( E ) Sensitivity assay was conducted with WT and HMCES KO cells upon treatment with AP Endonuclease inhibitor III ( APEi ) . Results are presented from two independent experiments and data are presented as mean ± s . e . m .

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(E) Sensitivity assay was conducted with WT and HMCES KO cells upon treatment with AP Endonuclease inhibitor III (APEi). Results are presented from two independent experiments and data are presented as mean±s.e.m.


( F ) Colony forming assay was performed with WT and HMCES KO cells upon either pemetrexed treatment alone or in combination with APEi . Data are presented as mean ± s . e . m ( n = 2 each with technical repeats ) .

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(F) Colony forming assay was performed with WT and HMCES KO cells upon either pemetrexed treatment alone or in combination with APEi. Data are presented as mean±s.e.m (n=2 each with technical repeats).


( G , H ) Western blotting revealed levels of POLH ( G ) and REV1 ( H ) in two independent clones upon selection following transfection with indicated shRNA .

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(G, H) Western blotting revealed levels of POLH (G) and REV1 (H) in two independent clones upon selection following transfection with indicated shRNA.


( I ) Colony formation assay was conducted to reveal proliferation of WT , POLH KD , REV1 KD , HMCES KO , HMCES KO + POLH KD and HMCES KO + REV1 KD cells .

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(I) Colony formation assay was conducted to reveal proliferation of WT, POLH KD, REV1 KD, HMCES KO, HMCES KO + POLH KD and HMCES KO +REV1 KD cells.


( J ) Representative images showing proliferation of respective shRNA resistant reconstitution of POLH in 293A and HMCES KO cells stably expressing POLH shRNA . Experiment was repeated two independent times in duplicates .

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(J) Representative images showing proliferation of respective shRNA resistant reconstitution of POLH in 293A and HMCES KO cells stably expressing POLH shRNA. Experiment was repeated two independent times in duplicates.


( K ) Mutagenesis frequency analysis was conducted in 293T scrambled and 293T HMCES shRNA cells using sup F shuttle vector - based assay system . Experiments were repeated three independent times and data are presented as mean ± s . e . m . Student ' s t - test was used to calculate statistical significance ( * * , p < 0 . 01 ) .

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(K) Mutagenesis frequency analysis was conducted in 293T scrambled and 293T HMCES shRNA cells using sup F shuttle vector-based assay system. Experiments were repeated three independent times and data are presented as mean±s.e.m. Student's t-test was used to calculate statistical significance (**, p<0.01).


( B ) Volcano plot showing DDR CRISPR - CAS9 screen for HMCES KO cells . - log10 ( p - value ) is plotted against log2 ( HMCES KO / WT ) fold change . Genes showing significant positive and negative enrichment are colored in red and top enriched candidates are also labeled .

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(B) Volcano plot showing DDR CRISPR-CAS9 screen for HMCES KO cells. -log10(p-value) is plotted against log2(HMCES KO/ WT) fold change. Genes showing significant positive and negative enrichment are colored in red and top enriched candidates are also labeled.


( C ) Plot showing number of sgRNAs that are negatively or positively enriched for each gene in DDR library .

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(C) Plot showing number of sgRNAs that are negatively or positively enriched for each gene in DDR library.


Western blots showing levels of CtIP ( D ) upon shRNA mediated knockdown in 293A cells .

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Western blots showing levels of CtIP (D) upon shRNA mediated knockdown in 293A cells.


Western blots showing levels of BRCA2 ( E ) upon shRNA mediated knockdown in 293A cells .

Western blots showing levels of BRCA2 (E) upon shRNA mediated knockdown in 293A cells.


Cell proliferation measured using CellTiter - Glo assay in 293A and HMCES KO cells without or with transfection with viruses encoding CtIP ( F ) shRNAs . Each experiment was repeated three independent times done in triplicates each time . Data was represented as mean ± s . e . m . Student ' s t - test was used to calculate statistical significance ( * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) .

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Cell proliferation measured using CellTiter-Glo assay in 293A and HMCES KO cells without or with transfection with viruses encoding CtIP (F) shRNAs. Each experiment was repeated three independent times done in triplicates each time. Data was represented as mean±s.e.m. Student's t-test was used to calculate statistical significance (***, p<0.001; ****, p<0.0001).


Cell proliferation measured using CellTiter - Glo assay in 293A and HMCES KO cells without or with transfection with viruses encoding BRCA2 ( G ) shRNAs . Each experiment was repeated three independent times done in triplicates each time . Data was represented as mean ± s . e . m . Student ' s t - test was used to calculate statistical significance ( * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) .

Cell proliferation measured using CellTiter-Glo assay in 293A and HMCES KO cells without or with transfection with viruses encoding BRCA2 (G) shRNAs. Each experiment was repeated three independent times done in triplicates each time. Data was represented as mean±s.e.m. Student's t-test was used to calculate statistical significance (***, p<0.001; ****, p<0.0001).


T cells were collected from age - and gender - matched WT , EGR1KO and EGR4KO mice . ( A ) CD4 + T cells were isolated from the spleen by negative selection before incubating with anti - CD3 / CD28 antibodies for the indicated time periods . Levels of expression of EGR1 , EGR2 , EGR3 and EGR4 were measured by qPCR .

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T cells were collected from age- and gender-matched WT, EGR1KO and EGR4KO mice. (A) CD4+ T cells were isolated from the spleen by negative selection before incubating with anti-CD3/CD28 antibodies for the indicated time periods. Levels of expression of EGR1, EGR2, EGR3 and EGR4 were measured by qPCR.


T cells were collected from age - and gender - matched WT , EGR1KO and EGR4KO mice . ( B ) WBC were collected from WT , EGR1KO and EGR4KO mice before staining and FACS analysis to determine composition ( gating strategies in Appendix Fig S1 ) . Subclasses of CD8 + , CD4 + and Treg populations were determined as shown .

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T cells were collected from age- and gender-matched WT, EGR1KO and EGR4KO mice. (B) WBC were collected from WT, EGR1KO and EGR4KO mice before staining and FACS analysis to determine composition (gating strategies in Appendix Fig S1). Subclasses of CD8+, CD4+ and Treg populations were determined as shown.


WT and EGR4 - / - CD4 + and CD8 + T cells were isolated from the spleen by cell sorting and stained with CFSE . A Cells were incubated under unstimulated conditions or stimulated with either anti - TCRβ or anti - CD3 / CD28 antibodies for 0 to 5 days ( mean ± SEM ; a minimum of 3 biological replicates were examined ; each biological replicate includes 2 technical replicates ) .

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WT and EGR4-/- CD4+ and CD8+ T cells were isolated from the spleen by cell sorting and stained with CFSE. A Cells were incubated under unstimulated conditions or stimulated with either anti-TCRβ or anti-CD3/CD28 antibodies for 0 to 5 days (mean±SEM; a minimum of 3 biological replicates were examined; each biological replicate includes 2 technical replicates).


WT and EGR4 - / - CD4 + and CD8 + T cells were isolated from the spleen by cell sorting and stained with CFSE . B Dose dependence of EGR4 - dependent differences in proliferation were also determined in both CD4 + and CD8 + cells aft er treatment with anti - TCRβ ( E , F ) or anti - CD3 / CD28 ( G , H ) at the indicated concentrations before FACS analysis ( Appendix Fig S2 ) ( mean ± SEM ; a minimum of 3 biological replicates were examined ; each biological replicate includes 2 technical replicates ) .

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WT and EGR4-/- CD4+ and CD8+ T cells were isolated from the spleen by cell sorting and stained with CFSE. B Dose dependence of EGR4-dependent differences in proliferation were also determined in both CD4+ and CD8+ cells aft er treatment with anti-TCRβ (E,F) or anti-CD3/CD28 (G,H) at the indicated concentrations before FACS analysis (Appendix Fig S2) (mean±SEM; a minimum of 3 biological replicates were examined; each biological replicate includes 2 technical replicates).


WT and EGR4 - / - CD4 + and CD8 + T cells were isolated from the spleen by cell sorting and stained with CFSE . C , D Generation analysis was then assessed in WT , EGR1 - / - and EGR4 - / - CD4 + ( C ) or CD8 + ( D ) T cells after a 4 day incubation , under unstimulated conditions , or stimulated with either anti - TCRβ or anti - CD3 / CD28 antibodies . Generation analysis was determined using FlowJo as depicted in Appendix figure S3 ( mean ± SEM ; a minimum of 3 biological replicates were examined ; each biological replicate includes 2 technical replicates ) . . EGR4 deletion significantly shifted the number of generations as determined by two - way ANOVA ; loss of either EGR1 or EGR4 significantly changed the number of generations under resting or stimulated conditions ( p < 0 . 0001 ) .

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WT and EGR4-/- CD4+ and CD8+ T cells were isolated from the spleen by cell sorting and stained with CFSE. C,D Generation analysis was then assessed in WT, EGR1-/- and EGR4-/- CD4+ (C) or CD8+ (D) T cells after a 4 day incubation, under unstimulated conditions, or stimulated with either anti-TCRβ or anti-CD3/CD28 antibodies. Generation analysis was determined using FlowJo as depicted in Appendix figure S3 (mean±SEM; a minimum of 3 biological replicates were examined; each biological replicate includes 2 technical replicates).. EGR4 deletion significantly shifted the number of generations as determined by two-way ANOVA; loss of either EGR1 or EGR4 significantly changed the number of generations under resting or stimulated conditions (p < 0.0001).


CD4 + and CD8 + T cells were isolated from the spleens of WT , EGR1KO and EGR4KO mice by cell sorting . ( A - F ) Cytokine secretion was measured by ELISA after incubating CD4 + ( A , C , E ) and CD8 + ( B , D , F ) T cells for 48 hrs in resting conditions ( A , B ) or stimulated with either anti - TCRβ ( C , D ) or anti - CD3 / CD28 antibodies ( E , F ) .

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CD4+ and CD8+ T cells were isolated from the spleens of WT, EGR1KO and EGR4KO mice by cell sorting. (A-F) Cytokine secretion was measured by ELISA after incubating CD4+ (A,C,E) and CD8+ (B,D,F) T cells for 48 hrs in resting conditions (A,B) or stimulated with either anti-TCRβ (C,D) or anti-CD3/CD28 antibodies (E,F).


CD4 + and CD8 + T cells were isolated from the spleens of WT , EGR4KO mice by cell sorting . ( G ) Representative FACS plots depicting IFNγ levels after intracellular staining in WT and EGR4KO CD4 + and CD8 + T cells .

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CD4+ and CD8+ T cells were isolated from the spleens of WT, EGR4KO mice by cell sorting. (G) Representative FACS plots depicting IFNγ levels after intracellular staining in WT and EGR4KO CD4+ and CD8+ T cells.


CD4 + and CD8 + T cells were isolated from the spleens of WT , and EGR4KO mice by cell sorting . ( H ) Comparison of IFNγ levels in WT vs . EGR4 - / - T cells .

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CD4+ and CD8+ T cells were isolated from the spleens of WT, and EGR4KO mice by cell sorting. (H) Comparison of IFNγ levels in WT vs. EGR4-/- T cells.


CD4 + T cells were isolated from the spleens of WT , EGR1KO and EGR4KO mice by cell sorting . ( I ) Representative FACS plots of CD4 + T cells incubated for 24 hrs under Th1 polarizing conditions before intracellular staining with IFNγ and IL - 17 antibodies .

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CD4+ T cells were isolated from the spleens of WT, EGR1KO and EGR4KO mice by cell sorting. (I) Representative FACS plots of CD4+ T cells incubated for 24 hrs under Th1 polarizing conditions before intracellular staining with IFNγ and IL-17 antibodies.


CD4 + T cells were isolated from the spleens of WT , EGR1KO and EGR4KO mice by cell sorting . ( J ) Comparison of the number of Th1 polarized cells between WT , EGR1 - / - and EGR4 - / - CD4 + T cells .

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CD4+ T cells were isolated from the spleens of WT, EGR1KO and EGR4KO mice by cell sorting. (J) Comparison of the number of Th1 polarized cells between WT, EGR1-/- and EGR4-/- CD4+ T cells.


CD4 + T cells were isolated from the spleens of WT , EGR1KO and EGR4KO mice by cell sorting . ( K ) CD4 + T cells were incubated for 5 days under polarizing conditions before analysis of cytokine production by ELISA .

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CD4+ T cells were isolated from the spleens of WT, EGR1KO and EGR4KO mice by cell sorting. (K) CD4+ T cells were incubated for 5 days under polarizing conditions before analysis of cytokine production by ELISA.


( A ) NFAT nuclear localization was measured was measured by immunocytochemistry in CD4 + and CD8 + T cells isolated by negative selection from WT , EGR1KO and EGR4KO mice . Cells were stimulated with anti - CD3 / CD28 antibodies for the indicated time periods before fixation and staining . Nuclear localization of NFATc1 in CD4 + and CD8 + T cells was determined based on colocalization of NFATc1 and DAPI measured by Pearson analysis ( mean ± SEM ; data are based upon 13 to 20 cells / data point from one experiment ; experiment was repeated 3 times ) . EGR4 - mediated differences in NFATc1 localization were determined by two - way ANOVA . NFAT localization was dependent upon EGR4 expression in both CD4 + ( p = 0 . 025 ) and CD8 + ( p = 0 . 039 ) T cells . Post - hoc analysis revealed EGR4 - dependent differences at specific timepoints * p < 0 . 05 ; * * * p < 0 . 001 .

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(A) NFAT nuclear localization was measured was measured by immunocytochemistry in CD4+ and CD8+ T cells isolated by negative selection from WT, EGR1KO and EGR4KO mice. Cells were stimulated with anti-CD3/CD28 antibodies for the indicated time periods before fixation and staining. Nuclear localization of NFATc1 in CD4+ and CD8+ T cells was determined based on colocalization of NFATc1 and DAPI measured by Pearson analysis (mean±SEM; data are based upon 13 to 20 cells/data point from one experiment; experiment was repeated 3 times). EGR4-mediated differences in NFATc1 localization were determined by two-way ANOVA. NFAT localization was dependent upon EGR4 expression in both CD4+ (p=0.025) and CD8+ (p=0.039) T cells. Post-hoc analysis revealed EGR4-dependent differences at specific timepoints * p < 0.05; *** p < 0.001.


( B ) WT , EGR1KO and EGR4KO CD4 + T cells were isolated from the spleen by negative selection before plating on poly - lysine ( control ) or CD3 / CD28 and loading with fura2 . Ca2 + levels shown are from representative single cells measured from multiple experiments . ( C ) Scatter plots showing area under the curve ( AUC ) and variance for each cell measured under all conditions .

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(B) WT, EGR1KO and EGR4KO CD4+ T cells were isolated from the spleen by negative selection before plating on poly-lysine (control) or CD3/CD28 and loading with fura2. Ca2+ levels shown are from representative single cells measured from multiple experiments. (C) Scatter plots showing area under the curve (AUC) and variance for each cell measured under all conditions.


CD4 + and CD8 + T cells were isolated by negative selection from WT or EGR4 - / - mice . ( A ) CD4 + and CD8 + T cells were loaded with Fura - 2AM and plated on poly - D - lysine ( 2 hrs ) before treating with biotinylated anti - CD3ε , followed by streptavidin where indicated . The average response of the majority of CD4 + ( WT 74 . 2 ± 3 . 2 % ; EGR4 - / - 77 . 8 ± 2 . 9 % ) and CD8 + ( WT 79 . 4 ± 3 . 5 % ; EGR4 - / - 78 . 2 ± 2 . 5 % ) cells that responded to streptavidin crosslinking of CD3 ( mean ± SEM ; 40 - 60 cells / coverslip ; 7 to 8 separate experiments ) .

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CD4+ and CD8+ T cells were isolated by negative selection from WT or EGR4-/-mice. (A) CD4+ and CD8+ T cells were loaded with Fura-2AM and plated on poly-D-lysine (2 hrs) before treating with biotinylated anti-CD3ε, followed by streptavidin where indicated. The average response of the majority of CD4+ (WT 74.2±3.2%; EGR4-/- 77.8±2.9%) and CD8+ (WT 79.4±3.5%; EGR4-/- 78.2±2.5%) cells that responded to streptavidin crosslinking of CD3 (mean±SEM; 40-60 cells/coverslip; 7 to 8 separate experiments).


CD4 + and CD8 + T cells were isolated by negative selection from WT or EGR4 - / - mice . ( B ) Cells were plated on anti - CD3 / CD28 antibodies for 2 hrs ; BTP2 ( 10 μM ) was added where marked by the arrow .

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CD4+ and CD8+ T cells were isolated by negative selection from WT or EGR4-/-mice. (B) Cells were plated on anti-CD3/CD28 antibodies for 2 hrs; BTP2 (10 μM) was added where marked by the arrow.


CD4 + T cells were isolated by negative selection from WT or EGR4 - / - mice . ( C ) CD4 + T cells were incubated with anti - CD3 / CD28 antibodies for 6 hrs before measuring expression of IFNγ , IL - 2 , IL - 17 and IL - 4 by FACS analysis . Data are mean ± SEM ; a minimum of 3 biological replicates were examined ; each biological replicate includes 2 technical replicates .

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CD4+ T cells were isolated by negative selection from WT or EGR4-/-mice. (C) CD4+ T cells were incubated with anti-CD3/CD28 antibodies for 6 hrs before measuring expression of IFNγ, IL-2, IL-17 and IL-4 by FACS analysis. Data are mean±SEM; a minimum of 3 biological replicates were examined; each biological replicate includes 2 technical replicates.


CD4 + T cells were isolated by negative selection from WT , EGR4 - / - mice . ( A ) WT or EGR4 - / - cells were loaded with Fura - 2AM and plated on anti - CD3 / CD28 antibodies for 2 hrs . Senicapoc ( 4 μM ) or Tram - 34 ( 5 μM ) were added where marked by the arrow .

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CD4+ T cells were isolated by negative selection from WT, EGR4-/- mice. (A) WT or EGR4-/- cells were loaded with Fura-2AM and plated on anti-CD3/CD28 antibodies for 2 hrs. Senicapoc (4 μM) or Tram-34 (5 μM) were added where marked by the arrow.


CD4 + T cells were isolated by negative selection from WT , EGR4 - / - mice . ( B ) CD4 + T cells were isolated by negative selection from WT or EGR4 - / - mice and incubated with anti - CD3 / CD28 antibodies for 6 hrs in the presence or absence of either senicapoc , TRAM - 34 or Shk - Dap22 before measuring expression of IFNγ levels by FACS analysis . Data are mean ± SEM ; 3 biological replicates were examined ; each biological replicate includes 2 technical replicates . * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

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CD4+ T cells were isolated by negative selection from WT, EGR4-/- mice. (B) CD4+ T cells were isolated by negative selection from WT or EGR4-/- mice and incubated with anti-CD3/CD28 antibodies for 6 hrs in the presence or absence of either senicapoc, TRAM-34 or Shk-Dap22 before measuring expression of IFNγ levels by FACS analysis. Data are mean±SEM; 3 biological replicates were examined; each biological replicate includes 2 technical replicates. *** p < 0.001; **** p < 0.0001.


CD4 + T cells were isolated by negative selection from WT , EGR4 - / - mice . ( C ) To measure the effect of KCa3 . 1 inhibition of cell proliferation , WT and EGR4 - / - CD4 + T cells were isolated from the spleen by cell sorting and stained with CFSE . Cells were incubated with anti - TCRβ antibodies in the presence of vehicle , Senicapoc ( 5 µM ) or TRAM - 34 ( 5 µM ) for 4 days and then collected for FACS analysis . ( D ) Generation analysis was completed for all data using FlowJo software . Data are mean ± SEM ; 3 biological replicates were examined ; each biological replicate includes 2 technical replicates .

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CD4+ T cells were isolated by negative selection from WT, EGR4-/- mice. (C) To measure the effect of KCa3.1 inhibition of cell proliferation, WT and EGR4-/- CD4+ T cells were isolated from the spleen by cell sorting and stained with CFSE. Cells were incubated with anti-TCRβ antibodies in the presence of vehicle, Senicapoc (5 µM) or TRAM-34 (5 µM) for 4 days and then collected for FACS analysis. (D) Generation analysis was completed for all data using FlowJo software. Data are mean±SEM; 3 biological replicates were examined; each biological replicate includes 2 technical replicates.


B16N melanoma cells stably expressing GFP - luciferase were injected into syngeneic WT and EGR4 - / - mice by tail vein injection . ( A ) Luciferase expression was monitored by IVIS imaging at the indicated timepoints after luciferin injections .

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B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. (A) Luciferase expression was monitored by IVIS imaging at the indicated timepoints after luciferin injections.


B16N melanoma cells stably expressing GFP - luciferase were injected into syngeneic WT and EGR4 - / - mice by tail vein injection . ( B - D ) Mice were sacrificed on day 20 and tumors were counted . Data is presented as total / mouse ( B ) , N = 9 for each gender and genotype . Differences in luciferase activity measured by IVIS in Panel A were determined by two - way ANOVA . Changes in luciferase activity were significantly altered in EGR4 knockout mice ( P < 0 . 0112 ) .

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

B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. (B-D) Mice were sacrificed on day 20 and tumors were counted. Data is presented as total/mouse (B), N=9 for each gender and genotype. Differences in luciferase activity measured by IVIS in Panel A were determined by two-way ANOVA. Changes in luciferase activity were significantly altered in EGR4 knockout mice (P < 0.0112).


B16N melanoma cells stably expressing GFP - luciferase were injected into syngeneic WT and EGR4 - / - mice by tail vein injection . Mice were sacrificed on day 20 and tumors were counted . ( B ) , lung tumors ( C ) and metastatic tumors ( all tumors found outside of the lungs ; D ) . N = 9 for each gender and genotype . Differences in luciferase activity measured by IVIS in Panel A were determined by two - way ANOVA . Changes in luciferase activity were significantly altered in EGR4 knockout mice ( P < 0 . 0112 ) .

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

B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. Mice were sacrificed on day 20 and tumors were counted. (B), lung tumors (C) and metastatic tumors (all tumors found outside of the lungs; D). N=9 for each gender and genotype. Differences in luciferase activity measured by IVIS in Panel A were determined by two-way ANOVA. Changes in luciferase activity were significantly altered in EGR4 knockout mice (P < 0.0112).


B16N melanoma cells stably expressing GFP - luciferase were injected into syngeneic WT and EGR4 - / - mice by tail vein injection . All lung tumors were isolated from both WT and EGR4KO mice along with spleens and analysed for CD45 + cells . WT lung tumors were 5 . 407 ± 1 . 843 % CD45 + , while EGR4KO mice were 12 . 445 ± 2 . 239 % CD45 + . WT spleens were 83 . 92 ± 3 . 344 % CD45 + , while EGR4KO mice were 95 . 13 ± 1 . 856 % CD45 + . Relative distributions of T and myeloid cells within the CD45 + populations were determined by flow cytometric analysis . ( E , F ) Distributions of T and myeloid cells found within lung tumors ( E ) vs . the spleen ( F ) .

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

B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. All lung tumors were isolated from both WT and EGR4KO mice along with spleens and analysed for CD45+ cells. WT lung tumors were 5.407 ± 1.843% CD45+, while EGR4KO mice were 12.445 ± 2.239% CD45+. WT spleens were 83.92 ± 3.344% CD45+, while EGR4KO mice were 95.13 ± 1.856% CD45+. Relative distributions of T and myeloid cells within the CD45+ populations were determined by flow cytometric analysis. (E,F) Distributions of T and myeloid cells found within lung tumors (E) vs. the spleen (F).


B16N melanoma cells stably expressing GFP - luciferase were injected into syngeneic WT and EGR4 - / - mice by tail vein injection . All lung tumors were isolated from both WT and EGR4KO mice along with spleens and analysed for CD45 + cells . ( G ) Representative FACS plots depicting gating strategies for defining Treg cell populations in Panels E and

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

B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. All lung tumors were isolated from both WT and EGR4KO mice along with spleens and analysed for CD45+ cells. (G) Representative FACS plots depicting gating strategies for defining Treg cell populations in Panels E and F


Tumors collected and analysed for exhaustion / anergy . Cells were collected and enriched for the immune cell population by Ficoll - Hypaque . WT cells were 36 . 9 ± 4 . 68 % Thy1 . 2 + , EGR4KO cells were 48 . 0 ± 0 . 58 % Thy1 . 2 + . ( A ) CD4 + and CD8 + T cells were stimulated in vitro with ionomycin / PMA before intracellular staining for IFNγ , IL - 17 and IL - 9 and FACS analysis .

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

Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. (A) CD4+ and CD8+ T cells were stimulated in vitro with ionomycin/PMA before intracellular staining for IFNγ, IL-17 and IL-9 and FACS analysis.


Tumors collected and analysed for exhaustion / anergy . Cells were collected and enriched for the immune cell population by Ficoll - Hypaque . WT cells were 36 . 9 ± 4 . 68 % Thy1 . 2 + , EGR4KO cells were 48 . 0 ± 0 . 58 % Thy1 . 2 + . ( B , C ) Representative FACS plots depicting gating strategies for defining cytokine expression in panels A to

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

Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. (B,C) Representative FACS plots depicting gating strategies for defining cytokine expression in panels A to D


Tumors collected and analysed for exhaustion / anergy . Cells were collected and enriched for the immune cell population by Ficoll - Hypaque . WT cells were 36 . 9 ± 4 . 68 % Thy1 . 2 + , EGR4KO cells were 48 . 0 ± 0 . 58 % Thy1 . 2 + . CD4 + and CD8 + T cells were stained with CD44 , CD73 , FR4 and PD1 to monitor the formation of anergic / exhausted T cells . ( D ) Percentages of each population .

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

Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. CD4+ and CD8+ T cells were stained with CD44, CD73, FR4 and PD1 to monitor the formation of anergic/exhausted T cells. (D) Percentages of each population.


Tumors collected and analysed for exhaustion / anergy . Cells were collected and enriched for the immune cell population by Ficoll - Hypaque . WT cells were 36 . 9 ± 4 . 68 % Thy1 . 2 + , EGR4KO cells were 48 . 0 ± 0 . 58 % Thy1 . 2 + . CD4 + and CD8 + T cells were stained with CD44 , CD73 , FR4 and PD1 to monitor the formation of anergic / exhausted T cells . ( E ) Gating strategies .

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

Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. CD4+ and CD8+ T cells were stained with CD44, CD73, FR4 and PD1 to monitor the formation of anergic/exhausted T cells. (E) Gating strategies.


Tumors collected and analysed for exhaustion / anergy . Cells were collected and enriched for the immune cell population by Ficoll - Hypaque . WT cells were 36 . 9 ± 4 . 68 % Thy1 . 2 + , EGR4KO cells were 48 . 0 ± 0 . 58 % Thy1 . 2 + . ( F ) FR4 and PD1 levels in WT vs . EGR4 - / - T cells .

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

Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. (F) FR4 and PD1 levels in WT vs. EGR4-/- T cells.


a - c , Expression of CD45RO and CCR7 ( a ) , CD45RO and CD27 ( b ) , CD27 and CD28 ( c ) on peripheral blood CD8 T cells of three participants [ P1 ( heterozygous mutation ) , P2 ( homozygous mutation ) and P3 ( heterozygous ) for the c . 192G > C mutation in the DJ - 1 gene ] . d - g , Expression of CD57 ( d ) , PD - 1 ( e ) , EOMES ( f ) and T - bet ( g ) on peripheral blood CD8 T cells of three participants . The enlarged number in the corresponding gate represents the corresponding percentage out of the parent population .

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

a-c, Expression of CD45RO and CCR7 (a), CD45RO and CD27 (b), CD27 and CD28 (c) on peripheral blood CD8 T cells of three participants [P1 (heterozygous mutation), P2 (homozygous mutation) and P3 (heterozygous) for the c.192G>C mutation in the DJ-1 gene]. d-g, Expression of CD57 (d), PD-1 (e), EOMES (f) and T-bet (g) on peripheral blood CD8 T cells of three participants. The enlarged number in the corresponding gate represents the corresponding percentage out of the parent population. h


b - c , Percentages of CD44low CD62Lhigh ( Tn ) ( b ) and CD44high CD62Llow ( Tem ) ( c ) cells among total CD8 T cells of spleen and pLNs from young and 45 - wk - old Dj - 1 KO and WT littermates ( young KO , n = 5 ; young WT , n = 5 ; 45 - wk - old KO , n = 8 ; 45 - wk - old WT , n = 6 ; for 45 - wk - old mice , data pooled from 2 independent experiments ) .

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

b-c, Percentages of CD44low CD62Lhigh (Tn) (b) and CD44high CD62Llow (Tem) (c) cells among total CD8 T cells of spleen and pLNs from young and 45-wk-old Dj-1 KO and WT littermates (young KO, n=5; young WT, n=5; 45-wk-old KO, n=8; 45-wk-old WT, n=6; for 45-wk-old mice, data pooled from 2 independent experiments).


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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 % .

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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 .

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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 .

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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 .

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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 .

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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 . ) .

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

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 . ) .

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

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 . ) .

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

F) 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 . ) .

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

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 ) .

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

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 . ) .

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

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 . ) .

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

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 . ) .

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

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 .

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

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 . ) .

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

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 . ) .

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

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 . ) .

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

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 . ) .

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

D&E) 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 . ) .

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

F&G) 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 . ) .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) .

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

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 ) .

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

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 ) .

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 B-MEASURED_VAR I-MEASURED_VAR I-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

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 . ) .

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

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.).