( A , B ) Representative traces from adult control ( WT ) CAMDI - KO ( KO ) in open field . Locomotor activity ( A ) traveled ( B ) for 15 min . n = 8 for WT mice , n = 7 for KO mice . * , p < 0 . 05 , One - way ANOVA with Bonferroni ' s post hoc ( F ( 1 , 13 ) = 5 . 53 ) . Data are presented as mean ± .

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(A, B) Representative traces from adult control (WT) and CAMDI-KO (KO) in open field test. Locomotor activity (A) and distance traveled (B) for 15 min. n = 8 for WT mice, n = 7 for KO mice. *, p<0.05, One-way ANOVA with Bonferroni's post hoc test (F(1, 13) = 5.53). Data are presented as mean ± SEM.


( C ) Repetitive jumping behavior in open field . n = 8 for WT mice , n = 7 for KO mice . * , p < 0 . 05 , One - way ANOVA with Bonferroni ' s post hoc ( F ( 1 , 13 ) = 5 . 09 ) . Data are presented as mean ± .

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(C) Repetitive jumping behavior in open field test. n = 8 for WT mice, n = 7 for KO mice. *, p<0.05, One-way ANOVA with Bonferroni's post hoc test (F(1, 13) = 5.09). Data are presented as mean ± SEM.


( D ) Three - chamber social interaction . time spent sniffing with stranger mouse . n = 19 for WT mice , n = 15 for KO mice . * , p < 0 . 05 , * * * , p < 0 . 001 , Two - way ANOVA followed by Scheffe ' s post - hoc ( main effect of genotype F ( 1 , 63 ) = 5 . 79 , main effect of chamber F ( 1 , 63 ) = 90 . 47 , interaction F ( 1 , 63 ) = 5 . 29 ) . Data are presented as mean ± .

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(D) Three-chamber social interaction test. The time spent sniffing with stranger mouse. n = 19 for WT mice, n = 15 for KO mice. *, p<0.05, ***, p<0.001,Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 63)=5.79, main effect of chamber F(1, 63)=90.47, interaction F(1, 63)=5.29). Data are presented as mean ± SEM.


( E ) Resident - intruder ( social sniffing ) . n = 7 for WT mice , n = 8 for KO mice . * , p < 0 . 05 , One - way ANOVA with Bonferroni ' s post hoc ( F ( 1 , 13 ) = 5 . 15 ) . Data are presented as mean ± .

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(E) Resident-intruder test (social sniffing). n = 7 for WT mice, n = 8 for KO mice. *, p<0.05, One-way ANOVA with Bonferroni's post hoc test (F(1, 13) = 5.15). Data are presented as mean ± SEM.


( F ) Resident - intruder ( digging ) . n = 7 for WT mice , n = 8 for KO mice . * * , p < 0 . 01 , One - way ANOVA with Bonferroni ' s post hoc ( F ( 1 , 13 ) = 10 . 97 ) . Data are presented as mean ± .

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(F) Resident-intruder test (digging). n = 7 for WT mice, n = 8 for KO mice. **, p<0.01, One-way ANOVA with Bonferroni's post hoc test (F(1, 13) = 10.97). Data are presented as mean ± SEM.


( G ) Marble burying . n = 9 for WT mice , n = 25 for KO mice . * , p < 0 . 05 , One - way ANOVA with Bonferroni ' s post hoc ( F ( 1 , 32 ) = 5 . 39 ) . Data are presented as mean ± .

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(G) Marble burying test. n = 9 for WT mice, n = 25 for KO mice. *, p<0.05, One-way ANOVA with Bonferroni's post hoc test (F(1, 32) = 5.39). Data are presented as mean ± SEM.


( H ) Forced swimming . n = 10 for WT mice , n = 13 for KO mice . * * , p < 0 . 01 , * * * , p < 0 . 001 Two - way ANOVA followed by Scheffe ' s post - hoc ( main effect of genotype F ( 1 , 42 ) = 84 . 30 , main effect of session F ( 1 , 42 ) = 33 . 88 , interaction F ( 1 , 42 ) = 4 . 92 ) . Data are presented as mean ± .

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(H) Forced swimming test. n = 10 for WT mice, n = 13 for KO mice. **, p<0.01, ***, p<0.001 Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 42)=84.30, main effect of session F(1, 42)=33.88, interaction F(1, 42)=4.92). Data are presented as mean ± SEM.


( I , J ) - . Time in chamber ( I ) Latency to chamber ( J ) . n = 8 for WT mice , n = 7 for KO mice . * , p < 0 . 05 , * * , p < 0 . 01 , One - way ANOVA with Bonferroni ' s post hoc , ( ( I ) F ( 1 , 13 ) = 8 . 75 , ( J ) F ( 1 , 13 ) = 9 . 73 ) . Data are presented as mean ± .

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(I, J) Light-dark test. Time in light chamber (I) and Latency to light chamber (J). n = 8 for WT mice, n = 7 for KO mice. *, p<0.05, **, p<0.01, One-way ANOVA with Bonferroni's post hoc test, ((I) F(1, 13) = 8.75, (J) F(1, 13)=9.73). Data are presented as mean ± SEM.


( K ) Hot plate . . n = 8 for WT mice , n = 12 for KO mice . * * , p < 0 . 01 , One - way ANOVA with Bonferroni ' s post hoc ( F ( 1 , 18 ) = 17 . 51 ) . Data are presented as mean ± .

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(K) Hot plate test.. n = 8 for WT mice, n = 12 for KO mice. **, p<0.01, One-way ANOVA with Bonferroni's post hoc test (F(1, 18) = 17.51). Data are presented as mean ± SEM.


( A ) Co - expressed FLAG - CAMDI HDAC6 - HA interacted with each other in SH - SY5Y . n = 3 independent experiments .

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(A) Co-expressed FLAG-CAMDI and HDAC6-HA interacted with each other in SH-SY5Y cells. n=3 independent experiments.


( B ) Endogenous interaction of CAMDI with HDAC6 in mousebrain . Co - IP was performed with indicated antibodies using E16 mousebrain . n = 3 independent experiments .

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(B) Endogenous interaction of CAMDI with HDAC6 in the mousebrain. Co-IP assay was performed with the indicated antibodies using E16 mousebrain. n=3 independent experiments.


( C , D ) N - terminal region of CAMDI recognizes second HDAC domain of HDAC6 . SH - SY5Y were co - transfected with HDAC6 - HA each FLAG - CAMDI fragment ( C ) ( fragment 1 , 1 - 250 ; 2 , 251 - 500 ; 3 , 501 - 750 ; 4 , 751 - 1000 ; 5 , 1001 - 1451 ) or co - transfected with FLAG - CAMDI each HDAC6 - HA fragment ( D ) ( fragment 1 ( DD1 ) , 1 - 440 ; 2 ( DD2 ) , 441 - 840 ; 3 , 841 - 1215 ) . Co - IP was performed with indicated antibodies . n = 3 independent experiments . * * , p < 0 . 01 , * * * , p < 0 . 001 , One - way ANOVA with Bonferroni ' s post hoc . Data are presented as mean ± .

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

(C, D) N-terminal region of CAMDI recognizes the second HDAC domain of HDAC6. SH-SY5Y cells were co-transfected with HDAC6-HA and each FLAG-CAMDI fragment (C) (fragment 1, 1-250; 2, 251-500; 3, 501-750; 4, 751-1000; 5, 1001-1451 amino acids) or co-transfected with FLAG-CAMDI and each HDAC6-HA fragment (D) (fragment 1 (DD1), 1-440; 2 (DD2), 441-840; 3, 841-1215 amino acids). Co-IP assay was performed with the indicated antibodies. n=3 independent experiments. **, p<0.01, ***, p<0.001, One-way ANOVA with Bonferroni's post hoc test. Data are presented as mean ± SEM.


( A ) CAMDI CAMDI Fragment 1 overexpression enhanced α - tubulin acetylation . HeLa overexpressing FLAG - CAMDI FLAG - CAMDI frag . 1 were immunostained with anti - FLAG or anti - Ac - tubulin antibodies . - scans along ( from x - to y - axis ) from . Scale bar , 10 μm .

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(A) CAMDI and CAMDI Fragment 1 overexpression enhanced α-tubulin acetylation. HeLa cells overexpressing FLAG-CAMDI and FLAG-CAMDI frag.1 were immunostained with anti-FLAG or anti-Ac-tubulin antibodies. Ratio line-scans along the line (from x- to y-axis) from images. Scale bar, 10 μm.


( B - C ) CAMDI blocked HDAC6 - mediated α - tubulin deacetylation . ( B ) Centrosomal fraction was isolated from lysates of SH - SY5Y expressing each construct , Ac - tubulin level was assessed by immunoblot . n = 3 independent experiments . * * , p < 0 . 01 , * * * , p < 0 . 001 , Two - way ANOVA followed by Scheffe ' s post - hoc . Data are presented as mean ± .

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(B-C) CAMDI blocked HDAC6-mediated α-tubulin deacetylation. (B) Centrosomal fraction was isolated from cell lysates of SH-SY5Y cells expressing each construct, and Ac-tubulin level was assessed by immunoblot analysis. n = 3 independent experiments. **, p<0.01, ***, p<0.001, Two-way ANOVA followed by Scheffe's post-hoc test. Data are presented as mean ± SEM.


( B - C ) CAMDI blocked HDAC6 - mediated α - tubulin deacetylation . ( C ) CAMDI N - terminal domain alone increased Ac - tubulin level . n = 3 independent experiments . * * , p < 0 . 01 , One - way ANOVA with Bonferroni ' s post hoc . Data are presented as mean ± .

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(B-C) CAMDI blocked HDAC6-mediated α-tubulin deacetylation. (C) CAMDI N-terminal domain alone increased Ac-tubulin level. n=3 independent experiments. **, p<0.01, One-way ANOVA with Bonferroni's post hoc test. Data are presented as mean ± SEM.


( D ) Deletion of CAMDI N - terminal domain ( 1 - 250 ) failed to inhibit HDAC6 . n = 3 independent experiments . N . S . , not significant . Two - way ANOVA followed by Scheffe ' s post - hoc . Data are presented as mean ± .

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(D) Deletion of CAMDI N-terminal domain (1-250 amino acids) failed to inhibit HDAC6. n = 3 independent experiments. N.S., not significant. Two-way ANOVA followed by Scheffe's post-hoc test. Data are presented as mean ± SEM.


( A ) Effect of HDAC6 over - expression or CAMDI co - expression on level of acetylated α - tubulin in centrosome . HeLa co - transfected with Centrin2 - EGFP with / without HDAC6 - HA FLAG - CAMDI were immunostained with anti - EGFP ( green ) anti - acetylated α - tubulin antibodies ( red ) . Scale bar , 1 μm . ( B ) of of acetylated α - tubulin at centrosome . n = 25 , 30 , 30 . * , p < 0 . 05 , One - way ANOVA with Bonferroni ' s post hoc . Data are presented as mean ± .

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(A) Effect of HDAC6 over-expression or CAMDI co-expression on the level of acetylated α-tubulin in the centrosome. HeLa cells co-transfected with Centrin2-EGFP with/without HDAC6-HA and FLAG-CAMDI were immunostained with anti-EGFP (green) and anti-acetylated α-tubulin antibodies (red). Scale bar, 1 μm.(B) Quantification of intensity of acetylated α-tubulin at centrosome. n = 25, 30, 30 cells. *, p<0.05, One-way ANOVA with Bonferroni's post hoc test. Data are presented as mean ± SEM.


( C ) Effects of HDAC6 overexpression or CAMDI co - expression on centrosomal localization of γ - tubulin . HeLa co - transfected with Centrin2 - EGFP with / without HDAC6 - HA FLAG - CAMDI were immunostained with anti - EGFP ( green ) anti - γ - tubulin antibodies ( red ) . was stained with Hoechst 33258 . Scale bar , 1 μm . ( D ) of of γ - tubulin at centrosome . n = 39 , 16 , 17 . * , p < 0 . 05 , * * , p < 0 . 01 , One - way ANOVA with Bonferroni ' s post hoc . Data are presented as mean ± .

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(C) Effects of HDAC6 overexpression or CAMDI co-expression on the centrosomal localization of γ-tubulin. HeLa cells co-transfected with Centrin2-EGFP with/without HDAC6-HA and FLAG-CAMDI were immunostained with anti-EGFP (green) and anti-γ-tubulin antibodies (red). DNA was stained with Hoechst 33258. Scale bar, 1 μm.(D) Quantification of intensity of γ-tubulin at centrosome. n = 39, 16, 17 cells. *, p<0.05, **, p<0.01, One-way ANOVA with Bonferroni's post hoc test. Data are presented as mean ± SEM.


( E ) CAMDI stabilizes centrosomal γ - tubulin through HDAC6 inhibition . Centrosomal fraction was isolated from lysates of SH - SY5Y expressing each construct , γ - tubulin level was assessed by immunoblot . n = 3 independent experiments . * * , p < 0 . 01 , One - way ANOVA followed with Bonferroni ' s post hoc . Data are presented as mean ± .

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(E) CAMDI stabilizes centrosomal γ-tubulin through HDAC6 inhibition. Centrosomal fraction was isolated from cell lysates of SH-SY5Y cells expressing each construct, and γ-tubulin level was assessed by immunoblot analysis. n = 3 independent experiments. **, p<0.01, One-way ANOVA followed with Bonferroni's post hoc test. Data are presented as mean ± SEM.


( F ) Effects of CAMDI knockdown on centrosomal localization of γ - tubulin in vivo . Coronal sections through somatosensory cortex of E17 . 5 were analyzed following in utero electroporation of EGFP CAMDI - sh plasmid at E14 . 5 . sections were immunostained with anti - EGFP ( green ) anti - γ - tubulin antibodies ( red ) . Representative of EGFP at Cortical Plateneuron expressing CAMDI - sh stained for endogenous γ - tubulin with Hoechst ( blue ) . Z - stack number 6 indicates centrosome of EGFP - positive CAMDI - sh - expressing neuron . Z - stack number 19 indicates centrosome of a nearby non - electroporated neuron . of γ - tubulin . EGFP ( - ) = 12 , EGFP ( + ) ( CAMDI - sh ) = 12 . * , p < 0 . 05 , One - way ANOVA with Bonferroni ' s post hoc . Data are presented as mean ± . Scale bar , 5 μm .

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

(F) Effects of CAMDI knockdown on the centrosomal localization of γ-tubulin in vivo.Coronal sections through the somatosensory cortex of E17.5 were analyzed following in utero electroporation of EGFP and CAMDI-sh plasmid at E14.5. The sections were immunostained with anti-EGFP (green) and anti-γ-tubulin antibodies (red). Representative images of EGFP at Cortical Plateneuron expressing CAMDI-sh stained for endogenous γ-tubulin and with Hoechst (blue). Z-stack number 6 indicates centrosome of EGFP-positive CAMDI-sh-expressing neuron. Z-stack number 19 indicates centrosome of a nearby non-electroporated neuron. Quantification of γ-tubulin intensity. EGFP (-) = 12 cells, EGFP (+) (CAMDI-sh) = 12 cells. *, p<0.05, One-way ANOVA with Bonferroni's post hoc test. Data are presented as mean ± SEM. Scale bar, 5 μm.


( G ) Reduced γ - tubulin in centrosome fraction of CAMDI - KO brain . centrosomal fraction was isolated from brain lysates of WT CAMDI - KO mice γ - tubulin level was assessed by immunoblot . n = 3 independent experiments . * * , p < 0 . 01 , One - way ANOVA followed with Bonferroni ' s post hoc . Data are presented as mean ± .

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(G) Reduced γ-tubulin in the centrosome fraction of CAMDI-KO brain. The centrosomal fraction was isolated from brain lysates of WT and CAMDI-KO mice and γ-tubulin level was assessed by immunoblot analysis. n = 3 independent experiments. **, p<0.01, One-way ANOVA followed with Bonferroni's post hoc test. Data are presented as mean ± SEM.


( A ) Rescue of a delayed cortical migration in CAMDI - KO mice by Tubastatin A . Immunohistochemical of Cux1 expression in somatosensory cortex at P2 after intraperitoneal injections of Tubastatin A into pregnant CAMDI - KO mice from E12 . 5 - 17 . 5 . Scale bar , 100 μm . ( B ) of number of Cux1 - positive neurons . Note abnormal distribution of neurons in deep cortical layers of CAMDI - KO mice . n = 3 mice / genotype ( WT ( vehicle ) = 3 , 628 , KO ( vehicle ) = 2 , 816 , WT ( Tubastatin A ) = 1 , 847 , KO ( Tubastatin A ) = 2 , 364 ) . * , p < 0 . 05 , * * , p < 0 . 01 , Two - way ANOVA followed by Tukey HSD . Data are presented as mean ± .

O O O O O O O O O O B-GENEPROD O O B-ORGANISM O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD O O B-TISSUE I-TISSUE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O 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) Rescue of a delayed cortical migration in CAMDI-KO mice by Tubastatin A. Immunohistochemical analysis of Cux1 expression in the somatosensory cortex at P2 after intraperitoneal injections of Tubastatin A into pregnant CAMDI-KO mice from E12.5-17.5. Scale bar, 100 μm.(B) Quantification of the number of Cux1-positive neurons. Note the abnormal distribution of neurons in deep cortical layers of CAMDI-KO mice. n = 3 mice/genotype (WT (vehicle) = 3,628 cells, KO (vehicle) = 2,816 cells, WT (Tubastatin A) = 1,847 cells, KO (Tubastatin A) = 2,364 cells). *, p<0.05, **, p<0.01, Two-way ANOVA followed by Tukey HSD test. Data are presented as mean ± SEM.


( C ) Enhanced α - tubulin acetylation at P0 by Tubastatin A . Ac - tubulin level was assessed by immunoblot . ( D ) of α - tubulin acetylation . n = 3 independent experiments . * , p < 0 . 05 , Two - way ANOVA followed by Scheffe ' s post - hoc . Data are presented as mean ± .

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(C) Enhanced α-tubulin acetylation at P0 by Tubastatin A. Ac-tubulin level was assessed by immunoblot analysis.(D) Quantification of the α-tubulin acetylation. n=3 independent experiments. *, p<0.05, Two-way ANOVA followed by Scheffe's post-hoc test. Data are presented as mean ± SEM.


( A ) Representative traces from control ( WT ) CAMDI - KO ( KO ) mice in open field at P21 . Sample traces of locomotor activity were shown . ( B ) traveled for 10 min . n = 13 for WT ( vehicle ) mice , n = 13 for KO ( vehicle ) mice , n = 5 for WT ( Tubastatin A ) mice , n = 16 for KO ( Tubastatin A ) mice . * * , p < 0 . 01 , * * * , p < 0 . 001 . Two - way ANOVA followed by Scheffe ' s post - hoc ( main effect of genotype F ( 1 , 43 ) = 6 . 25 , main effect of drug F ( 1 , 43 ) = 2 . 14 , interaction F ( 1 , 43 ) = 12 . 68 ) . Data are presented as mean ± .

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(A) Representative traces from control (WT) and CAMDI-KO (KO) mice in the open field test at P21. Sample traces of locomotor activity were shown.(B) Distance traveled for 10 min. n = 13 for WT (vehicle) mice, n = 13 for KO (vehicle) mice, n = 5 for WT (Tubastatin A) mice, n = 16 for KO (Tubastatin A) mice. **, p<0.01, ***, p<0.001. Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 43)=6.25, main effect of drug F(1, 43)=2.14, interaction F(1, 43)=12.68). Data are presented as mean ± SEM.


( C ) Repetitive jumping behavior . n = 13 for WT ( vehicle ) mice , n = 27 for KO ( vehicle ) mice , n = 5 for WT ( Tubastatin A ) mice , n = 18 for KO ( Tubastatin A ) mice . * * , p < 0 . 01 , * * * , p < 0 . 001 . Two - way ANOVA followed by Scheffe ' s post - hoc ( main effect of genotype F ( 1 , 59 ) = 5 . 04 , main effect of drug F ( 1 , 59 ) = 10 . 13 , interaction F ( 1 , 59 ) = 4 . 98 ) . Data are presented as mean ± .

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

(C) Repetitive jumping behavior. n = 13 for WT (vehicle) mice, n = 27 for KO (vehicle) mice, n = 5 for WT (Tubastatin A) mice, n = 18 for KO (Tubastatin A) mice. **, p<0.01, ***, p<0.001. Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 59)=5.04, main effect of drug F(1, 59)=10.13, interaction F(1, 59)=4.98). Data are presented as mean ± SEM.


( D ) Grooming time in small open field . n = 13 for WT ( vehicle ) mice , n = 13 for KO ( vehicle ) mice , n = 5 for WT ( Tubastatin A ) mice , n = 16 for KO ( Tubastatin A ) mice . * * , p < 0 . 01 , * * * , p < 0 . 001 . Two - way ANOVA followed by Scheffe ' s post - hoc ( main effect of genotype F ( 1 , 43 ) = 11 . 18 , main effect of drug F ( 1 , 43 ) = 7 . 23 , interaction F ( 1 , 43 ) = 9 . 34 ) . Data are presented as mean ± .

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(D) Grooming time in small open field test. n = 13 for WT (vehicle) mice, n = 13 for KO (vehicle) mice, n = 5 for WT (Tubastatin A) mice, n = 16 for KO (Tubastatin A) mice. **, p<0.01, ***, p<0.001. Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 43)=11.18, main effect of drug F(1, 43)=7.23, interaction F(1, 43)=9.34). Data are presented as mean ± SEM.


( E ) traveled for 15 min . n = 10 for adult WT ( Vehicle ) mice , n = 5 for KO ( Vehicle ) mice , n = 6 for WT ( Tubastatin A ) mice , n = 11 for KO ( Tubastatin A ) mice . * * , p < 0 . 01 , Two - way ANOVA followed by Scheffe ' s post - hoc ( main effect of genotype F ( 1 , 28 ) = 1 . 73 , main effect of drug F ( 1 , 28 ) = 3 . 96 , interaction F ( 1 , 28 ) = 11 . 80 ) . Data are presented as mean ± .

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(E) Distance traveled for 15 min. n = 10 for adult WT (Vehicle) mice, n = 5 for KO (Vehicle) mice, n = 6 for WT (Tubastatin A) mice, n = 11 for KO (Tubastatin A) mice. **, p<0.01, Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 28)=1.73, main effect of drug F(1, 28)=3.96, interaction F(1, 28)=11.80). Data are presented as mean ± SEM.


( F ) - . Time in latency to chamber ( latency ) . n = 10 for adult WT ( Vehicle ) mice , n = 5 for KO ( Vehicle ) mice , n = 6 for WT ( Tubastatin A ) mice , n = 11 for KO ( Tubastatin A ) mice . * , p < 0 . 05 , Two - way ANOVA followed by Scheffe ' s post - hoc ( main effect of genotype F ( 1 , 28 ) = 2 . 34 , main effect of drug F ( 1 , 28 ) = 3 . 82 , interaction F ( 1 , 28 ) = 10 . 64 ) . Data are presented as mean ± .

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

(F) Light-dark test. Time in latency to the light chamber (latency). n = 10 for adult WT (Vehicle) mice, n = 5 for KO (Vehicle) mice, n = 6 for WT (Tubastatin A) mice, n = 11 for KO (Tubastatin A) mice. *, p<0.05, Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 28)=2.34, main effect of drug F(1, 28)=3.82, interaction F(1, 28)=10.64). Data are presented as mean ± SEM.


Detection of native GFP - fused TgAtg8 . extracts corresponding to 107 tachyzoites from parental RHΔHX transgenic GFP - TgAtg8 lines were separated by SDS - PAGE analysed by Western blot using anti - TgAtg8 or anti - GFP antibodies . Overexpressed GFP - TgAtg8 native TgAtg8 are indicated by arrows .

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Detection of native and GFP-fused TgAtg8.Protein extracts corresponding to 107 tachyzoites from parental RHΔHX and transgenic GFP-TgAtg8 cell lines were separated by SDS-PAGE and analysed by Western blot using anti-TgAtg8 or anti-GFP antibodies. Overexpressed GFP-TgAtg8 and native TgAtg8 are indicated by arrows.


A . Autophagic vesicles marker GFP - TgAtg8 was found in cytosol of tachyzoites occasionally in punctate vesicles in recently egressed .

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A. Autophagic vesicles marker GFP-TgAtg8 was found in the cytosol of tachyzoites and occasionally in punctate vesicles in recently egressed parasites.


B . Extracellular tachyzoites were put to starve in HBSS medium for increased time periods proportions of displaying punctate or GFP - TgAtg8 signals were assessed . Data are mean from n = 4 independent experiments ± .

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B. Extracellular tachyzoites were put to starve in HBSS medium for increased time periods and the proportions of cells displaying punctate or cytosolic GFP-TgAtg8 signals were assessed. Data are mean from n = 4 independent experiments ±SEM.


A . Extracellular tachyzoites were starved for 8 hours in HBSS medium observed ( 2 , 3 ) . Unstarved control is shown for comparison ( 1 ) . Several vacuoles ( V ) can be seen in starved , with also cytosol - organelle - sequestrating membranous structures resembling autophagosomes ( Ap ) , as well as vacuoles containing partially digested material corresponding to autophagic degradative vacuoles ( Av ) . 2 ′ , 3 ′ 3 " pictures are a magnification of squared regions . Scale bar = 1 µm in initial ( 1 , 2 , 3 ) 0 . 2 µm in magnifications .

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A. Extracellular tachyzoites were starved for 8 hours in HBSS medium and observed (2, 3). Unstarved control is shown for comparison (1). Several vacuoles (V) can be seen in starved parasites, with also cytosol- and organelle-sequestrating membranous structures resembling autophagosomes (Ap), as well as vacuoles containing partially digested material corresponding to autophagic degradative vacuoles (Av). 2′, 3′ and 3" pictures are a magnification of squared regions. Scale bar = 1 µm in initial micrographs (1, 2, 3) and 0.2 µm in magnifications.


B . Partial co - localisation of GFP - TgAtg8 - labeled autophagosomes Toxoplasma mitochondrion . Extracellular tachyzoites expressing GFP - TgAtg8 were incubated in complete medium with Mitotracker Red to label mitochondrion ; partial co - localisation could be observed in some displaying autophagic vesicles .

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B. Partial co-localisation of GFP-TgAtg8-labeled autophagosomes and Toxoplasma mitochondrion. Extracellular tachyzoites expressing GFP-TgAtg8 were incubated in complete medium with Mitotracker Red to label the mitochondrion; partial co-localisation could be observed in some parasites displaying autophagic vesicles.


C . Identification of autophagic vesicles in extracellular tachyzoites by immuno - electron microscopy . Extracellular tachyzoites expressing GFP - TgAtg8 were starved for 8 hours in HBSS medium , fixed , cryosectioned stained with an anti - GFP antibody , then with gold - conjugated secondary antibody observed . Gold particles can be seen around cytosol - sequestrating membranous structures resembling autophagosomes ( Ap ) , as well as larger vacuoles containing partially digested material corresponding to autophagic degradative vacuoles ( Av ) . Scale bar = 200 nm .

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C. Identification of autophagic vesicles in extracellular tachyzoites by immuno-electron microscopy. Extracellular tachyzoites expressing GFP-TgAtg8 were starved for 8 hours in HBSS medium, fixed, cryosectioned and stained with an anti-GFP antibody, then with gold-conjugated secondary antibody and observed. Gold particles can be seen around cytosol-sequestrating membranous structures resembling autophagosomes (Ap), as well as larger vacuoles containing partially digested material corresponding to autophagic degradative vacuoles (Av). Scale bar = 200 nm.


A . extracts corresponding to tachyzoites incubated in same starvation conditions as in Figure 2B were separated by urea SDS - PAGE analysed with an anti - GFP antibody to detect TgAtg8 lipidated TgAtg8 - PE form . GFP - TgAtg8 extracts were analysed with anti GFP antibody ( left ) , while parental was analysed with anti - TgAtg8 antibody ( right ) . Anti - ROP5 was used as a loading control .

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A. Protein extracts corresponding to tachyzoites incubated in the same starvation conditions as in Figure 2B were separated by urea SDS-PAGE and analysed with an anti-GFP antibody to detect TgAtg8 and the lipidated TgAtg8-PE form. GFP-TgAtg8 parasites extracts were analysed with anti GFP antibody (left), while parental cell line was analysed with anti-TgAtg8 antibody (right). Anti-ROP5 was used as a loading control.


B . lysates from GFP - TgAtg8 were subjected to a centrifugation at 100 , 000 g to separate soluble fraction ( high speed supernatant , HSS ) from fraction ( high speed pellet , HSP ) . faster migrating form of GFP - TgAtg8 is exclusively present in fraction as revealed by Western blot after urea SDS - PAGE using anti -

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B. Cell lysates from GFP-TgAtg8 parasites were subjected to a centrifugation at 100,000 g to separate the soluble fraction (high speed supernatant, HSS) from the membrane fraction (high speed pellet, HSP). The faster migrating form of GFP-TgAtg8 is exclusively present in the membrane fraction as revealed by Western blot analysis after urea SDS-PAGE using anti-GFP


C . HSP fraction described in B . was extracted with various agents new HSS HSP fractions were separated analysed by Western blot after urea SDS - PAGE . Only a treatment with a detergent ( DOC ) resulted in GFP - TgAtg8 solubilisation from HSP fraction .

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C. The HSP fraction described in B. was extracted with various agents and new HSS and HSP fractions were separated and analysed by Western blot after urea SDS-PAGE. Only a treatment with a detergent (DOC) resulted in GFP-TgAtg8 solubilisation from the HSP fraction.


D . GFP - TgAtg8 - expressing were grown in host in presence of 3H - ethanolamine then starved in HBSS for 8 hours , still in presence of radioactive PE precursor . Tachyzoites were then lysed GFP - TgAtg8 was immunoprecipitated using anti - TgAtg8 antibody analysed by urea SDS - PAGE . immunoprecipitated forms of GFP - TgAtg8 were detected by Western blot using anti - GFP antibody radioactive ethanolamine was found to be incorporated into immunoprecipitated GFP - TgAtg8 , as detected by fluorography ( 3H - etn ) .

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D. GFP-TgAtg8-expressing parasites were grown in host cells in the presence of 3H-ethanolamine and then starved in HBSS for 8 hours, still in the presence of the radioactive PE precursor. Tachyzoites were then lysed and GFP-TgAtg8 was immunoprecipitated using anti-TgAtg8 antibody and analysed by urea SDS-PAGE. The immunoprecipitated forms of GFP-TgAtg8 were detected by Western blot using anti-GFP antibody and radioactive ethanolamine was found to be incorporated into immunoprecipitated GFP-TgAtg8, as detected by fluorography (3H-etn).


A . microscopy of extracellular tachyzoites expressing GFP - TgAtg8 or its glycine mutant version , before or after induction of autophagy for 8 hours in HBSS . Autophagosomes are labelled by GFP - TgAtg8 in control ( arrowheads ) , but not by glycine mutant version of . B . Extracellular tachyzoites expressing GFP - TgAtg8 or its glycine mutant version were put to starve in HBSS medium for increased lengths of time proportions of displaying punctate or GFP signals were assessed . Data are mean from n = 4 independent experiments ± .

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A. Fluorescence microscopy analysis of extracellular tachyzoites expressing GFP-TgAtg8 or its glycine mutant version, before or after induction of autophagy for 8 hours in HBSS. Autophagosomes are labelled by GFP-TgAtg8 in control parasites (arrowheads), but not by the glycine mutant version of the protein. B. Extracellular tachyzoites expressing GFP-TgAtg8 or its glycine mutant version were put to starve in HBSS medium for increased lengths of time and the proportions of cells displaying punctate or cytosolic GFP signals were assessed. Data are mean from n = 4 independent experiments ±SEM.


C . extracts corresponding to tachyzoites expressing GFP - TgAtg8 glycine mutant version incubated in HBSS for up to 8 hours , were separated by urea SDS - PAGE analysed with an anti - GFP antibody to detect GFP - TgAtg8 lipidated GFP - TgAtg8 - PE forms . Anti - ROP5 was used as a loading control .

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C. Protein extracts corresponding to tachyzoites expressing GFP-TgAtg8 and glycine mutant version incubated in HBSS for up to 8 hours, were separated by urea SDS-PAGE and analysed with an anti-GFP antibody to detect GFP-TgAtg8 and lipidated GFP-TgAtg8-PE forms. Anti-ROP5 was used as a loading control.


A . Intracellular tachyzoites expressing GFP - TgAtg8 were used to invade host HFF proportions of displaying punctate or GFP signals were assessed along time during development . Data are mean from n = 5 independent experiments ± .

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A. Intracellular tachyzoites expressing GFP-TgAtg8 were used to invade host HFF and the proportions of cells displaying punctate or cytosolic GFP signals were assessed along time during development. Data are mean from n = 5 independent experiments ±SEM.


B . microscopy pictures of GFP - TgAtg8 - expressing tachyzoites during their intracellular development division . arrowhead indicates residual made of maternal material left after endodyogeny .

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B. Fluorescence microscopy pictures of GFP-TgAtg8-expressing tachyzoites during their intracellular development and division. The arrowhead indicates the residual body made of maternal material left after endodyogeny.


C . Detection of GFP - TgAtg8 - labelled autophagosomes in dividing . Progression of daughter division was followed by expression of an IMC1 - RFP construct .

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C. Detection of GFP-TgAtg8-labelled autophagosomes in dividing parasites. Progression of daughter cells division was followed by expression of an IMC1-RFP construct.


A . Schematic representation of strategy . Recombineered cosmid bearing chloramphenicol acetyl transferase ( CAT ) gene for selection of resistant , flanked by homologous regions from TgAtg3 , was used to replace endogenous genomic locus in expressing a regulatable extra copy of TgAtg3 ( imyc - sTgAtg3 ) . Mutants obtained after double homologous recombination events were selected with chloramphenicol . PCR amplifications Southern blot strategy used for subsequent characterisation of mutant are indicated in red green , respectively . B . PCR verification of removal of TgAtg3 in mutant ( imyc - sTgAtg3 - ΔAtg3 , A ) compared to parental ( imyc - sTgAtg3 , B )

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A. Schematic representation of the strategy. Recombineered cosmid bearing chloramphenicol acetyl transferase (CAT) gene for selection of resistant parasites, flanked by homologous regions from TgAtg3, was used to replace endogenous genomic locus in the cell line expressing a regulatable extra copy of TgAtg3 (imyc-sTgAtg3). Mutants obtained after double homologous recombination events were selected with chloramphenicol. PCR amplifications and Southern blot strategy used for subsequent characterisation of the mutant are indicated in red and green, respectively. B. PCR verification of the removal of TgAtg3 in the mutant (imyc-sTgAtg3-ΔAtg3, A) compared to the parental cell line (imyc-sTgAtg3, B)


C . Southern blot verification of TgAtg3 gene replacement . ∼ 4 µg of genomic from mutant ( A ) parental ( B ) tachyzoites were digested by HindIII / AvrII , transferred on a nylon probed with 32P - labeled 5 ′ 3 ′ probes located as schematised in A .

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C. Southern blot verification of the TgAtg3 gene replacement. ∼4 µg of genomic DNA from mutant (A) and parental (B) tachyzoites were digested by HindIII/AvrII, transferred on a nylon membrane and probed with 32P-labeled 5′ and 3′ DNA probes located as schematised in A.


A . vector for expression of ATc - regulatable sTgAtg3 extra copy allows myc - epitope tagging of this . Anti - myc detection of myc - tagged TgAtg3 in intracellular either without treatment with ATc , or with a continuous 2 days treatment with 1 . 5 µg / ml ATc , or after reinvasion following a continuous 4 days treatment . DIC : differential interference contrast .

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A. The vector for the expression of the ATc-regulatable sTgAtg3 extra copy allows myc-epitope tagging of this protein. Anti-myc detection of myc-tagged TgAtg3 protein in intracellular parasites either without treatment with ATc, or with a continuous 2 days treatment with 1.5 µg/ml ATc, or after reinvasion following a continuous 4 days treatment. DIC: differential interference contrast.


B . Western blot detection of myc - tagged sTgAtg3 ( arrowed ) in lysates corresponding to induction conditions described in A . Anti - SAG1 antibody was used as a loading control .

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B. Western blot detection of the myc-tagged sTgAtg3 protein (arrowed) in parasite lysates corresponding to the induction conditions described in A. Anti-SAG1 antibody was used as a loading control.


C . Western blot following urea SDS - PAGE of TgAtg3 - depleted extracts , showing an absence of upregulation of autophasome - bound TgAtg8 - PE form . Anti - ROP5 was used as a loading control .

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C. Western blot analysis following urea SDS-PAGE of TgAtg3-depleted parasites extracts, showing an absence of upregulation of the autophasome-bound TgAtg8-PE form. Anti-ROP5 was used as a loading control.


A . Confluent monolayers of fibroblasts were infected either with extra copy - expressing imyc - sTgAtg3 parental in presence of ATc ( a ) , imyc - sTgAtg3 - ΔAtg3 mutant without ATc ( b ) , mutant in presence of ATc ( c ) , or mutant pre - incubated with ATc before start of experiment maintained in presence of ATc . Plaques ( arrowheads ) resulting from lysis of host due to multiplication of are only visible when TgAtg3 is still expressed . B . Mean plaque comparisons between fibroblast layers infected with controls TgAtg3 - depleted ( a , b , c : legend as in A ) . Plaques observed with mutant in presence of ATc were significantly smaller than with control lines ( * p < 0 . 005 , Student ' s T ) . Data are mean from n = 3 independent experiments ± .

O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Confluent monolayers of fibroblasts were infected either with extra copy-expressing imyc-sTgAtg3 parental cell line in the presence of ATc (a), imyc-sTgAtg3-ΔAtg3 mutant cell line without ATc (b), mutant cell line in the presence of ATc (c), or mutant cell line pre-incubated with ATc before start of the experiment and maintained in the presence of ATc. Plaques (arrowheads) resulting from the lysis of host cells due to the multiplication of the parasites are only visible when TgAtg3 is still expressed. B. Mean plaque area comparisons between fibroblast layers infected with controls and TgAtg3-depleted parasites (a, b, c: legend as in A). Plaques observed with the mutant in the presence of ATc were significantly smaller than with control cell lines (* p<0.005, Student's T test). Data are mean from n = 3 independent experiments ±SEM.


C . Numbers of per vacuole are significantly lower in TgAtg3 - depleted ( pre - incubated for 4 days with ATc ) compared with controls 24 or 48 hours post invasion . Data are mean from n = 3 independent experiments ± .

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C. Numbers of parasites per vacuole are significantly lower in TgAtg3-depleted cell line (pre-incubated for 4 days with ATc) compared with controls 24 or 48 hours post invasion. Data are mean from n = 3 independent experiments ±SEM.


TgAtg3 - depleted show a defect in mitochondrion . Mitochondrion labelled with specific antibodies ( see results section ) was found as a reduced structure , or even absent , in TgAtg3 - depleted lines ( middle series of , kept for 2 days in presence of ATc or pre - incubated with ATc for 4 days before invasion , respectively ) . left to develop for two days in presence of ATc to progressively extinguish expression of TgAtg3 showed an accumulation of mitochondrial marker at residual . TgAtg3 depletion was verified by detecting myc - tagged regulatable extra - copy with specific antibody . TgAtg3 - expressing imyc - sTgAtg3cultivated in presence of ATc for 4 days was used as a control for mitochondrial ( bottom ) . was labelled with DAPI .

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

TgAtg3-depleted parasites show a defect in mitochondrion morphology.Mitochondrion labelled with specific antibodies (see results section) was found as a reduced structure, or even absent, in TgAtg3-depleted cell lines (middle series of micrographs, kept for 2 days in the presence of ATc or pre-incubated with ATc for 4 days before invasion, respectively). Parasites left to develop for two days in the presence of ATc to progressively extinguish the expression of TgAtg3 showed an accumulation of mitochondrial marker at the residual body. TgAtg3 depletion was verified by detecting myc-tagged regulatable extra-copy with specific antibody. TgAtg3-expressing cell line imyc-sTgAtg3cultivated in the presence of ATc for 4 days was used as a control for mitochondrial morphology (bottom). DNA was labelled with DAPI.


Altered mitochondrial ultrastructure in TgAtg3 - depleted tachyzoites observed by electron microscopy . Ultrathin section of a TgAtg3 - depleted ( 3 days of ATc treatment ) tachyzoite showing dramatic alteration of mitochondrion , where cristae remnants are still visible ( arrows ) , but which is filled up with membranous profiles ( arrowheads ) , whereas Golgi ( G ) , apicoplast ( A ) , rhoptries ( R ) dense granule ( D ) look normal .

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

Altered mitochondrial ultrastructure in TgAtg3-depleted tachyzoites observed by electron microscopy.Ultrathin section of a TgAtg3-depleted (3 days of ATc treatment) tachyzoite showing the dramatic alteration of the mitochondrion, where cristae remnants are still visible (arrows), but which is filled up with membranous profiles (arrowheads), whereas Golgi (G), apicoplast (A), rhoptries (R) and dense granule (D) look normal.


Relative fold repression for let - 7a reporter in RAW 264 . 7 during LPS treatment ( mean + / - s . e . m . , n = 3 ) ( B ) . schematic representation of constructs used for luciferase assays are shown .

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

Relative fold repression for let-7a reporter in RAW 264.7 cells during LPS treatment (mean+/- s.e.m., n=3) (B). The schematic representation of the constructs used for luciferase assays are shown.


Relative of pro - inflammatory cytokines TNF - ɑ IL - 6 mRNA , a target of let - 7a miRNA , have been plotted against time of LPS stimulation , 18S rRNA was used for normalization ( mean + / - s . e . m . , n = 2 ( IL - 6 ) , n = 3 ( TNF - α ) ) ( C ) .

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

Relative levels of pro-inflammatory cytokines TNF-ɑ and IL-6 mRNA, a target of let-7a miRNA, have been plotted against time of LPS stimulation, 18S rRNA was used for normalization (mean+/- s.e.m., n=2 (IL-6), n=3 (TNF-α)) (C).


Time dependent Ago2 phosphorylation its miRNA binding in LPS treated macrophage . RAW 264 . 7 were transfected with FH - Ago2 expression plasmid treated with LPS ( 1ng / ml ) for different time points . Ago2 was pulled down using anti - FLAG beads of Ago - associated let - 7a miRNA were measured by qRT - PCR . Quantity of immunoprecipitated Ago2 was detected in western blot with anti - HA antibody used for normalization of amounts of miRNA detected by qRT - PCR . of let - 7a were measured by Northern blot U6 was used as loading control . Relative of Ago2 bound let - 7a were plotted ( mean + / - s . e . m . , n = 3 ) ( D ) .

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

Time dependent Ago2 phosphorylation and its miRNA binding in LPS treated macrophage cells. RAW 264.7 cells were transfected with FH-Ago2 expression plasmid and treated with LPS (1ng/ml) for different time points. Ago2 was pulled down using anti-FLAG beads and the levels of Ago-associated let-7a miRNA were measured by qRT-PCR. Quantity of immunoprecipitated Ago2 was detected in western blot analysis with anti-HA antibody and used for normalization of amounts of miRNA detected by qRT-PCR. Levels of cellular let-7a were measured by Northern blot and U6 RNA was used as loading control. Relative levels of cellular and Ago2 bound let-7a were plotted (mean+/- s.e.m., n=3) (D).


Time dependent Ago2 phosphorylation its miRNA binding in LPS treated macrophage . RAW 264 . 7 were transfected with FH - Ago2 expression plasmid treated with LPS ( 1ng / ml ) for different time points . Phosphorylated Ago2 ( pY - Ago2 ) level was measured using 4G10 antibody specific for phosphorylated tyrosine . Phosphorylated Ago2 ( pY - Ago2 ) band intensities were normalized against total Ago2 detected with HA specific antibody that was also used for Ago2 pull down ( E , upper panel ) . Relative 4G10 intensities were plotted from three independent experiments ( mean + / - s . e . m ) ( E , lower panel ) .

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

Time dependent Ago2 phosphorylation and its miRNA binding in LPS treated macrophage cells. RAW 264.7 cells were transfected with FH-Ago2 expression plasmid and treated with LPS (1ng/ml) for different time points. Phosphorylated Ago2 (pY-Ago2) level was measured using 4G10 antibody specific for phosphorylated tyrosine. Phosphorylated Ago2 (pY-Ago2) band intensities were normalized against total Ago2 detected with HA specific antibody that was also used for Ago2 pull down (E, upper panel). Relative 4G10 intensities were plotted from three independent experiments (mean+/- s.e.m) (E, lower panel).


Time dependent Ago2 phosphorylation its miRNA binding in LPS treated macrophage . RAW 264 . 7 were transfected with FH - Ago2 expression plasmid treated with LPS ( 1ng / ml ) for different time points . ( PEC ) were isolated from BALB / c mice endogenous Ago2 was immunoprecipitated with anti - Ago2 ( eIF2C2 ) antibody phospho tyrosine level of Ago2 was measured using 4G10 antibody . Relative intensities of phospho - Ago2 against total amount of immunoprecipitated Ago2 were quantified mentioned below lanes ( mean + / - s . e . m . , n = 3 ) ( F , upper panel ) . Similar experiments were done with FH - Ago1 Ago3 of Phosphotyrosine in immunoprecipitated FH - Ago1 FH - Ago3 were measured in RAW 264 . 7 ( F , lower panel ) .

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

Time dependent Ago2 phosphorylation and its miRNA binding in LPS treated macrophage cells. RAW 264.7 cells were transfected with FH-Ago2 expression plasmid and treated with LPS (1ng/ml) for different time points. Primary cells (PEC) were isolated from BALB/c mice and endogenous Ago2 was immunoprecipitated with anti-Ago2 (eIF2C2) antibody and phospho tyrosine level of Ago2 was measured using 4G10 antibody. Relative intensities of phospho-Ago2 against total amount of immunoprecipitated Ago2 were quantified and mentioned below the lanes (mean+/- s.e.m., n=3) (F, upper panel). Similar experiments were done with FH-Ago1 and Ago3 and levels of Phosphotyrosine in immunoprecipitated FH-Ago1 and FH-Ago3 were measured in RAW 264.7 cells (F, lower panel).


Effect of knockdown of different phosphatases on miRNA - mediated repression in 24hrs LPS treated RAW 264 . 7 . Knockdown of PP2A , MKP1 , MKP3 or SHP1 / 2 was checked by western blot done with lysates of treated with respective siRNAs . β - Actin was used as loading control ( G ) .

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

Effect of knockdown of different phosphatases on miRNA-mediated repression in 24hrs LPS treated RAW 264.7 cells. Knockdown of PP2A, MKP1, MKP3 or SHP1/2 was checked by western blot analysis done with lysates of cells treated with respective siRNAs. β-Actin was used as loading control (G).


Effect of knockdown of different phosphatases on miRNA - mediated repression in 24hrs LPS treated RAW 264 . 7 . Luciferase let - 7a miRNA reporter was done in LPS - treated downregulated for specific phosphatases . at 0 hrs time point were taken as units ( mean + / - s . e . m . , n = 4 ) ( H ) .

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

Effect of knockdown of different phosphatases on miRNA-mediated repression in 24hrs LPS treated RAW 264.7 cells. Luciferase based let-7a miRNA reporter assay was done in LPS-treated cells downregulated for specific phosphatases. Levels at 0 hrs time point were taken as units (mean +/- s.e.m., n=4) (H).


Effect of knockdown of different phosphatases on miRNA - association of Ago2 in 24hrs LPS treated RAW 264 . 7 . Ago2 associated let - 7a was estimated by qRT - PCR in LPS treated depleted for specific phosphatases . let - 7a miRNA level was normalized against respective FH - Ago2 bands ( mean + / - s . e . m . , n = 3 ) ( I ) .

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

Effect of knockdown of different phosphatases on miRNA-association of Ago2 in 24hrs LPS treated RAW 264.7 cells. Ago2 associated let-7a was estimated by qRT-PCR in LPS treated cells depleted for specific phosphatases. let-7a miRNA level was normalized against respective FH-Ago2 bands (mean+/- s.e.m., n=3) (I).


Effect of PP2A knock - down on Ago2 phosphorylation its miRNA association . Phospho Ago2 were measured in siCon siPP2A transfected upon 24hrs of LPS treatment using Phospho Tyrosine specific 4G10 antibody . HA - Ago2 was detected with anti - HA antibody ( J ) . Immunoprecipitated Ago2 content was used for normalization for both miRNA mRNA in above mentioned experiments .

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

Effect of PP2A knock-down on Ago2 phosphorylation and its miRNA association. Phospho Ago2 levels were measured in siCon and siPP2A transfected cells upon 24hrs of LPS treatment using Phospho Tyrosine specific 4G10 antibody. HA-Ago2 was detected with anti-HA antibody (J). Immunoprecipitated Ago2 content was used for normalization for both miRNA and mRNA levels in above mentioned experiments.


Effect of PP2A knock - down on Ago2 phosphorylation its miRNA association . miRNA ( K , left panel mean + / - s . e . m . , n = 3 ) Ago2 associated miRNAs content in treated with siCon or siPP2A were measured using qRT - PCR plotted ( K , right panel , mean + / - s . e . m . , n = 3 ) .

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Effect of PP2A knock-down on Ago2 phosphorylation and its miRNA association. Cellular miRNA levels (K, left panel mean +/- s.e.m., n=3) and Ago2 associated miRNAs content in cells treated with siCon or siPP2A were measured using qRT-PCR and plotted (K, right panel, mean+/- s.e.m., n=3).


Effect of PP2A knock - down on Ago2 phosphorylation its miRNA association . Ago2 associated IL - 1β mRNA level was estimated in siCon siPP2A treated which were stimulated with LPS for 24hrs ( mean + / - s . e . m . , n = 3 ) ( L ) .

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

Effect of PP2A knock-down on Ago2 phosphorylation and its miRNA association. Ago2 associated IL-1β mRNA level was estimated in siCon and siPP2A treated cells which were stimulated with LPS for 24hrs (mean+/- s.e.m., n=3) (L).


Effect of Okadaic acid ( OA ; 100nM ) , PP2A inhibitor Expression of PP2A was detected in western blot done for extract from control OA treated RAW 264 . 7 after LPS stimulation ( A ) .

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Effect of Okadaic acid (OA; 100nM), the PP2A inhibitor Expression of PP2A was detected in western blot done for cell extract from control and OA treated RAW 264.7 cells after LPS stimulation (A).


Effect of Okadaic acid ( OA ; 100nM ) , PP2A inhibitor , on Ago2 phosphorylation amount of phosphorylated Ago2 was measured in OA treated before after LPS stimulation . amount of Tyr phosphorylated Ago2 was measured by densitometric of western blot data upon normalization against immunoprecipitated Ago2 amount ( B ) .

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

Effect of Okadaic acid (OA; 100nM), the PP2A inhibitor, on Ago2 phosphorylation The amount of phosphorylated Ago2 was measured in OA treated cells before and after LPS stimulation. The amount of Tyr phosphorylated Ago2 was measured by densitometric quantification of western blot data upon normalization against immunoprecipitated Ago2 amount (B).


Effect of Okadaic acid ( OA ; 100nM ) , PP2A inhibitor , on miRNA activity . Change in fold repression for a let - 7a reporter upon LPS exposure in control OA treated ( mean + / - s . e . m . , n = 3 ) ( C ) .

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

Effect of Okadaic acid (OA; 100nM), the PP2A inhibitor, on miRNA activity. Change in fold repression for a let-7a reporter upon LPS exposure in control and OA treated cells (mean+/- s.e.m., n=3) (C).


Schematic representation of in vitro phosphatase ( upper panel ) . PP2A was immunoprecipitated from 24hrs LPS treated RAW 264 . 7 were incubated in vitro with wild type or phosphorylation defective FH - Ago2 mutant ( Ago2Y529F ) isolated from HEK293 transfected with respective expression constructs . Phosphorylated Ago2 ( pYAgo2 ) were measured by western blot using anti - phospho Tyrosine specific 4G10 antibody . Relative intensities were quantified by densitometric mentioned below respective panels . FH - Ago2 was detected by anti - HA antibody ( Lower panel ) .

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

Schematic representation of the in vitro phosphatase assay (upper panel). PP2A was immunoprecipitated from 24hrs LPS treated RAW 264.7 cells and were incubated in vitro with wild type or phosphorylation defective FH-Ago2 mutant (Ago2Y529F) isolated from HEK293 cells transfected with respective expression constructs. Phosphorylated Ago2 (pYAgo2) levels were measured by western blot analysis using anti-phospho Tyrosine specific 4G10 antibody. Relative intensities were quantified by densitometric analysis and mentioned below the respective panels. FH-Ago2 was detected by anti-HA antibody (Lower panel).


Defective de novo miRNP formation in pre - treated with PP2A inhibitor . Ago2 associated ( F ) total ( G ) miR - 122 were measured by qRT - PCR normalized against immuneprecipitated FH - Ago2 content U6 respectively . Values obtained upon 14 hrs of DOX treatment were considered as unit relative values obtained upon 24 hrs of DOX treatment are plotted . samples were treated for 24 hrs with LPS ( mean + / - s . e . m . , n = 4 ) .

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

Defective de novo miRNP formation in cells pre-treated with PP2A inhibitor. Ago2 associated (F) and total (G) miR-122 levels were measured by qRT-PCR and normalized against immuneprecipitated FH-Ago2 content and U6 RNA respectively. Values obtained upon 14 hrs of DOX treatment were considered as unit and relative values obtained upon 24 hrs of DOX treatment are plotted. All samples were treated for 24 hrs with LPS (mean+/- s.e.m., n=4).


PTPA is an essential factor for PP2A mediated dephosphorylation of Ago2 . Expression of PTPA after LPS stimulation in RAW 264 . 7 as well as in PEC isolated from BALB / c mice . β - actin is used as loading control ( H ) .

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

PTPA is an essential factor for PP2A mediated dephosphorylation of Ago2. Expression of PTPA after LPS stimulation in RAW 264.7 cells as well as in PEC isolated from BALB/c mice. β-actin is used as loading control (H).


PTPA is an essential factor for PP2A mediated dephosphorylation of Ago2 . Effect of PTPA depletion on Ago2 phosphorylation level in RAW 264 . 7 ( I ) . Phosphorylated Ago2 level was measured by western blot in HA - immunoprecipitated materials isolated from either siCon or siPTPA transfected expressing FH - Ago2 upon 24 hrs of LPS treatment using a phosphotyrosine specific 4G10 antibody ( I , left panel ) . PTPA was detected with anti PTPA antibody , β - Actin was used as loading control ( I , right panel ) .

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

PTPA is an essential factor for PP2A mediated dephosphorylation of Ago2. Effect of PTPA depletion on Ago2 phosphorylation level in RAW 264.7 cells (I). Phosphorylated Ago2 level was measured by western blot in HA-immunoprecipitated materials isolated from either siCon or siPTPA transfected cells expressing FH-Ago2 upon 24 hrs of LPS treatment using a phosphotyrosine specific 4G10 antibody (I, left panel). The PTPA was detected with anti PTPA antibody, β-Actin was used as loading control (I, right panel).


Effect of PTPA depletion on Ago2 associated miRNAs mRNAs . Ago2 - associated miRNA ( J , left panel ) ( mean + / - s . e . m . , n = 3 ) mRNA ( J , right panel ) ( mean + / - s . e . m . , n = 2 ) were also quantified using qRT - PCR .

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

Effect of PTPA depletion on Ago2 associated miRNAs and mRNAs. Ago2-associated miRNA (J, left panel) (mean+/- s.e.m., n=3) and mRNA levels (J, right panel) (mean+/- s.e.m., n=2) were also quantified using qRT-PCR.


PP2A was immunoprecipitated from siCon or siPTPA treated RAW 264 . 7 treated with LPS for 24 hrs was incubated in vitro with FH - Ago2 isolated from FH - Ago2 stable HEK293 upon reaction , phospho - Ago2 level was measured by western blot using phosphotyrosine specific 4G10 antibody ( K , right panel ) .

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PP2A was immunoprecipitated from siCon or siPTPA treated RAW 264.7 cells treated with LPS for 24 hrs and was incubated in vitro with FH-Ago2 isolated from FH-Ago2 stable HEK293 cells and upon the assay reaction, the phospho-Ago2 level was measured by western blot analysis using phosphotyrosine specific 4G10 antibody (K, right panel).


Expression of different cytokines phosphatases in RAW 264 . 7 PEC infected with Leishmania donovani ( Ld ) . at different time points of infection were harvested of various phosphatases were checked by western blot using antibodies specific for different phosphatases in both RAW 264 . 7 ( B , left panel ) mouse peritoneal macrophage PEC ( B , right panel ) .

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

Expression of different cytokines and phosphatases in RAW 264.7 cells and PEC infected with Leishmania donovani (Ld) parasites. Cells at different time points of infection were harvested and levels of various phosphatases were checked by western blot analysis using antibodies specific for different phosphatases in both RAW 264.7 ( B, left panel) and mouse peritoneal macrophage cells PEC (B, right panel).


Cytokine mRNA like IL - 10 , TNF - α IL - 6 were quantified after 24 hrs of Ld infection in RAW 264 . 7 ( C , upper panel mean + / - s . e . m . , n = 3 ) mouse peritoneal macrophage PEC ( mean + / - s . e . m . , n = 3 ) ( C , Lower panel ) .

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

Cytokine mRNA levels like IL-10, TNF-α and IL-6 were quantified after 24 hrs of Ld infection in RAW 264.7 (C, upper panel mean +/- s.e.m., n=3) and mouse peritoneal macrophage PEC (mean+/- s.e.m., n=3) (C, Lower panel).


Effect of Ld infection on Ago2 phosphorylation its miRNA association . RAW 264 . 7 were transfected with FH - Ago2 expression construct infection was done for various time points followed by FH - Ago2 pull down using anti - FLAG beads . Phosphorylated Ago2 were checked during course of Ld infection ( D ) .

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

Effect of Ld infection on Ago2 phosphorylation and its miRNA association. RAW 264.7 cells were transfected with FH-Ago2 expression construct and infection was done for various time points followed by FH-Ago2 pull down using anti-FLAG beads. Phosphorylated Ago2 levels were checked during the course of Ld infection (D).


Phosphorylation of Ago2 were measured by doing endogenous Ago2 pull down using Ago2 specific antibody after 4 hrs of infection quantified along with Ago2 associated let - 7a level measurement in macrophages ( mean + / - s . e . m . , n = 2 ) ( E , left panel ) in RAW 264 . 7 ( mean + / - s . e . m . , n = 3 ) ( E , right panel ) .

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Phosphorylation levels of Ago2 were measured by doing endogenous Ago2 pull down using Ago2 specific antibody after 4 hrs of infection and quantified along with Ago2 associated let-7a level measurement in primary macrophages (mean+/- s.e.m., n=2) (E, left panel) and in RAW 264.7 cells (mean+/- s.e.m., n=3) (E, right panel) .


Schematic representation of in vitro phosphatase ( F , upper panel ) . PP2A , MKP1 , MKP3 , or SHP1 were immunoprecipitated individually from naïve or Leishmania infected macrophage were incubated in vitro with FH - Ago2 isolated from FH - Ago2 stable HEK293 . Phosphorylated Ago2 level was detected by western blot using phosphotyrosine specific 4G10 antibody ( F , lower panel ) measured by densitometry . BC ; Bead Control .

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

Schematic representation of in vitro phosphatase assay (F, upper panel). PP2A, MKP1, MKP3, or SHP1 were immunoprecipitated individually from naïve or Leishmania infected macrophage cells and were incubated in vitro with FH-Ago2 isolated from FH-Ago2 stable HEK293 cells. Phosphorylated Ago2 level was detected by western blot analysis using phosphotyrosine specific 4G10 antibody (F, lower panel) and measured by densitometry. BC; Bead Control.


PP2A MKP3 , or SHP1 were immunoprecipitated individually from naïve or Leishmania infected macrophage were incubated in vitro with FH - Ago2 isolated from FH - Ago2 stable HEK293 . Relative of 4G10 specific band against immunoprecipitated Ago2 was plotted ( mean + / - s . e . m . , n = 3 ) ( G ) . BC ; Bead Control .

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

PP2A MKP3, or SHP1 were immunoprecipitated individually from naïve or Leishmania infected macrophage cells and were incubated in vitro with FH-Ago2 isolated from FH-Ago2 stable HEK293 cells. Relative intensity of 4G10 specific band against immunoprecipitated Ago2 was plotted (mean+/- s.e.m., n=3) (G). BC; Bead Control.


Effect of PP2A knockdown on Ago2 phosphorylation miRNA - Ago2 binding in RAW 264 . 7 infected with Ld . were co - transfected with siRNA FH - Ago2 expression plasmid . Phosphorylated Ago2 were detected after 4 hrs of infection in siCon or siPP2A transfected ( H ) .

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

Effect of PP2A knockdown on Ago2 phosphorylation and miRNA-Ago2 binding in RAW 264.7 cells infected with Ld. Cells were co-transfected with siRNA and FH-Ago2 expression plasmid. Phosphorylated Ago2 levels were detected after 4 hrs of infection in siCon or siPP2A transfected cells (H).


Effect of PP2A knockdown on Ago2 phosphorylation miRNA - Ago2 binding in RAW 264 . 7 infected with Ld . were co - transfected with siRNA FH - Ago2 expression plasmid . Ago2 associated let - 7a were also estimated plotted ( mean + / - s . e . m . , n = 4 ) ( I ) .

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

Effect of PP2A knockdown on Ago2 phosphorylation and miRNA-Ago2 binding in RAW 264.7 cells infected with Ld. Cells were co-transfected with siRNA and FH-Ago2 expression plasmid. Ago2 associated let-7a levels were also estimated and plotted (mean+/- s.e.m., n=4) (I).


Effect of OA on Ago2 phosphorylation in Ld - infected RAW 264 . 7 of PP2A Ago2 at different time points of Ld infection with or without OA treatment ( 100 nM ) were detected . were pre - treated with OA ( 100 nM ) for 2 hrs before infection ( J ) . β - Actin was used as loading control .

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Effect of OA on Ago2 phosphorylation in Ld-infected RAW 264.7 cells Cellular levels of PP2A and Ago2 at different time points of Ld infection with or without OA treatment (100 nM) were detected. Cells were pre-treated with OA (100 nM) for 2 hrs before infection (J). β-Actin was used as loading control.


Effect of OA on Ago2 phosphorylation in Ld - infected RAW 264 . 7 mouse PEC . Phospho - Ago2 level was measured in OA pre - treated ; Ld infected RAW 264 . 7 expressing FH - Ago2 in PEC post 24 hrs of infection with or without OA pre - treatment . Phosphorylated Ago2 were also checked ( K ) .

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

Effect of OA on Ago2 phosphorylation in Ld-infected RAW 264.7 cells and mouse PEC. Phospho-Ago2 level was measured in OA pre-treated; Ld infected RAW 264.7 cells expressing FH-Ago2 and in PEC post 24 hrs of infection with or without OA pre-treatment. Phosphorylated Ago2 levels were also checked (K).


Subcellular compartmentalization of PP2A in Ld - infected RAW 264 . 7 . RAW 264 . 7 were infected with Ld for 6 hrs extract were analysed on a OptiprepR density gradient to separate subcellular organelles structures . Subcellular localization of Ago2 PP2A in fractions were detected by western blots . Alix was used at MVB marker , while Calnexin was used as ER marker ( L ) .

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

Subcellular compartmentalization of PP2A in Ld-infected RAW 264.7 cells. RAW 264.7 cells were infected with Ld for 6 hrs and cell extract were analysed on a OptiprepR density gradient to separate subcellular organelles and structures. Subcellular localization of Ago2 and PP2A in individual fractions were detected by western blots. Alix was used at MVB marker, while Calnexin was used as ER marker (L).


RAW 264 . 7 were infected with Ld for 6 hrs extract were analysed on a OptiprepR density gradient to separate subcellular organelles structures . OptiprepR fractions , positive for MVB ( fraction number 2 , 3 , 4 ) ER ( fraction number 7 , 8 , 9 ) markers were pooled separately Ago2 was immunoprecipitated using Anti - FLAG beads . Phosphorylated Ago2 were quantified in western blot done with phospho - tyrosine specific 4G10 antibody ( M ) .

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

RAW 264.7 cells were infected with Ld for 6 hrs and cell extract were analysed on a OptiprepR density gradient to separate subcellular organelles and structures. OptiprepR fractions, positive for MVB (fraction number 2,3,4) and ER (fraction number 7,8,9) markers were pooled separately and Ago2 was immunoprecipitated using Anti-FLAG beads. Phosphorylated Ago2 levels were quantified in western blot done with phospho-tyrosine specific 4G10 antibody (M).


Subcellular compartmentalization of PP2A in Ld - infected RAW 264 . 7 . RAW 264 . 7 were infected with Ld for 6 hrs extract were analysed on a OptiprepR density gradient to separate subcellular organelles structures . PP2A plot for each fraction was done for control infected samples ( N ) .

O O O B-GENEPROD O B-ORGANISM O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-ORGANISM O O O O O O O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O O O O O O O

Subcellular compartmentalization of PP2A in Ld-infected RAW 264.7 cells. RAW 264.7 cells were infected with Ld for 6 hrs and cell extract were analysed on a OptiprepR density gradient to separate subcellular organelles and structures. PP2A percentage intensity plot for each fraction was done for control and infected samples (N).


Effect of PP2A downregulation or inhibition on pro anti - inflammatory cytokine in Ld infected macrophage . IL - 10 were measured after Ld infection in PP2A knocked - down RAW 264 . 7 ( mean + / - s . e . m . , n = 3 ) in PEC ( mean + / - s . e . m . , n = 3 ) . IL - 6 was also measured in RAW after OA treatment ( mean + / - s . e . m . , n = 5 ) also in PEC pre - treated with OA ( mean + / - s . e . m . , n = 3 ) . Values obtained at 0 hrs ( non - infected ) or non - OA treated samples were considered as unit in each case . IL - 6 level ( n = 5 ) in supernatant was also measured by ELISA after Ld infection in PP2A knock - down RAW 264 . 7 .

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

Effect of PP2A downregulation or inhibition on pro and anti-inflammatory cytokine levels in Ld infected macrophage. IL-10 levels were measured after Ld infection in PP2A knocked-down RAW 264.7 cells (mean+/- s.e.m., n=3) and in PEC (mean+/- s.e.m., n=3). IL-6 was also measured in RAW cells after OA treatment (mean+/- s.e.m., n=5) and also in PEC pre-treated with OA (mean+/- s.e.m., n=3). Values obtained at 0 hrs (non-infected) or non-OA treated samples were considered as unit in each case. IL-6 protein level (n=5) in cell supernatant was also measured by ELISA after Ld infection in PP2A knock-down RAW 264.7 cells.


Effect of PP2A knock - down on internalization Internalized were imaged in RAW 264 . 7 stained with Phalloidin - Alexa 488 ( green ) for actin cytoskeleton detection Ld was detected by indirect immunofluorescence done for specific GP63 ( red ) ( B ) .

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

Effect of PP2A knock-down on parasite internalization Internalized parasites were imaged in RAW 264.7 cells stained with Phalloidin-Alexa 488 (green) for actin cytoskeleton detection and Ld was detected by indirect immunofluorescence done for parasite specific membrane protein GP63 (red) (B).


Effect of PP2A knock - down on internalization IL - 1β level . Number of Ld internalized was measured in control PP2A knock - down relative numbers of within infected were plotted ( C , left panel , ( mean + / - s . e . m . , n = 20 ) . IL - 1β mRNA were also quantified in those by qRT - PCR done for total [ C , right panel , ( mean + / - s . e . m . , n = 3 ) ] . Scale bar 20 µm . 4x Zoomed insets are shown .

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

Effect of PP2A knock-down on parasite internalization and cellular IL-1β level. Number of Ld internalized was measured in control and PP2A knock-down cells and relative numbers of parasites within infected cells were plotted (C, left panel, (mean+/- s.e.m., n=20 cells). IL-1β mRNA levels were also quantified in those cells by qRT-PCR done for total cellular RNA [C, right panel, (mean+/- s.e.m., n=3)]. Scale bar 20 µm. 4x Zoomed insets are shown.


Effect of PP2A knockdown on IL - 10 level in Ld - infected expressing phosphorylation defective mutant of Ago2 . In RAW 264 . 7 , depleted for endogeneous Ago2 ( using a specific siRNA against 3 ' UTR of Ago2 mRNA ) , either wild type or Y529F mutant of FH - Ago2 ( without having 3 ' UTR of Ago2 ) was expressed in siCon or siPP2A treated RAW 264 . 7 . IL - 10 mRNA level was measured subsequent to Ld infection . Relative IL - 10 were plotted . Expression in non - infected siCon treated in each case were designated as unit ( mean + / - s . e . m . , n = 3 ) .

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

Effect of PP2A knockdown on IL-10 level in Ld-infected cells expressing phosphorylation defective mutant of Ago2. In RAW 264.7 cells, depleted for endogeneous Ago2 (using a specific siRNA against the 3'UTR of Ago2 mRNA), either the wild type or Y529F mutant of FH-Ago2 (without having the 3'UTR of Ago2) was expressed in siCon or siPP2A treated RAW 264.7 cells. IL-10 mRNA level was measured subsequent to Ld infection. Relative IL-10 levels were plotted. Expression levels in non-infected siCon treated cells in each case were designated as unit (mean+/- s.e.m., n=3).


Effect of PP2A inhibitor OA on Ld internalization cytokine production in RAW 264 . 7 . In RAW 264 . 7 transfected with FH - Ago2 WT Y529F mutant pre - treated with OA , Leishmania internalization was measured microscopically . In obtained , Ago2 was detected with α - HA ( detected at 405nm ) Leishmania was stained for GP63 ( detected at 564nm ) ( E ) .

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

Effect of PP2A inhibitor OA on Ld internalization and cytokine production in RAW 264.7 cells. In RAW 264.7 cells transfected with FH-Ago2 WT and Y529F mutant and pre-treated with OA, Leishmania internalization was measured microscopically. In images obtained, Ago2 was detected with α-HA (detected at 405nm) and Leishmania was stained for GP63 (detected at 564nm) (E).


Effect of PP2A inhibitor OA on Ld internalization cytokine production in RAW 264 . 7 . In RAW 264 . 7 transfected with FH - Ago2 WT Y529F mutant pre - treated with OA , Leishmania internalization was measured microscopically . Quantitative measurement of GP63 positive structures was done quantitative data per 100 infected was plotted . Scale bar 10 µm . was isolated from different experimental sets TNF - α , IL - 1β IL - 10 mRNA were estimated ( mean + / - s . e . m . , n = 3 ) ( F ) .

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

Effect of PP2A inhibitor OA on Ld internalization and cytokine production in RAW 264.7 cells. In RAW 264.7 cells transfected with FH-Ago2 WT and Y529F mutant and pre-treated with OA, Leishmania internalization was measured microscopically. Quantitative measurement of GP63 positive structures was done and quantitative data per 100 infected cells was plotted. Scale bar 10 µm. RNA was isolated from different experimental sets and TNF-α, IL-1β and IL-10 mRNA levels were estimated (mean +/- s.e.m., n=3) (F).


Effect of PP2A inhibitor OA on Ld internalization cytokine production in RAW 264 . 7 . RAW 264 . 7 with without OA treatment were given Ld infection for 2 hrs 24 hrs . RAW 264 . 7 were stained with Phalloidin - Alexa 488 ( green ) for actin cytoskeleton Ld was stained for specific GP63 ( red ) ( G , left panel ) . Number of internalized per 100 macrophages were calculated relative values were plotted ( G , right panel ) ( mean + / - s . e . m . , n = 20 ) . Scale bar 20 µm .

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

Effect of PP2A inhibitor OA on Ld internalization and cytokine production in RAW 264.7 cells. RAW 264.7 cells with and without OA treatment were given Ld infection for 2 hrs and 24 hrs. RAW 264.7 cells were stained with Phalloidin-Alexa 488 (green) for actin cytoskeleton and Ld was stained for parasite specific protein GP63 (red) (G, left panel). Number of internalized parasites per 100 macrophages were calculated and relative values were plotted (G, right panel) (mean+/- s.e.m., n=20). Scale bar 20 µm.


LPS induced miRNA export from mammalian macrophage . A schematic diagram of experiments has been described in left panel of ( A ) . Characterization of exosomes derived from Control LPS - activated RAW 264 . 7 by Nanoparticle Tracking ( NTA ) done for isolated exosomes . Relative distributions are shown ( A , right panel ) .

B-SMALL_MOLECULE O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O O O O O O

LPS induced miRNA export from mammalian macrophage cells. A schematic diagram of the experiments has been described in left panel of (A). Characterization and quantification of exosomes derived from Control and LPS-activated RAW 264.7 cells by Nanoparticle Tracking Analysis (NTA) done for isolated exosomes. Relative size distributions are shown (A, right panel).


of HuR exosomal miRNA isolated from control 24 hrs of LPS treated RAW 264 . 7 . content was normalized against total exosomal ( B ) . β - Actin as loading control for HuR western blot .

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

Levels of HuR and exosomal miRNA isolated from control and 24 hrs of LPS treated RAW 264.7 cells. RNA content was normalized against total exosomal proteins (B). β-Actin as loading control for HuR western blot.


Binding of miRNAs with HuR after LPS exposure was measured . Amount of was normalized against HuR content . Anti GFP - antibody was used for immunoprecipitation control ( C ) . Values are mean + / - s . e . m . , n =

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

Binding of miRNAs with HuR after LPS exposure was measured. Amount of RNA was normalized against HuR content. Anti GFP-antibody was used for immunoprecipitation control (C). Values are mean+/- s.e.m. and, n=3


Effect of GW4869 , inhibitor of exosome mediated miRNA export , on exosomal miRNA content released by LPS stimulated . level of miRNAs , let - 7a miR - 155 were measured in exosomes ( EV ) released from LPS activated after GW4869 treatment . Values for EVs from naïve macrophages treated without LPS without GW4869 treatment was set as unit . Values are mean + / - s . e . m . , n =

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

Effect of GW4869, the inhibitor of exosome mediated miRNA export, on exosomal miRNA content released by LPS stimulated cells. The level of miRNAs, let-7a and miR-155 were measured in exosomes (EV) released from LPS activated cell after GW4869 treatment. Values for EVs from naïve macrophages treated without LPS without GW4869 treatment was set as unit. Values are mean+/- s.e.m. and, n=3


Effect of GW4869 on cytokine in LPS treated . effect of GW4869 on expression of pro - inflammatory cytokines IL - 1β TNF - α in LPS - activated macrophages were measured normalized against 18S rRNA . Values in naive RAW 264 . 7 without GW4869 LPS treatment were set as unit . Values are mean + / - s . e . m . , n =

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

Effect of GW4869 on cellular cytokine levels in LPS treated cells. The effect of GW4869 on cellular expression of pro-inflammatory cytokines IL-1β and TNF-α in LPS-activated macrophages were measured and normalized against 18S rRNA. Values in naive RAW 264.7 cells without GW4869 and LPS treatment were set as unit. Values are mean+/- s.e.m. and, n=3