A showing unsupervised consensus clustering of TCGA melanomas ( n = 445 : 356 metastases 89 melanomas ) . identified clusters are indicated below : cluster 1 ( n = 55 , red ) , cluster 2 ( n = 304 , blue ) cluster 3 ( n = 86 , green ) . Type of melanoma samples ( or metastatic ) of metastatic site ( subcutaneous , lymph node or distant ) within each cluster are indicated below above , respectively . B showing unsupervised consensus clustering of TCGA melanomas ( n = 89 ) using 1 , 000 most variable expressed genes in same dataset . identified signature clearly divides TCGA dataset in two groups : - A ( n = 34 , red ) - B ( n = 55 , pink ) , as indicated .

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A Heatmap showing the unsupervised consensus clustering of TCGA melanomas (n=445: 356 metastases and 89 primary melanomas). The identified clusters are indicated below the heatmap: cluster 1 (n=55, red), cluster 2 (n=304, blue) and cluster 3 (n=86, green). Type of melanoma samples (primary or metastatic) and of metastatic site (subcutaneous, lymph node or distant) within each cluster are indicated below and above the heatmap, respectively. B Heatmap showing the unsupervised consensus clustering of TCGA primary melanomas (n=89) using the 1,000 most variable expressed genes in the same dataset. The identified signature clearly divides the TCGA dataset in two groups: Primary-A (n=34, red) and Primary-B (n=55, pink), as indicated.


C Boxplots showing distributions of normalized rank - sums of up - regulated ( left panel ) down - regulated ( right panel ) genes in identified signature . Down - regulated genes in - B melanomas better discriminate two groups of melanoma samples ( p = 5 . 9e - 15 , Wilcoxon rank - sum ) . Median , minimum , maximum , 25th , 75th whiskers of ranksum distribution are shown .

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C Boxplots showing the distributions of the normalized rank-sums of the up-regulated (left panel) and down-regulated (right panel) genes in the identified signature. Down-regulated genes in Primary-B melanomas better discriminate the two groups of primary melanoma samples (p=5.9e-15, Wilcoxon rank-sum test). Median, minimum, maximum, 25th, 75th whiskers of ranksum distribution are shown.


A Box whiskers ( from 5th to 95th percentile ) plots depicting TINCR in TCGA SKCM - seq dataset of melanoma samples . TINCR expression is higher in A melanomas , its reduction in - B melanomas groups of metastatic samples is statistically significant ( Mann - Whitney , * * p < 0 . 01 ) . Median , minimum , maximum , 25th , 75th whiskers of expression values are shown .

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A Box and whiskers (from 5th to 95th percentile) plots depicting TINCR RNA levels in the TCGA SKCM RNA-seq dataset of melanoma samples. TINCR expression is higher in Primary A melanomas, and its reduction in Primary-B melanomas and all groups of metastatic samples is statistically significant (Mann-Whitney test, **p< 0.01). Median, minimum, maximum, 25th, 75th whiskers of expression values are shown.


B Scatter - plot showing correlation between expression of TINCR normalized rank - sums s of down - regulated ( upper panel ) up - regulated ( lower panel ) genes in melanoma signature . TINCR expression strongly correlates ( Spearman ' s correlation coefficient of 0 . 79 , Spearman rank p - value : * * p < 0 . 01 ) with expression of down - regulated genes .

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B Scatter-plot showing correlation between the expression of TINCR RNA and normalized rank-sums s of the down-regulated (upper panel) and up-regulated (lower panel) genes in the primary melanoma signature. TINCR expression strongly correlates (Spearman's correlation coefficient of 0.79, Spearman rank p-value: **p<0.01) with the expression of the down-regulated genes.


C Dot plot showing qRT - PCR of TINCR expression in ( 8 ) metastatic ( 8 ) melanoma samples from surgical specimens or - derived xenografts ( 4 8 metastatic PDXs ) . TINCR relative to L32P housekeeping gene are shown . Paired metastasis PDXs are marked with an asterisk . difference between metastatic samples is significant ( Mann - Whitney , * * p < 0 . 01 ) . Median , minimum , maximum , 25th , 75th whiskers of expression values are shown .

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C Dot plot showing qRT-PCR analysis of TINCR expression in primary (8) and metastatic (8) melanoma samples from surgical specimens or patient-derived xenografts (4 primary and 8 metastatic PDXs). TINCR RNA levels relative to the L32P housekeeping gene are shown. Paired primary and metastasis PDXs are marked with an asterisk. The difference between primary and metastatic samples is significant (Mann-Whitney test, **p< 0.01). Median, minimum, maximum, 25th, 75th whiskers of expression values are shown.


A qRT - PCR of TINCR expression in MMC70 WM902B lines , independently infected with two shRNA targeting TINCR expression ( shTINCR - 1 - 2 ) . transduced with shScamble ( shSCR ) were used as control . TINCR relative to L32P housekeeping gene control ( mean ± sd ) measured in three independent biological experiments are shown . In both lines TINCR knockdown efficiency is more than 70 % with two shRNAs . Statistical analyses were performed using Student t - ( * * : p < 0 . 01 ) .

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A qRT-PCR quantification of TINCR RNA expression in MMC70 and WM902B cell lines, independently infected with two shRNA targeting TINCR expression (shTINCR-1 and -2). Cells transduced with shScamble (shSCR) were used as control. TINCR RNA levels relative to L32P housekeeping gene and control (mean±sd) measured in three independent biological experiments are shown. In both cell lines TINCR knockdown efficiency is more than 70% with the two shRNAs. Statistical analyses were performed using Student t -test (**: p<0.01).


B In vitro migration of MMC70 WM902B transduced with shTINCR - 1 , shTINCR - 2 or shSCR . Relative migration ( mean ± sd ) from three independent experiments is indicated . Statistical analyses were performed using Student t - ( * * * : p < 0 . 001 ) .

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B In vitro migration assay of MMC70 and WM902B cells transduced with shTINCR-1, shTINCR-2 or shSCR. Relative migration (mean±sd) from three independent experiments is indicated. Statistical analyses were performed using Student t -test (***: p<0.001).


C In vitro proliferation of shSCR or shTINCR infected MMC70 WM902B assayed 72h after infection at indicated time points using CellTiter - Glo luminescent kit ( Promega ) . growth from three biological replicates is indicated as proliferation rate relative to control ( mean ± sd ) . difference between control TINCR - silenced samples at time points is not significant ( Student t - ) .

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C In vitro cell proliferation of shSCR or shTINCR infected MMC70 and WM902B cells assayed 72h after infection at the indicated time points using CellTiter-Glo luminescent kit (Promega). Cell growth from three biological replicates is indicated as proliferation rate relative to control cells (mean±sd). The difference between control and TINCR-silenced samples at all time points is not significant (Student t-test).


D Upper panel : in vivo growth of tumors derived from intradermal injection of shTINCR - 2 or shSCR WM902B in NSG mice . 600 , 000 WM902B per mouse were inoculated . volumes ( mean ± sd ) were measured 8 after transplantation . Results from two independent experiments ( four per group ) are shown . Statistical between control TINCR - silenced groups was performed using Student t - ( * * = p < 0 . 01 ) . Lower panel : qRT - PCR of TINCR expression in tumors derived from in vivo transplant of shSCR shTINCR WM902B ( 8 after injection ) . TINCR relative to L32P housekeeping gene control ( mean ± sd ) from four biological replicates ( ) from both experiments are shown .

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D Upper panel: in vivo growth of tumors derived from intradermal injection of shTINCR-2 or shSCR WM902B cells in NSG mice. 600,000 WM902B cells per mouse were inoculated. Tumor volumes (mean±sd) were measured 8 weeks after transplantation. Results from two independent experiments (four animals per group) are shown. Statistical analysis between control and TINCR-silenced groups was performed using the Student t -test (**=p<0.01). Lower panel: qRT-PCR analysis of TINCR RNA expression in tumors derived from in vivo transplant of shSCR and shTINCR WM902B cells (8 weeks after injection). TINCR RNA levels relative to L32P housekeeping gene and control (mean±sd) from four biological replicates (animals) from both experiments are shown.


E Drug - response curves showing increased resistance of shTINCR - WM902B after treatment with BRAF ( Vemurafenib ; from 10 to 10 , 000 nM ) MEK ( Trametinib ; from 4 to 2 , 000 nM ) inhibitors . shSCR - ( control ) shTINCR - WM902B were treated with increasing concentrations of drugs their viability assayed after 72 hours using CellTiter - Glo luminescent kit ( Promega ) . Error bars represent s . e . m . of three biological replicates . dose response curve was fit with nonlinear regression ( GraphPad Prism ) .

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E Drug-response curves showing increased resistance of shTINCR-WM902B cells after treatment with BRAF (Vemurafenib; from 10 to 10,000 nM) and MEK (Trametinib; from 4 to 2,000 nM) inhibitors. The shSCR- (control) and shTINCR-WM902B cells were treated with increasing concentrations of drugs and their viability assayed after 72 hours using CellTiter-Glo luminescent kit (Promega). Error bars represent s.e.m. of three biological replicates. The dose response curve was fit with nonlinear regression (GraphPad Prism).


G Western blot of a panel of that are specifically associated to proliferative or invasive phenotype . Total lysates were extracted from shSCR - shTINCR - WM902B ( lane 1 2 ) or shSCR - shTINCR - MMC70 ( lane 3 4 ) 10 days after infection analyzed by immunoblot using indicated antibodies . Vinculin was used as loading control .

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G Western blot analysis of a panel of proteins that are specifically associated to the proliferative or invasive phenotype. Total protein lysates were extracted from shSCR- and shTINCR-WM902B cells (lane 1 and 2) or shSCR- and shTINCR-MMC70 cells (lane 3 and 4) 10 days after infection and analyzed by immunoblot assay using the indicated antibodies. Vinculin was used as loading control.


A qRT - PCR of TINCR expression ( left ) of MM13 MM2 PDX infected with lentiviral vectors expressing full - TINCR cDNA . PDX infected with empty vector ( Empty ) were used as control . TINCR relative to L32P housekeeping gene control ( mean ± sd ) from three biological replicates are shown . Statistical analyses were performed using Student t - ( * * * : p < 0 . 001 ) .

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A qRT-PCR analysis of TINCR expression (left) of MM13 and MM2 PDX cells infected with lentiviral vectors expressing full-length TINCR cDNA. PDX cells infected with empty vector (Empty) were used as control. TINCR RNA levels relative to L32P housekeeping gene and control (mean±sd) from three biological replicates are shown. Statistical analyses were performed using Student t -test (***: p<0.001).


B In vitro relative migration ( mean ± sd ) from three independent experiments ( biological replicates ) is shown in MM13 MM2 TINCR overexpressing ( TINCR ) compared to control ( Empty ) . Statistical analyses were performed using Student t - ( * * * : p < 0 . 001 ) .

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B In vitro relative migration (mean±sd) from three independent experiments (biological replicates) is shown in MM13 and MM2 TINCR overexpressing (TINCR) compared to control (Empty) cells. Statistical analyses were performed using Student t -test (***: p<0.001).


C In vitro proliferation was assayed 72 hours post infection in Empty TINCR MM13 MM2 PDX at indicated time points ( 1 - 5 days ) using CellTiter - Glo viability . Relative proliferation rate ( mean ± sd ) of three independent experiments are shown .

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C In vitro cell proliferation was assayed 72 hours post infection in Empty and TINCR MM13 and MM2 PDX cells at the indicated time points (1-5 days) using CellTiter-Glo viability assay. Relative proliferation rate (mean±sd) of three independent experiments are shown.


D Drug - response curves showing sensitization to MEK inhibitor Trametinib of MM13 ( NRAS - mutant PDX ) to Trametinib BRAF inhibitor Vemurafenib of TINCR MM2 ( BRAF - mutant PDX ) . Empty ( control ) TINCR expressing MM13 were treated with increasing concentrations of drug ( ranging from 5 to 5000 nM ) viability was assayed after 72 hours by CellTiter - Glo viability . Error bars represent s . e . m . of three biological replicates . dose response curve was fit with nonlinear regression ( GraphPad Prism ) .

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D Drug-response curves showing sensitization to MEK inhibitor Trametinib of MM13 (NRAS-mutant PDX) and to Trametinib and BRAF inhibitor Vemurafenib of TINCR MM2 cells (BRAF-mutant PDX). The Empty (control) and TINCR expressing MM13 cells were treated with increasing concentrations of drug (ranging from 5 to 5000 nM) and cell viability was assayed after 72 hours by CellTiter-Glo viability assay. Error bars represent s.e.m. of three biological replicates. The dose response curve was fit with nonlinear regression (GraphPad Prism).


E Western blot of expression of invasive proliferative phenotype markers EPHA2 , EGFR , AXL , PDGFRβ , SOX10 , MITF in TINCR - overexpressing MM13 MM2 . Total lysates were extracted from PDX 7 days after infection analyzed by immunoblot using indicated antibodies . Vinculin was used as loading control . This experiment is representative of three independent experiments showing comparable of TINCR overexpression ( 8 - 10 folds ) .

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E Western blot analysis of the expression of the invasive and proliferative phenotype markers EPHA2, EGFR, AXL, PDGFRβ, SOX10, and MITF in TINCR-overexpressing MM13 and MM2 cells. Total protein lysates were extracted from PDX cells 7 days after infection and analyzed by immunoblot assay using the indicated antibodies. Vinculin was used as loading control. This experiment is representative of three independent experiments showing comparable levels of TINCR overexpression (8-10 folds).


F In vivo growth of Empty vector TINCR cDNA expressing MM13 following intradermal injection into NSG mice . Four ( 200 , 000 / mouse ) per group were used in each experiment . growth ( mean ± sd ) was monitored weekly volume ( mean ± sd ) measured at 4 for tumors ( originated in site of injection , left panel ) at 8 for spontaneous metastases which colonized lymph nodes ( right panel ) . results of two independent experiments are shown . Statistical analyses of control TINCR - overexpressing groups from both experiments were performed using Student t - ( * * : p < 0 . 01 ) .

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F In vivo growth of Empty vector and TINCR cDNA expressing MM13 cells following intradermal injection into NSG mice. Four animals (200,000 cells/mouse) per group were used in each experiment. Tumor growth (mean±sd) was monitored weekly and tumor volume (mean±sd) measured at 4 weeks for primary tumors (originated in the site of injection, left panel) and at 8 weeks for spontaneous metastases which colonized lymph nodes (right panel). The results of two independent experiments are shown. Statistical analyses of control and TINCR-overexpressing groups from both experiments were performed using Student t -test (**: p<0.01).


G Representative pictures of lymph node metastases derived from Empty TINCR expressing MM13 PDX are shown . Hematoxylin - eosin of sections of liver lung metastases derived from Empty TINCR MM13 PDX . Metastasis formation was evaluated 8 after transplantation into NSG mice . Pictures of representative lungs livers infiltrated with metastases are shown in inset . Scale bars : 200μm .

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G Representative pictures of lymph node metastases derived from Empty and TINCR expressing MM13 PDX cells are shown. Hematoxylin-eosin staining of sections of liver and lung metastases derived from Empty and TINCR MM13 PDX cells. Metastasis formation was evaluated 8 weeks after transplantation into NSG mice. Pictures of representative lungs and livers infiltrated with metastases are shown in the inset. Scale bars: 200μm.


A Western blot of integrated stress response activation in WM902B MMC70 . were harvested 96 h after shRNA infection western blot performed using indicated antibodies .

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A Western blot analysis of integrated stress response activation in WM902B and MMC70 cells. Cells were harvested 96 h after shRNA infection and western blot performed using the indicated antibodies.


B Representative comparison of translational efficiency ( TE ) in control ( shSCR ) shTINCR WM902B 72h post infection . genes with statistically significant TE up - downregulation ( FDR < 0 . 1 ) are highlighted in red blue respectively . ATF4 is circled among upregulated genes .

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B Representative comparison of translational efficiency (TE) in control (shSCR) and shTINCR WM902B cells 72h post infection. The genes with statistically significant TE up- and downregulation (FDR< 0.1) are highlighted in red and blue respectively. ATF4 is circled among the upregulated genes.


C representing top translationally upregulated down regulated genes in TINCR WM902B . plotted values are log2 fold TE change values in each biological replicate ( rep1 rep2 ) relative to control ( shSCR ) .

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C Heatmap representing top translationally upregulated and down regulated genes in TINCR WM902B cells. The plotted values are the log2 fold TE change values in each biological replicate (rep1 and rep2) relative to control (shSCR).


D Bar - graph representing ribosome protected fragments ( RPF ) normalized coverage ( mean ± sd ) mapping to ATF4 CDS in control ( shSCR ) TINCR ( shTINCR ) depleted WM902B from two biological replicates is shown .

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D Bar-graph representing ribosome protected fragments (RPF) normalized coverage (mean±sd) mapping to ATF4 CDS in control (shSCR) and TINCR (shTINCR) depleted WM902B cells from two biological replicates is shown.


E UCSC genome browser tracks of ribosome footprint data illustrating RPF density distribution across ATF4 mRNA in shSCR shTINCR WM902B .

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E UCSC genome browser tracks of ribosome footprint data illustrating RPF density distribution across ATF4 mRNA in shSCR and shTINCR WM902B cells.


B Q - PCR validation of TINCR ATF4 recovery in TINCR antisense pulldown . Enrichment relative to input ( mean ± sd ) from three independent pulldown experiments is shown . Statistical analyses were performed using Student t - ( * * : p < 0 . 01 , * * * : p < 0 . 001 ) .

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B Q-PCR validation of TINCR and ATF4 RNA recovery in TINCR antisense pulldown. Enrichment relative to input (mean±sd) from three independent pulldown experiments is shown. Statistical analyses were performed using Student t -test (**: p<0.01, ***: p<0.001).


C UCSC genome browser tracks of TINCR - RIA - seq sequencing reads illustrating coverage enrichment across ATF4 transcript in WM902B MMC70 .

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C UCSC genome browser tracks of TINCR-RIA-seq sequencing reads illustrating coverage and enrichment across ATF4 transcript in WM902B and MMC70 cells.


( A ) Remdesivir inhibition of SARS - CoV - 2 infections of Vero E6 . drugs , , were used at indicated concentrations . Viral level was determined by qRT - PCR 15 hours after inoculation of SARS - CoV - 2 ( Swedish isolate , 106 PFU ) .

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(A) Remdesivir inhibition of SARS-CoV-2 infections of Vero E6 cells. drugs, , were used at the indicated concentrations. Viral RNA level was determined by qRT-PCR 15 hours after inoculation of SARS-CoV-2 (Swedish isolate, 106 PFU).


( B ) hrsACE2 inhibition of SARS - CoV - 2 infections of Vero E6 . murine recombinant soluble ACE2 ( mrsACE2 , control treatment ) , were used at indicated concentrations . Viral level was determined by qRT - PCR 15 hours after inoculation of SARS - CoV - 2 ( Swedish isolate , 106 PFU ) .

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(B) hrsACE2 inhibition of SARS-CoV-2 infections of Vero E6 cells. murine recombinant soluble ACE2 (mrsACE2, control treatment), were used at the indicated concentrations. Viral RNA level was determined by qRT-PCR 15 hours after inoculation of SARS-CoV-2 (Swedish isolate, 106 PFU).


( B ) viability of isolated clones WT AN3 - 12 following 72h remdesivir treatment with indicated doses . Mean ± of 2 - 4 biological replicates is displayed .

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(B) Cell viability of isolated cell clones and WT AN3-12 cells following 72h remdesivir treatment with the indicated doses. Mean ±SEM of 2-4 biological replicates is displayed.


( A ) Treatment of SARS - CoV - 2 ( 106 PFU ) infected Vero - E6 with Human recombinant soluble ACE 2 ( hrsACE2 ) ( 200µg / ml ) remdesivir ( Remd . 4µM ) . Viral level was determined at 15 hours after inoculation .

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(A) Treatment of SARS-CoV-2 (106 PFU) infected Vero-E6 cells with Human recombinant soluble ACE 2 (hrsACE2) (200µg/ml) and remdesivir (Remd. 4µM). Viral RNA level was determined at 15 hours after virus inoculation.


( B ) Treatment of SARS - CoV - 2 ( 106 PFU ) infected human kidney organoids with hrsACE2 ( 200µg / ml ) / or remdesivir ( Remd . 4µM ) ) . Viral was determined by qRT - PCR 72 hours after inoculation of 106 PFU of SARS - CoV - 2 .

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(B) Treatment of SARS-CoV-2 (106 PFU) infected human kidney organoids with hrsACE2 (200µg/ml) and/or remdesivir (Remd.4µM)). Viral RNA was determined by qRT-PCR 72 hours after the inoculation of 106 PFU of SARS-CoV-2.


( C ) Treatment of SARS - CoV - 2 ( 106 PFU ) infected Vero - E6 with clinical doses of hrsACE2 ( 5 10µg / ml ) remdesivir ( Remd . 4µM ) ) .

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(C) Treatment of SARS-CoV-2 (106 PFU) infected Vero-E6 cells with clinical doses of hrsACE2 (5 and 10µg/ml) and remdesivir (Remd. 4µM)).


( D ) Treatment of SARS - CoV - 2 ( 106 PFU ) infected kidney organoids with hrsACE2 ( 10µg / ml ) remdesivir ( 4µM ) .

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(D) Treatment of SARS-CoV-2 (106 PFU) infected kidney organoids with hrsACE2 (10µg/ml) and remdesivir (4µM).


( E , F ) Progeny released from untreated Vero - E6 or Vero - E6 treated with clinical doses of hrsACE2 ( 5 10µg / ml ) remdesivir ( Remd . 4µM ) . Progeny was determined ( E ) 15 hours ( F ) 48 hours post - infection ( hpi ) .

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(E,F) Progeny virus released from untreated Vero-E6 cells or Vero-E6 cells treated with clinical doses of hrsACE2 (5 and 10µg/ml) and remdesivir (Remd. 4µM). Progeny was determined (E) 15 hours and (F) 48 hours post-infection (hpi).


( A , B ) A brightfield image showing zebrafish thyroid along with surrounding tissue . thyroid follicles reside in soft tissue surrounding ventral aorta , which extends from outflow tract of heart into gills towards basibranchial cartilage in lower jaw . thyroid follicular , or thyrocytes , are labeled in green in Tg ( tg : nls - mVenus - NTR ) transgenic ( B ' ) . Scale bars : 500

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(A , B) A brightfield image showing the zebrafish thyroid gland along with surrounding tissue. The thyroid follicles reside in the soft tissue surrounding the ventral aorta, which extends from the outflow tract of the heart into the gills towards the basibranchial cartilage in the lower jaw. The thyroid follicular cells, or thyrocytes, are labeled in green in the Tg(tg:nls-mVenus-NTR) transgenic line (B'). Scale bars: 500 µm


( C ) Maximum projection of 3D confocal stack obtained from dissected thyroid of Tg ( tg : nls - EGFP ) labeled with DAPI . ( D ) Confocal scan of a transverse section across dissected thyroid from Tg ( tg : nls - EGFP ) at 3 mpf . Sections were stained with DAPI to visualize surrounding thyroid follicles . ( E ) Boxplot depicting proportion of thyrocytes present in transverse sections obtained from three Tg ( tg : nls - EGFP ) at 3 mpf . Each dot represents a transverse section . Tukey boxplot marks 25th percentile , median 75th percentile with whiskers extending from smallest to largest values .

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(C) Maximum intensity projection of 3D confocal stack obtained from the dissected thyroid gland of Tg(tg:nls-EGFP) animal and labeled with DAPI. (D) Confocal scan of a transverse section across the dissected thyroid gland from Tg(tg:nls-EGFP) animal at 3 mpf. Sections were stained with DAPI to visualize cells surrounding thyroid follicles. (E) Boxplot depicting the proportion of thyrocytes present in transverse sections obtained from three Tg(tg:nls-EGFP) animals at 3 mpf. Each dot represents a transverse section. The Tukey boxplot marks the 25th percentile, median and 75th percentile with whiskers extending from smallest to largest values.


( F , G ) Representative FACS plot of single from Tg ( tg : nls - mVenus - T2A - NTR ) at 2 mpf ( F ) 8 mpf ( G ) . Calcein ( Pacific Blue ) labels live , while green ( FITC ) labels thyrocytes . values represent proportion of calcein + thyrocytes within total calcein + .

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(F , G) Representative FACS plot of single cells from Tg(tg:nls-mVenus-T2A-NTR) animals at 2 mpf (F) and 8 mpf (G). Calcein (Pacific Blue) labels live cells, while green fluorescence (FITC) labels thyrocytes. Percentage values represent proportion of calcein+ thyrocytes within total calcein+ cells.


( A ) A t - SNE plot displaying 6249 single - profiled in zebrafish thyroid atlas . colors represent clusters denoting a specific - type . ( B - D ) Cluster # 1 represents thyrocytes that express tg , slc5a5 ( NIS ) tpo . color scale represents normalized expression for each gene ranging from lowest ( grey ) to highest ( red ) .

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(A) A t-SNE plot displaying the 6249 single-cells profiled in the zebrafish thyroid gland atlas. The colors represent cell clusters denoting a specific cell-type. (B-D) Cluster #1 represents the thyrocytes that express tg, slc5a5 (NIS) and tpo. The color scale represents the normalized expression counts for each gene ranging from lowest (grey) to highest (red).


( A ) depicting five genes specifically expressed in each one of seven clusters of zebrafish thyroid atlas . color scale represents expression as a z - score ranging from - 2 ( purple ) to 2 ( yellow ) . NFE : Non - Follicular Epithelium .

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(A) Heatmap depicting five genes specifically expressed in each one of the seven clusters of the zebrafish thyroid gland atlas. The color scale represents expression as a z-score ranging from -2 (purple) to 2 (yellow). NFE: Non-Follicular Epithelium.


( B - G ) t - SNE plots overlaid with expression of a gene specific to each of cluster . endothelium cluster ( cluster # 2 ) is a mix of blood vessels lymphatic vessels while immune cluster ( cluster # 4 ) is a mix of macrophages , neutrophils lymphocytes color scale represents normalized expression for each gene ranging from lowest ( grey ) to highest ( blue ) .

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(B - G) t-SNE plots overlaid with the expression of a gene specific to each of the cluster. The endothelium cluster (cluster #2) is a mix of blood vessels and lymphatic vessels while the immune cell cluster (cluster #4) is a mix of macrophages, neutrophils and lymphocytes The color scale represents the normalized expression counts for each gene ranging from lowest (grey) to highest (blue).


( A - D ) show immunofluorescence labeling of thyroid from adult zebrafish . Transverse sections were utilized for imaging . organ was isolated from tissue - specific transgenic lines to allow marking of a particular - type adjacent to thyroid follicle . Blood vessels were marked using Tg ( kdrl : EGFP ) ( A ) , macrophages using Tg ( mpeg1 . 1 : mCherry ) ( B ) stroma using Tg ( col1a2 : mCherry ) ( C ) . Thyrocytes were labeled with pax2amKO2 expression in ( A ) Tg ( tg : nls - EGFP ) expression in ( B - C ) . ( D ) NFE was labeled using antibody against TP63 in sections of thyroid isolated from Tg ( tg : nls - EGFP ) . Gills are marked on their morphological appearance . DAPI labels nuclei . Scale bars : 10 µm ( A - B ) , 50 µm ( C - D ) .

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(A-D) Images show immunofluorescence labeling of thyroid gland from adult zebrafish. Transverse sections were utilized for imaging. The organ was isolated from tissue-specific transgenic lines to allow marking of a particular cell-type adjacent to the thyroid follicle. Blood vessels were marked using Tg(kdrl:EGFP) (A), macrophages using Tg(mpeg1.1:mCherry) (B) and stroma using Tg(col1a2:mCherry) (C). Thyrocytes were labeled with pax2amKO2 expression in (A) and Tg(tg:nls-EGFP) expression in (B - C). (D) NFE was labeled using antibody against TP63 in sections of the thyroid gland isolated from Tg(tg:nls-EGFP) animals. Gills are marked based on their morphological appearance. DAPI labels nuclei. Scale bars: 10 µm (A - B), 50 µm (C - D).


( C ) A dotplot depicting examples of paracrine autocrine signaling in thyroid . of dots represent of expressing gene in a particular cluster , while color scale represents average expression of gene in cluster after scaling .

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(C) A dotplot depicting examples of paracrine and autocrine signaling in the thyroid gland. The size of the dots represent percentage of cells expressing the gene in a particular cluster, while the color scale represents average expression of the gene in the cluster after scaling.


( A ) Dot plot depicting expression entropy on Y - axis against average gene expression on X - axis for thyrocyte population . Each dot depicts a gene , with red dots depicting genes that show statistically significant ( p - value < 0 . 05 ) difference in entropy from expected value . Expected value is represented by black regression . pax2a ctsba are marked on graph .

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(A) Dot plot depicting expression entropy on Y-axis against average gene expression on X-axis for the thyrocyte population. Each dot depicts a gene, with red dots depicting genes that show statistically significant (p-value < 0.05) difference in entropy from expected value. Expected value is represented by black regression line. pax2a and ctsba are marked on the graph.


( B ) t - SNE plot of thyrocyte cluster with expression of tg , pax2a ctsba . color scale represents normalized expression for each gene ranging from lowest ( yellow ) to highest ( red ) . ( B ' ) Histogram overlaid with density plots depicting distribution of normalized gene expression for tg , pax2a ctsba .

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(B) t-SNE plot of the thyrocyte cluster with expression of tg, pax2a and ctsba. The color scale represents the normalized expression counts for each gene ranging from lowest (yellow) to highest (red). (B') Histogram overlaid with density plots depicting the distribution of normalized gene expression for tg, pax2a and ctsba.


( B ) Whole mount immunofluorescence of 9 . 5hpf pax2amKO2 stained with anti - mKO2 antibody ( red ) anti - PAX2A antibody ( green ) . Anterior is to left , dorsal side is to top . Scale bar : 0 . 1 mm .

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(B) Whole mount immunofluorescence of 9.5hpf pax2amKO2 embryos stained with anti-mKO2 antibody (red) and anti-PAX2A antibody (green). Anterior is to the left, and dorsal side is to the top. Scale bar: 0.1 mm.


( C ) Whole mount immunofluorescence of 55 hpf pax2amKO2 ; Tg ( tg : nls - EGFP ) stained with PAX2A antibody ( PAX2A - Ab ) displays an overlap of mKO2 PAX2A - Ab signal . otic vesicle ( OV ) , mid - hindbrain barrier ( MHB ) , interneurons ( IN ) thyroid ( THY ) is labelled . dashed represents outline of zebrafish larvae . Scale bar : 100 µm . Anterior to right .

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(C) Whole mount immunofluorescence of 55 hpf pax2amKO2; Tg(tg:nls-EGFP) stained with PAX2A antibody (PAX2A-Ab) displays an overlap of mKO2 and PAX2A-Ab signal. The otic vesicle (OV), mid-hindbrain barrier (MHB), interneurons (IN) and thyroid gland (THY) is labelled. White dashed line represents the outline of the zebrafish larvae. Scale bar: 100 µm. Anterior to the right.


( D - F ) Confocal microscopy imaging of a sagittal section of a 55 hpf pax2amKO2 ( red ) ; Tg ( tg : nls - EGFP ) ( green ) immunostained with PAX2A antibody ( cyan ) showing co - localization of mKO2 pax2a in pronephros ( D ) , thyroid ( E ) mid - hindbrain barrier ( F ) . In thyroid , mKO2 ( red ) , PAX2A - Ab ( cyan ) thyrocyte - specific GFP ( green ) show co - localization . Scale bar : 50 µm .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-TISSUE O O O O B-TISSUE O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O B-TISSUE O B-GENEPROD O O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-CELL_TYPE O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O

(D - F) Confocal microscopy imaging of a sagittal section of a 55 hpf pax2amKO2 (red); Tg(tg:nls-EGFP) (green) embryos immunostained with PAX2A antibody (cyan) showing co-localization of mKO2 and pax2a in the pronephros (D), thyroid gland (E) and mid-hindbrain barrier (F). In the thyroid gland, mKO2 (red), PAX2A-Ab (cyan) and thyrocyte-specific GFP (green) show co-localization. Scale bar: 50 µm.


( G , H ) Snapshots from live imaging of 55 hpf pax2amKO2 ; Tg ( tg : nls - EGFP ) injected with sgRNA targeting pax2a coding . anterior part of a representative control ( G ) is shown alongside a representative crispant ( H ) . Crispants display a strong reduction of mKO2 , as well as an absence of GFP signal suggesting absence of thyroid ( THY ) tissue . dashed represents outline of zebrafish larvae . Scale bar : 100 µm . Anterior to right .

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(G , H) Snapshots from live imaging of 55 hpf pax2amKO2; Tg(tg:nls-EGFP) embryos injected with sgRNA targeting pax2a coding sequence. The anterior part of a representative control embryo (G) is shown alongside a representative crispant (H). Crispants display a strong reduction of mKO2 fluorescence, as well as an absence of GFP signal suggesting absence of thyroid (THY) tissue. White dashed line represents the outline of the zebrafish larvae. Scale bar: 100 µm. Anterior to the right.


( A - D ) of 3 mpf thyroid from pax2amKO2 zebrafish shows heterogeneity in pax2a reporter expression . ( A ) Whole mount confocal imaging of a 3 mpf pax2amKO2 thyroid labelled with mKO2 ( red ) , E - cadherin ( cyan , not shown in ' A ' for clarity reasons ) DAPI ( blue ) for localisation . ( B - D ) Optical sections of three follicles , with mKO2 - Low labelled with arrows . E - cadherin ( B ' - D ' ) DAPI ( B ' ' - D ' ' ) shows that absence of mKO2 signal does not correspond to an absence of . Anterior to bottom of pictures .

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(A-D) Analysis of 3 mpf thyroid gland from pax2amKO2 zebrafish shows heterogeneity in pax2a reporter expression. (A) Whole mount confocal imaging of a 3 mpf pax2amKO2 thyroid labelled with mKO2 (red), E-cadherin (cyan, not shown in 'A' for clarity reasons) and DAPI (dark blue) for nuclear localisation. (B - D) Optical sections of three follicles, with mKO2-Low cells labelled with arrows. E-cadherin (B' - D') and DAPI (B'' - D'') staining shows that absence of mKO2 signal does not correspond to an absence of cells. Anterior to the bottom of the pictures.


( E , F ) ( E ) Confocal image of thyroid section from Tg ( tg : nls - EGFP ) at 4 mpf stained with PAX2A antibody DAPI . dotted region is displayed at high magnification in ( F ) . Arrows marks thyrocytes displaying low PAX2A . Notably , PAX2A - Low thyrocytes display tg - driven EGFP expression , demonstrating their differentiated status .

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(E,F) (E) Confocal image of thyroid gland section from Tg(tg:nls-EGFP) at 4 mpf stained with PAX2A antibody and DAPI. The dotted region is displayed at high magnification in (F). Arrows marks thyrocytes displaying low PAX2A staining. Notably, PAX2A-Low thyrocytes display tg-driven EGFP expression, demonstrating their differentiated status.


( A - C ) from thyroid of 5 mpf Tg ( tg : nls - GFP ) ; pax2amKO2 were labelled with calcein ( live marker ) analysed using FACS . ( A ) A FACS plot showing calcein on Y - axis GFP on X - axis . box encompassing GFP + represents thyrocyte population , which was gated for further . ( B ) Histogram showing distribution of GFP in thyrocytes . ( C ) Histogram showing distribution of mKO2 in thyrocytes . Thyrocytes were selected by gating for GFP + population . Horizontal lines indicate mKO2 - Low mKO2 - High expression level , with values representing proportion of thyrocytes with mKO2 - Low mKO2 - High expression .

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(A - C) Cells from the thyroid gland of 5 mpf Tg(tg:nls-GFP); pax2amKO2 animals were labelled with calcein (live cell marker) and analysed using FACS. (A) A FACS plot showing calcein on Y-axis and GFP on X-axis. The box encompassing the GFP+ cells represents the thyrocyte population, which was gated for further analysis. (B) Histogram showing the distribution of GFP intensity in thyrocytes. (C) Histogram showing the distribution of mKO2 intensity in thyrocytes. Thyrocytes were selected by gating for GFP+ population. Horizontal lines indicate the mKO2-Low and mKO2-High expression level, with percentage values representing proportion of thyrocytes with mKO2-Low and mKO2-High expression.


( 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 .

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(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 B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O B-TISSUE I-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE 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 .

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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 B-TISSUE O B-DISEASE O B-DISEASE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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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 B-TISSUE O B-DISEASE O B-DISEASE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE 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 B-SMALL_MOLECULE O B-TISSUE O B-DISEASE O B-DISEASE O O O O O O O O O O O O O O B-DISEASE O O B-DISEASE O O O O O B-DISEASE I-DISEASE O O 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-DISEASE O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O 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 B-SMALL_MOLECULE O O B-TISSUE O B-DISEASE O B-DISEASE O O O O O O O O O O O O O O B-DISEASE O O B-DISEASE O O O O O O O O O O O O O O O O O O B-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O O O O O 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 B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-DISEASE I-DISEASE O O B-DISEASE I-DISEASE O O B-DISEASE I-DISEASE O O O O O O O O O B-DISEASE O B-DISEASE I-DISEASE I-DISEASE O O O O O O B-DISEASE O B-DISEASE I-DISEASE I-DISEASE O O O O O O B-DISEASE O B-DISEASE I-DISEASE I-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD B-EXP_ASSAY O B-SMALL_MOLECULE O O B-DISEASE O O O O O O O O O O O O O O O O O O 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-GENEPROD B-EXP_ASSAY O O B-SMALL_MOLECULE O B-DISEASE 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 B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-DISEASE O 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-DISEASE O B-DISEASE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-DISEASE O 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-DISEASE O B-DISEASE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY 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 .

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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 .

O O B-EXP_ASSAY I-EXP_ASSAY B-SMALL_MOLECULE O O B-DISEASE O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

B-EXP_ASSAY O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O 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-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O 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 .

B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O 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 .

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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-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O 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 B-SMALL_MOLECULE O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O B-SMALL_MOLECULE 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 .

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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 .

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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 ) .

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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 .

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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).