mean corpuscular hemoglobin ( MCH ) ( n = 5 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * P = 0 . 0048 , two - tailed unpaired Student ' s t - test ) in wild - type ( WT ) and Otud1 ─ / ─ mice with or without low - iron diets for indicated times .

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

mean corpuscular hemoglobin (MCH) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0048, two-tailed unpaired Student's t-test) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times.


mean corpuscular hemoglobin concentration ( MCHC ) ( n = 5 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * P = 0 . 0068 , two - tailed unpaired Student ' s t - test ) in wild - type ( WT ) and Otud1 ─ / ─ mice with or without low - iron diets for indicated times .

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

mean corpuscular hemoglobin concentration (MCHC) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0068, two-tailed unpaired Student's t-test) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times.


, red boold cells ( RBCs ) ( n = 5 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * P = 0 . 0049 , two - tailed unpaired Student ' s t - test ) in wild - type ( WT ) and Otud1 ─ / ─ mice with or without low - iron diets for indicated times .

, red boold cells (RBCs) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0049, two-tailed unpaired Student's t-test) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times.


hemoglobin ( HB ) ( n = 5 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * P = 0 . 0036 , * * * P = 0 . 0002 , two - tailed unpaired Student ' s t - test ) ( I ) in wild - type ( WT ) and Otud1 ─ / ─ mice with or without low - iron diets for indicated times .

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

hemoglobin (HB) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0036, ***P = 0.0002, two-tailed unpaired Student's t-test) (I) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times.


( A - B ) Macroscopic evaluation ( A ) and tumor volume ( mock , n = 7 biological replicates ; OTUD1 , n = 9 biological replicates ; OTUD1C320S , n = 9 biological replicates ; mean ± s . e . m . , * * * P < 0 . 001 , two - tailed unpaired Student ' s t - test ) ( B ) of mock , OTUD1 and OTUD1C320S expressing CT26 tumors in BALB / c mice .

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(A-B) Macroscopic evaluation (A) and tumor volume (mock, n = 7 biological replicates; OTUD1, n = 9 biological replicates; OTUD1C320S, n = 9 biological replicates; mean ± s.e.m., ***P < 0.001, two-tailed unpaired Student's t-test) (B) of mock, OTUD1 and OTUD1C320S expressing CT26 tumors in BALB/c mice.


Macroscopic evaluation of mock and OTUD1 expressing LLC tumors in C57BL / 6 mice .

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Macroscopic evaluation of mock and OTUD1 expressing LLC tumors in C57BL/6 mice.


tumor volume ( n = 6 biological replicates , mean ± s . e . m . , * * P < 0 . 01 , * * * P = 0 . 0007 , two - tailed unpaired Student ' s t - test ) of mock and OTUD1 expressing LLC tumors in C57BL / 6 mice .

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tumor volume (n=6 biological replicates, mean ± s.e.m., **P < 0.01, ***P = 0.0007, two-tailed unpaired Student's t-test) of mock and OTUD1 expressing LLC tumors in C57BL/6 mice.


survival analysis ( n = 5 biological replicates , * * P = 0 . 0021 , Log - rank ( Mantel - Cox ) test ) of mock and OTUD1 expressing LLC tumors in C57BL / 6 mice .

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survival analysis (n=5 biological replicates, **P = 0.0021, Log-rank (Mantel-Cox) test) of mock and OTUD1 expressing LLC tumors in C57BL/6 mice.


( F ) Endogenous IREB2 and TFRC protein levels in murine CT26 tumors were measured by anti - IREB2 antibody and anti - TFRC antibody .

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(F) Endogenous IREB2 and TFRC protein levels in murine CT26 tumors were measured by anti-IREB2 antibody and anti-TFRC antibody.


( G ) Flow cytometric analysis of TFRC expression in mock ( n = 4 biological replicates ) , OTUD1 ( n = 6 biological replicates ) and OTUD1C320S ( n = 5 biological replicates ) expressing CT26 tumors . MFI , mean fluorescence intensity ( mean ± s . e . m . , * * P < 0 . 01 , two - tailed unpaired Student ' s t - test ) .

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(G) Flow cytometric analysis of TFRC expression in mock (n = 4 biological replicates), OTUD1 (n = 6 biological replicates) and OTUD1C320S (n = 5 biological replicates) expressing CT26 tumors. MFI, mean fluorescence intensity (mean ± s.e.m., **P < 0.01, two-tailed unpaired Student's t-test).


( H ) Flow cytometric analysis of TFRC expression in mock or OTUD1 expressing LLC tumors . MFI , mean fluorescence intensity . ( n = 5 biological replicates , mean ± s . e . m . , * * P = 0 . 0011 , two - tailed unpaired Student ' s t - test ) .

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(H) Flow cytometric analysis of TFRC expression in mock or OTUD1 expressing LLC tumors. MFI, mean fluorescence intensity. (n = 5 biological replicates, mean ± s.e.m., **P = 0.0011, two-tailed unpaired Student's t-test).


( I ) Iron concentration of mock , OTUD1 and OTUD1C320S tumors was assessed by Inductively Coupled Plasma Mass Spectrometry ( n = 3 biological replicates , mean ± s . e . m . , * P = 0 . 0237 , * * P = 0 . 0098 , two - tailed unpaired Student ' s t - test ) .

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(I) Iron concentration of mock, OTUD1 and OTUD1C320S tumors was assessed by Inductively Coupled Plasma Mass Spectrometry (n = 3 biological replicates, mean ± s.e.m., *P = 0.0237, **P = 0.0098, two-tailed unpaired Student's t-test).


( J ) Analysis of tumor infiltrating CD8 + T cells or CD4 + T cells in mock ( n = 4 biological replicates ) , OTUD1 ( n = 6 biological replicates ) and OTUD1C320S ( n = 5 biological replicates ) expressing CT26 tumors in BALB / c mice ( mean ± s . e . m . , * * P < 0 . 01 , * * * P < 0 . 001 , two - tailed unpaired Student ' s t - test ) .

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

(J)Analysis of tumor infiltrating CD8+ T cells or CD4+ T cells in mock (n = 4 biological replicates), OTUD1 (n = 6 biological replicates) and OTUD1C320S (n = 5 biological replicates) expressing CT26 tumors in BALB/c mice (mean ± s.e.m., **P < 0.01, ***P < 0.001, two-tailed unpaired Student's t-test).


Flow cytometric analysis of CD8 + T cells , CD4 + T cells ( n = 6 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * * P = 0 . 0003 , two - tailed unpaired Student ' s t - test ) isolated from mock and OTUD1 overexpressing LLC tumors .

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

Flow cytometric analysis of CD8+ T cells, CD4+ T cells (n = 6 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), ***P = 0.0003, two-tailed unpaired Student's t-test) isolated from mock and OTUD1 overexpressing LLC tumors.


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 .

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

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 ) 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 .

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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 ) 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 ) .

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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 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 ) .

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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 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 ) .

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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 ) 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 ) .

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

(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 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 ) .

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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 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 .

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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 ) 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

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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 ) 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

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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 .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 analysis of protein levels of HMGB1 , HSP70 and HSP90 in tumor interstitial fluid of mock , OTUD1 and 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 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 ) .

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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 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 .

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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 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 ) .

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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 ) staining pictures of colon tumors ( The 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 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 ) .

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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 ) and 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 staining pictures of CD3 + T cells in colon tumors ( The 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 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 ) .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 staining of amyloid plaques ( 6E10 ) and 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 ) and ITPKB in AD prefrontal cortex . miR - 124 was used as a control . Scale bar , 50 μm .

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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 . 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 ) .

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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 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 .

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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 assay of saponin ( n ≥ 4 ) .

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


B Dose response curve of tip recordings using the 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 ) .

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


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 .

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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 assay with 26 Gr mutants for 5 % saponin ( n ≥ 4 ) . The 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 Concentration - dependent avoidance of saponin in control , Gr28bMi and 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 the indicated time points ( 15 , 30 , 60 , and 240 min ) using spectrophotogram analysis for control and 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 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 ) .

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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 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 ) .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 . 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 ) .

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

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 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

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 the cell death gene ( hid ) , under Gr28b . c - GAL4 ( n = 4 ) .

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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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O 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 O B-MEASURED_VAR 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 O O O O O O O O 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 and ( B ) Gr28bMi adult raised on control and the 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 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 .

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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 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 ) .

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

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 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 .

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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 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 .

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 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 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 ) .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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

(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 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 ) .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

(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 - tailed paired t - test , ( * p < 0 . 05 , * * p < 0 . 01 ) .

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

(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 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

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 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

(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 , and porcine oocytes Shown are percentages of miRNAs tailed at 3 ′ end with mono - and 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 the hSyn promoter ( AAV2 - hSyn : ReaChR - mCitrine ) ; transduced RGCs are visible as green puncta .

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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 and 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 ) 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 .

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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 ) Light sensitivity : Photocurrents of a ReaChR - expressing RGC stimulated with varying light 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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 ) .

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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 ) 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 ) .

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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 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 .

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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 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 ) .

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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 ± 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 ) .

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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 the retinal explant showing the macula ( yellow pigmented ) with the approximate location of the 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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 ) 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 .

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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 ± 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 ) .

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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 ) 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 .

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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 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 ) .

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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 ) 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 .

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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 the presence of the 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 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 .

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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 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 .

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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 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 .

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(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 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 ) .

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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 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 .

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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 light chains ( λ ) , while the kappa light chains ( κ ) showed prevalence in the 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 the first test ( 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 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 .

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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 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 .

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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 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 ) .

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

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).