 90 ) ) . Boxplot representation as in subfigure 2D .

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

Boxplots depict full length Alu RNA levels in cortex of AD and no AD patients separated into three age groups. No significant difference observed between the different age groups of either AD or no AD patients or for the comparisons between no AD and AD of each age group (unpaired non-directional t-test, with p<0.05 considered the threshold for statistical significance, n for no AD=19 (below 85), 19 (between 85 to 90), 13 (above 90); n for AD=8 (below 85), 21 (between 85 to 90), 38 (above 90)). Boxplot representation as in subfigure 2D.


Boxplots depict differences in SINE Alu processing in cortex of AD no AD separated into three groups . No significant difference observed between different groups of either AD or no AD . A p value 0 . 05 was considered as threshold for statistical significance for comparisons between no AD AD of each group ( unpaired non - directional t - , with p = 0 . 003 for comparison below 85 , p < 0 . 001 for other two comparisons n numbers as in ( E ) ) . Boxplot representation as in subfigure 2D .

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

Boxplots depict differences in SINE Alu RNA processing ratio in cortex of AD and no AD patients separated into three age groups. No significant difference observed between the different age groups of either AD or no AD patients. A p value 0.05 was considered as threshold for statistical significance for the comparisons between no AD and AD of each age group (unpaired non-directional t-test, with p=0.003 for the comparison below 85, and p < 0.001 for the other two comparisons and n numbers as in (E)). Boxplot representation as in subfigure 2D.


Boxplots depict differences in SINE Alu processing in MAP with regard to final clinical diagnosis ( p < 0 . 01 for comparison between NCI MCI , between NCI AD , between MCI AD , unpaired , non directional t - ) . NCI = no cognitive impairment ( n = 71 ) , MCI = mild cognitive impairment ( n = 67 ) , AD = Alzheimer ' s disease ( n = 87 ) . Boxplot representation as in subfigure 2D . Boxplots depict differences in SINE Alu processing in MAP ( n = 241 ) with regard to Braak stage classified into three categories ( p = 0 . 004 for comparison between 0 - 2 3 - 4 , p < 0 . 001 for comparisons between 3 - 4 5 - 6 between 0 - 2 5 - 6 , unpaired , non directional t - , n = 39 [ 0 - 2 ] , n = 148 [ 3 - 4 ] n = 54 [ 5 - 6 ] ) . Boxplot representation as in subfigure 2D .

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

Boxplots depict differences in SINE Alu RNA processing ratio in all MAP patients with regard to final clinical diagnosis (p < 0.01 for the comparison between NCI and MCI, between NCI and AD, and between MCI and AD, unpaired, non directional t-test). NCI=no cognitive impairment (n=71), MCI=mild cognitive impairment (n=67), AD=Alzheimer's disease (n=87). Boxplot representation as in subfigure 2D. Boxplots depict differences in SINE Alu RNA processing ratio in all MAP patients (n=241) with regard to Braak stage classified into three categories (p = 0.004 for the comparison between 0-2 and 3-4, and p < 0.001 for the comparisons between 3-4 and 5-6 and between 0-2 and 5-6, unpaired, non directional t-test, n=39[0-2],n=148[3-4] and n=54[5-6]). Boxplot representation as in subfigure 2D.


Scatterplot depicting positive correlation between TP53 expression values Alu processing in AD ( r = 0 . 65 , p < 0 . 001 ) . Statistical is on Pearson ' s product moment correlation coefficient follows a t distribution using cor . function in R package stats . Scatterplot depicting no correlation between TP53 expression values full Alu in AD ( r = 0 . 06 , no correlation , ns = non - significant ) . Statistical as in subfigure 5C .

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 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

Scatterplot depicting the positive correlation between TP53 expression values and Alu RNA processing ratio in AD patients (r=0.65, 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 no correlation between TP53 expression values and full length Alu RNA levels in AD patients (r=0.06, no correlation, ns=non-significant). Statistical test as in subfigure 5C.


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

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

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 Alu processing for AD upregulated genes ( Dataset EV3 ) ( upper panel ) a random set of non - differentially expressed genes ( Dataset EV5 ) ( lower panel ) . For every gene a correlation coefficient was calculated ( Pearson correlation ) together with respective p - value . Statistical is on Pearson ' s product moment correlation coefficient follows a t distribution using cor . function in R package stats . on r 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 ) strong correlation ( 0 .

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

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 ( RT - qPCR ) _ of selected genes from panel A that are either strongly correlated with Alu 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 - , error bars represent standard deviation from mean ) . Pearson correlation co - efficient for each gene is depicted below 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

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 Alu consensus sequences for different incubation periods . In vitro transcribed Alu ( 67nM ) incubated at 37oC with 250nM HSF1 in 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 B-CONTROLLED_VAR 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 ) no ( just TAP buffer ) with regard to Alu processing ( estimating relative full remaining ) ( two replicates ) . full gels are available as Source Data For Figure 8 . Relative full remaining was calculated using ImageJ under curve software over time . Error bars represent standard deviation from 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

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 ( three replicates ) , two control RNAs ( two replicates ) regarding full - remaining after in vitro incubation for 90 minutes at 37C with HSF1 . Sizes of control RNAs are control for # 1 , 143nt for control # 2 , 432nt . Incubation in absence of HSF1 but presence of same buffer ( TAP ) was used as control to take into account any non HSF1 specific 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 - ) for comparison between HSF1 no incubation ( n = 3 , for Alu ) . full gels are available as Source Data For Figure 8 . Error bars represent standard deviation from 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-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 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

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

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

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 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

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 existence of subspecies show wide phylogenetic spread according to NCBI taxonomy ( A ) include Methanobrevibacter smithii , main archaeal member of human gut microbiome , as well as representatives of 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 of circles on cladogram . Bars represent number of subspecies identified in each , with ' 1 ' indicating no subdivision . black portion of bar corresponds to subspecies for which no representative genome is available from NCBI . Geographic enrichments of subspecies are displayed as a heat map ( showing only significant enrichment , FDR corrected Fisher p - value < 0 . 05 , per country as maximum log - odds across con - specific subspecies ) . Subspecies with a restricted geographic range are predominantl y found in Chinese Kazakh population . 71 investigated species captured an average of 95 . 5 % of sequencing reads that were assigned to any reference genome . subset of 44 species with identified subspecies accounted for 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

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

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 is generally stable over time , with a change of dominant subspecies being rare even over a period of up to 1 , 000 days . An exception ( highlighted by purple ) is seen for an , in which one dominant subspecies is replaced by another one after antibiotic treatment . right panel summarizes subspecies frequency changes , underlining remarkable stability over time .

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(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 ) .

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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 ) .

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

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

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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 of propidium iodide ‐ stained after microinjection of buffer or nucleolar into α ‐ amanitin ‐ treated HeLa . Nucleoli were visualized by immunofluorescence of nucleophosmin ( NPM ) . graph represents average number of nucleolar domains ( ± 95 % CI ) on of 90 , 87 , 80 86 , respectively . * * P ‐ value 0 . 01 , t ‐ .

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

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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 average number of nucleolar bodies after microinjection of in vitro transcribed into HeLa 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 , respectively ) . Representative CLSM of propidium iodide ‐ stained are shown on right side .

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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 treated with aluRNAASO ( n = 143 ) showed irregular nucleoli or dispersed nucleoli ( 53 % of ) . Only 26 ± 9 % of treated with control ASO presented abnormal nucleoli , with none of them showing a dispersion of nucleolar domains ( n = 123 ) , P ‐ value 0 . 01 , t ‐ .

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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 showing NCL localization ( immunofluorescence ) , aluRNA ( FISH ) ( DAPI ) . Transfection of HeLa 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 were transfected as in ( A ) , nascent RNAs were visualized by labeling with ethynyl uridine ( EU ) for 30 min . Nuclei were counterstained with DAPI ( ) .

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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).


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

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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 were transfected with an empty vector or plasmids expressing aluRNA or L1 ‐ repeat GFP from a separate promoter . Transfected were identified by GFP , nucleoli volumes were evaluated on immunofluorescence signal of nucleolar marker NPM . graphs show that volume of nucleoli is increased by 30 % in presence of ectopic aluRNA ( ± 95 % CI , n = 20 ) . * * P ‐ value 0 . 01 , t ‐ . 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 ‐ imaging of RFP ‐ NCL in transfected with a plasmid expressing aluRNA imaged for 10 h . arrows indicate nucleolar domains displaying time ‐ dependent increase in nucleolar 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 were transfected as in ( A ) incubated for 24 h . Nascent was ‐ labeled for 30 min , 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 of nucleolar signals as of of signals reveal a 27 % increase in presence of ectopic aluRNA ( ± 95 % CI , n = 30 ) . * * P ‐ value 0 . 01 , * P ‐ value = 0 . 01 , t ‐ .

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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 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 localization of NPM ( immunofluorescence , red ) in mouse NIH 3T3 treated with ASO as indicated 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 of MS2 ‐ tagged aluRNA or MS2 ‐ tagged control ( a fragment from transcript of CDV3 gene ) . MS2 ‐ loop ‐ containing RNAs were visualized by 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).


top panel depicts experimental approach used to tether MS2 ‐ loop ‐ containing RNAs to lacO arrays via a GFP ‐ tagged LacI ‐ MS2 coat fusion . lacO arrays were stably integrated in genome of U2OS . In bottom panel , CLSM of U2OS are shown that were transfected with MS2 ‐ aluRNAR . They reveal localization of LacI ‐ GFP ‐ MS2 NCL ( immunofluorescence , red ) . arrow indicates 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 single stably integrated lacO array in U2OS F4IIB8 . propensity for nucleolar localization was evaluated as average number of lacO arrays with tethered detected in nucleoli ( ± 95 % CI ) is plotted in bar chart . Calculations are on of more than 100 . * * P ‐ value 0 . 01 , t ‐ , from of two independent biological replicates . As controls , transcripts of MS2 loops only , MS2 ‐ CDV3 , MS2 ‐ RepA , MS2 ‐ STARD7 MS2 ‐ CORO1C RNAs were used . inset shows a CLSM image with MS2 ‐ GFP ‐ LacI ( green ) 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 experimental approach used to tether GFP ‐ tagged LacI fusion via a GFP ‐ binding ( GBP ) to lacO arrays . lacO arrays are stably integrated in genome of U2OS . Bottom : CLSM showing localization of GBP ‐ LacI ‐ RFP , GFP ‐ NCL , GFP ‐ NPM or GFP ‐ TIP5 ‐ RBD fusion relative to aluRNA that was visualized by FISH . insets contain an enlarged image of one of 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 showing GBP ‐ LacI ‐ RFP GFP ‐ NCL ( green ) recruited to lacO arrays as indicated by arrows . Immunofluorescence of endogenous NPM ( red ) marks nucleoli . inset shows decondensation of lacO arrays in nucleolus . graph at bottom depicts average number of lacO loci tethered to nucleoli ( ± 95 % CI ) by indicated ( n = 72 ) . NCL ‐ ∆ is an NCL deletion mutant lacking RBD GAR domains required for binding . * * P ‐ value 0 . 01 , t ‐ .

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

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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 β - catenin ( B " ) in wild - type ( WT ) 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 performed on indicated lines treated with 10 μM CHIR , for 24 hrs . control FOP - flash reporter is not activated upon CHIR administration . data represent mean ± s . e . m . of averages of nine independent experiments ( N = 9 ) . dBcat , β - catenin knockout ; d4TCF , TCF / LEF quadruple knockout Transfection of TCF - expressing plasmids could restore ability of d4TCF 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 different 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 TCF - expressing plasmids could restore ability of d4TCF 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 upregulated genes in wild - type ( WT ) parental HEK 293T upon CHIR treatment , ranked on log2 fold change . Increased decreased transcription is depicted in red blue , respectively . Gene names are indicated along y - axis . Canonical Wnt targets are among most upregulated genes in WT ( red boxes ) . genotypes of clones used is indicated on bottom of heat map . Triplicates of each are included . b Hierarchical clustering of differentially expressed genes with at least a 2 - fold change in any comparison . Dendrogram clustering indicates relationships between different experimental conditions . c Volcano plot displays differentially regulated genes in d4TCF ( C ) dBcat ( C " ) compared to WT parental . Red dots indicate significantly regulated genes on adjusted p - value log - fold - change ( logFC ) . d Smear plots for every indicating behaviour of every gene after CHIR treatment . Differential gene expression is indicated by log2 fold change ( logFC ) , average logCPM ( logarithm of per million reads ) was used to estimate accuracy of

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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 - validation of three differentially expressed genes , upon CHIR stimulation in WT , dBcat or d4TCF lines . data represent mean ± s . e . m . of averages of three independent experiments ( N = 3 ) . Samples were compared using Student " s t - . Asterisks ( * ) indicate a p - value < 0 . 05 , ( * * ) 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 - validation of selected TCF - independent β - catenin - dependent target genes ( B ) upregulated or ( C ) downregulated in d4TCF ( TCF - independent ) treated with 10μM CHIR or DMSO for 24hr . Data represent mean ± s . e . m . of averages of three independent experiments ( N = 3 ) . Samples were compared using Student " s t - . 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 prototypical Wnt target genes , AXIN2 LEF1 ( A ) , at promoter regions of HOXC4 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 . data represent 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 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 - indicating that their regulation is dependent on β - catenin but can only occur in absence of TCF / LEF - β - catenin interaction . genotype treatment for each condition is indicated in 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 β - catenin - GHOST target GADD45 both in CHIR - treated WT ( black bars ) or d4TCF ( grey bars ) . Samples were compared using Student " s t - . 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 ) . Samples were compared using Student " s t - . 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 amount of β - catenin detected in 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 ; this positive transcriptional regulation is blocked by FOXO4 - specific siRNA but not control scrambled siRNA . experiments were done at least three times ( N = 3 ) . Samples were compared using Student " s t - . 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 were harvested at indicated time points after treatment with ionizing radiation ( 15 Gy ) followed by by SDS - PAGE immunoblotting with antibodies recognizing PALB2 Vinculin . ( NT , no treatment ) . lysate from 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 were left untreated or treated with ATM ( KU 55933 , 10 μM ) , ATR ( ETP - 464 , 1 μM ) , or - 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 were left untreated ( NT ) or exposed to HU ( 2 mM ) for indicated times . lysate from 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 or 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 lysates were prepared . 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 was performed with N - terminal PALB2 ( aa 1 - 560 ; GST - PALB2 - N ) as a substrate HA - tagged ATR as kinase visualized by Fujifilm Phosphoimager . 293T transfected with empty vector ( EV ) , GST , ATR inhibition ( AZ - 20 , 30 μM ) are controls . HA - ATR GST - PALB2 - N was controlled by immunoblotting with HA 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 Doxycycline - inducible lines expressing siRNA resistant wild type ( WT ) or mutant ( TMA or TMD ) FLAG / HA - tagged PALB2 . 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 ) characterization of FLAG / HA - tagged PALB2 localization 2 hours following exposure to 15 Gy of IR . Z - stack max image projection 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 was performed as described in Figure 1E except that SDS - PAGE gel was Coomassie stained following electrophoresis . WT was used at two different concentrations ( 1x 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 lines were left untreated or treated with Doxycycline / or IR ( 15 Gy , 2h recovery ) . Following extraction lysates were immunoprecipitated ( IP ) with IgG or antibody recognizing phosphorylated S / Q sites ( pS / Q ) 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 lines transfected with PALB2 siRNA followed by inducible expression of siRNA resistant PALB2 versions were exposed to 15 Gy of IR fixed for IF 2 hours later . Z - stack were acquired with a 60X oil objective ( Deltavision ) . Z stack max projection , background subtraction , foci was done using ImageJ / Fiji . ( * p < 0 . 0001 , unpaired Student ' s t - ) . Scale bar = 5μm .

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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 lines were transfected 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 were fixed processed for IF as in Figure 3A . in S - phase ( EdU + ) at time of HU treatment were Click - IT labeled with an Alexa Fluor 647 azide RAD51 foci in EdU positive were enumerated using ImageJ / Fiji . ( * p < 0 . 0001 , unpaired Student ' s t - ) . 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 was used to analyze homologous recombination activity . were transfected with PALB2 siRNA followed by transfection of I - SceI together with empty vector ( EV ) , WT - , TMA - , or TMD - PALB2 next day . 72 hours later were fixed analyzed by FACS . of with GFP signal from empty vector sample was set to one ( dotted ) . graph shows a representative image from three biological repeats ( error bars = ) .

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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 . were transfected with siPALB2 overnight followed by forward transfection of siFBH1 / siBLM for 6 hours then induced for expression of PALB2 ( WT TMA ) . 24 hours following induction were pulsed with EdU exposed to HU as in Figure 3B . in S - phase ( EdU + ) at time of HU treatment were Click - IT labeled with an Alexa Fluor 647 azide RAD51 foci in EdU positive were enumerated using ImageJ / Fiji ( * p < 0 . 0001 , unpaired Student ' s t - ) . Representative of displaying RAD51 ( green ) HA - PALB2 ( red ) localization in HU treated EdU positive ( not shown ) . 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 PALB2 lines were transfected with UNC ( Negative Control ) or PALB2 siRNA 24 hours later Doxycycline was added to PALB2 lines . Left panel , were treated following day with IR ( 5 Gy ) 2 hours later Nocodazole was added for 6 hours , or treated with only Nocodazole for 6 hours . Right panel , were treated with IR ( 5 Gy ) left to recover for 16 hours . were fixed stained with pMPM2 antibody to detect mitotic by flow cytometry . percentages of mitotic in IR + Nocodazole samples were normalized to Nocodazole samples ( left panel , n = 3 , error bars = ) . For samples with 16 hour recovery after IR of mitotic in WT - PALB2 expressing was set to one ( right panel ) . ( n = 3 , * * p < 0 . 005 , * p < 0 . 05 , unpaired Student ' s t - , error bars = ) .

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

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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 in TMA - PALB2 expressing G1 daughter . lines were prepared as above left untreated ( NT ) or exposed to IR ( 3 Gy ) , 16 hours later Cytochalasin B was added for 8 hours to block cytokinesis . irradiated bi - nucleated were scored for 53BP1 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 ) Foxa2 ( F ) synergistically downregulate SGK1 expression in cultured glia . ( A ) Up - downregulated genes in microarray data for cultured glia transduced with N + F ( vs . mock - transduced control ) . Sgk1 is marked with an arrow in 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 F on SGK1 expression in glial shown in microarray ( B ) , - seq ( C ) , Data in ( B C ) represent values of fold change ( FC ) or read ( RC ) with color intensities . n = 3 . One - way ANOVA . Data are represented as mean ± . 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 F on SGK1 expression in glial shown in RT - PCR analyses . One - way ANOVA . Data are represented as mean ± . 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 exhibiting N + F - induced inhibition of NFκB intracellular signaling in cultured VM - glia . 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 exhibiting N + F - induced inhibition of NFκB intracellular signaling in cultured VM - glia . N + F - mediated downregulation of NFκB signaling is attained by inhibiting IκB

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

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


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

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


D , Scatterplots for differentially expressed genes ( DEGs ) highlighting inflammatory / immune genes that are most significantly downregulated in 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 , for selected inflammatory genes in SGK1 inhibitor - treated - untreated glial cultures . Data represent RC values ( inside box ) log2 [ SGK1 inhibitor - treated / control ] ( color ) .

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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 astrocyte reactivation - specific marker expressions in - seq data .

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


mRNA expression of NLRP3 - inflammasome components Nlrp3 , Pycard ( Asc ) , Caspase1 . Decreased expression of components shown in - seq Data are expressed as mean ± . 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 NLRP3 - inflammasome components Nlrp3 , Pycard ( Asc ) , Caspase1 . Decreased expression of components shown in q - PCR analyses in other independent VM - glial cultures treated with inhibitor ( GSK - 650394 ) sh - Sgk1 si - Sgk1 ( vs . DMSO , sh - control , si - control , respectively ) ( B ) . n = 3 ; Student ' s t - . Data are expressed as mean ± . 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 to assess NLRP3 inflammasome activation . To activate inflammasome pathway , VM - derived glial were treated with LPS ( 0 . 25 µg / ml , 3h ) then ATP ( 2 . 5 mM , 30 min ) in presence or absence of SGK1 inhibitor GSK - 650394 . Two days later , intracellular released of pro - activated caspase - 1 IL - 1β were determined in culture media 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 - seq data . 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 demonstrating suppression of CGAS - STING signaling by SGK1 inhibitor in VM - glial cultures . Representative blots are shown on left . of active forms of CGAS - STING signaling molecules are quantified from 5 - 10 independent blots ( right ) . Data are represented as mean ± . 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 CGAS - STING pathway , determined by real - time PCR analyses . Data are represented as mean ± . n = 3 ; student ' s t - . 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 SGK1 inhibitors in - seq data Data are represented as mean ± . n = 3 ; Student ' s t - . 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 SGK1 inhibitors in q - PCR Data are represented as mean ± . n = 3 ; Student ' s t - . 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 expression . Data are represented as mean ± . n = 3 . Student ' s t - . 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 indicates newly synthesized mitochondria , red indicates old or damaged mitochondria . MFIs were estimated in 3 independent cultures ( > 5 randomly selected microscopic fields / culture ) . Data are represented as mean ± . 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 estimated using MitoSox red probe . was measured using ImageJ , ROS are presented as mean ( MFI ) . Mitochondrial damage was induced by treatment with 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 ± . 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.