D . Blood lactate to glucose at P200 ( n = 8 / group ) Bar graphs represent mean ± SD dat were analyzed using Kruskal - Wallis Mann - Whitney U for selected comparison . Significant differences between groups ( p value ) are indicated on

O O B-TISSUE B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Blood lactate to glucose ratio at P200 (n=8/group) Bar graphs represent mean±SD The dat were analyzed using Kruskal-Wallis and Mann-Whitney U tests for selected comparison. Significant differences between groups (p value) are indicated on graphs


Dual energy X - ray absorptiometry ( DEXA ) ( n = 10 / group ) of ( E ) lean mass , ( F ) fat mas at P98 Bar graphs represent mean ± SD . dat were analyzed using Kruskal - Wallis Mann - Whitney U for selected comparison . Significant differences between groups ( p value ) are indicated on

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Dual energy X-ray absorptiometry (DEXA) analysis (n=10/group) of (E) lean mass, (F) fat mas at P98 Bar graphs represent mean±SD. The dat were analyzed using Kruskal-Wallis and Mann-Whitney U tests for selected comparison. Significant differences between groups (p value) are indicated on graphs


Dual energy X - ray absorptiometry ( DEXA ) ( n = 10 / group ) o ( G ) bone mineral density at P98 Bar graphs represent mean ± SD . dat were analyzed using Kruskal - Wallis Mann - Whitney U for selected comparison . Significant differences between groups ( p value ) are indicated on

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Dual energy X-ray absorptiometry (DEXA) analysis (n=10/group) o (G) bone mineral density at P98 Bar graphs represent mean±SD. The dat were analyzed using Kruskal-Wallis and Mann-Whitney U tests for selected comparison. Significant differences between groups (p value) are indicated on graphs


Indirect calorimetric measurement ( n = 10 / group ) of ( H ) heat productio at P77 Bar graphs represent mean ± SD dat were analyzed using Kruskal - Wallis Mann - Whitney U for selected comparison . Significant differences between groups ( p value ) are indicated on

O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Indirect calorimetric measurement (n=10/group) of (H) heat productio at P77 Bar graphs represent mean±SD The dat were analyzed using Kruskal-Wallis and Mann-Whitney U tests for selected comparison. Significant differences between groups (p value) are indicated on graphs


Indirect calorimetric measurement ( n = 10 / group ) o ( I ) respiratory exchange at P77 Bar graphs represent mean ± SD dat were analyzed using Kruskal - Wallis Mann - Whitney U for selected comparison . Significant differences between groups ( p value ) are indicated on

O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Indirect calorimetric measurement (n=10/group) o (I) respiratory exchange ratio at P77 Bar graphs represent mean±SD The dat were analyzed using Kruskal-Wallis and Mann-Whitney U tests for selected comparison. Significant differences between groups (p value) are indicated on graphs


A . Hematoxylin - eosin - stained cross sections of heart kidney at P200 .

O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY O O O B-TISSUE I-TISSUE O O O

A. Hematoxylin-eosin-stained cross sections of heart and kidney at P200.


B . of heart kidney at P200 ( n = 4 / group ) Bar graphs represent mean ± SD Statistics : one - way ANOVA followed by Tukey "

O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O

B. Weight of heart and kidney at P200 (n=4/group) Bar graphs represent mean±SD Statistics: one-way ANOVA followed by Tukey"


Sirius Red ( C ) for fibrosis in liver , heart kidney at

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

Sirius Red staining (C) for fibrosis in liver, heart and kidney at P200


( D ) of fibrosis in myocardium , kidney cortex liver ( n = 5 - 7 ) Bar graphs represent mean ± SD Statistics : one - way ANOVA followed by Tukey "

O O O O B-DISEASE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O

(D) Quantification of fibrosis in myocardium, kidney cortex and liver (n=5-7) Bar graphs represent mean±SD Statistics: one-way ANOVA followed by Tukey"


E - F . Echocardiography data ( n = 4 - 6 / group ) showing ( E ) ejection fraction ( EF ) fractional shortening ( FS ) . ( F ) Systolic ( LV Vol ; S ) diastolic ( LV Vol ; D ) left ventricle volume in mice Bar graphs represent mean ± SD Statistics One - way ANOVA followed by unpaired t - with Welch " s correction for graph

O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O B-ORGANISM 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. Echocardiography data (n=4-6/group) showing (E) ejection fraction (EF) and fractional shortening (FS). (F) Systolic (LV Vol;S) and diastolic (LV Vol;D) left ventricle volume in mice Bar graphs represent mean±SD Statistics One-way ANOVA followed by unpaired t-test with Welch"s correction for graph E


G . Expression of cardiac hypertrophy fibrosis - associated genes in pre - symptomatic ( P150 ) heart ( n = 6 / group ) Bar graphs represent mean ± SD Statistics : one - way ANOVA followed by Tukey "

O O B-EXP_ASSAY O B-DISEASE I-DISEASE I-DISEASE O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O

G. Expression of cardiac hypertrophy and fibrosis-associated genes in the pre-symptomatic (P150) heart (n=6/group) Bar graphs represent mean±SD Statistics: one-way ANOVA followed by Tukey"


H , I . 24 h excretion of ( H ) albumin ( I ) creatinine in urine at P200 ( n = 4 / group ) Bar graphs represent mean ± SD Statistics : one - way ANOVA followed by Tukey " s tes Mann - Whitney U ( for graph I )

O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-TISSUE O O O O O O O O O O O O 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, I. 24 h excretion of (H) albumin (I) creatinine in urine at P200 (n=4/group) Bar graphs represent mean±SD Statistics: one-way ANOVA followed by Tukey"s tes and Mann-Whitney U tests (for graph I)


J , K . Liver enzymes ( J ) alanine aminotransferase ( ALT ) ( K ) alkaline phosphatase ( ALP ) in plasma at P200 ( n = 8 / group ) Bar graphs represent mean ± SD Statistics : one - way ANOVA followed by Tukey "

O O O O B-TISSUE O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O

J, K. Liver enzymes (J) alanine aminotransferase (ALT) and (K) alkaline phosphatase (ALP) in plasma at P200 (n=8/group) Bar graphs represent mean±SD Statistics: one-way ANOVA followed by Tukey"


A . GFAP for cerebral astrocytes , barrel field of somatosensory cortex ( S1BF ) highlighted with

O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O B-TISSUE I-TISSUE O B-TISSUE O O O

A. GFAP staining for cerebral astrocytes, the barrel field of the primary somatosensory cortex (S1BF) highlighted with brackets


B . Mitochondrial ultrastructure in cardiomyocytes , kidney tubular hepatocytes at P200 as visualized by electron microscopy . Scale bar 1 µm .

O O B-SUBCELLULAR O O B-CELL_TYPE O O O B-CELL_TYPE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

B. Mitochondrial ultrastructure in cardiomyocytes, kidney tubular cells and hepatocytes at P200 as visualized by electron microscopy. Scale bar 1 µm.


( C ) Average cross - sectional of mitochondrio ( n = 3mice / group Bar graphs represent mean ± SD . Statistics for graph One - way ANOVA followed by Tukey "

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O

(C) Average cross-sectional area of mitochondrio (n=3mice/group Bar graphs represent mean±SD. Statistics for graph One-way ANOVA followed by Tukey"s


( D number of cristae per mitochondrio ( n = 3mice / group ) Bar graphs represent mean ± SD . Statistics for graph ) : One - way ANOVA followed by Tukey "

O O B-EXP_ASSAY O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D number of cristae per mitochondrio (n=3mice/group) Bar graphs represent mean±SD. Statistics for graph ): One-way ANOVA followed by Tukey"s


( E ) cristae thickness , ( F ) average between cristae in mitochondria ( n = 3mice / group ) Bar graphs represent mean ± SD . Statistics for graph One - way ANOVA followed by Tukey "

O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O

(E) cristae thickness, and (F) average distance between cristae in mitochondria (n=3mice/group) Bar graphs represent mean±SD. Statistics for graph One-way ANOVA followed by Tukey"s


A - C . Principal component of transcriptome data from ( A ) heart , ( B ) kidney ( C ) liver . D - F . Heat map visualization of pathway enrichment ( Reactome database ) . Full pathway is provided in Appendix Table S1 . Benjamin - Hochberg FDR - corrected p values are color - labelled as indicated for three comparisons .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O B-TISSUE O O O O B-TISSUE O O O B-TISSUE O O O O O O O O O O O O O O O O O O O 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. Principal component analysis of transcriptome data from (A) heart, (B) kidney and (C) liver. D-F. Heat map visualization of pathway enrichment analysis (Reactome database). Full pathway analysis is provided in Appendix Table S1. Benjamin-Hochberg FDR-corrected p values are color-labelled as indicated for the three comparisons.


Gene expression of major transcriptional regulators of energy metabolism ( PPAR - α , mitochondrial biogenesis ( PGC - 1α , TFAM ) in heart at P150 . ( n = 6 / group ) Box plot whiskers represent quartiles , maximum minimum value ( relative fold change ' FC ' to WT ) . Statistics : one - way ANOVA followed by Tukey ' s .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Gene expression of major transcriptional regulators of energy metabolism (PPAR-α, mitochondrial biogenesis (PGC-1α, TFAM) in heart at P150. (n=6/group) Box plot whiskers represent quartiles, maximum and minimum value (relative fold change 'FC' to WT). Statistics: one-way ANOVA followed by Tukey's test.


Gene expression of major transcriptional regulators of energy metabolism HIF - 1α ) stress responses ( ATF4 ) in heart at P150 . ( n = 6 / group ) Box plot whiskers represent quartiles , maximum minimum value ( relative fold change ' FC ' to WT ) . Statistics : one - way ANOVA followed by Tukey ' s .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Gene expression of major transcriptional regulators of energy metabolism HIF-1α) and stress responses (ATF4) in heart at P150. (n=6/group) Box plot whiskers represent quartiles, maximum and minimum value (relative fold change 'FC' to WT). Statistics: one-way ANOVA followed by Tukey's test.


L . Expression of cIII stress signature genes in heart , kidney , liver . ( n = 6 / group )

O O B-EXP_ASSAY O B-SUBCELLULAR O O O O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O O

L. Expression of cIII stress signature genes in heart, kidney, and liver. (n=6/group)


A . Significantly altered metabolites ( FDR < 0 . 2 ; P < 0 . 05 , n = 5 / group ) in presymptomatic ( P150 ) heart tissue of GRAC ( red ) GROX ( green ) mice Bar graph represent mean ± S Statistics for graph one - way ANOVA followed by Tukey "

O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O B-DISEASE O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O

A. Significantly altered metabolites (FDR<0.2; P<0.05, n=5/group) in presymptomatic (P150) heart tissue of GRAC (red) and GROX (green) mice Bar graph represent mean±S Statistics for graph one-way ANOVA followed by Tukey"s


B . Significantly altered metabolites ( FDR < 0 . 2 ; P < 0 . 05 , n = 5 / group ) in liver tissue of GRAC ( red ) GROX ( green ) mice Bar graph represent mean ± S Statistics for graph one - way ANOVA followed by Tukey "

O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-TISSUE O O B-DISEASE O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O

B. Significantly altered metabolites (FDR<0.2; P<0.05, n=5/group) in liver tissue of GRAC (red) and GROX (green) mice Bar graph represent mean±S Statistics for graph one-way ANOVA followed by Tukey"s


C - F . Concentrations of TCA cycle intermediates ( C ) fumarate ( D ) malate , ( E ) adenylate energy charge ( F ) NADH / NAD + ( n = 5 / group ) Bar graph represent mean ± S Statistics for graph one - way ANOVA followed by Tukey "

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

C-F. Concentrations of the TCA cycle intermediates (C) fumarate and (D) malate, (E) adenylate energy charge and (F) NADH/NAD+ ratio (n=5/group) Bar graph represent mean±S Statistics for graph one-way ANOVA followed by Tukey"s


( G ) Proline concentratio in heart tissue at P200 ( # below detection limit Bar graph represent mean ±

O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-TISSUE O O O O O O O O O O O O O

(G) Proline concentratio in heart tissue at P200 (# below detection limit Bar graph represent mean±S


mRNA expression of proline synthesis - related genes ( n = 6 / group ) ) ( H ) Aldh18a1 ( I ) Pycr1 at P150 box plots of mRNA expressions represent quartiles , minimum , maximum value ( relative fold change " FC & qu Statistics for graph one - way ANOVA followed by Tukey "

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

mRNA expression of proline synthesis-related genes (n=6/group)) (H) Aldh18a1 and (I) Pycr1 at P150 box plots of mRNA expressions represent quartiles, minimum, and maximum value (relative fold change "FC&qu Statistics for graph one-way ANOVA followed by Tukey"s


A . Blue native PAGE of RISP CORE1 assembled into free cIII2 supercomplexes ( SCs )

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O

A. Blue native PAGE analysis of RISP and CORE1 assembled into free cIII2 and supercomplexes (SCs)


B - C . RISP per CORE1 in ( B ) SC1 ( C ) free cIII2 .

O O O O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O

B-C. RISP per CORE1 ratio in (B) SC1 and (C) free cIII2.


Spectrophotometric of ( D ) cI , ( E ) cII activity in isolated mitochondria at P200 ( n = 4 - 6 / group ) Bar graphs represent mean ± SD . Statistics : one - way ANOVA followed by Tukey " s . Significant differences between groups ( p value ) are indicated on

B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Spectrophotometric assay of (D) cI, (E) cII activity in isolated mitochondria at P200 (n=4-6/group) Bar graphs represent mean±SD. Statistics: one-way ANOVA followed by Tukey"s test. Significant differences between groups (p value) are indicated on g


Spectrophotometric o ( F ) cIII activity in isolated mitochondria at P200 ( n = 4 - 6 / group ) Bar graphs represent mean ± SD . Statistics : one - way ANOVA followed by Tukey " s . Significant differences between groups ( p value ) are indicated on

B-EXP_ASSAY O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Spectrophotometric assay o (F) cIII activity in isolated mitochondria at P200 (n=4-6/group) Bar graphs represent mean±SD. Statistics: one-way ANOVA followed by Tukey"s test. Significant differences between groups (p value) are indicated on g


G . CIV activity as measured using an oxygraph Bar graphs represent mean ± SD . Statistics : one - way ANOVA followed by Tukey " s . Significant differences between groups ( p value ) are indicated on

O O B-SUBCELLULAR O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. CIV activity as measured using an oxygraph Bar graphs represent mean±SD. Statistics: one-way ANOVA followed by Tukey"s test. Significant differences between groups (p value) are indicated on g


H . n - Propyl gallate - sensitive AOX mediated cI cII - linked state 3 respiration ( n = 7 / group , Mann - Whitney U ) Bar graphs represent mean ± SD . Statistics : one - way ANOVA followed by Tukey " s . Significant differences between groups ( p value ) are indicated on

O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. n-Propyl gallate-sensitive AOX mediated cI and cII-linked state 3 respiration (n=7/group, Mann-Whitney U test) Bar graphs represent mean±SD. Statistics: one-way ANOVA followed by Tukey"s test. Significant differences between groups (p value) are indicated on g


CI - linked ( I state 3 respiration in isolated heart , kidney liver mitochondria ( n = 8 / group ) We used malate , pyruvate glutamate to generate NADH for cI Bar graphs represent mean ± SD Statistics : one - way ANOVA followed by Tukey " s . Significant differences between groups ( p value ) are indicated on

B-SUBCELLULAR O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE B-SUBCELLULAR O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

CI-linked (I state 3 respiration in isolated heart, kidney and liver mitochondria (n=8/group) We used malate, pyruvate and glutamate to generate NADH for the cI Bar graphs represent mean±SD Statistics: one-way ANOVA followed by Tukey"s test. Significant differences between groups (p value) are indicated on g


cI - cII - linke state 3 respiration in isolated heart , kidney liver mitochondria ( n = 8 / group ) we added CII substrate , succinate , to obtain CI & CII - linked respiration in presence of saturating ADP . Bar graphs represent mean ± SD . Statistics : one - way ANOVA followed by Tukey " s . Significant differences between groups ( p value ) are indicated on

B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O B-TISSUE O B-TISSUE I-TISSUE B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

cI- and cII-linke state 3 respiration in isolated heart, kidney and liver mitochondria (n=8/group) we added the CII substrate, succinate, to obtain CI&CII-linked respiration in presence of saturating ADP. Bar graphs represent mean±SD. Statistics: one-way ANOVA followed by Tukey"s test. Significant differences between groups (p value) are indicated on g


A . Amplex Red - detection of H2O2 emission , a surrogate for respiratory chain - derived ROS , during cI cII - linked state 3 respiration ( n = 6 / group ) Bar graphs represent mean ± SD Statistics one - way ANOVA followed by Tukey "

O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Amplex Red-based detection of H2O2 emission, a surrogate for respiratory chain-derived ROS, during cI and cII-linked state 3 respiration (n=6/group) Bar graphs represent mean±SD Statistics one-way ANOVA followed by Tukey"


B. Gene expression related to ROS scavenging


C . Total glutathion in heart liver tissue ( n = 5 / group Bar graphs represent mean ± SD Statistics one - way ANOVA followed by Tukey "

O O O B-SMALL_MOLECULE O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O

C. Total glutathion in heart and liver tissue (n=5/group Bar graphs represent mean±SD Statistics one-way ANOVA followed by Tukey"


D . Urinary isoprostanes per creatinine as a measure of oxidative stress ( n = 4 / group ) Bar graphs represent mean ± SD Statistics : Mann - Whitney U ( graph

O O B-TISSUE B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

D. Urinary isoprostanes per creatinine as a measure of oxidative stress (n=4/group) Bar graphs represent mean±SD Statistics: Mann-Whitney U test (graph D


E F . Representative ( D ) of tissue sections immunostained with an antibody against 4 - hydroxynonenal ( 4 - HNE ) , a lipid peroxidation marker . ( E ) of 4 - HNE immunostaining .

E and F. Representative images (D) of tissue sections immunostained with an antibody against 4-hydroxynonenal (4-HNE), a lipid peroxidation marker. (E) Quantification of the 4-HNE immunostaining.


A . visualization of expressions of selected nitric oxide ( NO ) - related genes .

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

A. Heatmap visualization of the expressions of selected nitric oxide (NO)-related genes.


B - E . Gene expression of Ca2 + channel gene ( B ) ryanodin receptor , RYR2 , ( C ) sarco ( endo ) plasmic reticulum Ca2 + - ATPase ( SERCA2 or ATP2A2 ) , ( D ) phospholamban ( PLN ) , ( E ) endothelial nitric oxide synthase NOS3 ( n = 6 / group ) Box plots for mRNA expressio represent quartiles , minimum maximum value ( relative fold change " FC & qu Statistics : one - way ANOVA followed by Tukey " s for Significant differences between groups ( p value ) are indicated on

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B-E. Gene expression of Ca2+ channel gene (B) ryanodin receptor, RYR2, (C) sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2 or ATP2A2), (D) phospholamban (PLN), and (E) endothelial nitric oxide synthase NOS3 (n=6/group) Box plots for mRNA expressio represent quartiles, minimum and maximum value (relative fold change "FC&qu Statistics: one-way ANOVA followed by Tukey"s test for Significant differences between groups (p value) are indicated on graphs


Expression of neuronal nitric oxide synthase ( NOS1 ) mRNA ( F Bar graph represent mean ± SD Statistics : one - way ANOVA followed by Tukey " s for Significant differences between groups ( p value ) are indicated on

B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Expression of neuronal nitric oxide synthase (NOS1) mRNA (F Bar graph represent mean±SD Statistics: one-way ANOVA followed by Tukey"s test for Significant differences between groups (p value) are indicated on graphs


Expression of neuronal nitric oxide synthase ( NOS1 with a representative Western blot WT brain was used as positive control in 1st lane ( G )

B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O O O O O O

Expression of neuronal nitric oxide synthase (NOS1 protein with a representative Western blot WT brain was used as positive control in the 1st lane (G)


H . Cardiac gene expression of dimethylarginine dimethylaminohydrolase 1 ( DDAH1 ) ( n = 6 / group ) Box plots for mRNA expressio represent quartiles , minimum maximum value ( relative fold change " FC & qu Statistics : one - way ANOVA followed by Tukey " s for Significant differences between groups ( p value ) are indicated on

O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Cardiac gene expression of dimethylarginine dimethylaminohydrolase 1 (DDAH1) (n=6/group) Box plots for mRNA expressio represent quartiles, minimum and maximum value (relative fold change "FC&qu Statistics: one-way ANOVA followed by Tukey"s test for Significant differences between groups (p value) are indicated on graphs


I . Cardia citrulline to arginine ( n = 5 / group ) Bar graph represent mean ±

O O B-TISSUE B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O

I. Cardia citrulline to arginine ratio (n=5/group) Bar graph represent mean±SD


J . Cardiac nitrotyrosine quantified from Western

O O B-TISSUE B-SMALL_MOLECULE O O B-EXP_ASSAY

J. Cardiac protein nitrotyrosine quantified from Western blots


K . Nitrite in heart tissue ( n = 4 / group ) Bar graph represent mean ±

O O B-SMALL_MOLECULE O B-TISSUE O O O O O O O O O O O O O

K. Nitrite concentration in heart tissue (n=4/group) Bar graph represent mean±SD


( A ) of tRNA profiling of MCF10A at 8 24 hours post exposure to oxidative stress ( 200μM H2O2 ) . Biological triplicate data is depicted at each time point relative to control .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O

(A) Heatmap of tRNA profiling of MCF10A cells at 8 and 24 hours post exposure to oxidative stress (200μM H2O2). Biological triplicate data is depicted at each time point relative to control cells.


( B ) MCF10A were exposed to oxidative stress ( 200μM H2O2 ) processed for small - sequencing . log2 - fold induction for tRFs derived from distinct tRNA isoacceptor is plotted .

O O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O

(B) MCF10A cells were exposed to oxidative stress (200μM H2O2) and processed for small RNA-sequencing. The log2-fold induction levels for tRFs derived from distinct tRNA isoacceptor is plotted.


( A ) A northern blot depicting a time course experiment ranging from five minutes to 24 hours of MCF10A in response to oxidative stress . A single probe complementary to pre - tRNATyrGUA , mature tRNATyrGUA , tRFTyrGUA expression was 32P - labeled used for detection . ( B ) of pre - tRNATyrGUA ( left ) mature tRNATyrGUA ( right ) by northern blot from multiple independent experiments ( normalized to U6 ) are shown ( n = 6 ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O

(A) A northern blot depicting a time course experiment ranging from five minutes to 24 hours of MCF10A cells in response to oxidative stress. A single probe complementary to pre-tRNATyrGUA, mature tRNATyrGUA, and tRFTyrGUA expression was 32P-labeled and used for detection. (B) Quantification of pre-tRNATyrGUA (left) and mature tRNATyrGUA levels (right) by northern blot analysis from multiple independent experiments (normalized to U6 levels) are shown (n=6).


( C ) MCF10A were exposed to oxidative stress ( 200μM H2O2 ) once daily for five continuous days . ( D ) of mature tRNATyrGUA bands by northern blot after were treated once daily for five continuous days ( normalized to U6 ) from multiple independent experiments ( n = 12 ) .

O O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O

(C) MCF10A cells were exposed to oxidative stress (200μM H2O2) once daily for five continuous days. (D) Quantification of mature tRNATyrGUA bands by northern blot after cells were treated once daily for five continuous days (normalized to U6) from multiple independent experiments (n=12).


( E ) of northern blot for pre - tRNATyrGUA ( left ) tRNATyrGUA ( right ) after one hour 24 hours respectively in HBEC30 upon exposure to oxidative stress ( 200μM H2O2 ) as in ( A ) ( n = 6 ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O

(E) Quantification of northern blot analysis for pre-tRNATyrGUA (left) and tRNATyrGUA (right) after one hour and 24 hours respectively in HBEC30 cells upon exposure to oxidative stress (200μM H2O2) as in (A) (n=6).


( A ) Growth curves of MCF10A exposed to oxidative stress ( 200μM H2O2 ) relative to control ( n = 3 ) . Two - way ANOVA was used to for significance .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O

(A) Growth curves of MCF10A cells exposed to oxidative stress (200μM H2O2) relative to control cells (n=3). Two-way ANOVA was used to test for significance.


( B ) Northern blot of MCF10A expressing a control short - hairpin or a hairpin targeting tRNATyrGUA .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE O

(B) Northern blot of MCF10A cells expressing a control short-hairpin RNA or a hairpin targeting tRNATyrGUA.


( C ) Western blot of MCF10A expressing a control short - hairpin or a hairpin targeting tyrosine - tRNA synthetase , YARS ( red arrow ) . HSC70 was used as a loading control .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O

(C) Western blot of MCF10A expressing a control short-hairpin RNA or a hairpin targeting the tyrosine-tRNA synthetase, YARS (red arrow). HSC70 was used as a loading control.


( D ) Growth curves of MCF10A expressing RNAi targeting mature tRNATyrGUA or YARS relative to expressing a control hairpin ( n = 3 ) . Two - way ANOVA was used to for significance .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

(D) Growth curves of MCF10A cells expressing RNAi targeting mature tRNATyrGUA or YARS relative to cells expressing a control hairpin (n=3). Two-way ANOVA was used to test for significance.


( E ) growth of MCF10A transiently transfected with a tRNATyrGUA overexpression vector relative to an empty control vector ( n = 3 ) . A one - Mann - Whitney was used to for significance at day 3 .

O O O B-EXP_ASSAY O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Cell growth of MCF10A cells transiently transfected with a tRNATyrGUA overexpression vector relative to an empty control vector (n=3). A one-tailed Mann-Whitney test was used to test for significance at day 3.


( A ) depleted of tRNATyrGUA or YARS were processed for label free quantitation by mass spectrometry to identify that were reduced by a log2 - fold change of 0 . 5 or more . This set was overlapped with containing a higher than median abundance of Tyr codon content to identify candidate mediators of pleiotropic effects of tRNATyrGUA depletion .

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

(A) Cells depleted of tRNATyrGUA or YARS were processed for label free quantitation by mass spectrometry to identify proteins that were reduced by a log2-fold change of 0.5 or more. This set was overlapped with proteins containing a higher than median abundance of Tyr codon content to identify candidate mediators of the pleiotropic effects of tRNATyrGUA depletion.


( C ) Quantitative western blot validation depicting abundances of targets ( EPCAM , SCD , USP3 ) identified from ( A ) . HSC70 was used as a loading control is not modulated upon molecular perturbation of tRNATyrGUA . ( D ) of western blot in ( C ) ( n = 4 ) . A one - Mann - Whitney was used to for statistical significance .

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

(C) Quantitative western blot validation depicting abundances of protein targets (EPCAM, SCD, and USP3) identified from (A). HSC70 was used as a loading control and is not modulated upon molecular perturbation of tRNATyrGUA. (D) Quantification of western blot analysis in (C) (n=4). A one-tailed Mann-Whitney test was used to test for statistical significance.


( E ) Growth curves for MCF10A were transfected with either control siRNA or two independent siRNA targeting EPCAM , SCD , or USP3 . Note that control growth curve is same in graphs were plotted separately for clarity does not represent independent experiments . Two - way ANOVA was used to for significance ( n = 3 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Growth curves for MCF10A cells were transfected with either control siRNA or two independent siRNA targeting EPCAM, SCD, or USP3. Note that the control cell growth curve is the same in all graphs and were plotted separately for clarity and does not represent independent experiments. Two-way ANOVA was used to test for significance (n=3).


( A ) Quantitative western blot EPCAM , SCD , USP3 in MCF10A 24 hours after treatment with H2O2 ( 200μM ) . HSC70 was used as a loading control . ( B ) of western results in ( A ) ( n = 9 ) . A one - Mann - Whitney was used to for statistical significance .

O O O O B-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O

(A) Quantitative western blot EPCAM, SCD, and USP3 in MCF10A cells 24 hours after treatment with H2O2 (200μM). HSC70 was used as a loading control. (B) Quantification of western results in (A) (n=9). A one-tailed Mann-Whitney test was used to test for statistical significance.


( D ) Quantitative western blot for Myc - tag Luciferase ( top ) with normalized intensities ( bottom ) are shown ( n = 3 ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O

(D) Quantitative western blot for the Myc-tag and Luciferase (top) with normalized fluorescent intensities (bottom) are shown (n=3).


( E ) Ribosome occupancy of 20 - 22nt RPFs in tRNATyrGUA - depleted compared to control reveal greater occupancy at both tyrosine codons in tRNATyrGUA - depleted using a Wilcoxon .

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

(E) Ribosome occupancy of 20-22nt RPFs in tRNATyrGUA-depleted cells compared to control cells reveal greater occupancy at both tyrosine codons in tRNATyrGUA-depleted cells using a Wilcoxon test.


( A - B ) of tRFTyrGUA induction in response to oxidative stress as a function of time in MCF10A ( A ) ( n = 4 ) in HBEC30 ( B ) ( n = 6 ) with mean ± s . e . m shown for each .

O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O

(A-B) Quantification of tRFTyrGUA induction in response to oxidative stress as a function of time in MCF10A (A) (n=4) and in HBEC30 (B) (n=6) with mean ± s.e.m shown for each cell line.


( C ) Volcano plot of mass spectrometry results from a synthetic 5 ' - biotinylated tRFTyrGUA co - precipitation experiment with lysate . Log2 fold enrichment values of identified from tRFTyrGUA relative to scrambled tRF control samples .

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

(C) Volcano plot of mass spectrometry results from a synthetic 5'-biotinylated tRFTyrGUA co-precipitation experiment with cell lysate. Log2 fold enrichment values of proteins identified from tRFTyrGUA relative to scrambled tRF control samples.


( D ) Western blot validation of mass spectrometry results for three of top hits in ( A ) , showing co - precipitation of endogenous with transfected tRFTyrGUA relative to a scrambled control ( Scr ) .

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

(D) Western blot validation of mass spectrometry results for three of the top hits in (A), showing co-precipitation of endogenous proteins with transfected tRFTyrGUA relative to a scrambled control sequence (Scr).


( B ) IGV plots from an hnRNPA1 HITS - CLIP ( Huelga et al . , 2012 ) reveals interactions with tRFTyrGUA .

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

(B) IGV plots from an hnRNPA1 HITS-CLIP (Huelga et al., 2012) reveals interactions with tRFTyrGUA.


( C ) IGV plots representing SSB interacting with tRFTyrGUA in samples that were treated with low of RNase digestion . SSB bound tRFTyrGUA reads mapped to multiple loci encoding tRNATyrGUA . ( D ) Similar to IGV plots shown in ( C ) , but depicting SSB interactions with tRFTyrGUA loci in samples without RNase digestion .

O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O

(C) IGV plots representing SSB interacting with the tRFTyrGUA in samples that were treated with low levels of RNase digestion. SSB bound tRFTyrGUA reads mapped to multiple loci encoding tRNATyrGUA. (D) Similar to the IGV plots shown in (C), but depicting SSB interactions with tRFTyrGUA loci in samples without RNase digestion.


( E ) A cumulative distribution in control hnRNPA1 depleted of stability for mRNA transcripts with 3 ' UTR hnRNPA1 CLIP binding ( Huelga et al . , 2012 ) . Transfection of tRFTyrGUA led to a significant right - shift in expression of 3 ' UTR bound hnRNPA1 transcripts . Statistical significance was measured using Kolmogorov - Smirnov . ( F ) Cumulative distribution as in ( E ) . Transfection of locked nucleic acid against tRFTyrGUA treatment with 200µM H2O2 led to a significant left - shift in mRNA stability of 3 ' UTR bound hnRNPA1 transcripts . Statistical significance was assessed using Kolmogorov - Smirnov .

O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O

(E) A cumulative distribution in control and hnRNPA1 depleted cells of the stability levels for mRNA transcripts with 3' UTR hnRNPA1 CLIP binding (Huelga et al., 2012). Transfection of tRFTyrGUA led to a significant right-shift in the expression levels of 3' UTR bound hnRNPA1 transcripts. Statistical significance was measured using the Kolmogorov-Smirnov test. (F) Cumulative distribution as in (E). Transfection of locked nucleic acid against tRFTyrGUA and treatment with 200µM H2O2 led to a significant left-shift in mRNA stability of 3'UTR bound hnRNPA1 transcripts. Statistical significance was assessed using the Kolmogorov-Smirnov test.


( H ) UMAP maps color - coded for transcript of genes enriched in subsets of analyzed . scale bars represent color coded sequencing depth - corrected log transformed transcript .

O O O O O O O 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) UMAP maps color-coded for transcript counts of genes enriched in subsets of the analyzed cells. The scale bars represent color coded sequencing depth-corrected log transformed transcript counts.


( H ) Representative immunofluorescence of Iba1 ( green ) APOE ( red ) on cortical sections from SPF GF mice . Nuclei were stained with DAPI ( blue ) . Scale bar : 50 μm . arrowheads indicate APOE + / Iba1 + microglia arrows show APOE - / Iba1 + microglia . of APOE + / Iba1 + microglia . Each symbol represents one mouse . At least three slides were examined per mouse . Data are presented as mean ± s . e . m . Significant differences were determined by unpaired t - marked with asterisks ( * P < 0 . 05 ) . Data are representative of two independent experiments .

O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O B-TISSUE O O O O B-ORGANISM O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O 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) Representative immunofluorescence images of Iba1 (green) and APOE (red) on cortical sections from SPF and GF mice. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. White arrowheads indicate APOE+/Iba1+ microglia and arrows show APOE-/Iba1+ microglia. Quantification of APOE+/Iba1+ microglia. Each symbol represents one mouse. At least three slides were examined per individual mouse. Data are presented as mean ± s.e.m. Significant differences were determined by unpaired t-test and marked with asterisks (*P < 0.05). Data are representative of two independent experiments.


( H ) Representative Iba1 + immunohistochemistry in choroid plexus of SPF , GF , ABX - treated , ASF , recolonized ASF , sDMDMm2 , TLR2 , 3 , 4 , 7 , 9 - deficient , SCFA - treated GF mice or FFAR2 - deficient mice . Scale bar = 50 µm . ( I - O ) of stromal CPMΦ . Each symbol represents one mouse . Three to four sections per mouse were examined . Means ± s . e . m . are indicated . Significant differences were evaluated by unpaired t - or one - way ANOVA followed by Tukey ' s post - hoc comparison marked with asterisks ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Data are representative of at least two independent experiments .

O O O O B-GENEPROD O B-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O B-ORGANISM O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative Iba1+ immunohistochemistry in the choroid plexus of SPF, GF, ABX-treated, ASF, recolonized ASF, sDMDMm2, TLR2,3,4,7,9-deficient, SCFA-treated GF mice or FFAR2-deficient mice. Scale bar = 50 µm. (I-O) Quantification of stromal CPMΦ. Each symbol represents one mouse. Three to four sections per mouse were examined. Means ± s.e.m. are indicated. Significant differences were evaluated by unpaired t-test or one-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). Data are representative of at least two independent experiments.


( H ) Representative Iba1 + immunohistochemistry in perivascular space of SPF , GF , ABX - treated , ASF , recolonized ASF , sDMDMm2 , TLR2 , 3 , 4 , 7 , 9 - deficient , SCFA - treated GF mice or FFAR2 - deficient mice . Arrows indicate Iba1 + perivascular macrophages . Scale bar = 50 µm . ( I - O ) thereof . Each symbol represents one mouse . Three to four sections per mouse were examined . Means ± s . e . m . are indicated . No significant differences were determined by unpaired t - or one - way ANOVA followed by Tukey ' s post - hoc comparison . Data are representative of at least two independent experiments .

O O O O B-GENEPROD O B-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O B-ORGANISM O B-GENEPROD O O B-ORGANISM O O O B-GENEPROD O B-TISSUE B-CELL_TYPE O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative Iba1+ immunohistochemistry in the perivascular space of SPF, GF, ABX-treated, ASF, recolonized ASF, sDMDMm2, TLR2,3,4,7,9-deficient, SCFA-treated GF mice or FFAR2-deficient mice. Arrows indicate Iba1+ perivascular macrophages. Scale bar = 50 µm. (I-O) Quantification thereof. Each symbol represents one mouse. Three to four sections per mouse were examined. Means ± s.e.m. are indicated. No significant differences were determined by unpaired t-test or one-way ANOVA followed by Tukey's post-hoc comparison test. Data are representative of at least two independent experiments.


( H ) Representative Iba1 + immunohistochemistry in leptomeninges of SPF , GF , ABX - treated , ASF , recolonized ASF , sDMDMm2 , TLR2 , 3 , 4 , 7 , 9 - deficient , SCFA - treated GF mice or FFAR2 - deficient mice . Arrows indicate Iba1 + meningeal macrophages . Scale bar = 50 μm . ( I - O ) thereof . Each symbol represents one mouse . Three to four sections per mouse were examined . Means ± s . e . m . are indicated . No significant differences were determined by unpaired t - or one - way ANOVA followed by Tukey ' s post - hoc comparison . Data are representative of at least two independent experiments .

O O O O B-GENEPROD O B-EXP_ASSAY O B-TISSUE O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O B-ORGANISM O B-GENEPROD O O B-ORGANISM O O O B-GENEPROD O B-TISSUE B-CELL_TYPE O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative Iba1+ immunohistochemistry in the leptomeninges of SPF, GF, ABX-treated, ASF, recolonized ASF, sDMDMm2, TLR2,3,4,7,9-deficient, SCFA-treated GF mice or FFAR2-deficient mice. Arrows indicate Iba1+ meningeal macrophages. Scale bar = 50 μm. (I-O) Quantification thereof. Each symbol represents one mouse. Three to four sections per mouse were examined. Means ± s.e.m. are indicated. No significant differences were determined by unpaired t-test or one-way ANOVA followed by Tukey's post-hoc comparison test. Data are representative of at least two independent experiments.


( A ) Iba1 immunohistochemistry depicting cortical microglia , meningeal macrophages , perivascular macrophages choroid plexus macrophages 4 days after i . c . challenge with LCMV or vehicle controls . Arrows indicate Iba1 + meningeal or perivascular macrophages , respectively . ( B ) of Iba1 + microglia , choroid plexus macrophages , meningeal macrophages perivascular macrophages . Each symbol represents one mouse . Three to four sections per mouse were examined . Significant differences were evaluated by two - way ANOVA followed by Tukey ' s post - hoc comparison marked with asterisks ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Data are representative of two independent experiments .

O O O B-GENEPROD B-EXP_ASSAY O B-TISSUE B-CELL_TYPE O B-TISSUE B-CELL_TYPE O B-TISSUE B-CELL_TYPE B-TISSUE I-TISSUE B-CELL_TYPE O O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD O B-TISSUE O B-TISSUE B-CELL_TYPE O O O O O O O B-GENEPROD O B-CELL_TYPE O B-TISSUE I-TISSUE B-CELL_TYPE O B-TISSUE B-CELL_TYPE B-TISSUE B-CELL_TYPE O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Iba1 immunohistochemistry depicting cortical microglia, meningeal macrophages, perivascular macrophages and choroid plexus macrophages 4 days after i.c. challenge with LCMV or vehicle controls. Arrows indicate Iba1+ meningeal or perivascular macrophages, respectively. (B) Quantification of Iba1+ microglia, choroid plexus macrophages, meningeal macrophages and perivascular macrophages. Each symbol represents one mouse. Three to four sections per mouse were examined. Significant differences were evaluated by two-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). Data are representative of two independent experiments.


( C , D ) Representative immunofluorescence ( left ) of ( C ) Iba1 ( green ) KI67 ( red ) or ( D ) CD74 ( red ) on cortical sections from SPF GF mice . Nuclei were stained with DAPI ( blue ) . Scale bars : 25 μm . arrows indicate KI67 + / Iba1 + or CD74 + / Iba1 + macrophages arrowheads show KI67 - / Iba1 + or CD74 - / Iba1 + macrophages . thereof ( right ) . Each symbol represents one mouse . At least three slides were examined per mouse . Data are presented as mean ± s . e . m . Significant differences were evaluated by two - way ANOVA followed by Tukey ' s post - hoc comparison marked with asterisks ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Data are representative of two independent experiments .

O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-TISSUE O O O O B-ORGANISM O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D) Representative immunofluorescence images (left) of (C) Iba1 (green) and KI67 (red) or (D) CD74 (red) on cortical sections from SPF and GF mice. Nuclei were stained with DAPI (blue). Scale bars: 25 μm. White arrows indicate KI67+/Iba1+ or CD74+/Iba1+ macrophages and arrowheads show KI67-/Iba1+ or CD74-/Iba1+ macrophages. Quantification thereof (right). Each symbol represents one mouse. At least three slides were examined per individual mouse. Data are presented as mean ± s.e.m. Significant differences were evaluated by two-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). Data are representative of two independent experiments.


( A - C ) Representative immunofluorescence visualizing ( A ) cortical mMΦ , ( B ) cpMΦ located in lateral ventricle ( C ) hippocampal pvMΦ from SPF ( left ) , GF ( middle ) ABX - treated 5xFAD mice ( right ) . Iba1 ( green ) Aβ ( 6E10 , red ) . Nuclei were stained with DAPI ( blue ) . arrowheads indicate Iba1 + Aβ + MΦ arrows show Iba1 + Aβ - MΦ . Scale bar : 50 μm .

O O O O O O B-EXP_ASSAY O O O O B-TISSUE O O O O O B-TISSUE O O B-TISSUE I-TISSUE O O O B-TISSUE I-TISSUE O O O O O O O O O O B-SMALL_MOLECULE O O O B-ORGANISM O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O

(A-C) Representative immunofluorescence images visualizing (A) cortical mMΦ, (B) cpMΦ located in the lateral ventricle and (C) hippocampal pvMΦ from SPF (left), GF (middle) and ABX-treated 5xFAD mice (right). Iba1 (green) and Aβ (6E10, red). Nuclei were stained with DAPI (blue). White arrowheads indicate Iba1+Aβ+ MΦ and white arrows show Iba1+Aβ- MΦ. Scale bar: 50 μm.


( D ) of number of 6E10 + Aβ - plaques per mm2 . Each symbol represents one mouse . Means ± s . e . m . are indicated . Significant differences were evaluated by one - way ANOVA followed by Tukey ' s post - hoc comparison marked with asterisks ( * * * P < 0 . 001 ) . Data are representative of four independent experiments .

O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of the number of 6E10+ Aβ-plaques per mm2. Each symbol represents one mouse. Means ± s.e.m. are indicated. Significant differences were evaluated by one-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (***P < 0.001). Data are representative of four independent experiments.


( E - G ) of Iba1 + ( E ) mMΦ , ( F ) cpMΦ ( G ) pvMΦ per mm2 . Each symbol represents one mouse . At least three slides were examined per mouse . Data are presented as mean ± s . e . m . Significant differences were determined by two - way ANOVA followed by Tukey ' s post - hoc comparison marked with asterisks ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Data are representative of two independent experiments .

O O O O O O B-GENEPROD O O O O O O O O O B-TISSUE O O O B-TISSUE O O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) Quantification of Iba1+ (E) mMΦ, (F) cpMΦ and (G) pvMΦ per mm2. Each symbol represents one mouse. At least three slides were examined per individual mouse. Data are presented as mean ± s.e.m. Significant differences were determined by two-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). Data are representative of two independent experiments.


( H - J ) of Iba1 + Aβ + ( H ) cortical mMΦ , ( I ) cpMΦ ( J ) hippocampal pvMΦ of SPF , GF ABX - treated 5xFAD mice . Means ± s . e . m . are indicated . Significant differences were evaluated by one - way ANOVA followed by Tukey ' s post - hoc comparison marked with asterisks ( * P < 0 . 05 ) . Data are representative of two independent experiments .

O O O O O O B-GENEPROD O B-GENEPROD O O O O B-TISSUE O O O O O B-TISSUE O O O B-TISSUE I-TISSUE O O O O B-SMALL_MOLECULE O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J) Percentage of Iba1+Aβ+ (H) cortical mMΦ, (I) cpMΦ and (J) hippocampal pvMΦ of SPF, GF and ABX-treated 5xFAD mice. Means ± s.e.m. are indicated. Significant differences were evaluated by one-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (*P < 0.05). Data are representative of two independent experiments.


B RT - PCR on ( C ) ( N ) extracts showing subcellular localization of lnc - SMaRT . was isolated from C2C12 after two days of differentiation . GAPDH mRNA pre - mRNA ( pre - GAPDH ) were used respectively as controls . Representative results from three independent experiments are shown .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_LINE O O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O

B RT-PCR on cytoplasmic (C) and nuclear (N) extracts showing the subcellular localization of lnc-SMaRT. RNA was isolated from C2C12 cells after two days of differentiation. GAPDH mRNA and pre-mRNA (pre-GAPDH) were used respectively as cytoplasmic and nuclear controls. Representative results from three independent experiments are shown.


C qPCR of lnc - SMaRT expression in C2C12 undergoing differentiation at indicated time points . expression in qPCR analyses were normalized against GAPDH mRNA expressed as relative quantities with respect to GM samples set to a value of 1 . Data are presented as mean ± s . e . m . of three biological replicates ( dots ) .

O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C qPCR analysis of lnc-SMaRT RNA expression in C2C12 cells undergoing differentiation at the indicated time points. The RNA expression levels in qPCR analyses were normalized against GAPDH mRNA and expressed as relative quantities with respect to GM samples set to a value of 1. Data are presented as the mean ± s.e.m. of three biological replicates (dots).


D qPCR on extracts from C2C12 myoblasts treated with either control siRNAs ( si - SCR ) or with two different siRNAs against lnc - SMaRT ( si - SMaRT - 1 , si - SMaRT - 2 ) induced to differentiate for two days . expression in qPCR analyses were normalized against GAPDH mRNA expressed as relative quantities with respect to GM samples set to a value of 1 . Data are presented as mean ± s . e . m . of three biological replicates ( dots ) . Statistical was performed with ordinary of variance ( ANOVA ) followed by Dunnett ' s multiple comparison . * * * P < 0 . 001 .

O B-EXP_ASSAY O O O B-CELL_LINE B-CELL_TYPE O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 qPCR on RNA extracts from C2C12 myoblasts treated with either control siRNAs (si-SCR) or with two different siRNAs against lnc-SMaRT (si-SMaRT-1, si-SMaRT-2) and induced to differentiate for two days. The RNA expression levels in qPCR analyses were normalized against GAPDH mRNA and expressed as relative quantities with respect to GM samples set to a value of 1. Data are presented as the mean ± s.e.m. of three biological replicates (dots). Statistical analysis was performed with ordinary analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. ***P < 0.001.


E Representative immunofluorescence for Myosin Heavy Chain ( MHC in red ) in combination with DAPI ( in blue ) C2C12 murine myoblasts transfected with either control ( si - SCR ) or lnc - SMaRT siRNAs ( si - SMaRT - 1 , si - SMaRT - 2 ) fixed after at two days of differentiation . Histograms represent MHC - positive mononucleated / total MHC - positive fusion index ( F . I . ) . At least 5 randomly chosen microscope fields of two independent biological samples were analyzed ( n > 600 for each field ) . Data are presented as mean . of two biological replicates ( dots ) .

O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O B-CELL_LINE B-ORGANISM B-CELL_TYPE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O 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 Representative immunofluorescence for Myosin Heavy Chain (MHC in red) in combination with DAPI staining (in blue) C2C12 murine myoblasts transfected with either control (si-SCR) or lnc-SMaRT siRNAs (si-SMaRT-1, si-SMaRT-2) fixed after at two days of differentiation. Histograms represent MHC-positive mononucleated cells/total MHC-positive cells ratio and fusion index quantification (F.I.). At least 5 randomly chosen microscope fields of two independent biological samples were analyzed (n>600 cells for each field). Data are presented as the mean. of the two biological replicates (dots).


A Gene expression showing hierarchical clustering of - coding genes whose expression is altered upon lnc - SMaRT depletion . Expression values , calculated as RPKMs , were log2 - transformed mean centered . Only genes with an average RPKM between 10 1000 were plotted .

O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Gene expression heatmap showing the hierarchical clustering of protein-coding genes whose expression is altered upon lnc-SMaRT depletion. Expression values, calculated as RPKMs, were log2-transformed and mean centered. Only genes with an average RPKM between 10 and 1000 were plotted.


B Volcano plot describing differential gene expression between si - SCR si - SMaRT treated C2C12 samples . For each gene , log2 of fold - change versus - log10 of unadjusted p - value is plotted . Points in red represent differentially expressed genes ( FDR adjusted p - value < 0 . 05 ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

B Volcano plot describing the differential gene expression between si-SCR and si-SMaRT treated C2C12 samples. For each gene, log2 of fold-change versus -log10 of the unadjusted p-value is plotted. Points in red represent differentially expressed genes (FDR adjusted p-value < 0.05).


D Western blot on extract from control lnc - SMaRT overexpressing C2C12 stable lines collected after one day of differentiation . CASP3 antibody was used . ACTN was used as loading control . Representative results from three independent experiments are shown .

O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O

D Western blot on protein extract from control and lnc-SMaRT overexpressing C2C12 stable cell lines collected after one day of differentiation. CASP3 antibody was used. ACTN was used as loading control. Representative results from three independent experiments are shown.


A Localization on lnc - SMaRT transcript of two sets of biotinylated probes ( Set # 1 Set # 2 ) used for lnc - SMaRT pull - down experiment .

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

A Localization on the lnc-SMaRT transcript of the two sets of biotinylated probes (Set#1 and Set#2) used for lnc-SMaRT pull-down experiment.


B Left panel : qPCR of lnc - SMaRT enrichment in pull - down performed in C2C12 at day 2 of differentiation ( DM2 ) ; Set # 1 Set # 2 probes were used against lnc - SMaRT together with a control set of probes against LacZ mRNA ( LacZ ) . Data are expressed in of input presented as mean ± s . e . m . of three biological replicates ( dots ) . Right panel : Western blot showing specific enrichment of DHX36 helicase in lnc - SMaRT pull - down ; GAPDH was used as negative control . Representative results from three independent experiments are shown .

O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O

B Left panel: qPCR analysis of lnc-SMaRT enrichment in the RNA pull-down performed in C2C12 cells at day 2 of differentiation (DM2); Set#1 and Set#2 probes were used against lnc-SMaRT together with a control set of probes against LacZ mRNA (LacZ). Data are expressed in percentage of input and presented as the mean ± s.e.m. of three biological replicates (dots). Right panel: Western blot analysis showing the specific enrichment of the DHX36 helicase in lnc-SMaRT pull-down; GAPDH was used as the negative control. Representative results from three independent experiments are shown.


C Upper panel : Western blot with DHX36 antibodies on extracts from DHX36 immunoprecipitation . Input sample ( IN ) accounts for 2 . 5 % of extract . Representative results from three independent experiments are shown . Lower panel : qPCR of lnc - SMaRT enrichment in DHX36 RIP - derived extracts . WBP4 was used as positive control [ 31 ] , while Neat1 ( a lncRNA expressed at comparable level of lnc - SMaRT according to - sequencing ) Rps7 were used as negative controls . Data are expessed as of input normalized on IgG control , presented as mean ± s . e . m . of three biological replicates ( dots ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Upper panel: Western blot with DHX36 antibodies on protein extracts from DHX36 RNA immunoprecipitation. Input sample (IN) accounts for 2.5% of the extract. Representative results from three independent experiments are shown. Lower panel: qPCR analysis of lnc-SMaRT enrichment in DHX36 RIP-derived RNA extracts. WBP4 was used as positive control [31], while Neat1 (a lncRNA expressed at comparable level of lnc-SMaRT according to the RNA-sequencing) and Rps7 were used as negative controls. Data are expessed as percentage of input normalized on IgG control, and presented as the mean ± s.e.m. of three biological replicates (dots).


D RT - PCR validation of Mlx - α , Mlx - β MLX - γ mRNA enrichment upon lnc - SMaRT pull - down performed with Set # 1 Set # 2 probes ; a control set of probes against LacZ mRNA ( LacZ ) was used as negative control . GAPDH was used as negative control . Input sample ( IN ) accounts for 10 % of extract . Representative results from three independent experiments are shown .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

D RT-PCR validation of Mlx-α, Mlx-β and MLX-γ mRNA enrichment upon the lnc-SMaRT pull-down performed with Set#1 and Set#2 probes; a control set of probes against LacZ mRNA (LacZ) was used as negative control. GAPDH was used as negative control. Input sample (IN) accounts for 10% of the extract. Representative results from three independent experiments are shown.


E RT - PCR of Mlx - α , Mlx - β Mlx - γ mRNA enrichment in DHX36 RIP - derived extracts in samples treated with control siRNA ( siSCR , upper panel ) or siRNA against lnc - SMaRT ( siSMaRT , lower panel ) . GAPDH was used as negative control . Input sample ( IN ) accounts for 10 % of extract . Representative results from three independent experiments are shown .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

E RT-PCR analysis of Mlx-α, Mlx-β and Mlx-γ mRNA enrichment in DHX36 RIP-derived RNA extracts in samples treated with control siRNA (siSCR, upper panel) or siRNA against lnc-SMaRT (siSMaRT, lower panel). GAPDH was used as negative control. Input sample (IN) accounts for 10% of the extract. Representative results from three independent experiments are shown.


F Upper panel : Western blot with FLAG antibody on extracts from HeLa overexpressing indicated isoforms of FLAG - tagged MLX ( MLX - α , MLX - β MLX - γ ) in control condition ( CTRL ) or in overexpression of lncSMaRT ( OE - SMaRT ) . FLAG - tag has been inserted at N - terminus of MLX isoforms . HPRT was used as loading control . Representative results from three independent experiments are shown . Middle panel : of FLAG - tagged normalized on HPRT signals . Data are expressed presented as mean ± s . d . of three biological replicates ( dots ) . Statistical was performed with two - way ANOVA followed by Bonferroni ' s multiple comparisons . * * * P < 0 . 001 . Lower panel : RT - PCR of lnc - SMaRT Mlx expression on extracts from HeLa overexpressing indicated isoforms of flagged - MLX ( MLX - α , MLX - β MLX - γ ) in control condition ( CTRL ) or in overexpression of lncSMaRT ( OE - SMaRT ) . GAPDH was used as loading control . Representative results from three independent experiments are shown .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O

F Upper panel: Western blot with FLAG antibody on protein extracts from HeLa cells overexpressing the indicated isoforms of FLAG-tagged MLX (MLX-α, MLX-β and MLX-γ) in control condition (CTRL) or in overexpression of lncSMaRT (OE-SMaRT). FLAG-tag has been inserted at the N-terminus of MLX protein isoforms. HPRT was used as loading control. Representative results from three independent experiments are shown. Middle panel: Quantification of FLAG-tagged protein levels normalized on HPRT signals. Data are expressed presented as the mean ± s.d. of three biological replicates (dots). Statistical analysis was performed with two-way ANOVA followed by Bonferroni's multiple comparisons test. ***P < 0.001. Lower panel: RT-PCR analysis of lnc-SMaRT and Mlx expression on RNA extracts from HeLa cells overexpressing the indicated isoforms of flagged-MLX (MLX-α, MLX-β and MLX-γ) in control condition (CTRL) or in overexpression of lncSMaRT (OE-SMaRT). GAPDH was used as loading control. Representative results from three independent experiments are shown.


G Upper panel : Western blot with FLAG antibodies on extracts from N2a transfected with an empty vector ( CTRL ) or with a plasmid overexpressing FLAG - tagged MLX - γ ( MLX - γ ) in control conditions ( si - SCR ) or in knockdown of DHX36 ( si - DHX36 ) . ACTININ was used as loading control . Representative results from three independent experiments are shown . Lower panel : RT - PCR of Mlx - γ expression on described extracts . GAPDH was used as loading control . Representative results from three independent experiments are shown .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O

G Upper panel: Western blot with FLAG antibodies on protein extracts from N2a cells transfected with an empty vector (CTRL) or with a plasmid overexpressing FLAG-tagged MLX-γ (MLX-γ) in control conditions (si-SCR) or in knockdown of DHX36 (si-DHX36). ACTININ was used as loading control. Representative results from three independent experiments are shown. Lower panel: RT-PCR analysis of Mlx-γ expression on the described RNA extracts. GAPDH was used as loading control. Representative results from three independent experiments are shown.


A Immunofluorescence for total MLX detection ( red signal ) performed on C2C12 treated with siRNAs against Mlx - γ ( si - Mlx - γ ) , lnc - SMaRT ( si - SMaRT ) , DHX36 ( si - DHX36 ) or with a control siRNA ( si - SCR ) . were fixed two days after switch to differentiation medium . Representative binary of single confocal planes for MLX immunofluorescence are shown for each treatment . inserts on topside of sketch MLX signal peaks in nuclei ( stars ) to highlight variation of MLX in each condition . Dashed lines indicate edge of nucleus .

O B-EXP_ASSAY O O B-GENEPROD O O O O O O O B-CELL_LINE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O

A Immunofluorescence for total MLX protein detection (red signal) performed on C2C12 cells treated with siRNAs against Mlx-γ (si-Mlx-γ), lnc-SMaRT (si-SMaRT), DHX36 (si-DHX36) or with a control siRNA (si-SCR). Cells were fixed two days after the switch to differentiation medium. Representative binary images of single confocal planes for MLX immunofluorescence are shown for each treatment. The inserts on the topside of the images sketch the MLX signal peaks in the nuclei (white stars) to highlight the variation of MLX staining in each condition. Dashed lines indicate the edge of the nucleus.


B Histogram represents of MLX signals in ( Nuc ) ( Cyt ) compartment in indicated conditions . About 50 from two independent experiments were analyzed Data are presented as mean of two biological replicates ( dots ) .

O O O O B-GENEPROD O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Histogram represents the fluorescence intensity ratio of the MLX protein signals in the nuclear (Nuc) and cytoplasmic (Cyt) compartment in the indicated conditions. About 50 cells from two independent experiments were analyzed Data are presented as the mean of two biological replicates (dots).


A Left panel : schematic representation of luciferase constructs produced for Mlx . Mlx 5 ' - 3 ' UTR - interacting regions were cloned respectively upstream downstream Renilla luciferase coding region ( RLuc - Mlx 5 ' - 3 ' ) mutant derivatives devoid of 5 ' interacting region ( RLuc - Mlx - 3 ' ) or 3 ' UTR interacting region ( RLuc - Mlx - 5 ' ) were obtained . These constructs were co - transfected in C2C12 myoblasts in growth conditions together with plasmids expressing lnc - SMaRT ( + ) or with a control vector ( - ) . Right panel : Luciferase activity data , presented as mean ± s . e . m . of three biological replicates ( dots ) , are shown with respect to each RLuc control vector ( RLuc - Mlx 5 ' - 3 ' , RLuc - Mlx - 3 ' , RLuc - Mlx - 5 ' ) set to a value of 1 . Statistical was performed with ordinary of variance ( ANOVA ) followed by Dunnett ' s multiple comparison . * P < 0 . 05 .

O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-ORGANISM B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-CELL_LINE B-CELL_TYPE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Left panel: schematic representation of the luciferase constructs produced for Mlx. Mlx 5'- and 3'UTR-interacting regions were cloned respectively upstream and downstream the Renilla luciferase coding region (RLuc-Mlx 5'-3') and mutant derivatives devoid of the 5' interacting region (RLuc-Mlx-3') or the 3' UTR interacting region (RLuc-Mlx-5') were obtained. These constructs were co-transfected in C2C12 myoblasts in growth conditions together with plasmids expressing lnc-SMaRT (+) or with a control vector (-). Right panel: Luciferase activity data, presented as the mean ± s.e.m. of three biological replicates (dots), are shown with respect to each RLuc control vector (RLuc-Mlx 5'-3', RLuc-Mlx-3', RLuc-Mlx-5') set to a value of 1. Statistical analysis was performed with ordinary analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. *P < 0.05.


B RLuc - Mlx - 5 ' construct was co - transfected in N2a together with plasmids expressing lnc - SMaRT ( + ) or with a control vector ( - ) treated with control siRNA ( + ) or siRNA against DHX36 ( - ) . Luciferase activity data , presented as mean ± s . e . m . of three biological replicates ( dots ) , are shown with respect to RLuc - Mlx - 5 ' vector set to a value of 1 . Statistical was performed with ordinary of variance ( ANOVA ) followed by Dunnett ' s multiple comparison . * * P < 0 . 01 , * * * P < 0 . 001 .

O B-GENEPROD O B-GENEPROD O O O O O O O O O B-CELL_LINE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B The RLuc-Mlx-5' construct was co-transfected in N2a cells together with plasmids expressing lnc-SMaRT (+) or with a control vector (-) and treated with control siRNA (+) or siRNA against DHX36 (-). Luciferase activity data, presented as the mean ± s.e.m. of three biological replicates (dots), are shown with respect to RLuc-Mlx-5' vector set to a value of 1. Statistical analysis was performed with ordinary analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. **P < 0.01, ***P < 0.001.


C Left panel : schematic representation of luciferase constructs used . RLuc - Mlx - 5 ' construct its derived mutants ( RLuc - Mlxmut30 , RLuc - Mlx Δ75 ) were transfected in N2a in growth conditions together with lnc - SMaRT - expressing plasmids ( lnc - SMaRT ) or with its derived mutant ( lnc - SMaRTmut ) in indicated combinations . Right panel : Luciferase activity data , presented as mean ± s . e . m . of three biological replicates ( dots ) , are shown with respect to each RLuc control vector ( RLuc - Mlx - 5 ' , RLuc - Mlxmut30 , RLuc - Mlx Δ75 ) set to a value of 1 . Statistical was performed with ordinary of variance ( ANOVA ) followed by Dunnett ' s multiple comparison . * * * P < 0 . 001 .

O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-CELL_LINE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Left panel: schematic representation of the luciferase constructs used. The RLuc-Mlx-5' construct and its derived mutants (RLuc-Mlxmut30, RLuc-Mlx Δ75) were transfected in N2a cells in growth conditions together with lnc-SMaRT-expressing plasmids (lnc-SMaRT) or with its derived mutant (lnc-SMaRTmut) in the indicated combinations. Right panel: Luciferase activity data, presented as the mean ± s.e.m. of three biological replicates (dots), are shown with respect to each RLuc control vector (RLuc-Mlx-5', RLuc-Mlxmut30, RLuc-Mlx Δ75) set to a value of 1. Statistical analysis was performed with ordinary analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. ***P < 0.001.


A Upper panel : schematic representation of predicted base pairing between lnc - SMaRT ( region A ) Mlx γ mRNA ; predicted G - 4 structure is boxed in red while synthetic oligos sequences are underlined in red . Lower panel : Lane 1 2 : 5 pmol of lnc - SMaRT Mlx synthetic oligos were loaded on a denaturing polyacrylamide gel stained for total ( SYBR GOLD , indicated as SYBR in figure ) . Lane 3 4 : 5 pmol of lnc - SMaRT Mlx synthetic oligos were loaded on a native polyacrylamide gel stained for total ( SYBR GOLD ) . Lane 5 6 : 200 pmol of lnc - SMaRT Mlx synthetic oligos were loaded on a native polyacrylamide gel stained with selective G - quadruplex N - methyl mesoporphyrin IX ( NMM ) . Lane 7 8 : 5 pmol of Mlx oligo alone ( 7 ) or in combination with 5pmol of lnc - SMaRT oligo ( 8 ) were heated at 100 ° C for 10 min , after slow cooling , loaded on a native polyacrylamide gel stained for total ( SYBR GOLD ) . Lane 9 10 : 200 pmol of Mlx oligo alone ( 7 ) or in combination with 200 pmol of lnc - SMaRT oligo ( 8 ) were heated at 100 ° C for 10 min , after slow cooling , loaded on a native polyacrylamide gel stained with selective G - quadruplex N - methyl mesoporphyrin IX . Asterisk ( * ) indicates second NMM - positive band obtained upon SMaRT γ - oligo interaction . Data information : Representative gels from at least three independent experiments are shown .

O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O

A Upper panel: schematic representation of the predicted base pairing between lnc-SMaRT (region A) and Mlx γ mRNA; the predicted G-4 structure is boxed in red while the synthetic oligos sequences are underlined in red. Lower panel: Lane 1 and 2: 5 pmol of lnc-SMaRT and Mlx synthetic oligos were loaded on a denaturing polyacrylamide gel and stained for total RNA (SYBR GOLD, indicated as SYBR in the figure). Lane 3 and 4: 5 pmol of lnc-SMaRT and Mlx synthetic oligos were loaded on a native polyacrylamide gel and stained for total RNA (SYBR GOLD). Lane 5 and 6: 200 pmol of lnc-SMaRT and Mlx synthetic oligos were loaded on a native polyacrylamide gel and stained with the selective G-quadruplex staining N-methyl mesoporphyrin IX (NMM). Lane 7 and 8: 5 pmol of Mlx oligo alone (7) or in combination with 5pmol of lnc-SMaRT oligo (8) were heated at 100°C for 10 min and, after slow cooling, loaded on a native polyacrylamide gel and stained for total RNA (SYBR GOLD). Lane 9 and 10: 200 pmol of Mlx oligo alone (7) or in combination with 200 pmol of lnc-SMaRT oligo (8) were heated at 100°C for 10 min and, after slow cooling, loaded on a native polyacrylamide gel and stained with the selective G-quadruplex staining N-methyl mesoporphyrin IX. Asterisk (*) indicates the second NMM-positive band obtained upon SMaRT and γ-oligo interaction. Data information: Representative gels from at least three independent experiments are shown.


B 5 pmol of indicated oligos were loaded on a native polyacrylamide gel . Oligos were treated as described . ° C = oligos were heated ( + ) or were not heated ( - ) at 100 ° C for 10 min loaded after slow cooling . KCl = oligos were incubated ( + ) or were not ( - ) in a buffer containing 100mM KCl ( for G - quadruplex stabilization ) . gel was stained with total ( SYBR GOLD ) . Data information : Representative gels from at least three independent experiments are shown .

O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O

B 5 pmol of indicated oligos were loaded on a native polyacrylamide gel. Oligos were treated as described. °C = oligos were heated (+) or were not heated (-) at 100 °C for 10 min and loaded after slow cooling. KCl= oligos were incubated (+) or were not (-) in a buffer containing 100mM KCl (for G-quadruplex stabilization). The gel was stained with total RNA staining (SYBR GOLD). Data information: Representative gels from at least three independent experiments are shown.


C 200 pmol of indicated oligos treated as in ( B ) were loaded on a native polyacrylamide gel with selective G - quadruplex N - methyl mesoporphyrin IX . Asterisk ( * ) indicates second NMM - positive band obtained upon SMaRT γ - oligo interaction . Data information : Representative gels from at least three independent experiments are shown .

O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O

C 200 pmol of indicated oligos treated as in (B) were loaded on a native polyacrylamide gel with the selective G-quadruplex staining N-methyl mesoporphyrin IX. Asterisk (*) indicates the second NMM-positive band obtained upon SMaRT and γ-oligo interaction. Data information: Representative gels from at least three independent experiments are shown.


C ) ykt6ts pho8 ∆ 60 containing an empty plasmid , 3HA - Ykt6 or 3HA - tagged Ykt6 mutant variants as indicated were analyzed as in A ) .

O O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O

C) ykt6ts pho8∆60 cells containing an empty plasmid, 3HA-Ykt6 or 3HA-tagged Ykt6 mutant variants as indicated were analyzed as in A).


D ) ykt6ts pho8 ∆ 60 containing an empty plasmid , 3HA - Ykt6 , or ADH1 promoter driven 3HA - Ykt6 wild type or 3HA - tagged Ykt6 mutant variants as indicated were grown Pho8 ∆ 60 activity was monitored as described in A ) .

O O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O

D) ykt6ts pho8∆60 cells containing an empty plasmid, 3HA-Ykt6, or ADH1 promoter driven 3HA-Ykt6 wild type or 3HA-tagged Ykt6 mutant variants as indicated were grown and Pho8∆60 activity was monitored as described in A).


E ) Ykt6 were analyzed from strains shown in D ) corresponding extracts were analyzed by anti - HA anti - Pgk1 Western blotting .

O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O

E) Ykt6 levels were analyzed from the strains shown in D) and corresponding cell extracts were analyzed by anti-HA and anti-Pgk1 Western blotting.


A ) ykt6ts containing centromeric plasmids as indicated were grown at permissive ( 23 ºC ) , shifted to restrictive at 37 ºC for 1 hour before starvation for 1 hour at 37 ºC . were lysed total extract ( T ) separated into a ( S ) a 100 ' 000x g pellet ( P ) fraction . Fractions were analyzed by anti - HA , anti - Tom70 anti - Pgk1 Western blotting . One representative experiment out of three biological replicates is shown .

O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

A) ykt6ts cells containing centromeric plasmids as indicated were grown at permissive temperature (23 ºC), shifted to the restrictive temperature at 37 ºC for 1 hour before starvation for 1 hour at 37 ºC. Cells were lysed and the total extract (T) separated into a cytoplasmic (S) and a 100'000x g membrane pellet (P) fraction. Fractions were analyzed by anti-HA, anti-Tom70 and anti-Pgk1 Western blotting. One representative experiment out of three biological replicates is shown.


B ) vam3 ∆ or ykt6ts vam3 ∆ containing indicated centromeric plasmids were grown at permissive ( 23 ºC ) , shifted to restrictive at 37 ° C for 1 hour followed by 1 hour starvation in SD - N at 37 ºC . After lysis , samples were subjected to proteinase K ( ProtK ) Triton X - 100 ( TX100 ) treatment as indicated analyzed by anti - Ape1 Western blotting . Ape1 * : proteinase K - resistant fragment of Ape1 . One representative experiment out of three biological replicates is shown .

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

B) vam3∆ or ykt6ts vam3∆ cells containing the indicated centromeric plasmids were grown at permissive temperature (23 ºC), shifted to the restrictive temperature at 37 °C for 1 hour followed by 1 hour starvation in SD-N at 37 ºC. After cell lysis, samples were subjected to proteinase K (ProtK) and Triton X-100 (TX100) treatment as indicated and analyzed by anti-Ape1 Western blotting. Ape1*: proteinase K-resistant fragment of Ape1. One representative experiment out of three biological replicates is shown.


C ) Trichloroacetic acid extracts were prepared from ykt6ts containing an empty plasmid , 3HA - Ykt6 wild type , or 3HA - tagged Ykt6 mutant variants . were grown at permissive ( 23 ºC ) to late exponential phase in selective medium , shifted to restrictive ( 37 ° C ) for 1 h preincubation continued to grow at restrictive for another 4 hours before harvesting . Samples were analyzed by anti - CPY anti - Pgk1 Western blotting . One representative experiment out of three biological replicates is shown .

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

C) Trichloroacetic acid extracts were prepared from ykt6ts cells containing an empty plasmid, 3HA-Ykt6 wild type, or 3HA-tagged Ykt6 mutant variants. Cells were grown at the permissive temperature (23 ºC) to late exponential phase in selective medium, shifted to the restrictive temperature (37 °C) for 1 h preincubation and continued to grow at the restrictive temperature for another 4 hours before harvesting. Samples were analyzed by anti-CPY and anti-Pgk1 Western blotting. One representative experiment out of three biological replicates is shown.


D ) Exponentially growing ykt6ts at permissive ( 23 ºC ) containing centromeric plasmids as indicated were shifted to restrictive ( 37 ºC ) for 1 hour before starvation for 4 hours in SD - N . Trichloroacetic acid extracts were prepared . Lipidation of Atg8 was analyzed on a 15 % SDS - PAGE containing 6 M urea by anti - Atg8 anti - Pgk1 Western blotting . One representative experiment out of three biological replicates is shown .

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

D) Exponentially growing ykt6ts cells at permissive temperature (23 ºC) containing centromeric plasmids as indicated were shifted to the restrictive temperature (37 ºC) for 1 hour before starvation for 4 hours in SD-N. Trichloroacetic acid extracts were prepared. Lipidation of Atg8 was analyzed on a 15 % SDS-PAGE containing 6 M urea by anti-Atg8 and anti-Pgk1 Western blotting. One representative experiment out of three biological replicates is shown.


A B ) GFP - Atg8 atg1 ∆ , GFP - Atg8 atg1 ∆ vam3 ∆ , GFP - Atg8 wild type , GFP - Atg8 vam3 ∆ , GFP - Atg8 ykt6ts or GFP - Atg8 ykt6ts vam3 ∆ containing centromeric plasmids as indicated were grown at permissive ( 23 ºC ) , shifted to restrictive at 37 ºC for 2 hours before starvation for 1 hour at 37 ºC in SD - N . Representative microscopy are shown in A ) . GFP - Atg8 per were quantified from 3 biological replicates ( n ≥ 100 ) mean was plotted in B ) . Error bars are SD . Scale bar : 5 µm .

O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A and B) GFP-Atg8 atg1∆, GFP-Atg8 atg1∆ vam3∆, GFP-Atg8 wild type, GFP-Atg8 vam3∆, GFP-Atg8 ykt6ts or GFP-Atg8 ykt6ts vam3∆ cell containing centromeric plasmids as indicated were grown at permissive temperature (23 ºC), shifted to the restrictive temperature at 37 ºC for 2 hours before starvation for 1 hour at 37 ºC in SD-N. Representative microscopy images are shown in A). GFP-Atg8 puncta per cell were quantified from 3 biological replicates (n≥100 cells) and the mean was plotted in B). Error bars are SD. Scale bar: 5 µm.


Autophagosomes were prepared from GFP - Atg8 vam3 ∆ pep4 ∆ or GFP - Atg8 ykt6ts vam3 ∆ pep4 ∆ containing centromeric plasmids as indicated . were starved for 16 hours at permissive ( 23 ° C ) to allow formation of mature , closed autophagosomes shifted for 1 hour to restrictive before harvesting . Vacuoles were isolated from Vph1 - 4xmCherry atg15 ∆ pep4 ∆ grown under rich conditions at 30 ° C . Fusion reactions were incubated at restrictive ( 30 ° C ) for 2 hours . Representative are shown in C ) .

B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Autophagosomes were prepared from GFP-Atg8 vam3∆ pep4∆ or GFP-Atg8 ykt6ts vam3∆ pep4∆ cells containing centromeric plasmids as indicated. Cells were starved for 16 hours at the permissive temperature (23 °C) to allow formation of mature, closed autophagosomes and shifted for 1 hour to the restrictive temperature before harvesting. Vacuoles were isolated from Vph1-4xmCherry atg15∆ pep4∆ cells grown under rich conditions at 30 °C. Fusion reactions were incubated at the restrictive temperature (30 °C) for 2 hours. Representative images are shown in C).


Autophagosomes were prepared from GFP - Atg8 vam3 ∆ pep4 ∆ or GFP - Atg8 ykt6ts vam3 ∆ pep4 ∆ containing centromeric plasmids as indicated . were starved for 16 hours at permissive ( 23 ° C ) to allow formation of mature , closed autophagosomes shifted for 1 hour to restrictive before harvesting . Vacuoles were isolated from Vph1 - 4xmCherry atg15 ∆ pep4 ∆ grown under rich conditions at 30 ° C . Fusion reactions were incubated at restrictive ( 30 ° C ) for 2 hours . graph in D ) shows mean from at least three independent biological experiments . Error bars represent standard deviation .

B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Autophagosomes were prepared from GFP-Atg8 vam3∆ pep4∆ or GFP-Atg8 ykt6ts vam3∆ pep4∆ cells containing centromeric plasmids as indicated. Cells were starved for 16 hours at the permissive temperature (23 °C) to allow formation of mature, closed autophagosomes and shifted for 1 hour to the restrictive temperature before harvesting. Vacuoles were isolated from Vph1-4xmCherry atg15∆ pep4∆ cells grown under rich conditions at 30 °C. Fusion reactions were incubated at the restrictive temperature (30 °C) for 2 hours. The graph in D) shows the mean from at least three independent biological experiments. Error bars represent the standard deviation.


indicated strains were grown to late exponential phase at permissive ( 23 ºC ) , shifted to restrictive at 37 ° C for 1 hour followed by starvation in SD - N for 1 hour at 37 ºC . 3HA - Ykt6 its mutant variants expressed from a centromeric plasmid were immunoprecipitated ( IP ) analyzed by anti - HA Western blotting . Co - precipitating were analyzed by anti - Vam3 anti - Vti1 Western blotting . One representative experiment out of three 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 B-GENEPROD O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

The indicated strains were grown to late exponential phase at permissive temperature (23 ºC), shifted to the restrictive temperature at 37 °C for 1 hour followed by starvation in SD-N for 1 hour at 37 ºC. 3HA-Ykt6 and its mutant variants expressed from a centromeric plasmid were immunoprecipitated (IP) and analyzed by anti-HA Western blotting. Co-precipitating proteins were analyzed by anti-Vam3 and anti-Vti1 Western blotting. One representative experiment out of three biological replicates is shown.


X - ray of 6 - months - old H2 - c - fosLTR / Egfrwa / + H2 - c - fosLTR / Egfrwa / wa . Scale bars :

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O

X-ray analysis of 6-months-old H2-c-fosLTR/Egfrwa/+ and H2-c-fosLTR/Egfrwa/wa littermates. Scale bars: 1cm