microscopy shows Glutamate - induced ( Glut ) excitotoxic stress increases DPR in rat cortical neurons expressing C9 DPR reporter in iPS sMN derived from C9orf72 NRE . Glutamate - induced DPR are reduced by pretreating with AMPA NMDA specific antagonists NBQX MK801 , respectively . Relative demonstrates there is a significant increase in DPR when cortical neurons sMNs are challenged with excitotoxic stressors : glutamate , AMPA , NMDA , HC . Blocking either AMPA or NMDA receptors reduces DPR in presence of excitotoxic stressors . NES - mIFP or MAP2 were used to mark transfection positive rat cortical neurons or iPS sMNs , respectively , for . DPR in iPS sMN was measured using DPR - specific antibodies . Changes in DPR are relative to non - stressed ( CTRL ) Cortical neuron : n = 6 with m > 40 per n . IPSC sMN : n = 4 with m > 25 per n .

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

Fluorescent microscopy images shows Glutamate-induced (Glut) excitotoxic stress increases DPR levels in rat cortical neurons expressing the C9 DPR reporter and in iPS sMN derived from C9orf72 NRE patients. Glutamate-induced DPR levels are reduced by pretreating cells with the AMPA and NMDA specific antagonists NBQX and MK801, respectively. Relative fluorescent intensity quantification demonstrates there is a significant increase in DPR levels when primary cortical neurons and sMNs are challenged with the excitotoxic stressors: glutamate, AMPA, NMDA, and HC. Blocking either the AMPA or NMDA receptors reduces DPR levels in the presence of excitotoxic stressors. NES-mIFP or MAP2 were used to mark transfection positive rat cortical neurons or iPS sMNs, respectively, for quantification. DPR fluorescent intensity in iPS sMN was measured using DPR-specific antibodies. Changes in DPR levels are relative to non-stressed cells (CTRL) Cortical neuron: n = 6 with m > 40 cells per n. IPSC sMN: n = 4 with m > 25 cells per n.


Microscopy show increased DPR from medium high frequency stimulation of ChR2 in cortical neurons transfected with C9 DPR reporter .

Microscopy images show increased DPR fluorescent intensity levels from medium and high frequency stimulation of ChR2 in primary cortical neurons transfected with the C9 DPR reporter.


Relative shows a significant increase in DPR with increases in neuronal activity through medium high stimulation in cortical neurons . No stimulation low stimulation frequencies did not increase of DPR . C9 DPR reporter , monitoring GP DPR ( ORF2 ) , was cotransfected with ChR2 . Cortical neuron : n = 4 with m > 10 per n . ( * * * * P < 0 . 0001 ) .

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

Relative fluorescent intensity quantification shows a significant increase in DPR levels with increases in neuronal activity through medium and high stimulation in primary cortical neurons. No stimulation and low stimulation frequencies did not increase levels of DPR. The C9 DPR reporter, monitoring the GP DPR (ORF2), was cotransfected with ChR2. Cortical neuron: n = 4 with m > 10 cells per n. (****P < 0.0001).


Filter trap binding assays show that ISR are enhanced by stressors . Significant increases following stress treatments are seen in of phospho - eif2α to total eif2α as well as phospho - PERK ATF4 compared to untreated controls . Changes in ISR are normalized to GAPDH shown relative to non - stressed or DMSO - only treated ( CTRL ) depending on vehicle compounds were dissolved in . n = 4 .

Filter trap binding assays show that ISR proteins are enhanced by all stressors. Significant increases following cellular stress treatments are seen in the ratio of phospho-eif2α to total eif2α as well as phospho-PERK and ATF4 levels compared to untreated controls. Changes in ISR proteins are normalized to GAPDH and shown relative to non-stressed or DMSO-only treated cells (CTRL) depending on the vehicle compounds were dissolved in. n = 4.


measurements show of two ISR - related , PERK relative phospho - eif2α ( phospho - eif2α / total eif2α ) , are increased following different excitotoxic stress treatments in cortical neurons . Representative indicate differences for phospho - eif2α phospho - PERK 24 h post treatment with different excitotoxic stressor paradigms . Blocking of AMPA NMDA receptors using known antagonists significantly reduces ISR - related , which parallels reduced DPR ( Figure 3B ) . Changes in ISR are relative to non - stressed ( CTRL ) .

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

Fluorescent intensity measurements show the levels of two ISR-related proteins, PERK and relative phospho-eif2α (phospho-eif2α / total eif2α), are increased following different excitotoxic stress treatments in cortical neurons. Representative images indicate fluorescent intensity differences for phospho-eif2α and phospho-PERK levels 24 h post treatment with different excitotoxic stressor paradigms. Blocking of AMPA and NMDA receptors using known antagonists significantly reduces ISR-related protein levels, which parallels reduced DPR levels (Figure 3B). Changes in ISR proteins are relative to non-stressed cells (CTRL).


Relative shows a significant decrease in C9 DPR reporter when stressor challenged NSC34 were treated with compounds inhibiting ISR - related or translation - targeting pathways . Dibenzoylmethane ( 1 , 3DBM ; 20 µM ) , which inhibits eif2α - P activity , had most significant decreases in DPR production . Trazodone ( Traz ; 20 µM ) showed similar significant reduction in DPR as PERK inhibitor , GSK2606414 ( 500 nM ) .

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

Relative fluorescent intensity quantification shows a significant decrease in C9 DPR reporter levels when stressor challenged NSC34 cells were treated with compounds inhibiting ISR-related or translation-targeting pathways. Dibenzoylmethane (1,3DBM; 20 µM), which inhibits eif2α-P activity, had the most significant decreases in DPR production. Trazodone (Traz; 20 µM) showed similar significant reduction in DPR levels as the PERK inhibitor, GSK2606414 (500 nM).


Relative shows a significant decrease in C9 DPR reporter when glutamate stressed cortical neurons were treated with compounds Trazodone ( Traz ; 20 µM ) or Dibenzoylmethane ( 1 , 3DBM ; 20 µM ) . Similar to treatments in NSC34 , 1 , 3DBM shows most significant decrease in DPR production .

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

Relative fluorescent intensity quantification shows a significant decrease in C9 DPR reporter levels when glutamate stressed primary cortical neurons were treated with the compounds Trazodone (Traz; 20 µM) or Dibenzoylmethane (1,3DBM; 20 µM). Similar to the treatments in NSC34, 1,3DBM shows the most significant decrease in DPR production.


A , Western blotting showing INSL5 in immortalized NPEC NPC lines after inhibition of secretion by BFA , β - actin was used as a loading control .

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

A, Western blotting showing cellular INSL5 in the immortalized NPEC and NPC cell lines after inhibition of protein secretion by BFA, and β-actin was used as a loading control.


B , ELISA showing secreted INSL5 in supernatants of NPECs NPC lines . For NPECs , n = 3 ; for NPC , n = 7 .

O O O 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, ELISA assay showing secreted INSL5 in the supernatants of NPECs and NPC cell lines. For NPECs, n=3; for NPC, n=7.


C , of plasma INSL5 in healthy controls ( normal EBV ( - ) ) , non - individuals with VCA - IgA positive ( normal EBV ( + ) ) NPC , in training cohort .

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

C, The concentration of plasma INSL5 in healthy controls (normal EBV (-)), non-tumor individuals with VCA-IgA positive (normal EBV (+)) and NPC patients, in training cohort.


E , of plasma INSL5 in healthy controls ( normal EBV ( - ) ) , non - individuals with VCA - IgA positive ( normal EBV ( + ) ) NPC , in validation cohort 1 ( GZ ) . F , of plasma INSL5 in healthy controls ( normal EBV ( - ) ) , non - individuals with VCA - IgA positive ( normal EBV ( + ) ) NPC , in validation cohort 2 ( ZS ) .

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

E, The concentration of plasma INSL5 in healthy controls (normal EBV (-)), non-tumor individuals with VCA-IgA positive (normal EBV (+)) and NPC patients, in validation cohort 1(GZ). F, The concentration of plasma INSL5 in healthy controls (normal EBV (-)), non-tumor individuals with VCA-IgA positive (normal EBV (+)) and NPC patients, in validation cohort 2(ZS).


A - C , Kaplan - Meier curves showing overall survival ( OS ) curves of NPC with low or high INSL5 ( A ) , low ( ≤ 4000 copy / ml ) or high ( > 4000 copy / ml ) EBV copy number ( B ) , combination of INSL5 with EBV copy number ( C ) .

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

A-C, Kaplan-Meier curves showing the overall survival (OS) curves of NPC patients with low or high INSL5 concentration (A), low (≤4000 copy/ml) or high (>4000 copy/ml) EBV DNA copy number (B), and the combination of INSL5 concentration with EBV DNA copy number (C).


D - F , Kaplan - Meier curves showing disease - free survival ( DFS ) curves of NPC with low or high INSL5 ( D ) , low or high EBV copy number ( E ) , combination of INSL5 with EBV copy number ( F ) .

O O O O O O O O O O O O O O O O O O O O O O O O O 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-F, Kaplan-Meier curves showing the disease-free survival (DFS) curves of NPC patients with low or high INSL5 concentration (D), low or high EBV DNA copy number (E), and the combination of INSL5 concentration with EBV DNA copy number (F).


A . Exogenous expression of INSL5 in NPC . Representative immunoblotting ( upper panel ) immunofluorescent ( lower panel ) showed stable exogenous expression of INSL5 in both CNE1 , CNE2 HK1 NPC lines . Scale bars represent 20μm .

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

A. Exogenous expression of INSL5 in NPC cells. Representative immunoblotting (upper panel) and immunofluorescent staining (lower panel) showed stable exogenous expression of INSL5 in both CNE1, CNE2 and HK1 NPC cell lines. Scale bars represent 20μm.B


MTT of vector control or INSL5 overexpressing CNE1 , CNE2 HK1 NPC lines ( upper panel ) either transfected with control siRNA ( NC ) or GPCR142 siRNA ( # 1 # 2 ) ( lower panel ) . n = 4 biological replicates for CNE1 CNE2 , n = 6 biological replicates for HK1 .

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

MTT assay of vector control or INSL5 overexpressing CNE1, CNE2 and HK1 NPC cell lines (upper panel) either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2) (lower panel). n=4 biological replicates for CNE1 and CNE2 cell line, n=6 biological replicates for HK1.


colony formation ( C D ) of vector control or INSL5 overexpressing CNE1 , CNE2 HK1 NPC lines either transfected with control siRNA ( NC ) or GPCR142 siRNA ( # 1 # 2 ) . Representative are shown in ( C ) for colony formation Number of colonies per field of view were plotted in D results are from three different experiments .

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

colony formation (C and D) of vector control or INSL5 overexpressing CNE1, CNE2 and HK1 NPC cell lines either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2). Representative images are shown in (C) for colony formation Number of colonies per field of view were plotted in D The results are from three different experiments.


Brdu incorporation ( E F ) of vector control or INSL5 overexpressing CNE1 , CNE2 HK1 NPC lines either transfected with control siRNA ( NC ) or GPCR142 siRNA ( # 1 # 2 ) . Representative are shown in ( E ) Brdu incorporation of Brdu positive were plotted in results are from three different experiments . Scale bars represent 20μm in ( E )

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

Brdu incorporation (E and F) of vector control or INSL5 overexpressing CNE1, CNE2 and HK1 NPC cell lines either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2). Representative images are shown in (E) Brdu incorporation the percentage of Brdu positive cells were plotted in The results are from three different experiments. Scale bars represent 20μm in (E)


migration assays ( G H ) of vector control or INSL5 overexpressing CNE1 , CNE2 HK1 NPC lines either transfected with control siRNA ( NC ) or GPCR142 siRNA ( # 1 # 2 ) . Representative are shown in ( G ) for migration assays migrated per field of view were plotted in H , respectively . results are from three different experiments . Scale bars represent 100μm in ( G )

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

migration assays (G and H) of vector control or INSL5 overexpressing CNE1, CNE2 and HK1 NPC cell lines either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2). Representative images are shown in (G) for migration assays migrated cells per field of view were plotted in H, respectively. The results are from three different experiments. Scale bars represent 100μm in (G)


I , J , Xenograft growth of INSL5 over expression NPC HK1 stable lines in nude mice . . ( I ) n = 11 mice per group .

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

I, J, Xenograft tumor growth of INSL5 over expression NPC HK1 stable cell lines in nude mice.. Tumor size (I) n=11 mice per group.


Xenograft growth of INSL5 over expression NPC HK1 stable lines in nude mice . . of two groups . n = 11 mice per group .

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

Xenograft tumor growth of INSL5 over expression NPC HK1 stable cell lines in nude mice.. Tumor weight of two groups. n=11 mice per group.


A , Gene expression profiles from CNE2 overexpressed with INSL5 or vector control . genes are shaded with green , black or red in heat map to indicate low , intermediate or high expression , respectively .

A, Gene expression profiles from CNE2 cells overexpressed with INSL5 or the vector control. The genes are shaded with green, black or red in the heat map to indicate low, intermediate or high expression, respectively.


C , signal pathway of subset of genes involved in metabolism was assessed by Gene Set Enrichment .

C, The signal pathway of the subset of genes involved in metabolism was assessed by Gene Set Enrichment Analysis.


D , of glycolytic gene expression by immunoblot in INSL5 overexpressed or knock - down lines .

D, Analysis of glycolytic gene expression by immunoblot in INSL5 overexpressed or knock-down cell lines.


E , Metabolites were purified from five ( CNE1 HK1 ) or seven ( CNE2 ) independent samples of two vector INSL5 overexpressed lines analyzed by liquid chromatography - mass spectrometry ( LC - MS ) . Red : glycolytic intermediate metabolites . Green : tricarboxylic acid cycle ( TCA cycle ) intermediate metabolites . PPP : pentose phosphate pathway . color scale bar represents Z - score of metabolites ' level .

E, Metabolites were purified from five (CNE1 cell line and HK1 cell line) or seven (CNE2 cell line) independent samples of two vector and INSL5 overexpressed cell lines and analyzed by liquid chromatography-mass spectrometry (LC-MS). Red: glycolytic intermediate metabolites. Green: tricarboxylic acid cycle (TCA cycle) intermediate metabolites. PPP: pentose phosphate pathway. The color scale bar represents Z-score of the metabolites' level.


F , extracellular acidification rate ( ECAR ) was measured in with or without INSL5 overexpression using a Seahorse XF96 Extracellular Flux analyzer . Glu , glucose ; Oligo , oligomycin .

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

F, The extracellular acidification rate (ECAR) was measured in cells with or without INSL5 overexpression using a Seahorse XF96 Extracellular Flux analyzer. Glu, glucose; Oligo, oligomycin.


G , Statistical of effects of INSL5 overexpression on glycolytic activity .

G, Statistical analysis of the effects of INSL5 overexpression on glycolytic activity.


H , CNE1 - vector CNE1 - INSL5 were grown in presence of glucose analog 2 - NBDG ( Invitrogen ) for 15min , glucose uptake was then quantified using flow cytometry ( FACS ) ( left panel ) . Statistical of glucose uptake was showed in right panel .

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

H, CNE1-vector and CNE1-INSL5 cells were grown in the presence of the fluorescent glucose analog 2-NBDG (Invitrogen) for 15min, and glucose uptake was then quantified using flow cytometry (FACS) (left panel). Statistical analysis of the glucose uptake was showed in the right panel.


glucose consumption lactate production ( I ) in CNE1 - vector CNE1 - INSL5 .

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

glucose consumption and lactate production (I) in CNE1-vector and CNE1-INSL5 cells.


basic ATP level or ATP level of which was treated by 1uM oligomycin or 50uM 2 - DG for 24h ( J ) in CNE1 - vector CNE1 - INSL5 .

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

basic ATP level or the ATP level of cell which was treated by 1uM oligomycin or 50uM 2-DG for 24h (J) in CNE1-vector and CNE1-INSL5 cells.


HK2 enzyme activity ( K ) in CNE1 - vector CNE1 - INSL5 .

HK2 enzyme activity (K) in CNE1-vector and CNE1-INSL5 cells.


L , ATP , HK2 enzyme activity lactate production in INSL5 wide - type or knock down HNE1 - EBV .

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

L, ATP concentration, HK2 enzyme activity and lactate production in INSL5 wide-type or knock down HNE1-EBV cells.


B , phosphorylation of STAT5 under INSL5 stimulation . CNE1 were starved in FBS - free medium for 24h then treated with INSL5 peptide ( 50ng / mL ) in presence of an anti - INSL5 antibody at 100ug / ml , or an isotype - matched IgG control ( IgG ) for 30min .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, phosphorylation of STAT5 under INSL5 stimulation. CNE1 cells were starved in FBS-free medium for 24h and then treated with INSL5 peptide (50ng/mL) in the presence of an anti-INSL5 antibody at 100ug/ml, or an isotype-matched IgG control (IgG) for 30min.


C , Localization of STAT5 . A confocal of STAT5 was performed using CNE1 transfected vector or INSL5 . Scale bars represent

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

C, Localization of STAT5. A confocal analysis of STAT5 staining was performed using CNE1 cells transfected vector or INSL5. Scale bars represent 20μm


D , from vector or INSL5overexpressing were separated subjected to immunoblot as indicated . marker Lamin B1 marker GAPDH were used to demonstrate purity of fractions .

O O O 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, Cytoplasmic and nuclear proteins from vector or INSL5overexpressing cells were separated and subjected to immunoblot as indicated. The nuclear marker Lamin B1 and the cytoplasmic marker GAPDH were used to demonstrate the purity of fractions.


E , INSL5 overexpression activated AKT , ERK , JAK1 STAT5 phosphorylation , not STAT3 in HK1 . INSL5 overexpressing HK1 were treated with MK2206 ( 50uM ) , U0126 ( 20uM ) , MK2206 + U0126 , or Ruxolitinib ( 5uM ) for 24h . Western blotting was performed to evaluate effects of those inhibitors on STAT5 STAT3 phosphorylation level .

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

E, INSL5 overexpression activated AKT, ERK, JAK1 and STAT5 phosphorylation, not STAT3 in HK1 cells. INSL5 overexpressing HK1 cells were treated with MK2206 (50uM), U0126(20uM), MK2206+U0126, or Ruxolitinib (5uM) for 24h. Western blotting was performed to evaluate the effects of those inhibitors on STAT5 and STAT3 phosphorylation level.


F , ChIP - qPCR analyzed occupancy of potential E - boxes by STAT5 in genes of HK2 , Glut3 PFK1 in INSL5 - overexpressing CNE1 using IgG or anti - STAT5 antibodies .

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

F, ChIP-qPCR analyzed the occupancy of potential E-boxes by STAT5 in the genes of HK2, Glut3 and PFK1 in INSL5-overexpressing CNE1 cells using IgG or anti-STAT5 antibodies.


G , Luciferase activity of different glycolytic gene promoter reporters in 293T transfected with STAT5 or empty vector .

G, Luciferase activity of different glycolytic gene promoter reporters in 293T cells transfected with STAT5 or empty vector.


H , Glucose uptake assessed by 2 - NBDG incubation in INSL5 overexpressed CNE1 with STAT5 knock down .

H, Glucose uptake assessed by 2-NBDG incubation in INSL5 overexpressed CNE1 cells with STAT5 knock down.


I , Glucose uptake was assessed by 2 - BNDG incubation in INSL5 overexpressed HK1 treated with JAK kinase inhibitor ( Ruxolitinib ) .

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

I, Glucose uptake was assessed by 2-BNDG incubation in INSL5 overexpressed HK1 cells treated with JAK kinase inhibitor (Ruxolitinib).


J , MTT of vector control or INSL5 overexpressing HK1 either treated with STAT5 siRNAs or JAK kinase inhibitor .

J, MTT assay of vector control or INSL5 overexpressing HK1 cells either treated with STAT5 siRNAs or JAK kinase inhibitor.


A , Brdu for CNE1 stimulated with 100ng / ml INSL5 peptide in presence of an anti - INSL5 antibody at 50 or 100ug / ml , or an isotype - matched IgG control ( IgG ) for 24h . Scale bars represent 20μm .

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

A, Brdu assay for CNE1 cells stimulated with 100ng/ml INSL5 peptide in the presence of an anti-INSL5 antibody at 50 or 100ug/ml, or an isotype-matched IgG control (IgG) for 24h. Scale bars represent 20μm.


B , similar to ( A ) , migration invasion for CNE1 stimulated with 100ng / ml INSL5 peptide in presence of an anti - INSL5 antibody at 50 or 100ug / ml , or an isotype - matched IgG control ( IgG ) for 24h . Scale bars represent 100μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, similar to (A), migration and invasion assay for CNE1 cells stimulated with 100ng/ml INSL5 peptide in the presence of an anti-INSL5 antibody at 50 or 100ug/ml, or an isotype-matched IgG control (IgG) for 24h. Scale bars represent 100μm.


C - E Growth curves of lines stably expressing INSL5 or vector 48 h post - treatment with indicated dose of glycolysis inhibitor , 2 - DG . CNE1 ( C ) , CNE2 ( D ) HK1 ( E ) . n = 4 biological replicates for CNE1 CNE2 , n = 6 biological replicates for

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

C-E Growth curves of cell lines stably expressing INSL5 or vector 48 h post-treatment with the indicated dose of glycolysis inhibitor, 2-DG. CNE1 cells (C), CNE2 cells (D) and HK1 cells (E). n=4 biological replicates for CNE1 and CNE2 cell line, n=6 biological replicates for HK1


F , Growth curves of lines stably expressing INSL5 or vector 48 h post - treatment with indicated dose of cis - platinum ( DDP ) 2 - DG . n = 4 biological replicates per group .

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

F, Growth curves of cell lines stably expressing INSL5 or vector 48 h post-treatment with the indicated dose of cis-platinum (DDP) and 2-DG. n=4 biological replicates per group.


( G ) of nude mice burdened with NPC PDX intraperitoneally injected with anti - INSL5 , anti - GPCR142 or isotype - matched IgG control ( IgG ) every two days . n = 6 mice per group .

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

Tumor size (G) of nude mice burdened with NPC PDX tumor and intraperitoneally injected with anti-INSL5, anti-GPCR142 or isotype-matched IgG control (IgG) every two days. n=6 mice per group.


( H ) of nude mice burdened with NPC PDX intraperitoneally injected with anti - INSL5 , anti - GPCR142 or isotype - matched IgG control ( IgG ) every two days . n = 6 mice per group .

tumor weight (H) of nude mice burdened with NPC PDX tumor and intraperitoneally injected with anti-INSL5, anti-GPCR142 or isotype-matched IgG control (IgG) every two days. n=6 mice per group.


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 .

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

A 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 .

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

C 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 .

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

A 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

C 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 ) .

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

A 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 - ) .

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

C 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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

E 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 .

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

G 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 ) .

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

A 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

C 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 ) .

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

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 ) .

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 ) .

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

F 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 .

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

G Representative 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 .

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

A 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 .

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

B Representative 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 ) .

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

C 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 .

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

D Bar-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 .

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

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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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 ) .

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

(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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O 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) 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 .

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

(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 .

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

(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 ) ) .

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

(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 ) .

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

(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 ) .

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

(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

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

(A , 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 .

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

(C) 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 + .

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

(F , 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 ) .

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

(A) 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 .

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

(A) 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - 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 ) .

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

(A-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 .

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

(C) 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 .

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

(A) 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 .

(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 .

O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

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

(C) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - 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 .

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

(G , H) 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 .

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

(A-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 .

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

(E,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 .

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

(A - 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 .

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

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-MEASURED_VAR O O B-MEASURED_VAR 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 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

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

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-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 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 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 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

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 .

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 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

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

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

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 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 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 B-MEASURED_VAR 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

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 O 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

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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

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

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 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 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

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.