e , Percentages of Ki - 67 + cells among total CD8 T cells . f , Percentage of Ki - 67 + cells among splenic CD8 Tn , Tem and Tcm in 45 - wk - old Dj - 1 KO and age - and sex - matched WT mice .

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e, Percentages of Ki-67+ cells among total CD8 T cells. f, Percentage of Ki-67+ cells among splenic CD8 Tn, Tem and Tcm in 45-wk-old Dj-1 KO and age- and sex-matched WT mice. g


j , Percentages of Tn ( top ) , Tvm ( middle ) , Tmem ( bottom ) among 60 - wk - old or young mice ( young KO , n = 4 ; young WT , n = 3 ; 60 - wk - old KO , n = 4 ; 60 - wk - old WT , n = 5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Percentages of Tn (top), Tvm (middle), Tmem (bottom) among 60-wk-old or young mice (young KO, n=4; young WT, n=3; 60-wk-old KO, n=4; 60-wk-old WT, n=5).


b , Percentages of KLRG1 + CD8 T cells derived from 45 - wk - old Dj - 1 KO and WT BM cells within young Dj - 1 KO ( n = 4 ) or WT recipients ( n = 5 ) . c , Percentages of KLRG1 + among CD8 Tem derived from 45 - wk - old Dj - 1 KO and WT BM cells within young Dj - 1 KO or WT recipients . d d , Percentages of PD - 1 + population among CD8 T cells derived from 45 - wk - old Dj - 1 KO and WT BM cells within young Dj - 1 KO ( n = 4 ) or WT recipients ( n = 5 ) .

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

b, Percentages of KLRG1+ CD8 T cells derived from 45-wk-old Dj-1 KO and WT BM cells within young Dj-1 KO (n=4) or WT recipients (n=5). c, Percentages of KLRG1+ among CD8 Tem derived from 45-wk-old Dj-1 KO and WT BM cells within young Dj-1 KO or WT recipients. d d, Percentages of PD-1+ population among CD8 T cells derived from 45-wk-old Dj-1 KO and WT BM cells within young Dj-1 KO (n=4) or WT recipients (n=5).


j , Percentage of KLRG1 + population among total CD8 T cells ( 55 - wk - old KO , n = 4 ; 55 - wk - old WT , n = 5 ; young KO , n = 4 ; young WT , n = 4 ) . k , Percentage of KLRG1 + population among CD8 Tem cells . l , Percentage of KLRG1 + PD - 1 + population among total CD8 T cells ( 55 - wk - old KO , n = 4 ; 55 - wk - old WT , n = 5 ; young KO , n = 4 ; young WT , n = 4 ) . m , Percentage of KLRG1 + PD - 1 + population among CD8 Tem cells .

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

j, Percentage of KLRG1+ population among total CD8 T cells (55-wk-old KO, n=4; 55-wk-old WT, n=5; young KO, n=4; young WT, n=4). k, Percentage of KLRG1+ population among CD8 Tem cells. l, Percentage of KLRG1+PD-1+ population among total CD8 T cells (55-wk-old KO, n=4; 55-wk-old WT, n=5; young KO, n=4; young WT, n=4). m, Percentage of KLRG1+PD-1+ population among CD8 Tem cells. Dat


b , the percentages of proliferating cells among living CD8 singlets ( CD8 Tn were from young mice ) .

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b, the percentages of proliferating cells among living CD8 singlets (CD8 Tn were from young mice).


e , the percentages of proliferating cells among living CD8 singlets ( CD8 Tn were from 45 - wk - old mice ) .

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e, the percentages of proliferating cells among living CD8 singlets (CD8 Tn were from 45-wk-old mice).


e - h , Oxygen consumption rate ( OCR ) of unstimulated ( e ) and stimulated CD8 Tn ( f ) by 2ug / ml of anti - CD3 and 1ug / ml of anti - CD28 for 24 hours . The CD8 Tn were isolated from 45 - wk - old mice . OCR of unstimulated ( g ) and stimulated CD8 Tn ( h ) by anti - CD3 and - CD28 for 24 hours . The CD8 Tn were isolated from young mice . Left of each OCR measurement panel , representation plots following Mito stress test . Right of each panel , quantitation of basal and maximum respiration under Mito stress test conditions The dashed line indicates the time when the corresponding modulator Oligomycin ( Oligo ) , Carbonyl cyanide - 4 ( trifluoromethoxy ) phenylhydrazone ( FCCP ) or Rotenone and Antimycin ( AA / Rot ) was added .

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

e-h, Oxygen consumption rate (OCR) of unstimulated (e) and stimulated CD8 Tn (f) by 2ug/ml of anti-CD3 and 1ug/ml of anti-CD28 for 24 hours. The CD8 Tn were isolated from 45-wk-old mice. OCR of unstimulated (g) and stimulated CD8 Tn (h) by anti-CD3 and -CD28 for 24 hours. The CD8 Tn were isolated from young mice. Left of each OCR measurement panel, representation plots following Mito stress test. Right of each panel, quantitation of basal and maximum respiration under Mito stress test conditions The dashed line indicates the time when the corresponding modulator Oligomycin (Oligo), Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) or Rotenone and Antimycin (AA/Rot) was added.


i - k , Extracellular acidification rate ( ECAR ) of unstimulated and stimulated CD8 Tn by anti - CD3 and - CD28 antibodies for 24 hours ( i ) . The CD8 Tn were isolated from 45 - wk - old mice . ECAR of unstimulated ( j ) and stimulated ( k ) CD8 Tn by anti - CD3 and - CD28 for 24 hours . The CD8 Tn were isolated from young mice . Left of each ECAR measurement panel , representation plots following Glycolysis stress test . Right of each panel , quantitation of glycolysis and glycolytic capacity under Glycolysis stress test conditions . The dashed line indicates the time when the corresponding modulator glucose , Oligomycin ( Oligo ) or 2 - deoxy - glucose ( 2 - DG ) was added .

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

i-k, Extracellular acidification rate (ECAR) of unstimulated and stimulated CD8 Tn by anti-CD3 and -CD28 antibodies for 24 hours (i). The CD8 Tn were isolated from 45-wk-old mice. ECAR of unstimulated (j) and stimulated (k) CD8 Tn by anti-CD3 and -CD28 for 24 hours. The CD8 Tn were isolated from young mice. Left of each ECAR measurement panel, representation plots following Glycolysis stress test. Right of each panel, quantitation of glycolysis and glycolytic capacity under Glycolysis stress test conditions. The dashed line indicates the time when the corresponding modulator glucose, Oligomycin (Oligo) or 2-deoxy-glucose (2-DG) was added.


( C ) Levels of mucosal vtRNA2 - 1 , vtRNA1 - 1 , vtRNA1 - 2 , and vtRNA1 - 3 in the colon of patients with active ulcerative colitis ( UC ) . Values are the means ± SEM ( n = 6 per group ) . Unpaired , two - tailed Student ' s t test was used . * P < 0 . 05 compared with controls . ( D ) Levels of tissue vtRNAs in the ileal mucosa of patients with Crohn ' s disease ( CD ) . Values are the means ± SEM ( n = 6 per group ) . Unpaired , two - tailed Student ' s t test was used . * P < 0 . 05 compared with controls .

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

(C) Levels of mucosal vtRNA2-1, vtRNA1-1, vtRNA1-2, and vtRNA1-3 in the colon of patients with active ulcerative colitis (UC). Values are the means ± SEM (n = 6 per group). Unpaired, two-tailed Student's t test was used. * P < 0.05 compared with controls. (D) Levels of tissue vtRNAs in the ileal mucosa of patients with Crohn's disease (CD). Values are the means ± SEM (n = 6 per group). Unpaired, two-tailed Student's t test was used. * P < 0.05 compared with controls.


( B ) Expression levels of claudin 1 , occludin , E - cadherin , ZO - 1 , JAM - A , and MVP in cells described in A . Hsc70 immunoblotting was performed as an internal control for equal loading .

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(B) Expression levels of claudin 1, occludin, E-cadherin, ZO-1, JAM-A, and MVP in cells described in A. Hsc70 immunoblotting was performed as an internal control for equal loading.


( B ) Immunoblots of claudin 1 , occludin , E - cadherin , ZO - 1 , and JAM - A in cells treated as described in A . Hsc70 immunoblotting served as an internal control for equal loading . Three separate experiments were performed and showed similar results .

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

(B) Immunoblots of claudin 1, occludin, E-cadherin, ZO-1, and JAM-A in cells treated as described in A. Hsc70 immunoblotting served as an internal control for equal loading. Three separate experiments were performed and showed similar results.


( E ) Immunostaining ( left ) of Paneth ( lysozyme - positive ) cells and quantitative data ( right ) in intestinal organoids treated as described in A . Lysozyme : green . Scale bars , 100 µm . Values are means ± SEM ( n = 4 biological replicates ) . Unpaired , two - tailed Student ' s t test was used . * P < 0 . 05 compared with control vector .

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(E) Immunostaining (left) of Paneth (lysozyme-positive) cells and quantitative data (right) in intestinal organoids treated as described in A. Lysozyme: green. Scale bars, 100 µm. Values are means ± SEM (n = 4 biological replicates). Unpaired, two-tailed Student's t test was used. * P < 0.05 compared with control vector.


( B ) Immunoblots of claudin 1 , occludin , JAM - A , ZO - 1 , and E - cadherin in the mucosa from mice treated as described in A . GAPDH immunoblotting served as an internal control for equal loading .

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

(B) Immunoblots of claudin 1, occludin, JAM-A, ZO-1, and E-cadherin in the mucosa from mice treated as described in A. GAPDH immunoblotting served as an internal control for equal loading.


( C ) Newly synthesized claudin 1 and occludin proteins in cells treated as described in A . After cells were exposed to L - azidohomoalaine ( AHA ) , cell lysates were incubated with the reaction buffer containing biotin / alkyne reagent ; the biotin - alkyne - azide - modified protein complex was pulled down by paramagnetic streptavidin - conjugated dynabeads . ( D ) Quantitative data of immunoblots shown in C . Values are means ± SEM ( n = 3 biological replicates ) . Unpaired , two - tailed Student ' s t test was used . * P < 0 . 05 compared with control vector .

O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Newly synthesized claudin 1 and occludin proteins in cells treated as described in A. After cells were exposed to L-azidohomoalaine (AHA), cell lysates were incubated with the reaction buffer containing biotin/alkyne reagent; the biotin-alkyne-azide-modified protein complex was pulled down by paramagnetic streptavidin-conjugated dynabeads. (D) Quantitative data of immunoblots shown in C. Values are means ± SEM (n = 3 biological replicates). Unpaired, two-tailed Student's t test was used. * P < 0.05 compared with control vector.


( A ) Association of vtRNA2 - 1 with HuR in Caco - 2 cells as measured by biotin - labeled RNA pull - down assays . After cytoplasmic lysates were incubated with biotinylated vtRNA2 - 1 , levels of HuR and other proteins in the pull - down material were assessed . Three separate experiments showed similar results .

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

(A) Association of vtRNA2-1 with HuR in Caco-2 cells as measured by biotin-labeled RNA pull-down assays. After cytoplasmic lysates were incubated with biotinylated vtRNA2-1, levels of HuR and other proteins in the pull-down material were assessed. Three separate experiments showed similar results.


( D ) Changes in the expression levels of claudin - 1 , occludin , and E - cadherin proteins 48 h after cells were transfected with HuR expression vector alone or co - transfected with HuR and vtRNA2 - 1 expression vectors .

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

(D) Changes in the expression levels of claudin-1, occludin, and E-cadherin proteins 48 h after cells were transfected with HuR expression vector alone or co-transfected with HuR and vtRNA2-1 expression vectors.


A . Relative abundance of miR species in human naive CD4 T cells . Cell purity was > 97 % .

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

A. Relative abundance of miR species in human naive CD4 T cells. Cell purity was >97%.


B , C . Average miR expression levels in unstimulated naive CD4 T cells non - infected vs HIV - 1NL4 - 3 ( B ) or HIV - 2ROD ( C ) infected after 24h ( n = 2 , three pooled samples per library ) . Mean normalized read counts across libraries are shown . Library normalization was performed using the mean normalization method available in the Deseq package of Bioconductor ( Anders Huber , 2010 ) . Lines indicate changes of + / - 1 log2 fold between samples .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C. Average miR expression levels in unstimulated naive CD4 T cells non-infected vs HIV-1NL4-3 (B) or HIV-2ROD (C) infected after 24h (n=2, three pooled samples per library). Mean normalized read counts across libraries are shown. Library normalization was performed using the mean normalization method available in the Deseq package of Bioconductor (Anders Huber, 2010). Lines indicate changes of +/- 1 log2 fold between samples.


A . Average miR expression levels in naive CD4 T cells ( purity > 97 % ) before and after 72h TCR - stimulation . Lines indicate changes of + / - 1 log2 fold between samples ( n = 2 , paired pools of three and nine individual samples per library ) . Library normalization performed using the mean normalization method available in Deseq package of Bioconductor ( Anders Huber , 2010 ) . miRs with significant changes are highlighted ( black ) and with insets presenting mean read counts in unstimulated samples ( mean ) , log2 fold change values in stimulated samples ( log FC ) and corresponding adjusted p value ( p ; Fisher test implemented in Deseq ) . The two most highly expressed miRs ( mir - 21 - 5p and miR - 146b - 5p ) are also indicated .

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

A. Average miR expression levels in naive CD4 T cells (purity >97%) before and after 72h TCR-stimulation. Lines indicate changes of +/- 1 log2 fold between samples (n=2, paired pools of three and nine individual samples per library). Library normalization performed using the mean normalization method available in Deseq package of Bioconductor (Anders Huber, 2010). miRs with significant changes are highlighted (black) and with insets presenting mean read counts in unstimulated samples (mean), log2 fold change values in stimulated samples (log FC) and corresponding adjusted p value (p; Fisher test implemented in Deseq). The two most highly expressed miRs (mir-21-5p and miR-146b-5p) are also indicated.


B . Relative miR - 34c - 5p and miR - 155 - 5p expression levels ( 2deltaCt normalized to RNU48 ) in unstimulated and stimulated naive CD4 T cells of individual samples pooled to generate the sequencing libraries . mean ± SEM and p - value for differences in miR - 155 - 5p expression ( paired two tailed t - test ) are shown . Comparison could not be performed for miR - 34c - 5p as it is undetermined in unstimulated samples .

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

B. Relative miR-34c-5p and miR-155-5p expression levels (2deltaCt normalized to RNU48) in unstimulated and stimulated naive CD4 T cells of individual samples pooled to generate the sequencing libraries. mean ± SEM and p-value for differences in miR-155-5p expression (paired two tailed t-test) are shown. Comparison could not be performed for miR-34c-5p as it is undetermined in unstimulated samples.


C . Time course quantification of miR - 34c - 5p expression ( top panel , 2deltaCt values ) in three different donors .

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C. Time course quantification of miR-34c-5p expression (top panel, 2deltaCt values) in three different donors.


C . Time course quantification of cell proliferation ( middle panel ) and cell activation ( bottom panel ) markers by flow cytometry , in three different donors .

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C. Time course quantification of cell proliferation (middle panel) and cell activation (bottom panel) markers by flow cytometry, in three different donors.


A , B . Comparison of the mean miR expression level by in TCR - stimulated naive CD4 T cell small RNA - seq libraries from uninfected and HIV - 1NL4 - 3 ( A ) or HIV - 2ROD ( B ) infected samples ( 24h ) . Only miRNAs with a minimum of 10 normalized read counts . Library normalization was performed using the mean normalization method available in Deseq package of Bioconductor ( Anders Huber , 2010 ) . Lines indicate changes of + / - 1 log2 fold between samples ( n = 3 , paired pools of three individual samples per library ) . miR - 34c - 5p is highlighted in black .

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

A, B. Comparison of the mean miR expression level by in TCR-stimulated naive CD4 T cell small RNA-seq libraries from uninfected and HIV-1NL4-3 (A) or HIV-2ROD (B) infected samples (24h). Only miRNAs with a minimum of 10 normalized read counts. Library normalization was performed using the mean normalization method available in Deseq package of Bioconductor (Anders Huber, 2010). Lines indicate changes of +/- 1 log2 fold between samples (n=3, paired pools of three individual samples per library). miR-34c-5p is highlighted in black.


C . Quantification of miR - 34c - 5p expression levels of the individual samples pooled to generate the sequencing libraries by qPCR ( mean ± SEM 2deltaCt values and p value from a paired two tailed t test ; n = 5 ) .

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C. Quantification of miR-34c-5p expression levels of the individual samples pooled to generate the sequencing libraries by qPCR (mean±SEM 2deltaCt values and p value from a paired two tailed t test; n=5).


A . Cell - associated viral DNA levels ( left ) , Tat mRNA levels ( center ) , and RT activity levels ( right ) in cell culture supernatants in infected J34c cells vs . infected JØ cells at 24 and 48 hours post - infection . * p0 . 05 ( paired two tailed t test ) .

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A. Cell-associated viral DNA levels (left), Tat mRNA levels (center), and RT activity levels (right) in cell culture supernatants in infected J34c cells vs. infected JØ cells at 24 and 48 hours post-infection. * p0.05 (paired two tailed t test).


B . Representative flow cytometry plot of intracellular HIV - 1 Gag protein staining ( KC57 ) versus side scatter ( SSC ) in JØ and J34c cells , mock infected or infected with HIV - 1NL4 - 3 , after 48 hours of culture and corresponding fold change ( J34c / JØ ) plot in four biological replicate assays . Differences to control were found to be significant with a paired two tailed t test ( p0 . 05 ) .

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B. Representative flow cytometry plot of intracellular HIV-1 Gag protein staining (KC57) versus side scatter (SSC) in JØ and J34c cells, mock infected or infected with HIV-1NL4-3, after 48 hours of culture and corresponding fold change (J34c/JØ) plot in four biological replicate assays. Differences to control were found to be significant with a paired two tailed t test (p0.05).


C . Fold change levels for miR - 34c - 5p , cell - associated viral DNA level , Tat mRNA , and RT activity levels in J34c cells treated with miR - 34c - 5p antagomiR versus control antagomiR , 48 hours post - infection . Differences to control were found to be significant with a paired two tailed t test ( p0 . 05 ) .

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C. Fold change levels for miR-34c-5p, cell-associated viral DNA level, Tat mRNA, and RT activity levels in J34c cells treated with miR-34c-5p antagomiR versus control antagomiR, 48 hours post-infection. Differences to control were found to be significant with a paired two tailed t test (p0.05).


A . Hierarchical clustering of the 82 differentially expressed ( DE ) genes identified in Jurkat cell lines overexpressing miR - 34c - 5p ( J34c - 2 and J34c - 5 ) vs control ( JØ - 1 , 2 and 3 clones ) . Color scale represents mean centered log2 fold expression level across samples . Pie charts represent the relative proportion of predicted miR - 34c - 5p binding sites by the Miranda or PITA algorithms ( or both ) in down - regulated ( top ) and up - regulated ( bottom ) gene sets .

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A. Hierarchical clustering of the 82 differentially expressed (DE) genes identified in Jurkat cell lines overexpressing miR-34c-5p (J34c-2 and J34c-5) vs control (JØ-1, 2 and 3 clones). Color scale represents mean centered log2 fold expression level across samples. Pie charts represent the relative proportion of predicted miR-34c-5p binding sites by the Miranda or PITA algorithms (or both) in down-regulated (top) and up-regulated (bottom) gene sets.


B . qRT - PCR validation of selected genes . Plot represents the average percentage of target gene mRNA ( normalized to GAPDH ) in J34c vs JØ cells ( all differences were found to be significant with a paired two tailed t test , P < 0 . 05 ; n = 5 ) .

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B. qRT-PCR validation of selected genes. Plot represents the average percentage of target gene mRNA (normalized to GAPDH) in J34c vs JØ cells (all differences were found to be significant with a paired two tailed t test, P<0.05; n=5).


B . Western - blot of PCAF / KAT2B and histone H3 in JØ or J34c ( top ) , confirming decrease in protein levels .

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B. Western-blot of PCAF/KAT2B and histone H3 in JØ or J34c (top), confirming decrease in protein levels.


B . Bottom : antagomiR to miR - 34c - 5p results in increased KAT2B mRNA levels compared to control - treated cells . Plot presents relative expression values for mRNA in 3 biological replicates ; bars represent mean and SEM . Differences were found to be significant with p < 0 . 05 ( paired two tailed t test ) .

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B. Bottom: antagomiR to miR-34c-5p results in increased KAT2B mRNA levels compared to control-treated cells. Plot presents relative expression values for mRNA in 3 biological replicates; bars represent mean and SEM. Differences were found to be significant with p<0.05 (paired two tailed t test).


C . Predicted target sites for miR - 34c - 5p in the PCAF / KAT2B mRNA ( black bars ) and miR - target hybridization for the two strongest sites ( top ) ; Luciferase assay with the pEZX renilla / firefly - PCAF / KAT2B 3 ' UTR reporter in HEK293 cells co - transfected with either the miR - 34c - 5p overexpression vector or pcDNA3 empty control ( bottom ) . Differences were found to be significant with p < 0 . 05 ( paired two tailed t test , n = 5 ) .

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C. Predicted target sites for miR-34c-5p in the PCAF/KAT2B mRNA(black bars) and miR-target hybridization for the two strongest sites (top); Luciferase assay with the pEZX renilla/firefly-PCAF/KAT2B 3'UTR reporter in HEK293 cells co-transfected with either the miR-34c-5p overexpression vector or pcDNA3 empty control (bottom). Differences were found to be significant with p<0.05 (paired two tailed t test, n=5).


Components of the RLCco - precipitate with ER markers in density gradient fractionation . The post - nuclear supernatant of cortical neurons was loaded underneath a continuous Optiprep density gradient . ER markers Calnexin , Ribophorin I and Climp63 , co - sedimented with ribosomes ( RPS6 , ribosomal protein subunit 6 ) , mitochondria ( Tim23 ) and RLC proteins at fractions 3 - 5 . The bottom graph shows the density of each fraction .

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Components of the RLCco-precipitate with ER markers in density gradient fractionation. The post-nuclear supernatant of cortical neurons was loaded underneath a continuous Optiprep density gradient. ER markers Calnexin, Ribophorin I and Climp63, co-sedimented with ribosomes (RPS6, ribosomal protein subunit 6), mitochondria (Tim23) and RLC proteins at fractions 3-5. The bottom graph shows the density of each fraction.


B ) TRBP , PACT and Ago2 partially co - localize with the ER marker mCherry - CLIMP63 . Hippocampal neurons ( 10 DIV ) expressing mCherry - CLIMP63 were immunostained with TRBP , PACT or Ago2 antibodies . Boxed insets are magnifications of primary dendrites as depicted by the adjacent letters ( scale bars ; 10μm ) .

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B) TRBP, PACT and Ago2 partially co-localize with the ER marker mCherry-CLIMP63. Hippocampal neurons (10 DIV) expressing mCherry-CLIMP63 were immunostained with TRBP, PACT or Ago2 antibodies. Boxed insets are magnifications of primary dendrites as depicted by the adjacent letters (scale bars; 10μm).


C ) GFP - Dicer partially co - localizes with mCherry - CLIMP63 at the neuronal soma and primary dendrites of hippocampal neurons . Neurons were imaged at 10 DIV using total internal reflection fluorescence ( TIRF ) microscopy using a penetration depth of 150nm . Boxed insets are magnifications of primary dendrites as depicted by the adjacent letters ( scale bars ; 2μm or 10μm ) .

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C) GFP-Dicer partially co-localizes with mCherry-CLIMP63 at the neuronal soma and primary dendrites of hippocampal neurons. Neurons were imaged at 10 DIV using total internal reflection fluorescence (TIRF) microscopy using a penetration depth of 150nm. Boxed insets are magnifications of primary dendrites as depicted by the adjacent letters (scale bars; 2μm or 10μm).


D ) The majority of Dicer , TRBP and PACT are associated with neuronal membranes . Sequential detergent extraction was used to separate cytoplasmic from membrane fractions in 7 DIV cortical neurons . The relative quantity of depicted proteins was analyzed in three independent experiments ( p < 0 . 005 , t - test type 3 , n = 3 , error bars s . d . ) . Actin and ribophorin I were used as loading controls for cytoplasmic and membrane fractions , respectively .

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D) The majority of Dicer, TRBP and PACT are associated with neuronal membranes. Sequential detergent extraction was used to separate cytoplasmic from membrane fractions in 7 DIV cortical neurons. The relative quantity of depicted proteins was analyzed in three independent experiments (p<0.005, t-test type 3, n=3, error bars s.d.). Actin and ribophorin I were used as loading controls for cytoplasmic and membrane fractions, respectively.


E ) TRBP is cytoplasmic in primary astrocytes . Sequential detergent extraction was performed in primary astrocyte cultures . Actin and calnexin were used as loading controls for cytoplasmic and membrane fractions , respectively .

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E) TRBP is cytoplasmic in primary astrocytes. Sequential detergent extraction was performed in primary astrocyte cultures. Actin and calnexin were used as loading controls for cytoplasmic and membrane fractions, respectively.


F ) The majority of Dicer , TRBP and PACT proteins are present in cytoplasmic fractions in HEK293T cells , isolated using sequential detergent extraction .

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F) The majority of Dicer, TRBP and PACT proteins are present in cytoplasmic fractions in HEK293T cells, isolated using sequential detergent extraction.


A ) BDNF stimulation leads to a decrease in TRBP levels from ER microsomes . Cortical neurons were stimulated BDNF with or vehicle control and ER microsomes were isolated using differential centrifugation . Protein intensity values were normalized to calnexin in each of three independent experiments ( p = 0 . 008 , t - test type 3 , n = 3 , error bars ; s . d . ) .

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A) BDNF stimulation leads to a decrease in TRBP levels from ER microsomes. Cortical neurons were stimulated BDNF with or vehicle control and ER microsomes were isolated using differential centrifugation. Protein intensity values were normalized to calnexin in each of three independent experiments (p=0.008, t-test type 3, n=3, error bars; s.d.).


B ) Endogenous TRBP partially co - localizes with calnexin at the neuronal cell soma and primary dendrites . Hippocampal neurons were transfected with GFP at 7DIV and fixed at 10DIV following BDNF or control stimulation . Boxed insets are magnifications of the primary dendrite ( 15μm in length ) . Arrows point to overlapping puncta ( scale bars ; 20μm ( GFP ) or 10μm ) .

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B) Endogenous TRBP partially co-localizes with calnexin at the neuronal cell soma and primary dendrites. Hippocampal neurons were transfected with GFP at 7DIV and fixed at 10DIV following BDNF or control stimulation. Boxed insets are magnifications of the primary dendrite (15μm in length). Arrows point to overlapping puncta (scale bars; 20μm (GFP) or 10μm).


C ) BDNF leads to a decrease in co - localization between TRBP and calnexin at the soma and primary dendrites of hippocampal neurons . Manders co - localization co - efficients between TRBP and Calnexin were measured in the cell soma or primary dendrites ( 15μm ) using GFP as a morphological mask . Average co - localization values were normalized to controls in each experiment ( p < 0 . 008 , t - test type 3 , n = 4 , error bars ; s . d . ) .

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C) BDNF leads to a decrease in co-localization between TRBP and calnexin at the soma and primary dendrites of hippocampal neurons. Manders co-localization co-efficients between TRBP and Calnexin were measured in the cell soma or primary dendrites (15μm) using GFP as a morphological mask. Average co-localization values were normalized to controls in each experiment (p<0.008, t-test type 3, n=4, error bars; s.d.).


D ) BDNF decreases mCherry - CLIMP63 co - localization with TRBP , but not with PACT or Ago2 . Hippocampal neurons were transfected with GFP and mCherry - CLIMP63 at 7DIV and immunostained three days later , following BDNF or control stimulation . Manders co - localization was measured at the cell soma using GFP as a mask as indicated ( p = 0 . 01 , t - test type 3 , n = 4 , error bars ; s . d . ) .

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

D) BDNF decreases mCherry-CLIMP63 co-localization with TRBP, but not with PACT or Ago2. Hippocampal neurons were transfected with GFP and mCherry-CLIMP63 at 7DIV and immunostained three days later, following BDNF or control stimulation. Manders co-localization was measured at the cell soma using GFP as a mask as indicated (p=0.01, t-test type 3, n=4, error bars; s.d.).


E ) BDNF leads to an increase in the levels of TRBP ( p = 0 . 03 ) and PACT ( p = 0 . 004 ) in the cytoplasmic fraction . Cortical neurons ( 7 DIV ) were control - or BDNF - stimulated and cytoplasmic fractions were isolated using digitonin . Protein intensity values were normalized to actin in each experiment . The position of the star corresponds to the TRBP band used for analysis ( ~ 43kDa ) ( t - test type 3 , n = 4 , error bars ; s . d . ) .

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E) BDNF leads to an increase in the levels of TRBP (p=0.03) and PACT (p=0.004) in the cytoplasmic fraction. Cortical neurons (7 DIV) were control- or BDNF-stimulated and cytoplasmic fractions were isolated using digitonin. Protein intensity values were normalized to actin in each experiment. The position of the star corresponds to the TRBP band used for analysis (~43kDa) (t-test type 3, n=4, error bars; s.d.).


F ) The BDNF - induced increase in cytoplasmic TRBP is dependent on intracellular calcium . Cortical neurons ( 7 DIV ) were treated with vehicle control , BDNF and BAPTA - AM as indicated , and cytoplasmic fractions were isolated using digitonin . Protein intensity values were normalized to actin in each independent experiment ( p = 0 . 03 , n = 3 , error bars ; s . d . ) .

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F) The BDNF-induced increase in cytoplasmic TRBP is dependent on intracellular calcium. Cortical neurons (7 DIV) were treated with vehicle control, BDNF and BAPTA-AM as indicated, and cytoplasmic fractions were isolated using digitonin. Protein intensity values were normalized to actin in each independent experiment (p=0.03, n=3, error bars; s.d.).


BDNF stimulation causes a decrease in the TRBP - Dicer interaction . Young cortical neurons were treated with BDNF or vehicle control and co - immunoprecipitations ( co - IPs ) were performed using 2μg control IgG or polyclonal anti - TRBP antibodies . Dicer binding in co - IP was normalized to inputs in each experiment ( p = 0 . 001 , t - test type 2 , n = 3 , error bars represent s . d . ) .

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BDNF stimulation causes a decrease in the TRBP-Dicer interaction. Young cortical neurons were treated with BDNF or vehicle control and co-immunoprecipitations (co-IPs) were performed using 2μg control IgG or polyclonal anti-TRBP antibodies. Dicer binding in co-IP was normalized to inputs in each experiment (p=0.001, t-test type 2, n=3, error bars represent s.d.).


B ) Short BDNF stimulation changes the levels of only a few microRNAs , such as miR - 16 - 5p and miR - 551b - 5p . Volcano plot of small RNA sequencing data from BDNF - or control - treated cortical neurons ( 6DIV ) was based on differential expression analysis using the edgeR package . Individual points represent the average fold change obtained in three independent experiments for each miRNA , and plotted against obtained p - values . MiRNAs above the red dashed line are significant ( p < 0 . 05 , t - test type 2 , n = 3 ) .

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B) Short BDNF stimulation changes the levels of only a few microRNAs, such as miR-16-5p and miR-551b-5p. Volcano plot of small RNA sequencing data from BDNF- or control-treated cortical neurons (6DIV) was based on differential expression analysis using the edgeR package. Individual points represent the average fold change obtained in three independent experiments for each miRNA, and plotted against obtained p-values. MiRNAs above the red dashed line are significant (p<0.05, t-test type 2, n=3).


C ) BDNF stimulation leads to a transient decrease in mature miR - 16 - 5p levels . 6DIV cortical neurons were treated with BDNF for 10 or 20 minutes and mature miRNA levels were measured in rtPCR . Ct values for each miRNA were normalized to U6 and to 20 minutes of control stimulation ( n = 3 , p = 0 . 02 , t - test type 3 , error bars ; s . d . ) .

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C) BDNF stimulation leads to a transient decrease in mature miR-16-5p levels. 6DIV cortical neurons were treated with BDNF for 10 or 20 minutes and mature miRNA levels were measured in rtPCR. Ct values for each miRNA were normalized to U6 and to 20 minutes of control stimulation (n=3, p=0.02, t-test type 3, error bars; s.d.).


D ) BDNF stimulation leads to a transient increase in the levels of pre - miR16 after 10 minutes of BDNF stimulation . Ct values obtained in rtPCR were normalized to U6 and to 20 minutes control stimulation ( n = 4 , t - test type 3 , error bars ; s . d . ) .

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D) BDNF stimulation leads to a transient increase in the levels of pre-miR16 after 10 minutes of BDNF stimulation. Ct values obtained in rtPCR were normalized to U6 and to 20 minutes control stimulation (n=4, t-test type 3, error bars; s.d.).


E ) BDNF causes a decrease in pre - miR16 binding to TRBP . Cortical neurons were treated with BDNF or vehicle control and RNA IPs were performed using a mouse monoclonal anti - TRBP antibody . Ct values measured in rtPCR were normalized to respective inputs ( n = 3 , p = 0 . 002 , t - test type 3 , error bars represent s . d . ) .

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E) BDNF causes a decrease in pre-miR16 binding to TRBP. Cortical neurons were treated with BDNF or vehicle control and RNA IPs were performed using a mouse monoclonal anti-TRBP antibody. Ct values measured in rtPCR were normalized to respective inputs (n=3, p=0.002, t-test type 3, error bars represent s.d.).


A ) BDNF changes the canonical isomiR proportion of a subset of miRNAs . IsomiR levels were compared to the total amount of the corresponding miRNA . Shown are the miRNAs that had significant changes in the canonical isomiR ( annotated as ' 0 ' , p < 0 . 05 , t - test type 2 , n = 3 ) . Results are shown in order of decreasing significance . Shown in red are miRNAs of the miR16 family , miR - 16 - 5p and miR - 15b - 5p . Black squares correspond non - existing isomiRs . Numbers on the top of the table represent the trimming ( negative ) or addition ( positive ) of nucleotides to the 3 ' - or 5 ' - end of the canonical 5p or 3p isomiRs , respectively . Only templated nucleotide additions and trimmings , which might reflect Dicer - mediated pre - miRNA cleavage , were considered for analysis .

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A) BDNF changes the canonical isomiR proportion of a subset of miRNAs. IsomiR levels were compared to the total amount of the corresponding miRNA. Shown are the miRNAs that had significant changes in the canonical isomiR (annotated as '0', p<0.05, t-test type 2, n=3). Results are shown in order of decreasing significance. Shown in red are miRNAs of the miR16 family, miR-16-5p and miR-15b-5p. Black squares correspond non-existing isomiRs. Numbers on the top of the table represent the trimming (negative) or addition (positive) of nucleotides to the 3'- or 5'-end of the canonical 5p or 3p isomiRs, respectively. Only templated nucleotide additions and trimmings, which might reflect Dicer-mediated pre-miRNA cleavage, were considered for analysis.


B ) BDNF stimulation leads to a decrease in the canonical ( 22 nucleotides ) isomiRs of the miR16 family , miR - 16 - 5p ( p = 0 . 005 ) and miR - 15b - 5p ( p = 0 . 02 ) . In both cases , this is accompanied by an increase in the levels of shorter isomiRs ; the 19 - nucleotide isoform of miR - 16 - 5p ( p = 0 . 04 ) and the 21 - nucleotide isoform of miR - 15b - 5p ( p = 0 . 007 ) . The relative isomiRs proportion was calculated as described in A ( t - test type 2 , n = 3 , error bars ; s . d . ) .

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B) BDNF stimulation leads to a decrease in the canonical (22 nucleotides) isomiRs of the miR16 family, miR-16-5p (p=0.005) and miR-15b-5p (p=0.02). In both cases, this is accompanied by an increase in the levels of shorter isomiRs; the 19-nucleotide isoform of miR-16-5p (p=0.04) and the 21-nucleotide isoform of miR-15b-5p (p=0.007). The relative isomiRs proportion was calculated as described in A (t-test type 2, n=3, error bars; s.d.).


B ) Endogenous miR - 16 - 5p activity in developing hippocampal neurons . Hippocampal neurons were transfected with 500ng miR16 or control sensor at 4 DIV and with or without LNA control ( ' LNA Ctrl ' ) or the anti - sense miR - 16 - 5p inhibitor ( ' LNA - miR16 ' ) . Cells were fixed at 7 DIV and the number of neurons expressing GFP and not dsRed ( ' GFP - only ' ) was compared to the total number of transfected neurons . LNA - miR16 reduces the amount of repression of the miR16 sensor compared to LNA - Ctrl ( n = 4 , p = 0 . 02 , t - test type 2 , error bars represent s . d . ) .

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B) Endogenous miR-16-5p activity in developing hippocampal neurons. Hippocampal neurons were transfected with 500ng miR16 or control sensor at 4 DIV and with or without LNA control ('LNA Ctrl') or the anti-sense miR-16-5p inhibitor ('LNA-miR16'). Cells were fixed at 7 DIV and the number of neurons expressing GFP and not dsRed ('GFP-only') was compared to the total number of transfected neurons. LNA-miR16 reduces the amount of repression of the miR16 sensor compared to LNA-Ctrl (n=4, p=0.02, t-test type 2, error bars represent s.d.).


C ) Short - term BDNF stimulation inhibits endogenous miR - 16 - 5p activity . Hippocampal neurons were transfected at 4 DIV and treated with BDNF either 24h or 20 minutes before fixation and neurons were counted as in B . Repression index was obtained by normalizing the miR16 sensor to the control sensor condition in each experiment ( n = 4 , p = 0 . 02 , t - test type 3 , error bars ; s . d . ) .

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C) Short-term BDNF stimulation inhibits endogenous miR-16-5p activity. Hippocampal neurons were transfected at 4 DIV and treated with BDNF either 24h or 20 minutes before fixation and neurons were counted as in B. Repression index was obtained by normalizing the miR16 sensor to the control sensor condition in each experiment (n=4, p=0.02, t-test type 3, error bars; s.d.).


D & E ) Inhibition of miR - 16 - 5p increases dendritic complexity and occludes BDNF - induced dendritogenesis . D ) Hippocampal neurons were transfected with the control sensor ( A ) , and stimulated with control or BDNF 24 hours before fixation . Pictures were obtained on a confocal microscope and thresholds were set to dsRed signal fluorescence in order to distinguish individual dendrites ( scale bars are 10μm ) . E ) To quantify dendritic complexity the total number of dendritic intersections was calculated using Sholl analysis . Values were normalized to LNA - Ctrl and control treatment ( n = 3 , p < 0 . 04 , t - test type 3 , error bars represent s . d . ) .

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D&E) Inhibition of miR-16-5p increases dendritic complexity and occludes BDNF-induced dendritogenesis. D) Hippocampal neurons were transfected with the control sensor (A), and stimulated with control or BDNF 24 hours before fixation. Pictures were obtained on a confocal microscope and thresholds were set to dsRed signal fluorescence in order to distinguish individual dendrites (scale bars are 10μm). E) To quantify dendritic complexity the total number of dendritic intersections was calculated using Sholl analysis. Values were normalized to LNA-Ctrl and control treatment (n=3, p<0.04, t-test type 3, error bars represent s.d.).


F & G ) Overexpression of miR - 16 - 5p blocks BDNF - induced dendritogenesis . F ) Hippocampal neurons were transfected with control sensor and either control miRNA mimic ( miR - Ctrl ) or a miR - 16 - 5p mimic ( miR16 ) and treated two days later with BDNF or control vehicle . DsRed signal fluorescence was used to set a threshold on images obtained on a confocal microscope ( scale bars ; 10μm ) . G ) Total number of intersections was quantified in Sholl analysis and values were normalized to miR - Ctrl , control treatment in each experiment ( n = 4 , p = 0 . 02 , t - test type 3 , error bars ; s . d . ) .

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F&G) Overexpression of miR-16-5p blocks BDNF-induced dendritogenesis. F) Hippocampal neurons were transfected with control sensor and either control miRNA mimic (miR-Ctrl) or a miR-16-5p mimic (miR16) and treated two days later with BDNF or control vehicle. DsRed signal fluorescence was used to set a threshold on images obtained on a confocal microscope (scale bars; 10μm). G) Total number of intersections was quantified in Sholl analysis and values were normalized to miR-Ctrl, control treatment in each experiment (n=4, p=0.02, t-test type 3, error bars; s.d.).


H & I ) Overexpression of the Dicer - binding deficient deletion mutant of TRBP ( GFP - TRBP Δ ) occludes BDNF - induced dendritogenesis . H ) Hippocampal neurons were transfected with 200ng dsRed and either 50ng GFP or 200ng GFP - TRBP Δ , and treated two days later with BDNF or control vehicle . DsRed signal fluorescence was used to set a threshold on images obtained on a confocal microscope ( scale bars ; 10μm ) . I ) Total number of intersections was quantified in Sholl analysis and values were normalized to control treatment in each experiment ( n = 5 , p < 0 . 02 , t - test type 3 , error bars ; s . d . ) .

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H&I) Overexpression of the Dicer-binding deficient deletion mutant of TRBP (GFP-TRBP Δ) occludes BDNF-induced dendritogenesis. H) Hippocampal neurons were transfected with 200ng dsRed and either 50ng GFP or 200ng GFP-TRBP Δ, and treated two days later with BDNF or control vehicle. DsRed signal fluorescence was used to set a threshold on images obtained on a confocal microscope (scale bars; 10μm). I) Total number of intersections was quantified in Sholl analysis and values were normalized to control treatment in each experiment (n=5, p<0.02, t-test type 3, error bars; s.d.).


B ) MiR - 16 - 5p targets the 3 ' UTR of the BDNFmRNA . Cortical neurons were transfected with 100ng pmiRGlo dual - luciferase reporter consisting of the 3 ' UTR of BDNF ( 3 ' UTR WT ) or a miR - 16 - 5p - binding site mutant ( 3 ' UTR Mut ) and either miR - Ctrl or miR - 16 . The Firefly to Renilla luciferase ratio was measured and values were normalized to the respective reporter expression without miRNA mimic co - transfection ( n = 3 , p = 0 . 02 , t - test type 2 ) .

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B) MiR-16-5p targets the 3'UTR of the BDNFmRNA. Cortical neurons were transfected with 100ng pmiRGlo dual-luciferase reporter consisting of the 3'UTR of BDNF (3'UTR WT) or a miR-16-5p-binding site mutant (3'UTR Mut) and either miR-Ctrl or miR-16. The Firefly to Renilla luciferase ratio was measured and values were normalized to the respective reporter expression without miRNA mimic co-transfection (n=3, p=0.02, t-test type 2).


C ) BDNFstimulation leads to an increase in the translation of 3 ' UTR WT ( p = 0 . 02 , t - test type 3 ) but not 3 ' UTR Mut luciferase reporter , which is rescued by miR - 16 - 5p overexpression ( p = 0 . 002 ; t - test type 2 ) . Cortical neurons were transfected as in ( B ) , stimulated with BDNF or vehicle control for 20 minutes , and lysed two hours after stimulation ( n = 4 ) .

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C) BDNFstimulation leads to an increase in the translation of 3'UTR WT (p=0.02, t-test type 3) but not 3'UTR Mut luciferase reporter, which is rescued by miR-16-5p overexpression (p=0.002; t-test type 2). Cortical neurons were transfected as in (B), stimulated with BDNF or vehicle control for 20 minutes, and lysed two hours after stimulation (n=4).


D ) Pre - miR16 is present in the neuronal cell soma and processes of hippocampal neurons . Neurons were plated on compartmentalized chambers and RNA was isolated from the neuronal cell body and processes separately . Pre - miR137 and pre - miR341 were used to assay somatic and neurite enrichment , respectively . Raw Ct values were normalized to GAPDH mRNA in each experiment ( n = 3 ) .

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D) Pre-miR16 is present in the neuronal cell soma and processes of hippocampal neurons. Neurons were plated on compartmentalized chambers and RNA was isolated from the neuronal cell body and processes separately. Pre-miR137 and pre-miR341 were used to assay somatic and neurite enrichment, respectively. Raw Ct values were normalized to GAPDH mRNA in each experiment (n=3).


More Dicer binds to membrane - targeted GFP - TRBP ( mGFP - TRBP ) compared to GFP - TRBP . HEK293T cells were transfected with GFP , GFP - TRBP or mGFP - TRBP and cell lysates were processed for co - IP using anti - GFP antibody . Dicer binding was normalized to respective input lanes in each condition ( n = 4 , p = 0 . 02 ; t - test type 2 , error bars are s . d . ) .

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More Dicer binds to membrane-targeted GFP-TRBP (mGFP-TRBP) compared to GFP-TRBP. HEK293T cells were transfected with GFP, GFP-TRBP or mGFP-TRBP and cell lysates were processed for co-IP using anti-GFP antibody. Dicer binding was normalized to respective input lanes in each condition (n=4, p=0.02; t-test type 2, error bars are s.d.).


B ) mGFP - TRBP but not GFP - TRBP blocks the BDNF - induced decrease in miR - 16 - 5p activity in dual - fluorescence sensor assay . Hippocampal neurons were transfected with 200ng miR16 - or control - sensor ( see Fig 5A ) plus 50ng GFP , 400ng GFP - TRBP or 400ng mGFP - TRBP . Neurons were treated with BDNF or vehicle control prior to fixation and immunostained with anti - GFP antibody . BDNF led to a decrease in miR - 16 - 5p activity in GFP - transfected neurons ( p = 0 . 03 , n = 4 , t - test type 3 ) , which was significantly different from mGFP - TRBP - expressing neurons ( p = 0 . 04 , n = 4 , t - test type 2 , error bars are s . d . ) .

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B) mGFP-TRBP but not GFP-TRBP blocks the BDNF-induced decrease in miR-16-5p activity in dual-fluorescence sensor assay. Hippocampal neurons were transfected with 200ng miR16- or control-sensor (see Fig 5A) plus 50ng GFP, 400ng GFP-TRBP or 400ng mGFP-TRBP. Neurons were treated with BDNF or vehicle control prior to fixation and immunostained with anti-GFP antibody. BDNF led to a decrease in miR-16-5p activity in GFP-transfected neurons (p=0.03, n=4, t-test type 3), which was significantly different from mGFP-TRBP-expressing neurons (p=0.04, n=4, t-test type 2, error bars are s.d.).


C & D ) BDNF - induced dendritogenesis is blocked by mGFP - TRBP overexpression . C ) Hippocampal neurons ( 4DIV ) were transfected for three days with 200ng dsRed and 50ng GFP , 400ng GFP - TRBP or 400ng mGFP - BDNF . Neurons were treated with BDNF or vehicle control 24 hours before fixation . Pictures were obtained on a confocal microscope and threshold was set for dsRed to distinguish individual dendrites ( scale bars are 10μm ) . D ) The total number of intersections was calculated in Sholl analysis , and values were normalized to control treatment in each of three independent experiments . BDNF led to a significant increase in dendritic complexity ( p = 0 . 04 ; t - test type 3 ) , which was significantly different in cells transfected with mGFP - TRBP , but not GFP - TRBP ( n = 3 , p = 0 . 03 ; t - test type 2 , error bars ; s . d . ) .

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C&D) BDNF-induced dendritogenesis is blocked by mGFP-TRBP overexpression. C) Hippocampal neurons (4DIV) were transfected for three days with 200ng dsRed and 50ng GFP, 400ng GFP-TRBP or 400ng mGFP-BDNF. Neurons were treated with BDNF or vehicle control 24 hours before fixation. Pictures were obtained on a confocal microscope and threshold was set for dsRed to distinguish individual dendrites (scale bars are 10μm). D) The total number of intersections was calculated in Sholl analysis, and values were normalized to control treatment in each of three independent experiments. BDNF led to a significant increase in dendritic complexity (p=0.04; t-test type 3), which was significantly different in cells transfected with mGFP-TRBP, but not GFP-TRBP (n=3, p=0.03; t-test type 2, error bars; s.d.).


B - D Left ventricular LXRα expression in Wt and LXRα - Tg mice aged 12 weeks . ( B ) Western blot of LXRα in liver and LV tissue .

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B-D Left ventricular LXRα expression in Wt and LXRα-Tg mice aged 12 weeks. (B) Western blot of LXRα in liver and LV tissue.


B - D Left ventricular LXRα expression in Wt and LXRα - Tg mice aged 12 weeks . ( B ) Western blot of LXRα in liver and LV tissue . Quantification of ( C ) mRNA expression ( normalized to 36b4 ; n = 6 - 8 / group , * P < 0 . 00001 versus Wt ) and ( D ) protein expression ( normalized to GAPDH ; n = 4 / group , * P = 0 . 0001 versus Wt ) .

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B-D Left ventricular LXRα expression in Wt and LXRα-Tg mice aged 12 weeks. (B) Western blot of LXRα in liver and LV tissue. Quantification of (C) mRNA expression (normalized to 36b4; n = 6-8/group, *P < 0.00001 versus Wt) and (D) protein expression (normalized to GAPDH; n = 4/group, *P = 0.0001 versus Wt).


E LV weight to tibia length ratios ( LV / tibia ) ; n = 15 / group . * P = 0 . 02 .

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E LV weight to tibia length ratios (LV/tibia); n = 15/group. *P = 0.02.


F Assessment of mean arterial pressure ( MAP ) with in situ catheterization shows no significant differences .

F Assessment of mean arterial pressure (MAP) with in situ catheterization shows no significant differences.


G Representative Masson ' s trichrome and wheat germ agglutinin ( WGA ) - FITC staining in cross - sectional LV , scale bars = 100 μm and 10 μm , respectively .

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G Representative Masson's trichrome and wheat germ agglutinin (WGA)-FITC staining in cross-sectional LV, scale bars = 100 μm and 10 μm, respectively.


H Relative mRNA levels of known LXRα - associated and target genes assessed with RT - PCR ; n = 8 / group . * P = 0 . 0003 , # P = 0 . 0004 , § P = 0 . 043 , † P = 0 . 0009 versus Wt .

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H Relative mRNA levels of known LXRα-associated and target genes assessed with RT-PCR; n = 8/group. *P = 0.0003, #P = 0.0004, §P = 0.043, †P = 0.0009 versus Wt.


LV / tibia ratios in sham - and TAC - operated Wt and LXRα - Tgmice ; n = 21 Wt sham , n = 24 LXRα - Tg sham , n = 24 Wt TAC , n = 26 LXRα - TgTAC . * P < 0 . 00001 versus respective sham , # P = 0 . 00004 .

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LV/tibia ratios in sham- and TAC-operated Wt and LXRα-Tgmice; n = 21 Wt sham, n = 24 LXRα-Tg sham, n = 24 Wt TAC, n = 26 LXRα-TgTAC. *P < 0.00001 versus respective sham, #P = 0.00004.


Quantification of cardiomyocyte cross - sectional area from WGA - FITC - stained histological sections ; n = 5 Wt sham , n = 4 LXRα - Tg sham , n = 5 Wt TAC , n = 3 LXRα - TgTAC . * * P = 0 . 00001 versus Wt sham , * P = 0 . 01 versus LXRα - Tg sham , # P = 0 . 01 .

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Quantification of cardiomyocyte cross-sectional area from WGA-FITC-stained histological sections; n = 5 Wt sham, n = 4 LXRα-Tg sham, n = 5 Wt TAC, n = 3 LXRα-TgTAC. **P = 0.00001 versus Wt sham, *P = 0.01 versus LXRα-Tg sham, #P = 0.01.


Representative LV sections stained with WGA - FITC and Masson ' s trichrome to assess cardiomyocyte hypertrophy and fibrosis , respectively ; scale bars = 1 mm , 100 μm , 10 μm , from top to bottom .

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Representative LV sections stained with WGA-FITC and Masson's trichrome to assess cardiomyocyte hypertrophy and fibrosis, respectively; scale bars = 1 mm, 100 μm, 10 μm, from top to bottom.


Whole area percentage of fibrosis in Masson ' s trichrome - stained sections ; n = 8 per group , except n = 7 in Wt TAC group . * P = 0 . 02 versus Wt sham , # P = 0 . 02 .

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Whole area percentage of fibrosis in Masson's trichrome-stained sections; n = 8 per group, except n = 7 in Wt TAC group. *P = 0.02 versus Wt sham, #P = 0.02.


Measurement of mRNA levels with RT - PCR to assess induction of fetal gene program , as well as hypertrophy ( Rcan1 ) and fibrosis ( Col1a1 , Ctgf ) ; n = 8 per group , except n = 7 in Wt TAC group . Myh6 / Myh7 : * P = 0 . 0001 versus Wt sham , # P = 0 . 00001 ; Anp : * P = 0 . 0005 versus LXRα - Tg sham , # P = 0 . 02 ; Bnp : * P = 0 . 0006 versus Wt sham , * * P = 0 . 009 versus LXRα - Tg sham , # P = 0 . 03 ; Acta1 : § P < 0 . 00001 versus Wt sham , * P = 0 . 01 versus LXRα - Tg sham , # P = 0 . 001 ; Rcan1 : * * P = 0 . 00004 versus Wt sham , * P = 0 . 05 versus LXRα - Tg sham , # P = 0 . 04 ; Col1a1 : * P = 0 . 00003 versus Wt sham ; Ctgf : § P < 0 . 00001 versus Wt sham , # P = 0 . 00001 .

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Measurement of mRNA levels with RT-PCR to assess induction of fetal gene program, as well as hypertrophy (Rcan1) and fibrosis (Col1a1, Ctgf); n = 8 per group, except n = 7 in Wt TAC group. Myh6/Myh7: *P = 0.0001 versus Wt sham, #P = 0.00001; Anp: *P = 0.0005 versus LXRα-Tg sham, #P = 0.02; Bnp: *P = 0.0006 versus Wt sham, **P = 0.009 versus LXRα-Tg sham, #P = 0.03; Acta1: §P < 0.00001 versus Wt sham, *P = 0.01 versus LXRα-Tg sham, #P = 0.001; Rcan1: **P = 0.00004 versus Wt sham, *P = 0.05 versus LXRα-Tg sham, #P = 0.04; Col1a1: *P = 0.00003 versus Wt sham; Ctgf: §P < 0.00001 versus Wt sham, #P = 0.00001.


A Echocardiographic assessment of percent fractional shortening in mice subjected to 5 weeks of TAC ; n = 19 Wt sham , n = 22 LXRα - Tg sham , n = 24 Wt TAC , n = 26 LXRα - TgTAC . * * P < 0 . 00001 versus Wt sham , * P = 0 . 0005 versus LXRα - Tg sham , # P = 0 . 009 .

A Echocardiographic assessment of percent fractional shortening in mice subjected to 5 weeks of TAC; n = 19 Wt sham, n = 22 LXRα-Tg sham, n = 24 Wt TAC, n = 26 LXRα-TgTAC. **P < 0.00001 versus Wt sham, *P = 0.0005 versus LXRα-Tg sham, #P = 0.009.


B - D Hemodynamic measurements obtained in situ in sham - and TAC - operated mice ; n = 6 - 8 / group . ( B ) Mean arterial pressure ( MAP ) showed no significant differences . ( C ) LV end - systolic pressure ( LVESP ) . * * P < 0 . 00001 versus Wt sham , * P = 0 . 00004 versus LXRα - Tg sham , * P = 0 . 003 . ( D ) LV end - diastolic pressure ( LVEDP ) . * P = 0 . 02 versus Wt sham .

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B-D Hemodynamic measurements obtained in situ in sham- and TAC-operated mice; n = 6-8/group. (B) Mean arterial pressure (MAP) showed no significant differences. (C) LV end-systolic pressure (LVESP). **P < 0.00001 versus Wt sham, *P = 0.00004 versus LXRα-Tg sham, *P = 0.003. (D) LV end-diastolic pressure (LVEDP). *P = 0.02 versus Wt sham.


Representative 18F - FDG images with microPET in the coronal and axial planes in Wt and LXRα - Tg mice 5 weeks post - TAC .

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Representative 18F-FDG images with microPET in the coronal and axial planes in Wt and LXRα-Tg mice 5 weeks post-TAC.


Myocardial FDG uptake measured as standard uptake value ( SUV ) ; n = 4 - 6 / group . * P = 0 . 04 versus Wt sham , * * P = 0 . 02 versus LXRα - Tg sham , # P = 0 . 03 , # # P = 0 . 01 .

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Myocardial FDG uptake measured as standard uptake value (SUV); n = 4-6/group. *P = 0.04 versus Wt sham, **P = 0.02 versus LXRα-Tg sham, #P = 0.03, ##P = 0.01.


Quantification of myocardial glycogen content shows no significant differences ; n = 5 per sham group , n = 7 per TAC group .

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Quantification of myocardial glycogen content shows no significant differences; n = 5 per sham group, n = 7 per TAC group.


GLUT protein expression in LV tissue normalized to GAPDH ; n = 6 / group . Glut1 : * P = 0 . 01 versus LXRα - Tg sham , # P = 0 . 03 ; Glut4 : * P = 0 . 03 versus LXRα - Tg sham , # P = 0 . 01 .

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GLUT protein expression in LV tissue normalized to GAPDH; n = 6/group. Glut1: *P = 0.01 versus LXRα-Tg sham, #P = 0.03; Glut4: *P = 0.03 versus LXRα-Tg sham, #P = 0.01.


A LV / tibia ratios in sham - and TAC - operated WT and LXRα − / − mice ; n = 10 WT sham , n = 8 LXRα − / − sham , n = 12 WT TAC , n = 10 LXRα − / − TAC . * P = 0 . 006 versus WT sham , * * P = 0 . 00008 versus LXRα − / − sham .

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A LV/tibia ratios in sham- and TAC-operated WT and LXRα−/− mice; n = 10 WT sham, n = 8 LXRα−/− sham, n = 12 WT TAC, n = 10 LXRα−/− TAC. *P = 0.006 versus WT sham, **P = 0.00008 versus LXRα−/−sham.


B , C Cardiac functional assessment at 5 weeks post - TAC . ( B ) Percent fractional shortening determined with echocardiography ; n = 10 WT sham , n = 7 LXRα − / − sham , n = 12 WT TAC , n = 10 LXRα − / − TAC . * P < 0 . 00001 versus respective sham , § P = 0 . 06 . ( C ) LV end - diastolic pressure ( LVEDP ) recorded in situ ; n = 10 WT sham , n = 7 LXRα − / − sham , n = 10 WT TAC , n = 9 LXRα − / − TAC . * P = 0 . 001 versus WT sham , * * P = 0 . 00005 versus LXRα − / − sham .

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B, C Cardiac functional assessment at 5 weeks post-TAC. (B) Percent fractional shortening determined with echocardiography; n = 10 WT sham, n = 7 LXRα−/− sham, n = 12 WT TAC, n = 10 LXRα−/− TAC. *P < 0.00001 versus respective sham, §P = 0.06. (C) LV end-diastolic pressure (LVEDP) recorded in situ; n = 10 WT sham, n = 7 LXRα−/− sham, n = 10 WT TAC, n = 9 LXRα−/−TAC. *P = 0.001 versus WT sham, **P = 0.00005 versus LXRα−/− sham.


D - G Relative mRNA gene expression as determined by RT - PCR , normalized to 36b4 ; n = 8 / group . Anp : no significant differences ; Bnp : * P = 0 . 01 versus WT sham , * * P = 0 . 03 versus LXRα − / − sham ; Myh6 / Myh7 : * P = 0 . 0002 versus WT sham , * * P = 0 . 0003 versus LXRα − / − sham ; Acta1 : * P = 0 . 001 versus respective sham .

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D-G Relative mRNA gene expression as determined by RT-PCR, normalized to 36b4; n = 8/group. Anp: no significant differences; Bnp: *P = 0.01 versus WT sham, **P = 0.03 versus LXRα−/− sham; Myh6/Myh7: *P = 0.0002 versus WT sham, **P = 0.0003 versus LXRα−/− sham; Acta1: *P = 0.001 versus respective sham.


H Western blot detection of GLUT protein expression in LV tissue normalized to GAPDH , expressed as fold change , shows no significant differences among groups ; n = 6 - 8 / group .

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H Western blot detection of GLUT protein expression in LV tissue normalized to GAPDH, expressed as fold change, shows no significant differences among groups; n = 6-8/group.


I Representative 18F - FDG - microPET scans .

I Representative 18F-FDG-microPET scans.


J Myocardial FDG uptake quantified as standard uptake value ( SUV ) ; n = 6 / group . * P = 0 . 0001 versus WT sham , # P = 0 . 047 .

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J Myocardial FDG uptake quantified as standard uptake value (SUV); n = 6/group. *P = 0.0001 versus WT sham, #P = 0.047.


A Assessment of 2 - deoxyglucose ( 2 - DG ) uptake from 4 independent experiments . * P = 0 . 03 versus Ad - cont .

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A Assessment of 2-deoxyglucose (2-DG) uptake from 4 independent experiments. *P = 0.03 versus Ad-cont.


B , C Glut mRNA expression determined by

B, C Glut mRNA expression determined by RT


D Western blot indicating Ad - LXRα - and PE - induced increases in global protein O - GlcNAcylation , which was abrogated following inhibition of HBP with DON . LXRα protein expression is shown , and GAPDH served as a loading control .

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D Western blot indicating Ad-LXRα- and PE-induced increases in global protein O-GlcNAcylation, which was abrogated following inhibition of HBP with DON. LXRα protein expression is shown, and GAPDH served as a loading control.


E , F Modulation of Anp and Bnp mRNA levels by Ad - LXRα - induced O - GlcNAc signaling . Gene expression as determined by RT - PCR normalized to 36b4 , n = 5 per condition in the absence of PE , n = 4 per condition in the presence of PE . * P = 0 . 02 versus Ad - cont , * * P = 0 . 008 versus Ad - cont , # P = 0 . 03 , # # P = 0 . 02 .

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E, F Modulation of Anp and Bnp mRNA levels by Ad-LXRα-induced O-GlcNAc signaling. Gene expression as determined by RT-PCR normalized to 36b4, n = 5 per condition in the absence of PE, n = 4 per condition in the presence of PE. *P = 0.02 versus Ad-cont, **P = 0.008 versus Ad-cont, #P = 0.03, ##P = 0.02.


G Measurement of cell size , n = 5 per condition . * P = 0 . 008 versus Ad - cont , # P = 0 . 02 , # # P = 0 . 03 .

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G Measurement of cell size, n = 5 per condition. *P = 0.008 versus Ad-cont, #P = 0.02, ##P = 0.03.


H Representative images for the determination of cell size . Cells were stained with an antibody specific for LXRα ( green , indicated by arrow ) , DAPI for nuclei ( blue ) , and rhodamine - phalloidin for F - actin ( red ) ; scale bar = 50 μm .

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H Representative images for the determination of cell size. Cells were stained with an antibody specific for LXRα (green, indicated by arrow), DAPI for nuclei (blue), and rhodamine-phalloidin for F-actin (red); scale bar = 50 μm.


A Protein synthesis determined via leucine incorporation , n = 2 - 4 independent experiments . * P = 0 . 03 versus Ad - cont , § P = 0 . 06 versus Ad - cont .

A Protein synthesis determined via leucine incorporation, n = 2-4 independent experiments. *P = 0.03 versus Ad-cont, §P = 0.06 versus Ad-cont.


B Global O - GlcNAc protein expression with Western blot , GAPDH served as a control .

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B Global O-GlcNAc protein expression with Western blot, GAPDH served as a control.


C - F mRNA expression , normalized to 36b4 , n = 6 per condition , except n = 5 for si - LXRα control . Anp : * P = 0 . 009 versus Ad - cont , # P = 0 . 004 , # # P = 0 . 009 ; Bnp : * P = 0 . 004 versus Ad - cont , * * P = 0 . 002 versus Ad - cont ; Acta1 : * P = 0 . 002 versus Ad - cont , § P = 0 . 06 versus Ad - cont , # P = 0 . 02 ; Rcan1 : * P = 0 . 03 versus Ad - cont , * * P = 0 . 004 versus Ad - cont , # P = 0 . 004 .

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C-F mRNA expression, normalized to 36b4, n = 6 per condition, except n = 5 for si-LXRα control. Anp: *P = 0.009 versus Ad-cont, #P = 0.004, ##P = 0.009; Bnp: *P = 0.004 versus Ad-cont, **P = 0.002 versus Ad-cont; Acta1: *P = 0.002 versus Ad-cont, §P = 0.06 versus Ad-cont, #P = 0.02; Rcan1: *P = 0.03 versus Ad-cont, **P = 0.004 versus Ad-cont, #P = 0.004.


A , B Western blot analyses of global protein O - GlcNAc levels in left ventricles of mice with either ( A ) cardiac - specific LXRα overexpression or ( B ) LXRα deficiency and subjected to 5 weeks TAC .

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A, B Western blot analyses of global protein O-GlcNAc levels in left ventricles of mice with either (A) cardiac-specific LXRα overexpression or (B) LXRα deficiency and subjected to 5 weeks TAC.


C , D Nuclear protein extracts were precipitated with agarose WGA in the absence or presence of GlcNAc , a competitor , and analyzed by Western blot with antibodies against Mef2c , GATA4 , or Nkx - 2 . 5 , known transcription factors of natriuretic peptides ; bands represent 3 pooled hearts per Wt and LXRα - Tg lanes , and ( D ) quantification is for n = 2 , expressed as fold change .

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C, D Nuclear protein extracts were precipitated with agarose WGA in the absence or presence of GlcNAc, a competitor, and analyzed by Western blot with antibodies against Mef2c, GATA4, or Nkx-2.5, known transcription factors of natriuretic peptides; bands represent 3 pooled hearts per Wt and LXRα-Tg lanes, and (D) quantification is for n = 2, expressed as fold change.


C ) Results of a competitive uptake assay showing inhibition of 0 . 2 µM L - [ 3H ] Trp or L - [ 3H ] Leu uptake in MhsT - WT - expressing MQ614 cells , measured for 30 seconds in the absence ( - ) or presence of 12 µM of the natural amino acids ( the one - letter code is used ) or 4 - fluoro - phenylalanine ( 4FP ) . Data are the mean ± S . E . M . of three independent experiments each performed as technical triplicates .

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C) Results of a competitive uptake assay showing inhibition of 0.2 µM L-[3H]Trp or L-[3H]Leu uptake in MhsT-WT-expressing MQ614 cells, measured for 30 seconds in the absence (-) or presence of 12 µM of the natural amino acids (the one-letter code is used) or 4-fluoro-phenylalanine (4FP). Data are the mean ± S.E.M. of three independent experiments each performed as technical triplicates.


A ) Uptake of 0 . 1 μM L - [ 3H ] Leu was measured for 10 - second periods in the presence or absence of the indicated natural amino acids and 4 - fluorophenylalanine ( 4FP , all compounds tested at 100 μM final concentration ) in MQ614 expressing MhsT - WT or - M236F .

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A) Uptake of 0.1 μM L-[3H]Leu was measured for 10-second periods in the presence or absence of the indicated natural amino acids and 4-fluorophenylalanine (4FP, all compounds tested at 100 μM final concentration) in MQ614 expressing MhsT-WT or -M236F.


B , C ) Time course of 0 . 1 µM L - [ 3H ] Leu ( B ) or 0 . 1 µM L - [ 3H ] Trp ( C ) by MQ614 expressing MhsT - WT ( ) or - M236F ( ) , or MQ614 transformed with the control plasmid , pQE60 ( ) .

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B, C) Time course of 0.1 µM L-[3H]Leu (B) or 0.1 µM L-[3H]Trp (C) by MQ614 expressing MhsT-WT () or -M236F (), or MQ614 transformed with the control plasmid, pQE60 ().


D , E ) Kinetics of L - [ 3H ] Leu ( D ) or L - [ 3H ] Trp ( E ) uptake .

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D, E) Kinetics of L-[3H]Leu (D) or L-[3H]Trp (E) uptake.


( A ) EGFR degradation was followed in MSD , MPS ‐ IIIA and WT MEFs by treating the cells with EGF for the indicated time to stimulate EGFR internalization . The cells were then immediately lysed and subjected to anti ‐ EGFR blotting . The amount of remaining EGFR was quantified by densitometry analysis ( Image J ) of the blot and expressed in the chart as % of the EGFR amount present at time T0 ( 100 % ) . The values in the chart represent the mean ± s . e . m . values of three independent experiments .

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(A) EGFR degradation was followed in MSD, MPS‐IIIA and WT MEFs by treating the cells with EGF for the indicated time to stimulate EGFR internalization. The cells were then immediately lysed and subjected to anti‐EGFR blotting. The amount of remaining EGFR was quantified by densitometry analysis (Image J) of the blot and expressed in the chart as % of the EGFR amount present at time T0 (100%). The values in the chart represent the mean±s.e.m. values of three independent experiments.


( B ) MSD , MPS ‐ IIIA and WT MEFs cells loaded with dextran ( alexafluor ‐ 594 conjugated ) were labelled with anti ‐ LAMP1 antibody and the percentage of dextran co ‐ localizing with LAMP1 was evaluated . The chart displays merge values ( means ± s . e . m . ) that represent the percentage of dextran co ‐ localizing with LAMP1 measured in 15 different cells of triplicate experiments .

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(B) MSD, MPS‐IIIA and WT MEFs cells loaded with dextran (alexafluor‐594 conjugated) were labelled with anti‐LAMP1 antibody and the percentage of dextran co‐localizing with LAMP1 was evaluated. The chart displays merge values (means±s.e.m.) that represent the percentage of dextran co‐localizing with LAMP1 measured in 15 different cells of triplicate experiments.


( C ) The rate of lysosome fusion with autophagosomes was monitored in MSD , MPS ‐ IIIA and WT MEFs transfected with a tandem fluorescently tagged LC3 ( Kimura et al , 2007 ) . The rate of autophagosome maturation reflected the percentage of the LC3 ' unfused ' ( green / red fluorescence ratio ) at each time ( 1 and 3 h ) after bafilomycin removal ( T0 ) . The percentage of LC3 ' unfused ' was displayed versus the value at T0 ( assumed to be 100 % ) . Values are represented as means ± s . e . m . of triplicate experiments . * P0 . 05 , Student ' s t ‐ test : ( A ) : WT versus MSD and WT versus MPS ‐ IIIA ; ( B , C ) : WT versus MSD and WT versus MPS ‐ IIIA at each time point . Scale bar : 10 μm ( B , C ) .

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(C) The rate of lysosome fusion with autophagosomes was monitored in MSD, MPS‐IIIA and WT MEFs transfected with a tandem fluorescently tagged LC3 ( Kimura et al, 2007). The rate of autophagosome maturation reflected the percentage of the LC3 'unfused' (green/red fluorescence ratio) at each time (1 and 3 h) after bafilomycin removal (T0). The percentage of LC3 'unfused' was displayed versus the value at T0 (assumed to be 100%). Values are represented as means±s.e.m. of triplicate experiments. *P0.05, Student's t‐test: (A): WT versus MSD and WT versus MPS‐IIIA; (B, C): WT versus MSD and WT versus MPS‐IIIA at each time point. Scale bar: 10 μm (B, C).


( A ) Cholesterol levels were measured in the indicated endolysosomal membrane samples containing equal amount of proteins and expressed as ng of cholesterol per μg of protein ( see Materials and methods section for details ) .

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(A) Cholesterol levels were measured in the indicated endolysosomal membrane samples containing equal amount of proteins and expressed as ng of cholesterol per μg of protein (see Materials and methods section for details).


( B ) Filipin staining showing cholesterol accumulation in the endolysosomal compartment of MSD and MPS ‐ IIIA MEFs ( arrowheads and enlarged images ) .

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(B) Filipin staining showing cholesterol accumulation in the endolysosomal compartment of MSD and MPS‐IIIA MEFs (arrowheads and enlarged images).


( C , D ) Total lipids extracted from the indicated endolysosomal membrane samples ( 30 μg of proteins ) were either ( C ) subjected to a phosphate assay to quantify the bulk of phospholipids or ( D ) separated by TLC . Phospholipids and cholesterol on TLC plates were revealed by molybdenum blue staining . CHOL , cholesterol ; LBPA lysobisphosphatidic acid ; PC , phosphatidylcoline ; PE phosphatidylethanolamine ; PI phosphatidyinositol ; SM sphingomyelin . ( A , C ) Values represent the mean ± s . e . m . values of three independent experiments . * P0 . 05 , Student ' s t ‐ test : ( A , C ) : WT versus MSD and WT versus MPS ‐ IIIA . Scale bar , 10 μm ( B ) .

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(C, D) Total lipids extracted from the indicated endolysosomal membrane samples (30 μg of proteins) were either (C) subjected to a phosphate assay to quantify the bulk of phospholipids or (D) separated by TLC. Phospholipids and cholesterol on TLC plates were revealed by molybdenum blue staining. CHOL, cholesterol; LBPA lysobisphosphatidic acid; PC, phosphatidylcoline; PE phosphatidylethanolamine; PI phosphatidyinositol; SM sphingomyelin. (A, C) Values represent the mean±s.e.m. values of three independent experiments. *P0.05, Student's t‐test: (A, C): WT versus MSD and WT versus MPS‐IIIA. Scale bar, 10 μm (B).


Cholesterol accumulation inhibits lysosomal fusion . Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either ( A ) WT MEFs loaded with cholesterol or in

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Cholesterol accumulation inhibits lysosomal fusion. Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either (A) WT MEFs loaded with cholesterol or in MSD


Cholesterol accumulation inhibits lysosomal fusion . Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either ( A ) WT MEFs loaded with cholesterol or in MSD and ( D ) MPS ‐ IIIA MEFs treated with MβCD . Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol ‐ loaded WT MEFs . After treatments the rate of autophagosome maturation ( B , E ) and the transport of fluorescent dextran to lysosomes ( C , F ) were also analysed as in Figure 1 . WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1 . ( A - F ) Values represent the mean ± s . e . m . values of three independent experiments . * P0 . 05 , Student ' s t ‐ test : ( A , C ) : WT versus WT + cholesterol ; ( B ) : WT versus WT + cholesterol for each time point ; ( D , F ) : MSD versus MSD + MβCD and MPS ‐ IIIA versus MPS ‐ IIIA + MβCD ; ( E ) : MSD versus MSD + MβCD and MPS ‐ IIIA versus MPS ‐ IIIA + MβCD for each time point . Scale

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

Cholesterol accumulation inhibits lysosomal fusion. Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either (A) WT MEFs loaded with cholesterol or in MSD and (D) MPS‐IIIA MEFs treated with MβCD. Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol‐loaded WT MEFs. After treatments the rate of autophagosome maturation (B, E) and the transport of fluorescent dextran to lysosomes (C, F) were also analysed as in Figure 1. WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1. (A-F) Values represent the mean±s.e.m. values of three independent experiments. *P0.05, Student's t‐test: (A, C): WT versus WT+cholesterol; (B): WT versus WT+cholesterol for each time point; (D, F): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD; (E): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD for each time point. Scale bar


Cholesterol accumulation inhibits lysosomal fusion . Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either ( A ) WT MEFs loaded with cholesterol or in MSD and ( D ) MPS ‐ IIIA MEFs treated with MβCD . Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol ‐ loaded WT MEFs . After treatments the rate of autophagosome maturation ( B , E ) and the transport of fluorescent dextran to lysosomes ( C , F ) were also analysed as in Figure 1 . WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1 . ( A - F ) Values represent the mean ± s . e . m . values of three independent experiments . * P0 . 05 , Student ' s t ‐ test : ( A , C ) : WT versus WT + cholesterol ; ( B ) : WT versus WT + cholesterol for each time point ; ( D , F ) : MSD versus MSD + MβCD and MPS ‐ IIIA versus MPS ‐ IIIA + MβCD ; ( E ) : MSD versus MSD + MβCD and MPS ‐ IIIA versus MPS ‐ IIIA + MβCD for each time point . Scale bar : 10 μm ( A , C , D , F ) .

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

Cholesterol accumulation inhibits lysosomal fusion. Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either (A) WT MEFs loaded with cholesterol or in MSD and (D) MPS‐IIIA MEFs treated with MβCD. Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol‐loaded WT MEFs. After treatments the rate of autophagosome maturation (B, E) and the transport of fluorescent dextran to lysosomes (C, F) were also analysed as in Figure 1. WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1. (A-F) Values represent the mean±s.e.m. values of three independent experiments. *P0.05, Student's t‐test: (A, C): WT versus WT+cholesterol; (B): WT versus WT+cholesterol for each time point; (D, F): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD; (E): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD for each time point. Scale bar: 10 μm (A, C, D, F).


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


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