PD - 1 expression in T ( n = 6 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * * P < 0 . 0001 , two - unpaired Student ' s t - ) isolated from mock OTUD1 overexpressing LLC tumors . MFI , mean .

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PD-1 expression in T cells (n = 6 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), ***P < 0.0001, two-tailed unpaired Student's t-test) isolated from mock and OTUD1 overexpressing LLC tumors. MFI, mean fluorescence intensity.


A ) Quantitative proteomics study of wild - type ( WT ) OTUD1 ─ / ─ NCM460 . expression of TFRC IREB2 were highlighted with red color . indicates label - free ( LFQ ) of in mass spectrometry .

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A) Quantitative proteomics study of wild-type (WT) and OTUD1─/─ NCM460 cells. The expression of TFRC and IREB2 were highlighted with red color. Intensity indicates label-free quantification (LFQ) intensity of proteins in mass spectrometry.


( B ) Wild - type ( WT ) OTUD1 ─ / ─ NCM460 were treated with indicated of H2O2 for 24 hours viability was measured by PI ( n = 3 biological replicates , mean ± s . e . m . , * P < 0 . 05 , * * P < 0 . 01 , two - unpaired Student ' s t - ) .

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(B) Wild-type (WT) and OTUD1─/─ NCM460 cells were treated with indicated concentration of H2O2 for 24 hours and cell viability was measured by PI staining (n = 3 biological replicates, mean ± s.e.m., *P < 0.05, **P < 0.01, two-tailed unpaired Student's t-test).


( C ) CT26 stably expressing mock or OTUD1 were treated with 200 μM H2O2 together with various inhibitors containing Fer - 1 ( 10 μM ) , Z - VAD ( 25 μM ) nec - 1 ( 20 μM ) . viability was measured by PI n = 3 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * P = 0 . 0119 , * * P = 0 . 0015 , * * * P < 0 . 001 , two - unpaired Student ' s t - ) .

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(C) CT26 cells stably expressing mock or OTUD1 were treated with 200 μM H2O2 together with various cell death inhibitors containing Fer-1 (10 μM), Z-VAD (25 μM) and nec-1 (20 μM). Cell viability was measured by PI staining n = 3 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), *P = 0.0119, **P = 0.0015, ***P < 0.001, two-tailed unpaired Student's t-test).


( D ) CT26 stably expressing mock , OTUD1 or OTUD1C320S were treated with Erastin ( 10 μM ) RSL3 ( 5 μM ) , viability was measured by PI ( n = 3 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * P < 0 . 01 , two - unpaired Student ' s t - ) .

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(D) CT26 cells stably expressing mock, OTUD1 or OTUD1C320S were treated with Erastin (10 μM) and RSL3 (5 μM), and cell viability was measured by PI staining (n = 3 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P < 0.01, two-tailed unpaired Student's t-test).


( E ) Wild - type ( WT ) OTUD1 ─ / ─ NCM460 were treated with Erastin ( 10 μM ) RSL3 ( 5 μM ) for 24 hours viability was measured by PI ( n = 3 biological replicates , mean ± s . e . m . , * * P = 0 . 0082 , * * * P < 0 . 001 , two - unpaired Student ' s t - ) .

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(E) Wild-type (WT) and OTUD1─/─ NCM460 cells were treated with Erastin (10 μM) and RSL3 (5 μM) for 24 hours and cell viability was measured by PI staining (n = 3 biological replicates, mean ± s.e.m., **P = 0.0082, ***P < 0.001, two-tailed unpaired Student's t-test).


F ) Intracellular ROS in mock , OTUD1 OTUD1C320S overexpressing CT26 treated with Erastin ( 10 μM ) , AFC ( 50 μM ) or H2O2 ( 100 μM ) were detected by DCFDA . MFI , mean ( n = 2 biological replicates ) .

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F) Intracellular ROS levels in mock, OTUD1 and OTUD1C320S overexpressing CT26 cells treated with Erastin (10 μM), AFC (50 μM) or H2O2 (100 μM) were detected by DCFDA staining. MFI, mean fluorescence intensity (n = 2 biological replicates).


( G ) Intracellular ROS in mock ( n = 4 biological replicates ) , OTUD1 ( n = 6 biological replicates ) or OTUD1C320S ( n = 5 biological replicates ) expressing CT26 tumors ( mean ± s . e . m . , * P = 0 . 0189 , * * P = 0 . 0086 , two - unpaired Student ' s t - ) were detected by DCFDA . MFI , mean .

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(G) Intracellular ROS levels in mock (n = 4 biological replicates), OTUD1 (n = 6 biological replicates) or OTUD1C320S (n = 5 biological replicates) expressing CT26 tumors (mean ± s.e.m., *P = 0.0189, **P = 0.0086, two-tailed unpaired Student's t-test) were detected by DCFDA staining. MFI, mean fluorescence intensity.


( I ) volume ( mock , n = 4 biological replicates ; mock + NP - VE , n = 6 biological replicates ; OTUD1 , n = 4 biological replicates ; OTUD1 + NP - VE , n = 6 biological replicates

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(I) Tumor volume (mock, n = 4 biological replicates; mock + NP-VE, n = 6 biological replicates; OTUD1, n = 4 biological replicates; OTUD1 + NP-VE, n = 6 biological replicates


( I ) volume ( mock , n = 4 biological replicates ; mock + NP - VE , n = 6 biological replicates ; OTUD1 , n = 4 biological replicates ; OTUD1 + NP - VE , n = 6 biological

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Flow cytometric of DCFDA ( n = 4 biological replicates , mean ± s . e . m . , * * * P < 0 . 001 , two - unpaired Student ' s t - ) of mock or OTUD1 expressing CT26 tumors treated with or without NP - VE . MFI , mean .

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Flow cytometric analysis of DCFDA (n = 4 biological replicates, mean ± s.e.m., ***P < 0.001, two-tailed unpaired Student's t-test) of mock or OTUD1 expressing CT26 tumors treated with or without NP-VE. MFI, mean fluorescence intensity.


Flow cytometric of of infiltrating CD8 + T ( n = 4 biological replicates , mean ± s . e . m . , * P = 0 . 0433 , * * * P = 0 . 0001 , two - unpaired Student ' s t - ) of mock or OTUD1 expressing CT26 tumors treated with or without NP - VE .

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Flow cytometric analysis of the percentage of tumor infiltrating CD8+ T cells (n = 4 biological replicates, mean ± s.e.m., *P = 0.0433, ***P = 0.0001, two-tailed unpaired Student's t-test) of mock or OTUD1 expressing CT26 tumors treated with or without NP-VE.


CT26 stably expressing mock or OTUD1 were treated with 5 μM RSL3 , medium was collected to measure ATP level ( n = 4 biological replicates , mean ± s . e . m . , * * * P < 0 . 0001 , two - unpaired Student ' s t - ) .

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CT26 cells stably expressing mock or OTUD1 were treated with 5 μM RSL3, and medium was collected to measure ATP level (n = 4 biological replicates, mean ± s.e.m., ***P < 0.0001, two-tailed unpaired Student's t-test).


wild - type ( WT ) or OTUD1 ─ / ─ NCM460 were treated with 5 μM RSL3 , medium was collected to measure ATP level ( n = 4 biological replicates , mean ± s . e . m . , * * * P < 0 . 0001 , two - unpaired Student ' s t - ) .

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wild-type (WT) or OTUD1─/─ NCM460 cells were treated with 5 μM RSL3, and medium was collected to measure ATP level (n = 4 biological replicates, mean ± s.e.m., ***P < 0.0001, two-tailed unpaired Student's t-test).


( C ) Volcano plots of genes differentially expressed in interstitial fluid ( TIF ) of mock OTUD1 expressing CT26 tumors . Left , example of genes downregulated in OTUD1 overexpressing interstitial fluid ; right , example of genes upregulated in OTUD1 overexpressing interstitial fluid .

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(C) Volcano plots of genes differentially expressed in tumor interstitial fluid (TIF) of mock and OTUD1 expressing CT26 tumors. Left, example of genes downregulated in OTUD1 overexpressing tumor interstitial fluid; right, example of genes upregulated in OTUD1 overexpressing tumor interstitial fluid.


( D ) Immunoblot of of HMGB1 , HSP70 HSP90 in interstitial fluid of mock , OTUD1 OTUD1C320S expressing CT26 tumors .

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(D) Immunoblot analysis of protein levels of HMGB1, HSP70 and HSP90 in tumor interstitial fluid of mock, OTUD1 and OTUD1C320S expressing CT26 tumors.


( E ) ATP level in interstitial fluid ( TIF ) of mock or OTUD1 overexpressing CT26 ( n = 5 mice , mean ± s . e . m . , * * * P < 0 . 001 , two - unpaired Student ' s t - ) .

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(E) ATP level in tumor interstitial fluid (TIF) of mock or OTUD1 overexpressing CT26 cells (n = 5 mice, mean ± s.e.m., ***P < 0.001, two-tailed unpaired Student's t-test).


( F G ) BALB / c mice were immunized with 1 × 106 OTUD1 expressing or mock CT26 in left flank challenged with 2 × 106 wild - type CT26 in right flank 10 days later . Macroscopic evaluation ( F ) volume ( n = 7 mice , mean ± s . e . m . , * * P = 0 . 0053 , two - unpaired Student ' s t - ) ( G ) of tumors in right flank were shown .

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(F and G) BALB/c mice were immunized with 1×106 OTUD1 expressing or mock CT26 cells in the left flank and challenged with 2×106 wild-type CT26 cells in the right flank 10 days later. Macroscopic evaluation (F) and tumor volume (n=7 mice, mean ± s.e.m., **P = 0.0053, two-tailed unpaired Student's t-test) (G) of tumors in the right flank were shown.


( A ) Representative pictures of colon tumors in AOM / DSS - induced Colitis - Associated Cancer ( CAC ) model ( n = 6 biological replicates ) .

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(A) Representative pictures of colon tumors in AOM/DSS-induced Colitis-Associated Cancer (CAC) model (n = 6 biological replicates).


( B C ) load ( B ) or number ( C ) in whole colon was measured ( n = 12 biological replicates , mean ± s . e . m . , * P = 0 . 0431 , * * P = 0 . 003 , two - unpaired Student ' s t - ) .

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(B and C) The tumor load (B) or tumor number (C) in the whole colon was measured (n = 12 biological replicates, mean ± s.e.m., *P = 0.0431, **P = 0.003, two-tailed unpaired Student's t-test).


( D ) Representative H & E ( Hematoxylin - eosin ) pictures of colon tumors ( scale bars represent 1000 μm ) .

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(D) Representative H&E (Hematoxylin-eosin) staining pictures of colon tumors (The scale bars represent 1000 μm).


( E ) Flow cytometric of TFRC expression in intestinal epithelial ( IECs ) from wild - type ( WT ) Otud1 ─ / ─ mice treated with AOM / DSS . MFI , mean . ( n = 4 biological replicates , mean ± s . e . m . , * * P = 0 . 0062 , two - unpaired Student ' s t - ) .

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(E) Flow cytometric analysis of TFRC expression in intestinal epithelial cells (IECs) from wild-type (WT) and Otud1─/─ mice treated with AOM/DSS. MFI, mean fluorescence intensity. (n = 4 biological replicates, mean ± s.e.m., **P = 0.0062, two-tailed unpaired Student's t-test).


( F ) GSEA of genes expressed in colon tissues from wild - type ( WT ) Otud1 ─ / ─ mice treated with AOM / DSS ( n = 3 biological replicates ) . ES , enrichment score ; NES , normalized enrichment score .

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(F) GSEA of genes expressed in colon tissues from wild-type (WT) and Otud1─/─ mice treated with AOM/DSS (n = 3 biological replicates). ES, enrichment score; NES, normalized enrichment score.


( G ) Representative immunohistochemistry pictures of CD3 + T in colon tumors ( scale bars represent 400 μm ) .

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(G) Representative immunohistochemistry staining pictures of CD3+ T cells in colon tumors (The scale bars represent 400 μm ).


( H ) Flow cytometric of various T subsets in lamina propria mononuclear ( LPMC ) isolated from wild - type ( WT ) Otud1 ─ / ─ mice treated with AOM / DSS ( n = 5 biological replicates , mean ± s . e . m . , * * P = 0 . 0025 , * * * P = 0 . 0005 , two - unpaired Student ' s t - ) .

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(H) Flow cytometric analysis of various T cell subsets in lamina propria mononuclear cells (LPMC) isolated from wild-type (WT) and Otud1─/─ mice treated with AOM/DSS (n = 5 biological replicates, mean ± s.e.m., **P = 0.0025, ***P = 0.0005, two-tailed unpaired Student's t-test).


( I ) Intracellular ROS in colon tissues from wild - type ( WT ) or Otud1 ─ / ─ mice treated with AOM / DSS ( n = 4 biological replicates , mean ± s . e . m . , * * P = 0 . 0072 , two - unpaired Student ' s t - ) were detected by DCFDA . MFI , mean .

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(I) Intracellular ROS levels in colon tissues from wild-type (WT) or Otud1─/─ mice treated with AOM/DSS (n = 4 biological replicates, mean ± s.e.m., **P = 0.0072, two-tailed unpaired Student's t-test) were detected by DCFDA staining. MFI, mean fluorescence intensity.


( J ) ATP level in interstitial fluid ( TIF ) of mice treated with AOM / DSS ( n = 4 biological replicates , mean ± s . e . m . , * P = 0 . 0292 , two - unpaired Student ' s t - ) .

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(J) ATP level in tumor interstitial fluid (TIF) of mice treated with AOM/DSS (n = 4 biological replicates, mean ± s.e.m., *P = 0.0292, two-tailed unpaired Student's t-test).


B . Semi - quantitative PCR of miR - 132 negative control miRNAs in hippocampus of antagomiR - 132 - injected mice ( ant - 132 ) in comparison to control - injected ( aCSF scramble ) at six months of . Sample , n = 9 per group .

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B. Semi-quantitative PCR of miR-132 and negative control miRNAs in the hippocampus of antagomiR-132-injected mice (ant-132) in comparison to control-injected animals (aCSF and scramble) at six months of age. Sample size, n=9 per group.


C . FISH of miR - 132 negative control miR - 124 in hippocampus of ant - 132 - scramble - injected mice . Scramble probes were used as FISH negative controls . Scale bar , 500μm .

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C. FISH of miR-132 and negative control miR-124 in the hippocampus of ant-132- and scramble-injected mice. Scramble probes were used as FISH negative controls. Scale bar, 500μm.


D . Semi - quantitative PCR of miR - 132 miR - 212 in hippocampus of 3 - month old miR - 132 mimic - injected mice ( miR - 132 ) compared to injected with a negative control oligonucleotide ( Ctr ) . Sample , n = 6 per group .

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D. Semi-quantitative PCR of miR-132 and miR-212 in the hippocampus of 3-month old miR-132 mimic-injected mice (miR-132) compared to animals injected with a negative control oligonucleotide (Ctr). Sample size, n=6 per group.


A . Amyloid ( 6E10 ) combined with miR - 132FISH in ant - 132 - scramble - injected mice at six months of . Scale bar , 500 μm . B . of amyloid burden in hippocampus cortex of ant - 132 - injected control ( see Materials Methods ) . Sample , n = 4 per group .

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A. Amyloid staining (6E10) combined with miR-132FISH in ant-132- and scramble-injected mice at six months of age. Scale bar, 500 μm. B. Quantification of amyloid burden in hippocampus and cortex of ant-132-injected and control animals (see Materials and Methods). Sample size, n=4 per group.


C , D . ELISA of soluble ( TBS ) insoluble ( Formic acid ) Aβ40 Aβ42 in hippocampus of ant - 132 - ( C ) miR - 132 - injected ( D ) at six three months of , respectively . Sample , n = 6 per group .

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C, D. ELISA of soluble (TBS) and insoluble (Formic acid) Aβ40 and Aβ42 levels in the hippocampus of ant-132- (C) and miR-132-injected (D) animals at six and three months of age, respectively. Sample size, n=6 per group.


Western blot of total TAU pTAU ( AT8 , AT270 ) upon miR - 132 knockdown ( A ) or overexpression ( B ) at six three months of , respectively . Sample , n = 9 per group ( A ) n = 6 per group ( B ) . Values were normalized to respective control groups presented as mean ± . Student ' s t - was used .

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Western blot analysis of total TAU and pTAU (AT8, AT270) levels upon miR-132 knockdown (A) or overexpression (B) at six and three months of age, respectively. Sample size, n=9 per group (A) and n=6 per group (B). Values were normalized to the respective control groups and presented as mean ± SEM. Student's t-test was used.


B . Positive hits were validated by semi - quantitative qPCR in ant - 132 - injected mousehippocampus . Black indicates mean of scramble control set to 1 . Dashed indicates chosen threshold of 1 . 3 - fold . Sample , n = 9 per group . Values were normalized to scramble - injected group presented as mean ± .

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B. Positive hits were validated by semi-quantitative qPCR in ant-132-injected mousehippocampus. Black line indicates mean of scramble control set to 1. Dashed line indicates chosen threshold of 1.3-fold. Sample size, n=9 per group. Values were normalized to scramble-injected group and presented as mean ± SEM.


A . Luciferasereporter of wild - type ( wt ) mutant ( mut ) ITPKB 3 ' UTR in HEK293 co - transfected with a synthetic miR - 132 ( miR - 132 ) or a negative control ( Neg Ctr ) oligonucleotide .

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A. Luciferasereporter assay of wild-type (wt) and mutant (mut) ITPKB 3'UTR in HEK293 cells co-transfected with a synthetic miR-132 (miR-132) or a negative control (Neg Ctr) oligonucleotide.


B . ELISA of Aβ40 Aβ42 in HEK293 - APPswecells transfected with a miR - 132 antisense oligonucleotide ( miR - 132 inh ) , an siRNA against ITPKB ( ITPKB siRNA ) or both . assays in A B were performed in three independent experiments , each in triplicates .

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B. ELISA of Aβ40 and Aβ42 levels in HEK293-APPswecells transfected with a miR-132 antisense oligonucleotide (miR-132 inh), an siRNA against ITPKB (ITPKB siRNA) or both. The assays in A and B were performed in three independent experiments, each in triplicates.


C . Western blot of ITPKB knock down in APPPS1hippocampus using an siRNA oligonucleotide against ITPKB . Sample , n = 6 per group . Values were normalized to control - injected group ( Ctr ) presented as mean ± .

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C. Western blot analysis of ITPKB knock down in APPPS1hippocampus using an siRNA oligonucleotide against ITPKB. Sample size, n=6 per group. Values were normalized to control-injected group (Ctr) and presented as mean ± SEM.


D . ELISA of insoluble ( Formic acid - soluble ) Aβ40 Aβ42 in hippocampus of ITPKB siRNA - control - injected at three months of . Sample , n = 6 per group . Values were normalized to control group presented as mean ± .

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D. ELISA of insoluble (Formic acid-soluble) Aβ40 and Aβ42 levels in the hippocampus of ITPKB siRNA- and control-injected animals at three months of age. Sample size, n=6 per group. Values were normalized to control group and presented as mean ± SEM.


E , F . Western blot of ITPKB upon miR - 132 down - ( E ) or up - regulation ( F ) at six three months of , respectively . Sample , n = 9 per group for miR - 132 down regulation n = 6 per group for miR - 132 overexpression . Values were normalized to control groups presented as mean ± .

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

E, F. Western blot analysis of ITPKB levels upon miR-132 down- (E) or up-regulation (F) at six and three months of age, respectively. Sample size, n=9 per group for miR-132 down regulation and n=6 per group for miR-132 overexpression. Values were normalized to control groups and presented as mean ± SEM.


Western blot of CTFβ ( A ) upon miR - 132 down - ( ant - 132 ) ( left panel ) or upregulation ( miR - 132 ) ( right panel ) in APPPS1hippocampus at six three months of , respectively . Sample , n = 9 per group for miR - 132 down regulation n = 6 per group for miR - 132 overexpression . Values were normalized to respective control groups presented as mean ± . Student ' s t - was used .

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 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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

Western blot analysis of CTFβ (A) levels upon miR-132 down- (ant-132) (left panel) or upregulation (miR-132) (right panel) in APPPS1hippocampus at six and three months of age, respectively. Sample size, n=9 per group for miR-132 down regulation and n=6 per group for miR-132 overexpression. Values were normalized to respective control groups and presented as mean ± SEM. The Student's t-test was used.


Western blot of sAPPβ - swe ( B ) upon miR - 132 down - ( ant - 132 ) ( left panel ) or upregulation ( miR - 132 ) ( right panel ) in APPPS1hippocampus at six three months of , respectively . Sample , n = 9 per group for miR - 132 down regulation n = 6 per group for miR - 132 overexpression . Values were normalized to respective control groups presented as mean ± . Student ' s t - was used .

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 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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

Western blot analysis of sAPPβ-swe (B) levels upon miR-132 down- (ant-132) (left panel) or upregulation (miR-132) (right panel) in APPPS1hippocampus at six and three months of age, respectively. Sample size, n=9 per group for miR-132 down regulation and n=6 per group for miR-132 overexpression. Values were normalized to respective control groups and presented as mean ± SEM. The Student's t-test was used.


Western blot of phosphorylated ERK1 / 2 ( pERK1 / 2 ) ( C ) upon miR - 132 down - ( ant - 132 ) ( left panel ) or upregulation ( miR - 132 ) ( right panel ) in APPPS1hippocampus at six three months of , respectively . Sample , n = 9 per group for miR - 132 down regulation n = 6 per group for miR - 132 overexpression . Values were normalized to respective control groups presented as mean ± . Student ' s t - was used .

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 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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

Western blot analysis of phosphorylated ERK1/2 (pERK1/2) (C) levels upon miR-132 down- (ant-132) (left panel) or upregulation (miR-132) (right panel) in APPPS1hippocampus at six and three months of age, respectively. Sample size, n=9 per group for miR-132 down regulation and n=6 per group for miR-132 overexpression. Values were normalized to respective control groups and presented as mean ± SEM. The Student's t-test was used.


A . Double immunofluorescence of amyloid plaques ( 6E10 ) ITPKB in AD prefrontal cortex . Scale bar , 50 μm . Arrowheads indicate ITPKB immunopositivity .

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A. Double immunofluorescence staining of amyloid plaques (6E10) and ITPKB in AD prefrontal cortex. Scale bar, 50 μm. Arrowheads indicate ITPKB immunopositivity.


B . miR - 132FISH coupled with double immunofluorescence against hyperphosphorylated TAU ( AT8 ) - containing neurofibrillary tangles ( NFTs ) ITPKB in AD prefrontal cortex . miR - 124 was used as a control . Scale bar , 50 μm .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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

B. miR-132FISH coupled with double immunofluorescence against hyperphosphorylated TAU (AT8)-containing neurofibrillary tangles (NFTs) and ITPKB in AD prefrontal cortex. miR-124 was used as a control. Scale bar, 50 μm.


C . of miR - 132 , ITPKB hyperphosphorylated TAU ( pTAU ) signal in single neurons . Integrated values of each signal per were normalized to mean integrated density of each signal across analyzed ( see Materials Methods ) . Sample ( AD ) , n = 3 . Values are presented as mean ± . Two - way Anova was used . Quantifications are summarized in table provided . Arrow directions refer to comparison of each normalized signal to same signal in other group ( in " low miR - 132 " group comparisons are made to " high miR - 132 " group vice versa ) .

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

C. Quantification of miR-132, ITPKB and hyperphosphorylated TAU (pTAU) signal in single neurons. Integrated intensity values of each signal per cell were normalized to the mean integrated density of each signal across all the cells analyzed (see Materials and Methods). Sample size (AD patients), n=3. Values are presented as mean ± SEM. Two-way Anova was used. Quantifications are summarized in the table provided. Arrow directions refer to the comparison of each normalized signal to the same signal in the other group (in the "low miR-132" group comparisons are made to the "high miR-132" group and vice versa).


Semi - quantitative PCR of miR - 132 western blot of ITPKB , phosphorylated ( pERK1 / 2 ) total ERK1 / 2 , phosphorylated ( AT8 , AT270 ) total TAU , phosphorylated sphingosine kinase 1 ( pSphK1 ) , BACE1 , full APP ( flAPP ) , APP CTFs sAPPβ in human AD hippocampi ( AD ) compared to non demented control samples ( ND ) . Sample , n = 39 for AD n = 15 for ND . Values were normalized to ND presented as mean ± . Student ' s t - was used .

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

Semi-quantitative PCR of miR-132 and western blot analysis of ITPKB, phosphorylated (pERK1/2) and total ERK1/2, phosphorylated (AT8 , AT270) and total TAU, phosphorylated sphingosine kinase 1 (pSphK1), BACE1, full length APP (flAPP), APP CTFs and sAPPβ in human AD hippocampi (AD) compared to non demented control samples (ND). Sample size, n=39 for AD and n=15 for ND. Values were normalized to ND and presented as mean ± SEM. The Student's t-test was used.


A Dose - response binary food choice of saponin ( n ≥ 4 ) .

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A Dose-response binary food choice assay of saponin (n ≥ 4).


B Dose response curve of tip recordings using indicated concentrations of saponin on S6 sensilla ( n ≥ 8 ) .

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B Dose response curve of tip recordings using the indicated concentrations of saponin on S6 sensilla (n ≥ 8).


C Sample traces of saponin - induced action potentials in ( B ) .

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

C Sample traces of saponin-induced action potentials in (B).


D Dose response curve for sugar inhibition using indicated concentrations of saponin with 100 mM sucrose on L4 sensilla ( n ≥ 8 ) . E Sample traces from ( D ) . asterisks indicate significant differences from that of control detected by a single - factor ANOVA with Scheffe ' s as a post hoc to compare two sets of data ( * * P < 0 . 01 ) . error bars represent SEMs .

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D Dose response curve for sugar inhibition using the indicated concentrations of saponin with 100 mM sucrose on L4 sensilla (n ≥ 8). E Sample traces from (D). The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis as a post hoc test to compare two sets of data (**P < 0.01). The error bars represent SEMs.


A Binary food choice with 26 Gr mutants for 5 % saponin ( n ≥ 4 ) . error bars represent SEMs .

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A Binary food choice assay with 26 Gr mutants for 5% saponin (n ≥ 4). The error bars represent SEMs.


B - dependent avoidance of saponin in control , Gr28bMi Gr28b Df / Gr28bMi ( n ≥ 4 ) .

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B Concentration-dependent avoidance of saponin in control, Gr28bMi and Gr28b Df/ Gr28bMi (n ≥ 4).


C Amount of ingestion calculated at indicated time points ( 15 , 30 , 60 , 240 min ) using spectrophotogram for control 5 % saponin - containing food ( n = 8 ) .

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C Amount of ingestion calculated at the indicated time points (15, 30, 60, and 240 min) using spectrophotogram analysis for control and 5% saponin-containing food (n = 8).


D , E PER for control Gr28bMi flies in ( D ) legs or ( E ) proboscis . Flies were initially given 2 % sucrose , then 2 % sucrose in combination with 5 % saponin ( n = 4 ) . error bars represent SEMs . asterisks indicate significant differences from that of control detected by a single - factor ANOVA with Scheffe ' s as a post hoc to compare two sets of data ( * P < 0 . 05 , * * P < 0 . 01 ) .

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D, E PER assay for control and Gr28bMi flies in the (D) legs or (E) proboscis. Flies were initially given 2% sucrose, then 2% sucrose in combination with 5% saponin (n = 4). The error bars represent SEMs. The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis as a post hoc test to compare two sets of data (*P < 0.05, **P < 0.01).


A Mapping of sensilla following stimulation with 5 % saponin in control Gr28bMi flies ( we followed Tanimura ' s nomenclature ) ( n ≥ 8 ) . error bars represent SEMs . asterisks indicate significant differences from that of control detected by a single - factor ANOVA with Scheffe ' s to compare two sets of data ( * P < 0 . 05 , * * P < 0 . 01 ) .

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A Mapping analysis of all the sensilla following stimulation with 5% saponin in control and Gr28bMi flies (we followed Tanimura's nomenclature) (n ≥ 8). The error bars represent SEMs. The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis to compare two sets of data (*P < 0.05, **P < 0.01).


B Sample traces of saponin - induced action potentials on S6 sensilla .

B Sample traces of saponin-induced action potentials on S6 sensilla.


C Tip recording of S6 sensilla from control Gr28bMi flies . Stimuli used are 0 . 2 % DEET , 1 . 0 mM coumarin , 0 . 5 mM quinine , 10 mM umbelliferone , 10 mM caffeine , 0 . 3 mM strychnine , 0 . 3 mM lobeline , 0 . 1 mM chloroquine , 0 . 2 mM denatonium , 0 . 5 mM papaverine , 0 . 1 mM berberine 0 . 1 % SDS ( n ≥ 7 ) . error bars represent SEMs .

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C Tip recording analysis of S6 sensilla from control and Gr28bMi flies. Stimuli used are 0.2% DEET, 1.0 mM coumarin, 0.5 mM quinine, 10 mM umbelliferone, 10 mM caffeine, 0.3 mM strychnine, 0.3 mM lobeline, 0.1 mM chloroquine, 0.2 mM denatonium, 0.5 mM papaverine, 0.1 mM berberine and 0.1% SDS (n ≥ 7). The error bars represent SEMs.


D Binary food choice assays were conducted after GRNs were ablated by expressing gene ( hid ) , under Gr33a - GAL4 or Gr66a - GAL4 . heterozygote controls ( UAS - hid / + , Gr33a - GAL4 / + , Gr66a - GAL4 / + ) are shown ( n ≥ 4 ) . error bars represent SEMs . asterisks indicate significant differences from that of control detected by a single - factor ANOVA with Scheffe ' s to compare two sets of data ( * P < 0 . 05 , * * P < 0 . 01 ) .

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

D Binary food choice assays were conducted after GRNs were ablated by expressing the cell death gene (hid), under Gr33a-GAL4 or Gr66a-GAL4. All heterozygote controls (UAS-hid/+, Gr33a-GAL4/+, and Gr66a-GAL4/+) are shown (n ≥ 4). The error bars represent SEMs. The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis to compare two sets of data (*P < 0.05, **P < 0.01).


E Tip recording of indicated S - type sensilla genotypes ( n ≥ 7 ) . error bars represent SEMs . asterisks indicate significant differences from that of control detected by a single - factor ANOVA with Scheffe ' s to compare two sets of data ( * P < 0 . 05 , * * P < 0 . 01 ) .

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E Tip recording analysis of the indicated S-type sensilla and genotypes (n ≥ 7). The error bars represent SEMs. The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis to compare two sets of data (*P < 0.05, **P < 0.01).


B RT - PCR with indicated primers in A for control Gr28bMi flies with tubulin as a control . From left to right , band of tubulin of wild - type , tubulin of Gr28bMi , ladder , Gr28b of wild - type , Gr28b of Gr28bMi .

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B RT-PCR analysis with the indicated primers in A for control and Gr28bMi flies with tubulin as a control. From left to right, the band of tubulin of wild-type, tubulin of Gr28bMi, DNA ladder, Gr28b of wild-type, and Gr28b of Gr28bMi.


Each isoform from Gr28b . a to Gr28b . e was driven by Gr28b . a - GAL4 or Gr28b . c - GAL4 in a Gr28bMi background . asterisks indicate significant differences from that of Gr28b mutant detected by a single - factor ANOVA with Scheffe ' s ( * * P < 0 . 01 ) . C Binary food choice for recovery experiments with each of five alternative Gr28b isoforms using GAL4 / UAS systems . ( n ≥ 4 ) .

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

Each isoform from Gr28b.a to Gr28b.e was driven by Gr28b.a-GAL4 or Gr28b.c-GAL4 in a Gr28bMi background. The asterisks indicate significant differences from that of the Gr28b mutant detected by a single-factor ANOVA with Scheffe's analysis (**P < 0.01). C Binary food choice assay for recovery experiments with each of the five alternative Gr28b isoforms using the GAL4/UAS systems. (n ≥ 4).


D Each isoform from Gr28b . a to Gr28b . e was driven by Gr28b . a - GAL4 or Gr28b . c - GAL4 in a Gr28bMi background . asterisks indicate significant differences from that of Gr28b mutant detected by a single - factor ANOVA with Scheffe ' s ( * * P < 0 . 01 ) . D Saponin - induced action potential frequencies from indicated transgenic flies on S6 sensilla ( n = 7 ) .

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

D Each isoform from Gr28b.a to Gr28b.e was driven by Gr28b.a-GAL4 or Gr28b.c-GAL4 in a Gr28bMi background. The asterisks indicate significant differences from that of the Gr28b mutant detected by a single-factor ANOVA with Scheffe's analysis (**P < 0.01). D Saponin-induced action potential frequencies from the indicated transgenic flies on S6 sensilla (n = 7).


A Binary food choice assays were conducted after GRNs were ablated by expressing gene ( hid ) , under Gr28b . c - GAL4 ( n = 4 ) .

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

A Binary food choice assays were conducted after GRNs were ablated by expressing the cell death gene (hid), under Gr28b.c-GAL4 (n = 4).


B Tip recording of indicated S - type sensilla genotypes ( n ≥ 7 ) . error bars represent SEMs . asterisks indicate significant differences from that of control detected by a single - factor ANOVA with Scheffe ' s to compare two sets of data ( * * P < 0 . 01 ) .

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

B Tip recording analysis of the indicated S-type sensilla and genotypes (n ≥ 7). The error bars represent SEMs. The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis to compare two sets of data (**P < 0.01).


C Expression of Gr28b . c - GAL4 reporter in subsets of Gr66a - positive GRNs . First panel shows that bitter responsive GRNs are labeled by Gr66a - I - GFP ( anti - GFP , green ) . Second panel shows expression of Gr28b . c - GAL4 , crossed with UAS - DsRed ( anti - DsRed , red ) . Third panel shows merged image of first second panels . Forth panel shows a schematic diagram of sensilla arrangement . Red - marked sensilla indicate expression of Gr28b . c - GAL4 reporter in second panel . Scale bars represent 40 μm .

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

C Expression of the Gr28b.c-GAL4 reporter in subsets of Gr66a-positive GRNs. First panel shows that all bitter responsive GRNs are labeled by Gr66a-I-GFP (anti-GFP, green). Second panel shows the expression of the Gr28b.c-GAL4, crossed with UAS-DsRed (anti-DsRed, red). Third panel shows merged image of first and second panels. Forth panel shows a schematic diagram of sensilla arrangement. Red-marked sensilla indicate the expression of Gr28b.c-GAL4 reporter in second panel. Scale bars represent 40 μm.


A , B Survival rate of ( A ) control ( B ) Gr28bMi adult raised on control indicated concentrations of saponin - containing cornmeal diet ( n = 4 ) .

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A, B Survival rate of (A) control and (B) Gr28bMi adult raised on control and the indicated concentrations of saponin-containing cornmeal diet (n = 4).


C , D Survival rate on binary food choice . control Gr28bMi were allowed to feed 100 mM fructose versus 100 mM ( C ) or 200 mM fructose ( D ) mixed with 5 % or 7 . 5 % saponin .

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C, D Survival rate based on binary food choice. control and Gr28bMi were allowed to feed 100 mM fructose versus 100 mM (C) or 200 mM fructose (D) mixed with 5% or 7.5% saponin.


E , F Cumulative pupation rate of ( E ) control ( F ) Gr28bMi larvae raised on indicated saponin - containing food as well as control food ( n = 4 ) . error bars represent SEMs . asterisks indicate significant differences from normal diet fed which were detected by a single - factor ANOVA with Scheffe ' s ( * P < 0 . 05 , * * P < 0 . 01 ) .

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

E, F Cumulative pupation rate of (E) control and (F) Gr28bMi larvae raised on the indicated saponin-containing food as well as control food (n = 4). The error bars represent SEMs. The asterisks indicate significant differences from normal diet fed which were detected by a single-factor ANOVA with Scheffe's analysis (*P < 0.05, **P < 0.01).


( A ) miRNA abundance in - sequencing samples from murine , porcine , bovine oocytes Each graph shows on y - axis reads per million of 19 - 32 nt reads for miRNAs on x - axis miRNAs ordered by name .

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(A) miRNA abundance in RNA-sequencing samples from murine, porcine, and bovine oocytes Each graph shows on the y-axis reads per million of 19-32 nt reads for miRNAs and on the x-axis miRNAs ordered by name.


( C ) qPCR of ssc - miR - 205 bta - miR - 10b miRNAs in murine ( M . m . ) , porcine ( S . s . ) , bovine ( B . t . ) oocytes , respectively . Shown are a mean values from three independent experiments . Error bars = SD . Detailed information concerning miRNA is provided in Methods section .

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

(C) qPCR quantification of ssc-miR-205 and bta-miR-10b miRNAs in murine (M.m.), porcine (S.s.), and bovine (B.t.) oocytes, respectively. Shown are a mean values from three independent experiments. Error bars = SD. Detailed information concerning miRNA quantification is provided in the Methods section.


( A ) bta - miR - 10b ssc - miR - 205 miRNAs efficiently repress microinjected NanoLuc luciferase reporters carrying a perfectly complementary ( 1x perfect ) miRNA binding site or four partially complementary ( bulged ) miRNA binding sites . Error bars = SD . Each data point represents a value obtained from five microinjected oocytes . Data were collected in two microinjection sessions , each microinjection session used a new batch of in vitro transcribed reporter mRNAs . Data information : asterisks indicate statistical significance ( p - value ) of one - t - , ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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

(A) bta-miR-10b and ssc-miR-205 miRNAs efficiently repress microinjected NanoLuc luciferase reporters carrying a perfectly complementary (1x perfect) miRNA binding site or four partially complementary (bulged) miRNA binding sites. Error bars = SD. Each data point represents a value obtained from five microinjected oocytes. Data were collected in two microinjection sessions, each microinjection session used a new batch of in vitro transcribed reporter mRNAs. Data information: asterisks indicate statistical significance (p-value) of one-tailed t-test, (*p < 0.05, **p < 0.01, ***p < 0.001).


( B ) De - repression of predicted ssc - miR - 205 targets . In three independent microinjection experiments , ssc - miR - 205 was inhibited by a microinjected antisense oligonucleotide inhibitor target were assessed 24 hours after microinjection by RT - qPCR . Error bars = SD . Data information : asterisks indicate statistical significance ( p - value ) of one - t - , ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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(B) De-repression of predicted ssc-miR-205 targets. In three independent microinjection experiments, ssc-miR-205 was inhibited by a microinjected antisense oligonucleotide inhibitor and target levels were assessed 24 hours after microinjection by RT-qPCR. Error bars = SD. Data information: asterisks indicate statistical significance (p-value) of one-tailed t-test, (*p < 0.05, **p < 0.01, ***p < 0.001).


( C ) Inhibition of ssc - miR - 205 by a microinjected antisense oligonucleotide inhibitor results in reduced development to blastocyst stage . Shown is development of microinjected oocytes to blastocyst stage relative to water - injected oocytes . Five independent experiments ( represented by data points ) were performed , ~ 50 porcine oocytes were microinjected in each group in each experiment . Difference between let - 7a miR - 205 inhibitors effects is statistically significant ( two - paired t - p - value = 0 . 016 ) . Error bars = SD . Data information : asterisks indicate statistical significance ( p - value ) of one - t - , ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O O O O O 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) Inhibition of ssc-miR-205 by a microinjected antisense oligonucleotide inhibitor results in reduced development to the blastocyst stage. Shown is development of microinjected oocytes to the blastocyst stage relative to water-injected oocytes. Five independent experiments (represented by individual data points) were performed, ~50 porcine oocytes were microinjected in each group in each experiment. Difference between let-7a and miR-205 inhibitors effects is statistically significant (two-tailed paired t-test p-value = 0.016). Error bars = SD. Data information: asterisks indicate statistical significance (p-value) of one-tailed t-test, (*p < 0.05, **p < 0.01, ***p < 0.001).


( A ) ssc - miR - 205 turnover . Fully - grown transcriptionally quiescent oocytes were cultured for indicated periods of time selected miRNAs were quantified by RT - qPCR in four ( ssc - let - 7a ) , five ( ssc - miR - 22 ) or six ( ssc - mir10b ssc - miR - 205 ) independent experiments . Error bars = SD .

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(A) ssc-miR-205 turnover. Fully-grown transcriptionally quiescent oocytes were cultured for indicated periods of time and selected miRNAs were quantified by RT-qPCR in four (ssc-let-7a), five (ssc-miR-22) or six (ssc-mir10b and ssc-miR-205) independent experiments. Error bars = SD.


( B ) Comparison of mean relative changes of miRNA level at 20 h of culture ssc - miR - 205 in oocytes is less than ssc - miR - 10b , ssc - miR - 22 , or ssc - let - 7a . Error bars = SD . Asterisks indicate statistical significance ( p - value ) of one - paired t - , ( * p < 0 . 05 , * * p < 0 . 01 ) .

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(B) Comparison of mean relative changes of miRNA level at 20 h of culture ssc-miR-205 in oocytes is less than ssc-miR-10b, ssc-miR-22, or ssc-let-7a. Error bars = SD. Asterisks indicate statistical significance (p-value) of one-tailed paired t-test, (*p < 0.05, **p < 0.01).


( C ) Treatment by sodium periodate followed by qPCR does not support 2 ' - OH modification of either ssc - miR - 205 or bta - miR - 10b miRNA . Shown are relative of sodium periodate - treated samples to non - treated samples , which were set to one . experiment was performed three times . Error bars = SD . Efficiency of periodate treatment was confirmed using non - methylated methylated miR - 221

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(C) Treatment by sodium periodate followed by qPCR does not support 2'-OH modification of either ssc-miR-205 or bta-miR-10b miRNA. Shown are relative levels of the sodium periodate-treated samples to non-treated samples, which were set to one. The experiment was performed three times. Error bars = SD. Efficiency of periodate treatment was confirmed using non-methylated and methylated miR-221 RNA oligonucleotides


( D ) 3 ′ tailing of small RNAs in murine , bovine , porcine oocytes Shown are percentages of miRNAs at 3 ′ end with mono - oligonucleotides .

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(D) 3′ tailing of small RNAs in murine, bovine, and porcine oocytes Shown are percentages of miRNAs tailed at 3′ end with mono- and oligonucleotides.


( A ) In vivo fundusimage of an AAV - injected rd1mouse expressing ReaChR - mCitrine driven by hSyn promoter ( AAV2 - hSyn : ReaChR - mCitrine ) ; transduced RGCs are visible as green .

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(A) In vivo fundusimage of an AAV-injected rd1mouse expressing ReaChR-mCitrine driven by the hSyn promoter (AAV2-hSyn:ReaChR-mCitrine); transduced RGCs are visible as green puncta.


( B ) Live 2 - photon image showing that ReaChR - mCitrine expression is restricted to RGC - membranes their dendritic processes .

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(B) Live 2-photon image showing that ReaChR-mCitrine expression is restricted to RGC-membranes and their dendritic processes.


( C ) Confocal image of a vertical retinal section expressing ReaChR - mCitrine . Endogenous mCitrine ( mCit ) , is localized to RGCmembranes as well as RGCdendritic processes ( see also Fig EV1A ) ; blue : DAPI ( to visualize retinalnuclei ) ; inner layer ( INL ) ; inner plexiform layer ( IPL ) ganglion layer ( GCL ) ; scale bars = 10 µm .

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(C) Confocal image of a vertical retinal section expressing ReaChR-mCitrine. Endogenous mCitrine (mCit) fluorescence, is localized to RGCmembranes as well as RGCdendritic processes (see also Fig EV1A); blue: DAPI (to visualize retinalnuclei); inner nuclear layer (INL); inner plexiform layer (IPL) and ganglion cell layer (GCL); scale bars = 10 µm.


( A ) sensitivity : Photocurrents of a ReaChR - expressing RGC stimulated with varying intensities at 590 nm ( ~ 1013 − 1018 photons cm - 2 s - 1 ) .

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(A) Light sensitivity: Photocurrents of a ReaChR-expressing RGC stimulated with varying light intensities at 590 nm (~1013 − 1018 photons cm-2 s-1).


( B ) ReaChR - induced photocurrents as a function of ( 590 nm , n = 7 , mean photocurrent amplitude at 4 . 2 × 1017 photons cm - 2 s - 1 was − 349 ± 192 pA ) . At bottom , sensitivity range for wild - type cones ( Nikonov et al , 2006 ) is shown in comparison to sensitivity range of ReaChR expressing RGCs . dashed blue orange indicate maximum intensities allowed in human eye at 470nm 590 nm , respectively ( EU - Directive , 2006 ; ICNIRP , 2013 ) .

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(B) ReaChR-induced photocurrents as a function of light intensity (590 nm, n = 7 cells, mean photocurrent amplitude at 4.2 × 1017 photons cm-2 s-1 was −349 ± 192 pA). At the bottom, the sensitivity range for wild-type cones (Nikonov et al, 2006) is shown in comparison to the sensitivity range of ReaChR expressing RGCs. The dashed blue and orange line indicate the maximum light intensities allowed in the human eye at 470nm and 590 nm, respectively (EU-Directive, 2006; ICNIRP, 2013).


( C ) Photocurrent action spectrum of a ReaChR - expressing RGC ( 1 . 2 × 1016 photons cm - 2 s - 1 ) . ( D ) Normalized action spectrums of ReaChR - RGCs . Solid represents patch - clamp data ( n = 8 ; 1 . 2 × 1016 photons cm - 2 s - 1 ) . mean photocurrent amplitude at 550 nm was − 324 ± 97 pA . Dashed represents multi - electrode array data ( n = 168 ; ~ 1016 photons cm - 2 s - 1 ) .

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(C) Photocurrent action spectrum of a ReaChR-expressing RGC (1.2 × 1016 photons cm-2 s-1). (D) Normalized action spectrums of ReaChR-RGCs. Solid line represents patch-clamp data (n = 8 cells; 1.2 × 1016 photons cm-2 s-1). The mean photocurrent amplitude at 550 nm was −324 ± 97 pA. Dashed line represents multi-electrode array data (n = 168 cells; ~1016 photons cm-2 s-1).


( E ) Temporal properties . Modulation of ReaChR - induced photocurrents at increasing stimulation frequencies ( patch - clamp recording from a ReaChR - expressing RGC ; at 22 Hz magnified trace is shown ; 1 . 3 × 1016 photons cm - 2 s - 1 ) . ( F ) Spiking modulation of ReaChR - RGCs quantified from multi - electrode array demonstrates reliable spike triggering at frequencies up to 30 Hz ( 297 , 1 . 3 × 1016 photons cm - 2 s - 1 ) .

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(E) Temporal properties. Modulation of ReaChR-induced photocurrents at increasing stimulation frequencies (patch-clamp recording from a ReaChR-expressing RGC; at 22 Hz magnified trace is shown; 1.3×1016 photons cm-2 s-1). (F) Spiking modulation ratio of ReaChR-RGCs quantified from multi-electrode array demonstrates reliable spike triggering at frequencies up to 30 Hz (297 cells, 1.3 × 1016 photons cm-2 s-1).


( A ) Examples of local field potential ( LFP ) traces in response to flashes at 595 nm ( stimulus is shown as orange bar ; 1 . 25 × 1017 photons cm - 2 s - 1 ) . ( B ) Spike raster plots in response to 200 repetitions of same stimulus . ( C ) Averaged spike responses shown as peristimulus time histograms ( PSTH , bin : 5 ms ) on spike raster plots . Top - row : WT - mice , middle row : rd1 control mice , bottom - row : ReaChR - treated rd1 mice . ( D ) ReaChR - induced cortical responses ( spikes LFP ) as a function of at ( 595 nm ) demonstrating that ReaChR - treated rd1 mice ( n = 4 ) can detect in visual cortex at intensities well below safety threshold allowed for humanretina . Data presented as mean ± .

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(A) Examples of local field potential (LFP) traces in response to light flashes at 595 nm (light stimulus is shown as orange bar; 1.25 × 1017 photons cm-2 s-1). (B) Spike raster plots in response to 200 repetitions of the same light stimulus. (C) Averaged spike responses shown as peristimulus time histograms (PSTH, bin size: 5 ms) based on the spike raster plots. Top-row: WT-mice, middle row: rd1 control mice, bottom-row: ReaChR-treated rd1 mice. (D) ReaChR-induced cortical responses (spikes and LFP) as a function of light intensity at (595 nm) demonstrating that ReaChR-treated rd1 mice (n = 4) can detect light in the visual cortex at intensities well below the safety threshold allowed for the humanretina. Data presented as mean ± SEM.


( A ) Locomotory behavior of a representative rd1 ReaChR - treated mouse during illumination with at 590 nm in an open field arena ( irradiance measured at level of mice ' s eyes was ~ 1015 photons cm - 2 s - 1 , cumulative activity trajectories are shown as red traces after 2 min of illumination ) , see also EV Movie 1 EV Movie 2 . ( B ) Mean velocities of ReaChR - treated rd1mice analyzed over a duration of 2 min at onset . mean velocity of ReaChR - treated mice were significantly reduced than rd1mice ( P = 0 . 006 , one - t - ) upon illumination . Mean ± are shown for each group . ReaChR - treated rd1 ( n = 11 ) , rd1 ( n = 12 ) .

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(A) Locomotory behavior of a representative rd1 and ReaChR-treated mouse during illumination with light at 590 nm in an open field arena (irradiance measured at the level of the mice's eyes was ~1015 photons cm-2 s-1, cumulative activity trajectories are shown as red traces after 2 min of illumination), see also EV Movie 1 and EV Movie 2. (B) Mean velocities of the ReaChR-treated and rd1mice analyzed over a duration of 2 min at light onset. The mean velocity of ReaChR-treated mice were significantly reduced than rd1mice (P = 0.006, one-tailed t-test) upon illumination. Mean ± SEM are shown for each group. ReaChR-treated rd1 (n = 11), rd1 (n = 12).


( C ) / box . of time mice spent in compartment as a function of ( WT , n = 9 ; rd1 , n = 9 ; ReaChR - treated n = 7 ) . Mean ± are shown for each group . ( D ) of time rd1 ( n = 9 ) , WT ( n = 9 ) ReaChR - treated ( n = 7 ) mice spent in compartment for an illumination of 2 . 11 × 1015 photons cm - 2 s - 1 . Both WT as well as ReaChR - treated mice spent significantly less time in compartment than rd1mice ( P < 0 . 0001 , ordinary one - way ANOVA ) . * * * * * * * indicates significance determined by Tukey ' s multiple comparisons for rd1 vs . WT rd1 vs . ReaChR - treated . See also EV Movie 3 for response of a representative ReaChR - treated rd1 mouse plotted in ( D ) .

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(C) Light/dark box test. Percentage of time mice spent in the light compartment as a function of light intensity (WT, n = 9; rd1, n = 9; ReaChR-treated n = 7). Mean ± SEM are shown for each group. (D) Percentage of time rd1 (n = 9), WT (n = 9) and ReaChR-treated (n = 7) mice spent in the light compartment for an illumination intensity of 2.11 × 1015 photons cm-2 s-1. Both WT as well as ReaChR-treated mice spent significantly less time in the light compartment than the rd1mice (P < 0.0001, ordinary one-way ANOVA). *** and **** indicates significance levels determined by Tukey's multiple comparisons test for rd1 vs. WT and rd1 vs. ReaChR-treated. See also EV Movie 3 for the response of a representative ReaChR-treated rd1 mouse plotted in (D).


( A ) Vertical section of macaqueretinal explant transfected with AAV2 hSyn : ReaChR - mCit after 10 days of incubation exhibiting mCitrine expresssion densely in neuronal processes in GCL . ( B ) Vertical section of macaqueretinal explant transfected with AAV8 Y447 733F - CAG : ReaChR - GFP after 3 days of incubation . GFP expression was primarily observed in bodies but also in processes of RGCs ; green : endogenous GFP ; blue : DAPI ; outer layer ( ONL ) . scale bars = 10 µm .

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(A) Vertical section of macaqueretinal explant transfected with AAV2 hSyn:ReaChR-mCit after 10 days of incubation exhibiting mCitrine expresssion densely in neuronal processes in the GCL. (B) Vertical section of macaqueretinal explant transfected with AAV8 Y447 733F-CAG:ReaChR-GFP after 3 days of incubation. GFP expression was primarily observed in the cell bodies but also in processes of RGCs; green: endogenous GFP fluorescence; blue: DAPI; outer nuclear layer (ONL). scale bars = 10 µm.


( C ) Averaged spike responses obtained from multi - electrode array recordings shown as PSTH of 4 , 8 2 respectively of three different macaques ( 580 nm ; 1 . 24 × 1017 photons cm - 2 s - 1 ) . PSTHs are presented as mean ± ( dashed lines , calculated over responding ) . Raster plots of a representative from each macaque are shown below ( 10 stimulus - repetitions ) .

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(C) Averaged spike responses obtained from multi-electrode array recordings shown as PSTH of 4, 8 and 2 cells respectively of three different macaques (580 nm; 1.24 × 1017 photons cm-2 s-1). PSTHs are presented as mean ± SEM (dashed lines, calculated over responding cells). Raster plots of a representative cell from each individual macaque are shown below (10 stimulus-repetitions).


( D ) Action spectrum of ReaChR in primate RGCs from multi - electrode array recordings . Data is shown as mean ± of normalized firing rate , averaged from three macaques ( 28 , error bars calculated over ) , see also Fig EV2B for action spectrums of three macaques obtained from multi - electrode array recordings . ( E ) Responses ( Hz ) of a ReaChR - expressing RGC ( patch clamp recording , in - attached mode ) to flicker stimulations at increasing frequencies ( 2 − 22 Hz ; 1 . 3 × 1016 photons cm - 2 s - 1 ) .

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(D) Action spectrum of ReaChR in primate RGCs from multi-electrode array recordings. Data is shown as mean ± SEM of the normalized firing rate, averaged from three individual macaques (28 cells, error bars calculated over cells), see also Fig EV2B for action spectrums of three individual macaques obtained from multi-electrode array recordings. (E) Responses (Hz) of a ReaChR-expressing RGC (patch clamp recording, in cell-attached mode) to flicker stimulations at increasing frequencies (2−22 Hz; 1.3 × 1016 photons cm-2 s-1).


( B ) Brightfield image of retinal explant showing macula ( yellow pigmented ) with approximate location of multi - electrode array chip indicated .

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(B) Brightfield image of the retinal explant showing the macula (yellow pigmented) with the approximate location of the multi-electrode array chip indicated.


( C ) Infrared image of explant pressed against multi - electrode array ; black dashed depicts ' electrophysiological boundary ' of para - fovea on absence of RGC - spike trains ( scale bar : 100 µm ; see also Fig EV3A B ) . Voltage trace of a para - foveal RGC stimulated with orange ( 600 nm ; 1 . 1 × 1017 photons cm - 2 s - 1 ) is shown on right . electrodes that recorded ReaChR - responding are indicated in green .

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(C) Infrared image of the explant pressed against the multi-electrode array; the black dashed line depicts the 'electrophysiological boundary' of the para-fovea based on the absence of RGC-spike trains (scale bar: 100 µm; see also Fig EV3A and B). Voltage trace of a para-foveal RGC stimulated with orange light (600 nm; 1.1 × 1017 photons cm-2 s-1) is shown on the right. Individual electrodes that recorded ReaChR-responding cells are indicated in green.


( D ) Action spectrum of ReaChR constructed from ( 6 , mean ± , error bars calculated over ) exhibiting peak activity at 550 nm ( 1 . 1 × 1017 photons cm - 2 s - 1 ) similar to what was observed in mice in macaqueretinae .

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(D) Action spectrum of ReaChR constructed from (6 cells, mean ± SEM, error bars calculated over cells) exhibiting peak activity at 550 nm (1.1 × 1017 photons cm-2 s-1) similar to what was observed in mice and in the macaqueretinae.


( E ) Peristimulus time histograms constructed for wavelengths 400 to 650 nm ( 1 . 1 × 1017 photons cm - 2 s - 1 ) demonstrate ReaChR - evoked spike trains ( under L - AP4 ) in humanretina ( 6 , mean ± , error bars calculated over ) , bin : 100 ms ; Bottom : Raster plots of responses of a representative spike - sorted .

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(E) Peristimulus time histograms constructed for wavelengths 400 to 650 nm (1.1 × 1017 photons cm-2 s-1) demonstrate ReaChR-evoked spike trains (under L-AP4) in the humanretina (6 cells, mean ± SEM, error bars calculated over cells), bin size: 100 ms; Bottom: Raster plots of the light responses of a representative spike-sorted cell.


( A ) Confocal image of central retina , exhibiting endogenous ReaChR - mCitrine ( mCit ) in ganglion , located within ~ 500 µm from center of foveal pit , a part of humanretina where midget density is estimated to be ~ 95 % of total RGC population ( Dacey , 1993 ) ( arrowhead : soma ; arrow : dendritic arbor ) . dashed illustrates beginning of foveal slope leading towards ' cone - only ' foveal pit . ( B ) Confocal image of same explant at a higher retinal eccentricity ( 3 - 4 mm from foveal center ) showing ReaChR - mCitrine in RGCs with larger dendritic arbors . endogenous mCitrine was not amplified . Scale bars : 10 µm . PR : Photoreceptors , GC : Ganglion , DAPI ( blue ) .

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(A) Confocal image of the central retina, exhibiting endogenous ReaChR-mCitrine fluorescence (mCit) in ganglion cells, located within ~500 µm from the center of the foveal pit, a part of the humanretina where midget cell density is estimated to be ~95% of total RGC population (Dacey, 1993) (arrowhead: soma; arrow: dendritic arbor). The dashed line illustrates the beginning of the foveal slope leading towards the 'cone-only' foveal pit. (B) Confocal image of the same explant at a higher retinal eccentricity (3-4 mm from the foveal center) showing ReaChR-mCitrine fluorescence in RGCs with larger dendritic arbors. The endogenous mCitrine was not amplified. Scale bars: 10 µm. PR: Photoreceptors, GC: Ganglion cells, DAPI (blue).


( A ) Humanretinal explant , prepared from far periphery , infected with an AAV8 Y447 733F - CAG : ReaChR - GFP , placed on a multi - electrode array chip ( left ) ; GFP epifluorescence of same explant following 12 days of incubation is shown on right . Scale bars : 100 µm . location of two that responded to repeated 2 s full field stimulations of orange ( 600 nm ) are highlighted .

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(A) Humanretinal explant, prepared from the far periphery, infected with an AAV8 Y447 733F-CAG :ReaChR-GFP, placed on a multi-electrode array chip (left); GFP epifluorescence of the same explant following 12 days of incubation is shown on the right. Scale bars: 100 µm. The location of two cells that responded to repeated 2 s full field stimulations of orange light (600 nm) are highlighted.


( B ) corresponding voltage traces show that responses in far peripheral humanretina are more transient , compared to experiments from human para - fovea ( see Fig 6C E , Fig EV3B ) where only sustained responses were evoked . response of ( 2 ) was restricted to a burst of action potentials fired within initial ~ 300 ms in response to a 2 s full field 600 nm stimulus .

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(B) The corresponding voltage traces show that the responses in the far peripheral humanretina are more transient, compared to the experiments from the human para-fovea (see Fig 6C and E, Fig EV3B) where only sustained light responses were evoked. The light response of cell (2) was restricted to a burst of action potentials fired within the initial ~300 ms in response to a 2 s full field 600 nm light stimulus.


( C ) Peristimulus time histograms constructed for wavelengths from 400 to 650 nm ( 1 . 1 × 1017 photons cm - 2 s - 1 ) demonstrate AAV - ReaChR - evoked spike trains ( mean ± , error bars calculated over 3 - 4 , bin : 100 ms ) Bottom : Raster plots of responses of a representative spike - sorted . recordings were performed under pharmacological blockade of photoreceptor input ( 50 µM L - AP4 ) .

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(C) Peristimulus time histograms constructed for wavelengths from 400 to 650 nm (1.1 × 1017 photons cm-2 s-1) demonstrate AAV-ReaChR-evoked spike trains (mean ± SEM, error bars calculated over 3-4 cells, bin size: 100 ms) Bottom: Raster plots of the light responses of a representative spike-sorted cell. All recordings were performed under pharmacological blockade of photoreceptor input (50 µM L-AP4).


( A ) ELISA titration curves ( OD at 450nm ) of Fab3 ( blue ; left panel ) Fab71 ( blue ; right panel ) against mouse recPrP23 ‑ 231 compared to their best affinity - matured versions , Fab83 Fab101 Curves were fitted by non - linear least - squares regression for statistical : log ( agonist ) vs . response .

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(A) ELISA titration curves (OD at 450nm) of Fab3 (blue; left panel) and Fab71 (blue; right panel) against mouse recPrP23‑231 compared to their best affinity-matured versions, Fab83 and Fab101 Curves were fitted by non-linear least-squares regression analysis for statistical analysis: log(agonist) vs. response.


( B - E ) FT - peptide competition ELISA to map epitopes of indicated Fabs . Peptides that strongly inhibit binding of Fabs to recPrP23 ‑ 231 are indicated by their residue numbers in PrP reflect respective binding epitopes . Positive control : recPrP23 ‑ 231 ; negative controls : Neu , BSA , secondary antibody ( 2 ° Ab ) .

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(B-E) FT-peptide competition ELISA to map the epitopes of the indicated Fabs. Peptides that strongly inhibit the binding of the Fabs to recPrP23‑231 are indicated by their residue numbers in the PrP sequence and reflect the respective binding epitopes. Positive control: recPrP23‑231; negative controls: Neu, BSA, and the secondary antibody (2° Ab).


( A ) of tga20 slices chronically exposed to prions cultured in presence of either Fab3 ( CC123 ‑ 50 ) , Fab71 ( OR51 ‑ 91 ) or Fab1 ( GD ) for 45 days . Scale bar : 500 μm .

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(A) Fluorescence micrographs of tga20 slices chronically exposed to prions and cultured in the presence of either Fab3 (CC123‑50), Fab71 (OR51‑91) or Fab1 (GD) for 45 days. Scale bar: 500 μm.


( B ) Same as ( A ) , but in presence of affinity - maturated antibodies .

(B) Same as (A), but in the presence of the affinity-maturated antibodies.


( C ) NeuN immunofluorescence coverage of prion - infected slices cultured in presence of Fabs . Antibodies targeting CC123 ‑ 50 OR51 ‑ 91 afforded protection against neurodegeneration . NBH : non - infectious brain homogenate . Each dot represents a cerebellar slice for treatment groups .

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(C) NeuN immunofluorescence coverage of prion-infected slices cultured in the presence of the Fabs. Antibodies targeting the CC123‑50 and the OR51‑91 afforded protection against neurodegeneration. NBH: non-infectious brain homogenate. Each dot represents a cerebellar slice for all treatment groups.


( D ) Western blot of COCS lysates . Prion - infected slices treated with Fab100 , but not with Fab83 Fab25 showed reduced of PrPSc . n = 3 biological replicates .

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(D) Western blot analysis of COCS lysates. Prion-infected slices treated with Fab100, but not with Fab83 and Fab25 showed reduced levels of PrPSc. n = 3 biological replicates.


( C ) Left : Heat map showing binding specificities ( Z - scores ) of selected Fab71 human analogous antibodies to human recPrP23 ‑ 230 compared to negative controls ( BSA Neu ) . Right : Heat map representing reactivity ( - logEC50 ) obtained from dose - dependent ELISA binding curves of analogous antibodies to human recPrP23 ‑ 230 . Red : low reactivity ; blue : high reactivity .

(C) Left: Heat map showing the binding specificities (Z-scores) of selected Fab71 human analogous antibodies to human recPrP23‑230 compared to the negative controls (BSA and Neu). Right: Heat map representing the reactivity (-logEC50) obtained from dose-dependent ELISA binding curves of the analogous antibodies to human recPrP23‑230. Red: low reactivity; blue: high reactivity.


( C ) Boxplot with half - violin plot displaying distribution of autoantibody reactivity in 48 , 718 plasma samples . Twenty - seven samples over reactivity threshold of - log ( EC50 ) ≥ 2 fitting error < 20 % were considered as hits in screen . Black dots correspond to single samples . boxplot divides data set into three quartiles . box extends from first to third quartile values of data set , with a at median . Whiskers show range of data ( minimum maximum ) .

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(C) Boxplot with half-violin plot displaying the distribution of autoantibody reactivity in 48,718 plasma samples. Twenty-seven samples over the reactivity threshold of -log(EC50) ≥ 2 and fitting error < 20% were considered as hits in the primary screen. Black dots correspond to single samples. The boxplot divides the data set into three quartiles. The box extends from the first to the third quartile values of the data set, with a line at the median. Whiskers show the range of the data (minimum and maximum).


( E ) Heat map representation for validation of hits from screen ( 21 out of 27 hits ) . Confirmed hits displayed selective reactivity against human recPrP23 ‑ 230 . Controls : # 11 - 13 , ApoE4 BSA . Red : high reactivity ; blue : low reactivity .

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(E) Heat map representation for the validation of the hits from the primary screen (21 out of 27 hits). Confirmed hits displayed selective reactivity against human recPrP23‑230. Controls: Patients #11-13, ApoE4 and BSA. Red: high reactivity; blue: low reactivity.


( F ) Heat maps representing autoantibody clonality of hits analyzed by ELISA . Autoantibodies were mostly constituted by lambda chains ( λ ) , while kappa chains ( κ ) showed prevalence in total IgG fraction .

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(F) Heat maps representing autoantibody clonality of hits analyzed by ELISA. Autoantibodies were mostly constituted by lambda light chains (λ), while the kappa light chains (κ) showed prevalence in the total IgG fraction.


( G ) Determination of antibody reactivity ( - log ( EC50 ) ) for three individuals at two different time points . Individuals were selected by displaying strong anti - PrP IgG reactivity in first ( Time 1 ) . Reactivity was maintained for even more than one year ( Time 2 ) .

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(G) Determination of antibody reactivity (-log(EC50)) for three individuals at two different time points. Individuals were selected by displaying strong anti-PrP IgG reactivity in the first test (Time 1). Reactivity was maintained for even more than one year (Time 2).


( H ) Purified IgGs from with strong anti - PrP reactivity were tested against PrP variants BSA ( negative control ) in dose - dependent ELISAs . # 5 # 6 showed binding mostly to human recPrP121 ‑ 230 . # 1 displayed a polyreactive pattern with binding to human recPrP121 ‑ 230 mouse recPrP23 - 110 .

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(H) Purified IgGs from patients with strong anti-PrP reactivity were tested against PrP variants and BSA (negative control) in dose-dependent ELISAs. Patient #5 and #6 showed binding mostly to the human recPrP121‑230. Patient #1 displayed a polyreactive pattern with binding to the human recPrP121‑230 and mouse recPrP23-110.


RT - qPCR of mRNA expression of indicated genes from MoLC or MoDC treated with TGF - β for 16h normalized to MoDC . Data represent mean values ± SD of triplicates from two donors .

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RT-qPCR analysis of mRNA expression of indicated genes from MoLC or MoDC treated with TGF-β for 16h normalized to MoDC. Data represent mean values ± SD of triplicates from two donors.


Subcellular localization of S100A9 S100A8 in MoLC , MoDC MoDC - TGF - β . S100A9 mean intensities ( MFI ) were normalized to MoLC represented in upper graph . same was done for S100A9 / MFI shown in lower graph . Scale bar : 5µm . Graphs show normalized percentages ± SD of data from four different donors for MoLC ( n = 4 ) MoDC ( n = 4 ) three different donors for TGF - β - treated MoDC ( n = 3 ) . When applicable , student t - was performed to determine statistical significance * * P < 0 . 01 ) .

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Subcellular localization analysis of S100A9 and S100A8 in MoLC, MoDC and MoDC-TGF-β. S100A9 mean fluorescence intensities (MFI) were normalized to MoLC and represented in the upper graph. The same was done for S100A9 cytoplasmic/nuclear MFI and shown in the lower graph. Scale bar: 5µm. Graphs show normalized percentages ± SD of data from four different donors for MoLC (n=4) and MoDC (n=4) and three different donors for TGF-β-treated MoDC (n=3). When applicable, student t-test was performed to determine statistical significance **P<0.01).


MoLC from six different donors ( n = 6 ) were left untreated ( NT ) or incubated with LPS for 48h analysed for S100A9 expression by RT - qPCR ( left graph ) . MoLC treated as before were then transduced with HIV - GFP vectors for 48h GFP + were analysed by flow cytometry data were graphically represented as fold relative to non - treated MoLC ( right graph ) . Black horizontal correspond to mean . When applicable , student t - was performed to determine statistical significance ( * * * P < 0 . 001 ) .

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MoLC from six different donors (n=6) were left untreated (NT) or incubated with LPS for 48h and analysed for S100A9 expression by RT-qPCR (left graph). MoLC treated as before were then transduced with HIV-GFP vectors for 48h and GFP+ cells were analysed by flow cytometry and data were graphically represented as fold relative to non-treated MoLC (right graph). Black horizontal line correspond to the mean. When applicable, student t-test was performed to determine statistical significance (***P<0.001).


transfected as above were pre - treated or not with nevirapine transduced with HIV - GFP vectors for 48h . of GFP + MoLC was analysed by flow cytometry . Data represent means values ± SD from two different donors .

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Cells transfected as above were pre-treated or not with nevirapine and transduced with HIV-GFP vectors for 48h. The percentage of GFP+ MoLC was analysed by flow cytometry. Data represent means values ± SD from two different donors.


MoLC , dermal epidermal were subjected to confocal immunofluorescence microscopy for langerin S100A9 expression . Scale bar : 5µm .

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MoLC, dermal and epidermal cells were subjected to confocal immunofluorescence microscopy for langerin and S100A9 expression analysis. Scale bar: 5µm.


Human epidermal treated as before were challenged for 48h with 20 200ng of p24 of HIV - Luc . When indicated were pre - treated with 10μM Nevirapine for 30 min before viral challenge . Luciferase activities measured in duplicates for each condition from two ( infection with 20 ng P24 ) or three ( infection with 200 ng P24 ) different skin samples were represented on a graph as means of relative luminescence units ( RLU ) ± SD .

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Human epidermal cells treated as before were challenged for 48h with 20 and 200ng of p24 of HIV-Luc. When indicated cells were pre-treated with 10μM Nevirapine for 30 min before viral challenge. Luciferase activities measured in duplicates for each condition from two (infection with 20 ng P24) or three (infection with 200 ng P24) different skin samples were represented on a graph as means of relative luminescence units (RLU) ± SD.


experiment shown is representative of four donors for HIV - 1 - R5 ( n = 4 ) three donors for HIV - 1 isolates ( n = 3 ) . Data collected from donors were compiled represented as bar charts for challenged with HIV - 1 - R5 HIV - 1 isolates . Nevirapine ( Nev ) ( 10μM ) was used as control when indicated data were represented as means ± SD of HIV - Gag + from at least 2 ( for siS100A9 - MoLC infected by HIV - 1 isolates ) to 3 donors . When applicable , student t - was performed to determine statistical significance ( * P < 0 . 1 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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The experiment shown is representative of four donors for HIV-1-R5 (n=4) and three donors for HIV-1 primary isolates (n=3). Data collected from all donors were compiled and represented as bar charts for cells challenged with HIV-1-R5 and HIV-1 primary isolates. Nevirapine (Nev) (10μM) was used as control when indicated and data were represented as means ± SD of HIV-Gag+ cells from at least 2 (for siS100A9-MoLC infected by HIV-1 primary isolates) to 3 donors. When applicable, student t-test was performed to determine statistical significance (*P<0.1;**P<0.01;***P<0.001).


E2C - S100A9 - expressing HEK293T were infected with indicated MOI of HIV - GFP for 48h GFP + were analysed by flow cytometry . Mean values ± SD from four independent experiments ( n = 4 ) were normalized to E2C - HEK293T control represented in graph .

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E2C- and S100A9-expressing HEK293T cells were infected with the indicated MOI of HIV-GFP for 48h and GFP+ cells were analysed by flow cytometry. Mean values ± SD from four independent experiments (n=4) were normalized to the E2C-HEK293T control and represented in graph.


of HIV - Gag p24 + from four independent experiments for CEM ( n = 4 ) two for Jurkat ( n = 2 ) analysed by flow cytometry were graphically represented as means of HIV - Gag + ± SD . When applicable , student t - was performed to determine statistical significance ( * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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The percentage of HIV-Gag p24+ cells from four independent experiments for CEM (n=4) and two for Jurkat (n=2) analysed by flow cytometry were graphically represented as means of HIV-Gag+ cells ± SD. When applicable, student t-test was performed to determine statistical significance (**P<0.01;***P<0.001).


E2C - S100A9 - expressing CHO were challenged with indicated MOI of MMLV - YFP vectors for 24h or 48h . percentages of YFP + CHO infected analysed by flow cytometry data were represented on graphs .

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E2C- and S100A9-expressing CHO cells were challenged with indicated MOI of MMLV-YFP vectors for 24h or 48h. The percentages of YFP+ CHO infected cells analysed by flow cytometry and data were represented on graphs.


E2C - or S100A9 - expressing THP1 were left uninfected ( / ) or infected for 3 h with indicated p24 amounts of HIV - 1 NL4 - 3 / Nef - IRES - Renilla containing BlaM - VPR . were processed for Blam - Vpr of CCF2 was analysed by flow cytometry . mock condition corresponds to unstained E2C - expressing . Dot - plots from a representative experiment are shown .

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E2C- or S100A9-expressing THP1 were left uninfected (/) or infected for 3 h with the indicated p24 amounts of HIV-1 NL4-3/Nef-IRES-Renilla viruses containing BlaM-VPR. Cells were processed for Blam-Vpr assay and the percentage of cleaved CCF2 was analysed by flow cytometry. The mock condition corresponds to unstained E2C-expressing cells. Dot-plots from a representative experiment are shown.


Schematic description of fate - of - capsid is displayed above . HEK293T expressing E2C or S100A9 were transduced with HIV - Luc ( 10ng p24 ) for 4 h . were then subjected to fate of capsid ( see methods ) in order to pull - down intact cores ( CA cores ) ( recovered in pellet ) . Equal volumes from soluble ( top fraction ) pelleted ( down fraction ) materials were analyzed by western blotting in order to evaluate presence of p24 ( CA ) , CypA , S100A9 E2C .

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Schematic description of fate-of-capsid assay is displayed above. HEK293T expressing E2C or S100A9 were transduced with HIV-Luc (10ng p24) for 4 h. Cells were then subjected to the fate of capsid assay (see methods) in order to pull-down intact cores (CA cores) (recovered in the pellet). Equal volumes from the soluble (top fraction) and pelleted (down fraction) materials were analyzed by western blotting in order to evaluate the presence of p24 (CA), CypA, S100A9 and E2C proteins.


Colorimetric HIV - 1 RTase activity was performed with 10µg of immunoprecipitated eluted E2C or S100A9 in presence of increasing MgCl2 concentrations . Means ± SD colorimetric data obtained from at least three independent experiments were normalized to E2C - containing conditions shown on graphs .

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Colorimetric HIV-1 RTase activity was performed with 10µg of immunoprecipitated and eluted E2C or S100A9 in presence of increasing MgCl2 concentrations. Means ± SD colorimetric data obtained from at least three independent experiments were normalized to E2C-containing conditions and shown on graphs.


TBH cytotoxicity in vivo of ScAhp1 lysine mutants in yeast . Growth of yap1Δahp1Δ carrying empty vector ( ev ) , wild type peroxiredoxin gene ( AHP1 ) or indicated cysteine or lysine substitutions was monitored without or with 1 mM TBH . ScAhp14KR : ScAhp1C31S K47 / 48R K124R K156R ; ScAhp15KR : ScAhp1C31S K32R K47 / 48R K124R K156R .

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TBH cytotoxicity assay in vivo of the ScAhp1 lysine mutants in yeast. Growth of yap1Δahp1Δ cells carrying empty vector (ev), wild type peroxiredoxin gene (AHP1) or indicated cysteine or lysine substitutions was monitored without or with 1 mM TBH. ScAhp14KR: ScAhp1C31S K47/48R K124R K156R; ScAhp15KR: ScAhp1C31S K32R K47/48R K124R K156R.


In vivo conjugation analyses of ScAhp1 lysine mutants with extracts obtained from indicated yeast strains expressing HA - URM1 . Unconjugated Urm1 Urm1 - Ahp1 conjugates were detected by anti - HA ( top panels ) Western blots . Anti - Ahp1 blots ( middle ) detect unmodified Ahp1 . Anti - Cdc19 ( bottom panels ) served as loading control . ScAhp14KR : ScAhp1C31S K47 / 48R K124R K156R ; ScAhp15KR : ScAhp1C31S K32R K47 / 48R K124R K156R .

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In vivo conjugation analyses of ScAhp1 lysine mutants with protein extracts obtained from the indicated yeast strains expressing HA-URM1. Unconjugated Urm1 and Urm1-Ahp1 conjugates were detected by anti-HA (top panels) Western blots. Anti-Ahp1 blots (middle) detect unmodified Ahp1. Anti-Cdc19 (bottom panels) served as loading control. ScAhp14KR: ScAhp1C31S K47/48R K124R K156R; ScAhp15KR: ScAhp1C31S K32R K47/48R K124R K156R.


TBH cytotoxicity in vivo . Basic growth synthetic effect in wild type , ahp1Δ , urm1Δ , tum1Δ , ncs6Δ , cys3Δ , cys4Δ , cys3Δ urm1Δ , cys4Δ urm1Δ strains was monitored without or with 1 mM TBH .

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TBH cytotoxicity assay in vivo. Basic growth and the synthetic effect in wild type, ahp1Δ, urm1Δ, tum1Δ, ncs6Δ, cys3Δ, cys4Δ, cys3Δ urm1Δ, and cys4Δ urm1Δ strains was monitored without or with 1 mM TBH.


H2S production using Biggy agar plates - Wild type , ahp1Δ , urm1Δ , tum1Δ , ncs6Δ , cys3Δ , cys4Δ , cys3Δ urm1Δ , cys4Δ urm1Δ strains were grown on Biggy medium , H2S production was

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

H2S production using Biggy agar plates- Wild type, ahp1Δ, urm1Δ, tum1Δ, ncs6Δ, cys3Δ, cys4Δ, cys3Δ urm1Δ, and cys4Δ urm1Δ strains were grown on Biggy medium, and H2S production was recorded