D . SDS - PAGE of co - sedimentation assays monitoring factor ( EF - G1 EF - G2 or EF - G2 variant EF - G2 ( ∆ 516 - 527 ) or EF - G2 ( ∆ 514 - 539 ) ) binding to E . coli 70S ribosomes . Binding reactions were centrifuged through a sucrose cushion to recover ribosome associated factor in pellets . positions of EF - G ribosomal molecular markers are indicated .

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

D. SDS-PAGE analysis of co-sedimentation assays monitoring factor (EF-G1 EF-G2 or EF-G2 variant EF-G2 (∆516-527) or EF-G2 (∆514-539)) binding to E. coli 70S ribosomes. Binding reactions were centrifuged through a sucrose cushion to recover ribosome and associated factor in the pellets. The positions of EF-G proteins and ribosomal proteins and molecular weight markers are indicated.


E . Western blot of reporter HisC - FLAG synthesized in vitro using a custom - made PURExpress ® system with EF - G1 , EF - G2 or EF - G2 ( S84H ) . Shown is a representative from at least two independent experiments .

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E. Western blot analysis of reporter HisC-FLAG protein synthesized in vitro using a custom-made PURExpress® system with the EF-G1, EF-G2 or EF-G2(S84H) proteins. Shown is a representative from at least two independent experiments.


Western blot of B . thetaiotaomicron strains expressing C - terminally FLAG - tagged EF - G1 ( WH405 ) ( A ) , or EF - G2 ( GT1301 ) ( B ) , from their normal promoters chromosomal locations , grown in Tryptone Yeast Extract Glucose liquid medium ( TYG ) sampled at indicated times .

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Western blot analysis of B. thetaiotaomicron strains expressing C-terminally FLAG-tagged EF-G1 (WH405) (A), or EF-G2 (GT1301) (B), from their normal promoters and chromosomal locations, grown in Tryptone Yeast Extract Glucose liquid medium (TYG) and sampled at the indicated times.


E - F . Western blot of crude extracts from wild - type B . thetaiotaomicron harvested from ceca of mice ( representative blot for 2 out of 4 mice tested ) or corresponding to stationary phase cultures harvested following growth in TYG for 24 h or purified EF - G1 EF - G2 at indicated amounts ( E ) . Blots were developed with anti - EF - G1 or EF - G2 polyclonal antibodies ; anti - GroEL antibodies were used as loading controls . Western blot quantifications of four biological replicates their average are shown in ( F ) , where EF - G1 EF - G2 abundances were estimated by normalizing signal of sample to closest signal of known amount of purified , derived absolute amount ( pmol ) was normalized to signal of GroEL .

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

E-F. Western blot of crude extracts from wild-type B. thetaiotaomicron harvested from the ceca of mice (representative blot for 2 out of 4 mice tested) or corresponding to stationary phase cultures harvested following growth in TYG for 24 h or purified EF-G1 and EF-G2 proteins at the indicated amounts (E). Blots were developed with anti-EF-G1 or EF-G2 polyclonal antibodies; anti-GroEL antibodies were used as loading controls. Western blot quantifications of all four biological replicates and their average are shown in (F), where EF-G1 and EF-G2 abundances were estimated by normalizing the signal of bacterial sample to the closest signal of known amount of purified protein, and the derived absolute amount (pmol) was normalized to the signal of GroEL.


Three - dimensional structure of 2019 - nCoV Mpro , in two different views . One protomer of dimer is shown in blue , other one in orange . - acid residues of catalytic site are indicated as yellow blue spheres , for Cys145 His41 , respectively . Black spheres indicate positions of Ala285 of each of two domains III ( see text ) . Chain termini are labeled N C for molecule A ( blue ) N * C * for molecule B ( orange ) .

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

Three-dimensional structure of 2019-nCoV Mpro, in two different views. One protomer of the dimer is shown in light blue, the other one in orange. Amino-acid residues of the catalytic site are indicated as yellow and blue spheres, for Cys145 and His41, respectively. Black spheres indicate the positions of Ala285 of each of the two domains III (see text). Chain termini are labeled N and C for molecule A (light blue) and N* and C* for molecule B (orange).


Compound 13b in substrate - binding cleft located between domains I II of Mpro , in monoclinic crystal form ( space group C2 ) . 2Fo - Fc electron density is shown for inhibitor ( contouring level : 1σ ) . Carbon atoms of inhibitor are magenta , oxygens red , nitrogens blue , sulfur yellow . Note interaction between N - terminal residue of chain B , S1 * , E166 of chain A .

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Compound 13b in the substrate-binding cleft located between domains I and II of the Mpro, in the monoclinic crystal form (space group C2). 2Fo-Fc electron density is shown for the inhibitor (contouring level: 1σ). Carbon atoms of the inhibitor are magenta, oxygens red, nitrogens blue, and sulfur yellow. Note the interaction between the N-terminal residue of chain B, S1*, and E166 of chain A.


( C ) Western blot of STAT2 in mock - , WT - RCMV - E - , ΔE27 - RCMV - E - or UV - inactivated RCMV - E - infected REF . 24 h p . i . , were either left untreated or treated for 5 . 5 h with DMSO , 2 . 5 µM MLN4924 for blocking CRL activity or 10 µM MG132 for proteasome inhibition . IE1 β - tubulin served as infection loading controls , respectively .

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

(C) Western blot analysis of STAT2 protein levels in mock-, WT-RCMV-E-, ΔE27-RCMV-E- or UV-inactivated RCMV-E-infected REF. 24 h p.i., cells were either left untreated or treated for 5.5 h with DMSO, 2.5 µM MLN4924 for blocking CRL activity or 10 µM MG132 for proteasome inhibition. IE1 and β-tubulin served as infection and loading controls, respectively.


( A , B ) GF of in vitro reconstitutions containing indicated combinations . Coomassie blue - stained SDS - PAGE analyses of fractions collected during GF runs are shown next to chromatograms . red * indicates a contaminant , which we identified by cryo - EM as E . coli ArnA , a notorious contaminant in E . coli Ni - NTA purifications ( Andersen et al . , 2013 ) ( Appendix Fig S2 , S3 ) . Note that ArnA is present in GF fraction 6 ( Ve ~ 10 . 2 ml ) of runs containing E27 , migrates similarly to E27 on SDS - PAGE . * - additional contaminant from E27 preparation ; * * - contaminant from STAT2 preparation .

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

(A, B) GF analysis of in vitro reconstitutions containing the indicated protein combinations. Coomassie blue-stained SDS-PAGE analyses of fractions collected during the GF runs are shown next to the chromatograms. The red * indicates a contaminant, which we identified by cryo-EM analysis as E. coli ArnA, a notorious contaminant in E. coli Ni-NTA protein purifications (Andersen et al., 2013) (Appendix Fig S2, S3). Note that ArnA is present in GF fraction 6 (Ve ~ 10.2 ml) of all runs containing E27, and migrates similarly to E27 on the SDS-PAGE. * - additional contaminant from E27 preparation; ** - contaminant from STAT2 preparation.


( E ) Pre - formed DDB1 / DCAF1 was incubated with indicated amounts of E27 analysed by GF . SDS - PAGE analyses of GF fractions are shown next to chromatogram . red * indicates ArnA contaminant ( see Fig 2 ) . * - additional contaminant from E27 preparation .

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

(E) Pre-formed DDB1/DCAF1 protein complex was incubated with indicated amounts of E27 and analysed by GF. SDS-PAGE analyses of GF fractions are shown next to the chromatogram. The red * indicates the ArnA contaminant (see Fig 2). * - additional contaminant from E27 preparation.


( B ) 293T were transfected with expression plasmids encoding E27 - HA or indicated variants . 24 h after transfection , were lysed E27 - HA was precipitated using an HA - specific antibody . Precipitates were separated by SDS - PAGE , blotted , probed using indicated antibodies .

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(B) 293T cells were transfected with expression plasmids encoding E27-HA or indicated variants. 24 h after transfection, cells were lysed and E27-HA was precipitated using an HA-specific antibody. Precipitates were separated by SDS-PAGE, blotted, and probed using the indicated antibodies.


( E ) 293T were transfected with expression plasmids encoding M27 - FLAG or indicated M27 - FLAG variants . 24 h after transfection , were lysed M27 - FLAG was precipitated using a FLAG - specific antibody . Precipitates were separated by SDS - PAGE , blotted probed using indicated antibodies .

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

(E) 293T cells were transfected with expression plasmids encoding M27-FLAG or the indicated M27-FLAG variants. 24 h after transfection, cells were lysed and M27-FLAG was precipitated using a FLAG-specific antibody. Precipitates were separated by SDS-PAGE, blotted and probed using the indicated antibodies.


( F ) Mouse fibroblasts were infected with MCMV or indicated MCMV mutants . At indicated time points , were lysed , were separated by SDS - PAGE blotted . Blots were probed using indicated antibodies . pIE1 GAPDH were used as infection loading controls .

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

(F) Mouse fibroblasts were infected with MCMV or the indicated MCMV mutants. At the indicated time points, cells were lysed, proteins were separated by SDS-PAGE and blotted. Blots were probed using the indicated antibodies. pIE1 and GAPDH were used as infection and loading controls.


( G ) NIH3T3 reporter stably expressing ISRE - Luc were infected with indicated MCMV strains , treated with IFNβ or IFNλ , luciferase activity was measured . error bars represent standard deviation , N = 4 biological replicates , * * * - P ≤ 0 . 001 ( ANOVA ) .

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

(G) NIH3T3 reporter cells stably expressing ISRE-Luc were infected with the indicated MCMV strains, treated with IFNβ or IFNλ, and luciferase activity was measured. The error bars represent the standard deviation, N=4 biological replicates, *** - P ≤ 0.001 (ANOVA).


( H ) BALB / c mice were infected i . p . with WT - MCMV indicated MCMV mutants . At 3 21 d p . i . , salivary were collected frozen . titres were determined from organ homogenates by plaque titration . Titrations were performed in quadruplicates . Bars depict geometric mean , dots show titres of mice ( N = 5 biological replicates ) . DL - detection limit .

O O O O O O B-ORGANISM O O O O O O O O O B-ORGANISM O B-ORGANISM O O O O O O O O O O O B-TISSUE O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O

(H) BALB/c mice were infected i.p. with WT-MCMV and indicated MCMV mutants. At 3 and 21 d p.i., salivary glands were collected and frozen. Virus titres were determined from organ homogenates by plaque titration. Titrations were performed in quadruplicates. Bars depict the geometric mean, dots show titres of individual mice (N=5 biological replicates). DL - detection limit.


A . Western blot of mTORC1 activity in C2C12 - Vevtor ( VC ) Aster - C KO ( KO ) in complete medium ( CM ) in response to nutrient starvation or stimulation with for 30 min . B C . Statistical of phosphorylated level of S6K ( B ) 4E - BP1 ( C ) in C2C12 - Vector Aster - C KO in CM , under nutrient starvation ( - AA ) in response to stimulation with ( + AA ) using ImageJ software . Data are represented as mean ± SD ( n = 3 biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 by Student ' s t .

O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O B-CELL_LINE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O 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. Western blot analysis of mTORC1 activity in C2C12-Vevtor (VC) and Aster-C KO (KO) cells in complete medium (CM) and in response to nutrient starvation or stimulation with amino acids for 30 min. B and C. Statistical analysis of phosphorylated level of S6K (B) and 4E-BP1 (C) in C2C12-Vector and Aster-C KO cells in CM, under nutrient starvation (-AA) and in response to stimulation with amino acids (+AA) using ImageJ software. Data are represented as mean ± SD (n=3 biological replicates). *p < 0.05, **p<0.01 by Student's t test.


D . Immunostaining of endogenous mTOR LAMP1 in C2C12 - Vector Aster - C KO under nutrient starvation ( - AA ) in response to AA stimulation ( + AA ) . LAMP1 was immunostained as a lysosome marker . Arrows highlight co - localization of mTOR with LAMP1 . Scale bar , 40 μm ( Row 1 ) 10 μm ( Rows 2 , 3 , 4 ) .

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

D. Immunostaining of endogenous mTOR and LAMP1 in C2C12-Vector and Aster-C KO cells under nutrient starvation (-AA) and in response to AA stimulation (+AA). LAMP1 was immunostained as a lysosome marker. Arrows highlight the co-localization of mTOR with LAMP1. Scale bar, 40 μm (Row 1) and 10 μm (Rows 2, 3, 4).


E . Statistical of Pearson ' s correlation coefficient of mTOR LAMP1 in Figure 1F ( n = 12 - 15 per group ) . Data are represented as mean ± SD . * * * p < 0 . 001 by one way ANOVA .

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

E. Statistical analysis of the Pearson's correlation coefficient of mTOR and LAMP1 in Figure 1F (n=12-15 cells per group). Data are represented as mean ± SD. ***p<0.001 by one way ANOVA.


F . Western blot of mTORC1 activity in Aster - C KO stably re - expressing exogenous GFP - Aster - C under nutrient starvation AA stimulation . Aster - C KO were transfected with GFP - Aster - C selected with G418 for 2 .

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

F. Western blot analysis of mTORC1 activity in Aster-C KO cells stably re-expressing exogenous GFP-Aster-C under nutrient starvation and AA stimulation. Aster-C KO cells were transfected with GFP-Aster-C and selected with G418 for 2 weeks.


G . Immunostaining of endogenous mTOR LAMP1 in C2C12 - Vector Aster - C KO under cholesterol depletion by treatment with 0 . 5 % MCD for 2 hrs ( - CHOL ) or cholesterol ( 50 µM ) re - stimulation for 1 hr ( + CHOL ) . Arrows highlight co - localization of mTOR with LAMP1 . Scale bar , 40 μm ( Row 1 ) 10 μm ( Rows 2 , 3 , 4 ) .

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

G. Immunostaining of endogenous mTOR and LAMP1 in C2C12-Vector and Aster-C KO cells under cholesterol depletion by treatment with 0.5% MCD for 2 hrs (-CHOL) or cholesterol (50 µM) re-stimulation for 1 hr (+CHOL). Arrows highlight the co-localization of mTOR with LAMP1. Scale bar, 40 μm (Row 1) and 10 μm (Rows 2, 3, 4).


H . Western blot of mTORC1 activity in C2C12 - Vector Aster - C KO in CM , under starvation , cholesterol depletion by treatment with 0 . 5 % MCD for 2 hrs or cholesterol ( 50 µM ) re - stimulation for indicated time .

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

H. Western blot analysis of mTORC1 activity in C2C12-Vector and Aster-C KO cells in CM, under starvation, cholesterol depletion by treatment with 0.5% MCD for 2 hrs or cholesterol (50 µM) re-stimulation for the indicated time.


A . Confocal image depicting co - localization of Aster - C with ER in C2C12 transiently expressing GFP - Aster - C DsRed - ER under starvation in response to AA stimulation . Scale bar , 5 μm .

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

A. Confocal image analysis depicting the co-localization of Aster-C with the ER in C2C12 cells transiently expressing GFP-Aster-C and DsRed-ER under starvation and in response to AA stimulation. Scale bar, 5 μm.


B . Subcellular fractionation of mTOR , GATOR2 Aster - C localization in HEK293T transiently expressing FLAG - Aster - C under starvation in response to AA stimulation . LAMP1 , Sec63 GAPDH were used as lysosomal ( Lyso ) , rough ER ( RER ) cytosol ( Cyto ) markers , respectively . Microsomal fraction ( Micro ) was also used in blot .

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B. Subcellular fractionation analysis of mTOR, GATOR2 complex and Aster-C localization in HEK293T cells transiently expressing FLAG-Aster-C under starvation and in response to AA stimulation. LAMP1, Sec63 and GAPDH were used as lysosomal (Lyso), rough ER (RER) and cytosol (Cyto) protein markers, respectively. Microsomal fraction (Micro) was also used in the blot.


C . Subcellular fractionation of mTOR GATOR2 in C2C12 - Vector Aster - C KO under starvation in response to AA stimulation . LAMP1 , Sec63 GAPDH were used as lysosomal ( Lyso ) , rough ER ( RER ) cytosol ( Cyto ) markers , respectively .

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

C. Subcellular fractionation analysis of mTOR and GATOR2 complex in C2C12-Vector and Aster-C KO cells under starvation and in response to AA stimulation. LAMP1, Sec63 and GAPDH were used as lysosomal (Lyso), rough ER (RER) and cytosol (Cyto) protein markers, respectively.


D . Co - IP of interaction of Aster - C with exogenous mTOR in response to starvation AA or insulin stimulation in HEK293T transiently expressing indicated . Anti - Myc antibody was used for immunoprecipitation .

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

D. Co-IP analysis of the interaction of Aster-C with exogenous mTOR in response to starvation and AA or insulin stimulation in HEK293T cells transiently expressing the indicated proteins. Anti-Myc antibody was used for immunoprecipitation.


E . Co - IP of interaction of Aster - C with endogenous mTOR GATOR2 using FLAG - Sestrin2 ( SESN2 ) as a positive control in HEK293T transiently expressing indicated . Anti - FLAG antibody was used for immunoprecipitation .

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

E. Co-IP analysis of the interaction of Aster-C with endogenous mTOR and the GATOR2 complex using FLAG-Sestrin2 (SESN2) as a positive control in HEK293T cells transiently expressing the indicated proteins. Anti-FLAG antibody was used for immunoprecipitation.


F . Co - IP of interaction of Aster - C with mTOR , GATOR2 , GATOR1 Ragulator - Rag using FLAG - DEPDC5 as a positive control in HEK293T transiently expressing indicated . Anti - FLAG antibody was used for immunoprecipitation . Black arrow indicates RagC .

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

F. Co-IP analysis of the interaction of Aster-C with mTOR, the GATOR2 complex, GATOR1 complex and the Ragulator-Rag complex using FLAG-DEPDC5 as a positive control in HEK293T cells transiently expressing the indicated proteins. Anti-FLAG antibody was used for immunoprecipitation. Black arrow indicates the RagC protein.


A . Co - IP of interaction of Aster - C with COPA COPG in response to starvation AA stimulation using FLAG - DEPDC5 as a negative control in HEK293T transiently expressing indicated . Anti - FLAG antibody was used for immunoprecipitation . Black arrow indicates COPG .

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

A. Co-IP analysis of the interaction of Aster-C with COPA and COPG in response to starvation and AA stimulation using FLAG-DEPDC5 as a negative control in HEK293T cells transiently expressing the indicated proteins. Anti-FLAG antibody was used for immunoprecipitation. Black arrow indicates the COPG protein.


B . Co - IP of interaction of COPA with mTOR in HEK293T transiently expressing GFP - COPA in response to starvation AA stimulation . Anti - GFP antibody was used for immunoprecipitation . Black arrow indicates COPG .

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

B. Co-IP analysis of the interaction of COPA with mTOR in HEK293T cells transiently expressing GFP-COPA in response to starvation and AA stimulation. Anti-GFP antibody was used for immunoprecipitation. Black arrow indicates the COPG protein.


C . Co - IP of interaction of COPA with mTOR in C2C12 - Vector Aster - C KO in response to starvation AA stimulation . Anti - COPA antibody was used for immunoprecipitation .

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

C. Co-IP analysis of the interaction of COPA with mTOR in C2C12-Vector and Aster-C KO cells in response to starvation and AA stimulation. Anti-COPA antibody was used for immunoprecipitation.


D . Confocal imaging depicting co - localization of YFP - mTOR ( yellow ) with GFP - COPA ( green ) lysosomes ( red ) in live C2C12 - Vector Aster - C KO in response to nutrient starvation re - stimulation by AA . Arrows highlight co - localization of mTOR , COPA lysosomes . Scale bar , 20 μm . E F . Statistical of Pearson ' s correlation coefficient of mTOR / Lysosome ( E ) COPA / Lysosome ( F ) in Figure 3D ( n = 10 - 12 per group ) . Data are represented as mean ± SD . * * * p < 0 . 001 by one - way ANOVA .

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

D. Confocal imaging analysis depicting the co-localization of YFP-mTOR (yellow) with GFP-COPA (green) and lysosomes (red) in live C2C12-Vector and Aster-C KO cells in response to nutrient starvation and re-stimulation by AA. Arrows highlight co-localization of mTOR, COPA and lysosomes. Scale bar, 20 μm. E and F. Statistical analysis of the Pearson's correlation coefficient of mTOR/Lysosome (E) and COPA/Lysosome (F) in Figure 3D (n=10-12 cells per group). Data are represented as mean ± SD. ***p<0.001 by one-way ANOVA.


Time - lapse confocal imaging of co - localization of YFP - mTOR ( red ) with GFP - COPA ( green ) lysosomes ( blue ) in live C2C12 under nutrient starvation ( 0 min ) , in response to AA re - stimulation for indicated time ( 0 . 5 min , 2 min , 6 min 8 min ) . Arrows highlight COPA that were co - localized with mTOR or COPA alone prior to their association with mTOR lysosomes . . Scale bar , 5 μm .

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

Time-lapse confocal imaging analysis of the co-localization of YFP-mTOR (red) with GFP-COPA (green) and lysosomes (blue) in live C2C12 cells under nutrient starvation (0 min), and in response to AA re-stimulation for indicated time (0.5 min, 2 min, 6 min and 8 min). Arrows highlight COPA puncta that were co-localized with mTOR or COPA puncta alone prior to their association with mTOR and lysosomes.. Scale bar, 5 μm.


A . Confocal imaging depicting lysosomal association of mTOR in response to AA stimulation in presence or absence of BFA ( 10 μM ) or Exo - 2 ( 10 μM ) in live C2C12 transiently expressing YFP - mTOR stained with Lysotracker Red . Scale bar , 20 μm .

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

A. Confocal imaging analysis depicting the lysosomal association of mTOR in response to AA stimulation in the presence or absence of BFA (10 μM) or Exo-2 (10 μM) in live C2C12 cells transiently expressing YFP-mTOR and stained with Lysotracker Red. Scale bar, 20 μm.


Western blot of mTORC1 activity in C2C12 in CM , in response to nutrient starvation AA stimulation in presence or absence of BFA ( 10 μM ) ( B )

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

Western blot analysis of mTORC1 activity in C2C12 cells in CM, in response to nutrient starvation and AA stimulation in the presence or absence of BFA (10 μM) (B)


C . Western blot of mTORC1 activity in C2C12 in CM , in response to nutrient starvation AA stimulation in presence or absence of Exo - 2 ( 10 μM ) ( C ) .

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

C. Western blot analysis of mTORC1 activity in C2C12 cells in CM, in response to nutrient starvation and AA stimulation in the presence or absence of Exo-2 (10 μM) (C).


D . Western blot of mTORC1 activity in C2C12 - Vector ARF1 KO in CM , in response to nutrient starvation AA stimulation .

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

D. Western blot analysis of mTORC1 activity in C2C12-Vector and ARF1 KO cells in CM, in response to nutrient starvation and AA stimulation.


E . Confocal imaging depicting COPA ( green ) formation co - localization with lysosomes ( red ) in live C2C12 transiently expressing GFP - COPA stained with Lysotracker Red in response to nutrient starvation AA stimulation in presence or absence of BFA ( 10 μM ) or Exo - 2 ( 10 μM ) . Arrows highlight co - localization of COPA with lysosomes . Scale bar , 5 μm .

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

E. Confocal imaging analysis depicting COPA puncta (green) formation and co-localization with lysosomes (red) in live C2C12 cells transiently expressing GFP-COPA and stained with Lysotracker Red in response to nutrient starvation and AA stimulation in the presence or absence of BFA (10 μM) or Exo-2 (10 μM). Arrows highlight co-localization of COPA with lysosomes. Scale bar, 5 μm.


F . Statistical of Pearson ' s correlation coefficient of COPA lysosomes in Figure 5E ( n = 10 - 15 per group ) . Data are represented as mean ± SD . * * * p < 0 . 001 by one - way ANOVA .

O O O O O O O O O O B-GENEPROD B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Statistical analysis of the Pearson's correlation coefficient of COPA and lysosomes in Figure 5E (n=10-15 cells per group). Data are represented as mean ± SD. ***p<0.001 by one-way ANOVA.


A . Co - IP of interaction of Aster - C with COPA , MYH10 , WDR24 Mios in HEK293T transiently expressing indicated in response to treatment with brefeldin A ( BFA , 10 μM ) / or blebbistatin ( BBS , 10 μM ) , inhibitors of ARFs MYH10 , respectively . Anti - FLAG antibody was used for immunoprecipitation .

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

A. Co-IP analysis of the interaction of Aster-C with COPA, MYH10, WDR24 and Mios in HEK293T cells transiently expressing the indicated proteins in response to treatment with brefeldin A (BFA, 10 μM) and/or blebbistatin (BBS, 10 μM), inhibitors of ARFs and MYH10, respectively. Anti-FLAG antibody was used for immunoprecipitation.


B . Western blot of mTORC1 activity in wild type ( WT ) MYH10 KO MEF cultured in CM , or in response to starvation AA stimulation .

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

B. Western blot analysis of mTORC1 activity in wild type (WT) and MYH10 KO MEF cells cultured in CM, or in response to starvation and AA stimulation.


C . Western blot of mTORC1 activity in MYH10 KO MEF with stably re - expressed exogenous mCherry - MYH10 in response to starvation AA stimulation . MYH10 KO MEF were transfected with mCherry - MYH10 selected with G418 for 2 .

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

C. Western blot analysis of mTORC1 activity in MYH10 KO MEF cells with stably re-expressed exogenous mCherry-MYH10 in response to starvation and AA stimulation. MYH10 KO MEF cells were transfected with mCherry-MYH10 and selected with G418 for 2 weeks.


D . Confocal imaging depicting COPA ( green ) formation co - localization with mTOR ( red ) in live C2C12 transiently expression GFP - COPA YFP - mTOR in response to nutrient starvation in presence or absence of BBS ( 10 µM ) . Arrows highlight co - localization of mTOR with COPA . Scale bar , 20 μm .

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

D. Confocal imaging analysis depicting COPA puncta (green) formation and co-localization with mTOR (red) in live C2C12 cells transiently expression GFP-COPA and YFP-mTOR in response to nutrient starvation in the presence or absence of BBS (10 µM). Arrows highlight the co-localization of mTOR with COPA. Scale bar, 20 μm.


E . Statistical of Pearson ' s correlation coefficient of mTOR COPA in Figure 6D ( n = 10 - 12 per group ) . Data are represented as mean ± SD . * * * p < 0 . 001 by student ' s t .

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

E. Statistical analysis of the Pearson's correlation coefficient of mTOR and COPA in Figure 6D (n=10-12 cells per group). Data are represented as mean ± SD. ***p<0.001 by student's t test.


A . Confocal imaging depicting mTOR ( green ) formation on actin filaments ( red ) in COS - 7 transiently expressing YFP - mTOR in response to AA stimulation in presence or absence of latrunculin B ( LA - B , 100 nM ) . Actin filaments were stained using BODIPY ™ 558 / 568 - Phalloidin . Arrows indicated association of mTOR with actin filaments . Scale bar , 5 μm .

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

A. Confocal imaging analysis depicting mTOR puncta (green) formation on actin filaments (red) in COS-7 cells transiently expressing YFP-mTOR in response to AA stimulation in the presence or absence of latrunculin B (LA-B, 100 nM). Actin filaments were stained using BODIPY™ 558/568-Phalloidin. Arrows indicated the association of mTOR with actin filaments. Scale bar, 5 μm.


B . Immunostaining of endogenous mTOR LAMP1 in C2C12 under starvation ( - AA ) , or in response to AA stimulation ( + AA ) in presence or absence of LA - B ( 100 nM ) . Scale bar , 20 μm .

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

B. Immunostaining of endogenous mTOR and LAMP1 in C2C12 cells under amino acids starvation (-AA), or in response to AA stimulation (+AA) in the presence or absence of LA-B (100 nM). Scale bar, 20 μm.


C . Western blot of mTORC1 activity in C2C12 in CM , in response to nutrient starvation AA stimulation in presence or absence of LA - B ( 100 nM ) .

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

C. Western blot analysis of mTORC1 activity in C2C12 cells in CM, in response to nutrient starvation and AA stimulation in the presence or absence of LA-B (100 nM).


D . Co - IP of interaction of Aster - C with COPA MYH10 in presence or absence of LA - B ( 100 nM ) or cytochalasin D ( Cyto - D , 250 nM ) in HEK293T transiently expressing FLAG - Aster - C . Anti - FLAG antibody was used for immunoprecipitation .

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

D. Co-IP analysis of the interaction of Aster-C with COPA and MYH10 in the presence or absence of LA-B (100 nM) or cytochalasin D (Cyto-D, 250 nM) in HEK293T cells transiently expressing FLAG-Aster-C. Anti-FLAG antibody was used for immunoprecipitation.


A - Distribution of Casp3 + in rostral ventral domains of an E11 . 5 Rx - Dicer mutant . NCx , neocortex ; OB , olfactory bulb ; Spt , septum .

O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE O

A - Distribution of Casp3+ cells in rostral and ventral domains of an E11.5 Rx-Dicer mutant embryo. NCx, neocortex; OB, olfactory bulb; Spt, septum.


B , C - Marker of Casp3 + in rostral telencephalon of E12 . 5 Rx - Dicer mutant . Most apoptotic are Pax6 + RGCs ( solid arrowheads ) not Tbr1 + neurons ( open arrowheads ) . N = 3 replicates per marker .

O O O O O O B-GENEPROD O O B-TISSUE I-TISSUE O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-CELL_TYPE O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O

B,C - Marker analysis of Casp3+ cells in the rostral telencephalon of E12.5 Rx-Dicer mutant embryos. Most apoptotic cells are Pax6+ RGCs (solid arrowheads) and not Tbr1+ neurons (open arrowheads). N = 3 replicates per marker.


D , E - Distribution abundance of apoptotic ( Casp3 + ) in rostral telencephalic primordium of control Rx - Dicer mutant at indicated ages . Dotted indicates basal surface , dashed apical surface . N = 3 replicates per genotype .

O O O O O O O O O B-GENEPROD O O O B-TISSUE I-TISSUE I-TISSUE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

D,E - Distribution and abundance of apoptotic cells (Casp3+) in the rostral telencephalic primordium of control and Rx-Dicer mutant embryos at the indicated ages. Dotted line indicates basal surface, dashed line apical surface. N = 3 replicates per genotype and age.


F , G - Distribution abundance of mitoses ( PH3 + ) neurons ( Tuj1 ) in rostral telencephalic primordium of control Rx - Dicer mutant at indicated ages . Dotted indicates basal surface , dashed apical surface . N = 3 - 4 replicates per genotype . No significant differences were found between control mutant in apical nor basal mitoses .

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

F,G - Distribution and abundance of mitoses (PH3+) and neurons (Tuj1) in the rostral telencephalic primordium of control and Rx-Dicer mutant embryos at the indicated ages. Dotted line indicates basal surface, dashed line apical surface. N = 3-4 replicates per genotype and age. No significant differences were found between control and mutant embryos in apical nor basal mitoses.


A - DAPI stain of sagittal sections through rostral telencephalon of E17 . 5 control Rx - Dicer mutant showing neocortex ( NCx ) olfactory bulb ( OB ; dashed ) .

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

A - DAPI stain of sagittal sections through the rostral telencephalon of E17.5 control and Rx-Dicer mutant littermates showing the neocortex (NCx) and olfactory bulb (OB; dashed line).


B - Expression pattern of Grm1 mRNA in OB ( dotted ) of control Rx - Dicer mutants . C - of OB perimeter ( mean ± ; symbols indicate values for ) ; t - , * * * P < 0 . 001 . N = 14 replicates per genotype .

O O B-EXP_ASSAY O O B-GENEPROD O O B-TISSUE O O O O O B-GENEPROD O B-GENEPROD O O O O O B-TISSUE O O O O O O 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 - Expression pattern of Grm1 mRNA in OB (dotted line) of control and Rx-Dicer mutants. C - Quantification of OB perimeter (mean ± SEM; symbols indicate values for individual embryos); t-test, ***P < 0.001. N = 14 replicates per genotype.


D - Immunostains of E17 . 5 control Rx - Dicer mutant brains showing distribution of progenitor ( Ki67 , red ) neurons ( Tuj1 , green ) . Arrowheads indicate rosettes . Sp , septum .

O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O B-TISSUE O O O O O B-GENEPROD O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O B-TISSUE O B-TISSUE O

D - Immunostains of E17.5 control and Rx-Dicer mutant brains showing the distribution of progenitor cells (Ki67, red) and neurons (Tuj1, green). Arrowheads indicate rosettes. Sp, septum.


E - Detail of a rosette from an E17 . 5 Rx - Dicer mutant displaying typical features : closed apical surface ( dotted ) with PH3 + apical mitoses ( arrowhead ) basal mitoses , surrounded by Tuj1 + neurons ( green ) . Dashed indicates basal border of rosette .

O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O

E - Detail of a rosette from an E17.5 Rx-Dicer mutant embryo displaying typical features: closed apical surface (dotted line) with PH3+ apical mitoses (white arrowhead) and basal mitoses, surrounded by Tuj1+ neurons (green). Dashed line indicates the basal border of the rosette.


F - Rostral half of an Rx - Dicer mutant E17 . 5 brain immunostained for Pax6 , clarified segmented to reveal rosettes ( yellow ) . C , caudal ; R , rostral .

O O O O O O B-GENEPROD O B-GENEPROD O O O O B-TISSUE B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O

F - Rostral half of an Rx-Dicer mutant E17.5 brain immunostained for Pax6, clarified and segmented to reveal rosettes (yellow). C, caudal; R, rostral.


G , H - BrdU incorporation cycle re - entry with progenitor marker Ki67 at E17 . 5 , in rostral cortex of control compared to rostral rosettes of Rx - Dicer mutants . Dotted lines indicate apical surface , dashed lines indicate basal border of VZ . Arrowheads indicate double - positive . Data in histograms are mean ± , symbols indicate values for ; n ≥ 3 ; X2 - , * P < 0 . 05 , * * * P < 0 . 001 .

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

G,H - BrdU incorporation and cell cycle re-entry analysis with the progenitor cell marker Ki67 at E17.5, in the rostral cortex of control embryos compared to rostral rosettes of Rx-Dicer mutants. Dotted lines indicate apical surface, dashed lines indicate basal border of VZ. Arrowheads indicate double-positive cells. Data in histograms are mean ± SEM, symbols indicate values for individual embryos; n ≥ 3 embryos; X2-test, *P < 0.05, ***P < 0.001.


I - Apical lumen of rosettes immunostained against apical ( Par3 , β - Catenin ) , cilia ( Arl13b ) , apical mitoses ( PhVim ) neurons ( Tuj1 ) .

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

I - Apical lumen of rosettes immunostained against apical complex proteins (Par3, β-Catenin), primary cilia (Arl13b), apical mitoses (PhVim) and neurons (Tuj1).


J - Coronal section through rostral telencephalon of an E14 . 5 Rx - Dicer mutant illustrating high abundance location of rosettes ( arrows ) , as revealed by distribution of mitoses ( PH3 ) neurons ( Tuj1 ) . K - S - of regional identity of rosettes in E14 . 5 Rx - Dicer mutants . ( K ) Schema of normal transcription factor expression patterns defining telencephalic regional identity . Expression of Ngn2 , Tbr2 Pax6 identifies rosettes in rostro - dorsal telencephalon as having dorsal identity ( L - N , P ) ; Gsx2 , Dlx2 Dlx5 identify rosettes in ventral telencephalon as having ventral identity ( O , Q ) ; Nkx2 . 1 identifies MGE rosettes as being normotopic ( R ) ; absence of Gsx2 in LGE rosette identifies them as ectopic ( S ) . Tuj1 labels neurons . In ( L , M ) , arrowheads indicate border between dorsal ventral territories , dotted indicates outer border of telencephalon . LGE , lateral ganglionic eminence ; MGE , medial ganglionic eminence ; P , pallium ; SP , subpallium .

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

J - Coronal section through the rostral telencephalon of an E14.5 Rx-Dicer mutant embryo illustrating the high abundance and location of rosettes (arrows), as revealed by the distribution of mitoses (PH3) and neurons (Tuj1). K-S - Analysis of the regional identity of rosettes in E14.5 Rx-Dicer mutants. (K) Schema of normal transcription factor expression patterns defining telencephalic regional identity. Expression of Ngn2, Tbr2 and Pax6 identifies rosettes in the rostro-dorsal telencephalon as having dorsal identity (L-N,P); Gsx2, Dlx2 and Dlx5 identify rosettes in the ventral telencephalon as having ventral identity (O,Q); Nkx2.1 identifies MGE rosettes as being normotopic (R); absence of Gsx2 in LGE rosette cells identifies them as ectopic (S). Tuj1 labels neurons. In (L,M), arrowheads indicate the border between dorsal and ventral territories, and dotted line indicates the outer border of the telencephalon. LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; P, pallium; SP, subpallium.


A - Coronal section through rostral telencephalon of an E14 . 5 Rx - Dicer mutant displaying three types of rosettes .

O O O O O B-TISSUE I-TISSUE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O

A - Coronal section through the rostral telencephalon of an E14.5 Rx-Dicer mutant embryo displaying the three types of rosettes.


B - High magnification of rosettes in Rx - Dicer mutants at E12 . 5 ( Type 1 ) , E14 . 5 ( Type 2 ) E17 . 5 ( Type 3 ) , immunostained for PH3 Tuj1 . Dashed indicates ventricular surface ; arrowheads indicate basal mitoses ; arrows indicate a stream of apical mitoses connecting lumen of rosette with lumen of telencephalic ventricle .

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

B - High magnification of rosettes in Rx-Dicer mutants at E12.5 (Type 1), E14.5 (Type 2) and E17.5 (Type 3), immunostained for PH3 and Tuj1. Dashed line indicates ventricular surface; arrowheads indicate basal mitoses; arrows indicate a stream of apical mitoses connecting the lumen of the rosette with the lumen of the telencephalic ventricle.


C , D - Abundance of rosette types at indicated ages ( C ) , rostro - caudal distribution of total rosette abundance per , independent of type ( D ) . n ≥ 2 brains per .

O O O O O O O O O O O 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-TISSUE O O

C,D - Abundance of rosette types at the indicated ages (C), and rostro-caudal distribution of total rosette abundance per age, independent of type (D). n ≥ 2 brains per age.


F , G - Distribution of neurons ( Tuj1 + ) apical adherens junction Par3 in OB primordium of control Rx - Dicer mutant at indicated ages . Dotted lines indicate basal border , dashed lines indicate apical border . arrowheads indicate ectopic neurons / or absence of Par3 . Arrow in ( G ) indicates accumulation of Par3 at lumen of a nascent rosette .

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

F,G - Distribution of neurons (Tuj1+ cells) and apical adherens junction protein Par3 in the OB primordium of control and Rx-Dicer mutant embryos at the indicated ages. Dotted lines indicate basal border, dashed lines indicate apical border. White arrowheads indicate ectopic neurons and/or absence of Par3. Arrow in (G) indicates accumulation of Par3 at the lumen of a nascent rosette.


A , B - Changes in expression of miRNAs between control Rx - Dicer mutants at E11 . 5 in rostral telencephalon . Red dots indicate statistically differentially expressed miRNAs according to FDR correction of P values on Wald statistic ( DESeq2 ) ( B ) . Dashes lines indicate stringency limits in evaluation of results .

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

A,B - Changes in expression levels of miRNAs between control and Rx-Dicer mutants at E11.5 in the rostral telencephalon. Red dots indicate statistically differentially expressed miRNAs according to FDR correction of P values based on the Wald statistic (DESeq2 analysis) (B). Dashes lines indicate stringency limits in evaluation of results.


D , E - Changes in mRNA expression between control Rx - Dicer mutants at E11 . 5 in rostral telencephalon . Statistically differentially expressed mRNAs according to FDR correction of P values on Wald statistic ( DESeq2 ; Adj . P < 0 . 1 FC > 1 . 25 ) are highlighted in red , three with highest Adj . P value are identified by name . Blue dot indicates Irs2 : FC = 1 . 748 , Adj . P = 5 . 95e - 08 .

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

D,E - Changes in mRNA expression levels between control and Rx-Dicer mutants at E11.5 in the rostral telencephalon. Statistically differentially expressed mRNAs according to FDR correction of P values based on the Wald statistic (DESeq2 analysis; Adj. P < 0.1 and FC > 1.25) are highlighted in red, and the three with the highest Adj. P value are identified by name. Blue dot indicates Irs2: FC=1.748, Adj. P = 5.95e-08.


G - Heatmaps of relative expression of DEGs related to apoptosis ( top ) proliferation ( bottom ) . Rows correspond to independent biological replicates . Indicated are some example genes .

O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O

G - Heatmaps of relative expression levels of DEGs related to apoptosis (top) and proliferation (bottom). Rows correspond to independent biological replicates. Indicated are some example genes.


H - Enrichment plots from GSEA for MSigDB Hallmark Neurogenesis ( NES = 2 . 98 ; p = 0 ; Adj . P = 0 . 00045 ) , Regulation of proliferation ( NES = 2 . 40 ; p = 0 ; Adj . P = 0 . 0033 ) , Positive regulation of adhesion ( NES = 2 . 06 ; p = 0 . 0058 ; Adj . P = 0 . 013 ) , Regulation of response to stress ( NES = 2 . 50 ; p = 0 ; Adj . P = 0 . 0023 ) , Positive regulation of ( NES = 1 . 84 ; p = 0 . 0179 ; Adj . P = 0 . 0326 ) , Apoptotic signaling pathway ( NES = 1 . 59 ; p = 0 . 047 ; Adj . P = 0 . 084 ) .

O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O

H - Enrichment plots from GSEA for MSigDB Hallmark Neurogenesis (NES = 2.98; p = 0; Adj. P = 0.00045), Regulation of cell proliferation (NES = 2.40; p=0; Adj. P = 0.0033), Positive regulation of cell adhesion (NES = 2.06; p = 0.0058; Adj. P = 0.013), Regulation of response to stress (NES = 2.50; p = 0; Adj. P = 0.0023), Positive regulation of cell death (NES = 1.84; p = 0.0179; Adj. P = 0.0326), Apoptotic signaling pathway (NES = 1.59; p= 0.047; Adj. P = 0.084).


I , J - ISH stains of Irs2 mRNA , qPCR for Irs2 mRNA let - 7 - 5p miRNA , in rostral telencephalon of control Rx - Dicer mutant . Dashed indicates basal border , dotted indicates apical surface . Arrowheads indicate with greatest increase in Irs2 expression . BG , basal ganglia ; H , hippocampus ; NCx , neocortex ; OB , olfactory bulb ; Spt , septum . Histograms represent mean ± ( logarithmic scale ) ; symbols in plots indicate values for ; t - , * P < 0 . 05 , * * P < 0 . 01 . N = 6 - 9 replicates per group . Scale bar , 100 µm .

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

I,J - ISH stains of Irs2 mRNA, and qPCR for Irs2 mRNA and let-7-5p miRNA, in the rostral telencephalon of control and Rx-Dicer mutant embryos. Dashed line indicates basal border, dotted line indicates apical surface. Arrowheads indicate area with the greatest increase in Irs2 expression. BG, basal ganglia; H, hippocampus; NCx, neocortex; OB, olfactory bulb; Spt, septum. Histograms represent mean ± SEM (logarithmic scale); symbols in plots indicate values for individual embryos; t-test, *P <0.05, **P <0.01. N = 6-9 replicates per group. Scale bar, 100 µm.


A - C - Immunostains for activated p53 ( red ) in rostral telencephalon of control Rx - Dicer mutant at E12 . 5 , of positive ( C ) . Dashed lines indicate apical side , dotted lines indicate basal side . N = 4 replicates per genotype . Scale bar , 50µm .

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

A-C - Immunostains for activated p53 (red) in the rostral telencephalon of control and Rx-Dicer mutant embryos at E12.5, and quantification of positive cells (C). Dashed lines indicate apical side, dotted lines indicate basal side. N = 4 replicates per genotype. Scale bar, 50µm.


D - L - Rostral telencephalon of E12 . 5 of indicated genotypes immunostained for detection of apoptotic ( Casp3 ) , neurons ( Tuj1 ) apical Par3 , of positive for Casp3 ( H ) . N = 4 - 7 replicates per genotype . Scale bar , 50 µm .

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

D-L - Rostral telencephalon of E12.5 embryos of the indicated genotypes immunostained for the detection of apoptotic cells (Casp3), neurons (Tuj1) and the apical complex protein Par3, and quantification of cells positive for Casp3 (H). N = 4-7 replicates per genotype. Scale bar, 50 µm.


M - O - Sagittal sections through rostral telencephalon of E17 . 5 of indicated genotypes . Note rosettes general disorganization in Rx - Dicer single mutants , complete rescue of this phenotype in Rx - Dicer - p53 double mutants . NCx , neocortex ; OB , olfactory bulb . Scale bar , 1 mm .

O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O

M-O - Sagittal sections through the rostral telencephalon of E17.5 embryos of the indicated genotypes. Note the rosettes and general disorganization in Rx-Dicer single mutants, and complete rescue of this phenotype in Rx-Dicer-p53 double mutants. NCx, neocortex; OB, olfactory bulb. Scale bar, 1 mm.


A - Relative Irs2 mRNA in HEK upon transfection with Gfp - or Irs2 - encoding plasmids . Mean ± ; t - , * * * * P < 0 . 0001 . N = 6 replicates per group .

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

A - Relative Irs2 mRNA levels in HEK cells upon transfection with Gfp- or Irs2-encoding plasmids. Mean ± SEM; t-test, ****P < 0.0001. N = 6 replicates per group.


B - D - Distribution of neurons ( Tuj1 , red ) , GFP ( green ) mitoses ( PH3 , ) in rostral telencephalon of wild - type E14 . 5 or E17 . 5 mouse , as indicated , electroporated at E12 . 5 with indicated plasmid combinations . Dashed indicates apical surface . Insets show same field of view , with location extent of electroporations . Note overlap of rosettes ( arrows ) with ectopic neurons at apical surface ( arrowheads ) increased basal mitoses , upon overexpression of Irs2 ( C ) . Asterisk indicates absence of malformation in nearby cortex . At E17 . 5 ( D ) GFP + rosettes remain in deep cortical layers ( arrowheads ) .

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

B-D - Distribution of neurons (Tuj1, red), GFP (green) and mitoses (PH3, white) in the rostral telencephalon of wild-type E14.5 or E17.5 mouse embryos, as indicated, electroporated at E12.5 with the indicated plasmid combinations. Dashed line indicates the apical surface. Insets show the same field of view, with location and extent of electroporations. Note the overlap of rosettes (arrows) with ectopic neurons at the apical surface (arrowheads) and increased basal mitoses, upon overexpression of Irs2 (C). Asterisk indicates absence of malformation in nearby cortex. At E17.5 (D) GFP+ rosettes remain in deep cortical layers (arrowheads).


E - H - Details of rosettes ( large arrows in E ; dashed lines in F - H ) from Irs2 + Gfp - overexpressing E14 . 5 immunostained as indicated . Small arrows indicate apical mitoses , arrowheads indicate basal mitoses ( F , G ) Par3 + apical surface at centre of rosettes ( H ) , dotted lines indicate apical surface , dashed lines indicate basal side of rosettes . Inset in ( H ) is at same scale .

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

E-H - Details of rosettes (large arrows in E; dashed lines in F-H) from Irs2+Gfp-overexpressing E14.5 embryos immunostained as indicated. Small arrows indicate apical mitoses, arrowheads indicate basal mitoses (F,G) and Par3+ apical surface at the centre of rosettes (H), dotted lines indicate apical surface, dashed lines indicate basal side of rosettes. Inset in (H) is at the same scale.


I - M - Distribution of neurons ( Tuj1 , red ) , GFP ( green ) mitotic ( PH3 , ) or apoptotic ( Casp3 , ) in rostral telencephalon of wild - type E13 . 5 electroporated at E12 . 5 with indicated plasmids , of Casp3 + ( M ) . Dashed indicates apical surface , dotted lines indicate borders of cortical plate ( CP ) . Nascent rosettes ( arrows ) are next to ectopic ventricular neurons ( arrowheads in K ' ) basal mitoses , without significant apoptosis in VZ ( L ' , M ) . Mean ± ; t - ; ns , not significant , * P < 0 . 05 . N = 3 - 6 replicates per group . N - Distribution of neurons ( Tuj1 , red ) in rostral telencephalon of wild - type E14 . 5 electroporated at E12 . 5 with a combination of plasmids encoding Irs2 , let - 7a , let - 7b let - 7c , Gfp . Dashed indicates apical surface . Formation of rosettes in Irs2 - expressing is rescued by overexpression of let - 7 . Inset shows same field of view , with location extent of electroporation .

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

I-M - Distribution of neurons (Tuj1, red), GFP (green) and mitotic (PH3, white) or apoptotic (Casp3, white) cells in the rostral telencephalon of wild-type E13.5 embryos electroporated at E12.5 with the indicated plasmids, and quantification of Casp3+ cells (M). Dashed line indicates apical surface, dotted lines indicate borders of the cortical plate (CP). Nascent rosettes (arrows) are next to ectopic ventricular neurons (arrowheads in K') and basal mitoses, without significant apoptosis in VZ (L',M). Mean ± SEM; t-test; ns, not significant, *P < 0.05. N = 3-6 replicates per group. N - Distribution of neurons (Tuj1, red) in the rostral telencephalon of wild-type E14.5 embryos electroporated at E12.5 with a combination of plasmids encoding Irs2, let-7a, let-7b and let-7c, and Gfp. Dashed line indicates apical surface. Formation of rosettes in Irs2-expressing embryos is rescued by overexpression of let-7. Inset shows the same field of view, with location and extent of electroporation.


A - Frequency of rosette formation in experimental conditions indicated . n , number of per condition .

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

A - Frequency of rosette formation in the experimental conditions indicated. n, number of embryos per condition.


B - G - Distribution of neurons ( Tuj1 , red ) , GFP ( green ) mitoses ( PH3 , ) in rostral telencephalon of wild - type E14 . 5 mouse electroporated at E12 . 5 with indicated plasmid combinations . Inset shows same field of view , with location extent of electroporations . In ( C ) ( E ) , arrows indicate rosettes arrowheads indicate neuronal ventricular ectopias . ( D ) ( F ) are high magnification details of rosettes ( dashed lines ) , where arrowheads indicate apical basal mitoses . Loss of endogenous let - 7 drives formation of rosettes ventricular neuronal ectopias ( C ) , which is rescued with additional loss of Irs2 ( G ) .

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

B-G - Distribution of neurons (Tuj1, red), GFP (green) and mitoses (PH3, white) in the rostral telencephalon of wild-type E14.5 mouse embryos electroporated at E12.5 with the indicated plasmid combinations. Inset shows the same field of view, with location and extent of electroporations. In (C) and (E), arrows indicate rosettes and arrowheads indicate neuronal ventricular ectopias. (D) and (F) are high magnification details of individual rosettes (dashed lines), where arrowheads indicate apical and basal mitoses. Loss of endogenous let-7 drives the formation of rosettes and ventricular neuronal ectopias (C), which is rescued with the additional loss of Irs2 (G).


B - G - Stains quantifications of human cerebral organoids electroporated with indicated construct combinations , to reveal frequency of electroporated remaining as progenitors ( % GFP + Ki67 + / GFP + ) . Histograms represent mean ± ; symbols in plots indicate values for organoids ; X2 - , * P < 0 . 05 . N = 8 - 11 replicates per group . Dashed lines indicate ventricular surface , solid arrowheads indicate Ki67 + / GFP + , open arrowheads indicate Ki67 - / GFP + . Scale bars , 50 µm .

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

B-G - Stains and quantifications of human cerebral organoids electroporated with the indicated construct combinations, to reveal the frequency of electroporated cells remaining as progenitors (%GFP+Ki67+/GFP+). Histograms represent mean ± SEM; symbols in plots indicate values for individual organoids; X2-test, *P < 0.05. N = 8-11 replicates per group. Dashed lines indicate ventricular surface, solid arrowheads indicate Ki67+/GFP+ cells, open arrowheads indicate Ki67-/GFP+ cells. Scale bars, 50 µm.


q - RT - PCR reveals that miR - 181a / b silencing leads to upregulation of miR - 181a / b predicted targets in SH - SY5Y . N = 3 independent experiments .

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

q-RT-PCR reveals that miR-181a/b silencing leads to upregulation of miR-181a/b predicted targets in SH-SY5Y cells. N=3 independent experiments.


miR - 181 - mimic transfection specifically inhibits Luciferase activity of constructs containing WT 3 ' - UTR predicted target sequences . Point mutations ( mut ) in miR - 181a / b binding sites abolish Luciferase repression in cases apart from PPARGC1A . Data are normalized to negative mimic transfection ( dashed ) . N = 6 independent experiments .

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

miR-181-mimic transfection specifically inhibits Luciferase activity of constructs containing WT 3'-UTR predicted target sequences. Point mutations (mut) in miR-181a/b binding sites abolish Luciferase repression in all cases apart from PPARGC1A. Data are normalized to negative mimic transfection (dashed line). N=6 independent experiments.


q - RT - PCR reveals upregulation of miR - 181a / b targets in eyes of miR - 181a / b - 1 - / - vs . miR - 181a / b - 1 + / + . n ≥ 5 / genotype .

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

q-RT-PCR reveals upregulation of miR-181a/b targets in the eyes of miR-181a/b-1-/- vs. miR-181a/b-1+/+ animals. n≥5 animals/genotype.


miR - 181a / b - 1 - / - mice show increased mtDNA content vs . miR - 181a / b - 1 + / + mice as measured by q - PCR . N = 4 / genotype .

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

miR-181a/b-1-/- mice show increased mtDNA content vs. miR-181a/b-1+/+ mice as measured by q-PCR. N=4 animals/genotype.


WB ( left panel ) reveals increased of mitochondrial in eyes of miR - 181a / b - 1 - / - ( - / - ) vs . miR - 181a / b - 1 + / + ( + / + ) mice ( quantified in right panel ) . Data are normalized to either p115 or Gapdh . N ≥ 3 / genotype . Please note that compared bands from + / + - / - samples are from same blots which were cropped shown split for sake of data presentation clarity .

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

WB analysis (left panel) reveals increased levels of mitochondrial proteins in the eyes of miR-181a/b-1-/- (-/-) vs. miR-181a/b-1+/+ (+/+) mice (quantified in the right panel). Data are normalized to either p115 or Gapdh. N≥3 animals/genotype. Please note that all compared bands from +/+ and -/- samples are from the same blots which were cropped and shown split for the sake of data presentation clarity.


WB on mitochondrial fractions ( left panel ) show increased of p62 Parkin in mitochondrial fraction from eye of - / - vs . + / + mice ( quantified in right panel ) . Data are normalized to Ndufb8 or Gapdh for mitochondrial fractions , respectively . N = 3 / genotype .

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

WB analysis on mitochondrial and cytosolic fractions (left panel) show increased levels of p62 and Parkin in mitochondrial fraction from the eye of -/- vs. +/+ mice (quantified in the right panel). Data are normalized to Ndufb8 or Gapdh for mitochondrial and cytosolic fractions, respectively. N=3 animals/genotype.


shows that miR - 181a / b silencing protects SH - SY5Y from FCCP treatment . N ≥ 7 independent experiments .

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

Cell death analysis shows that miR-181a/b silencing protects SH-SY5Y cells from FCCP treatment. N≥7 independent experiments.


TUNEL assays on eye brain sections of stage ( st ) 30 medakafish control MO - injected reveal a decrease in number of apoptotic in both hccs - MO / miR - 181a / b - MO - cox7b - MO / miR - 181a / b - MO - injected compared to hccs - MO - cox7b - MO - injected . Scale bars are 20μm .

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

TUNEL assays on eye and brain sections of stage (st)30 medakafish control and MO-injected embryos reveal a decrease in the number of apoptotic cells in both hccs-MO/miR-181a/b-MO- and cox7b-MO/miR-181a/b-MO-injected compared to hccs-MO- and cox7b-MO-injected embryos. Scale bars are 20μm.


TUNEL - positive / eye . n ≥ 5 eyes for each model .

B-EXP_ASSAY O O O B-TISSUE O O O O B-TISSUE O O O O

TUNEL-positive cells/eye. n≥5 eyes for each model.


Caspase assays show restored of caspase - 3 caspase - 9 activities in hccs - MO / miR - 181a / b - MO - cox7b - MO / miR - 181a / b - MO - injected with respect to hccs - MO - cox7b - MO - injected . n ≥ 5 for each model .

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

Caspase assays show restored levels of caspase-3 and caspase-9 activities in hccs-MO/miR-181a/b-MO- and cox7b-MO/miR-181a/b-MO-injected embryos with respect to hccs-MO- and cox7b-MO-injected embryos. n≥5 embryos for each model.


Representative of st30 medaka injected with hccs - MO ( B ) alone or co - injected with miR - 181a / b - MOs Co - injection of miR - 181a / b - MOs rescues microphthalmia microcephaly in both hccs - MO . hccs - MO / miR - 181a / b - MO - b - MO - injected were treated with Baf - A1 , PD98059 or HA14 - 1 . Baf - A1 treatment counteracts protective effect of miR - 181a / b downregulation in hccs - MO / miR - 181a / b - MO PD98059 treatment does not interfere with modulation of MLS phenotype mediated by miR - 181a / b downregulation HA14 - 1 treatment counteracts effect of miR - 181a / b downregulation in hccs - MO / miR - 181a / b - MO model Scale bars are 100μm .

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

Representative images of st30 medaka embryos injected with hccs-MO (B) alone or co-injected with miR-181a/b-MOs Co-injection of miR-181a/b-MOs rescues microphthalmia and microcephaly in both hccs-MO embryos. hccs-MO/miR-181a/b-MO- b-MO-injected embryos were treated with Baf-A1, PD98059 or HA14-1. Baf-A1 treatment counteracts the protective effect of miR-181a/b downregulation in hccs-MO/miR-181a/b-MO embryos PD98059 treatment does not interfere with the modulation of the MLS phenotype mediated by miR-181a/b downregulation HA14-1 treatment counteracts the effect of miR-181a/b downregulation in the hccs-MO/miR-181a/b-MO model Scale bars are 100μm.


Representative of st30 medaka injected with cox7b - MO ( H ) alone or co - injected with miR - 181a / b - MOs Co - injection of miR - 181a / b - MOs rescues microphthalmia microcephaly in cox7b - MO . cox7b - MO / miR - 181a / b - MO - injected were treated with Baf - A1 , PD98059 or HA14 - 1 . Baf - A1 treatment counteracts protective effect of miR - 181a / b downregulation in cox7b - MO / miR - 181a / b - MO PD98059 treatment does not interfere with modulation of MLS phenotype mediated by miR - 181a / b downregulation Scale bars are 100μm .

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

Representative images of st30 medaka embryos injected with cox7b-MO (H) alone or co-injected with miR-181a/b-MOs Co-injection of miR-181a/b-MOs rescues microphthalmia and microcephaly in cox7b-MO embryos. cox7b-MO/miR-181a/b-MO-injected embryos were treated with Baf-A1, PD98059 or HA14-1. Baf-A1 treatment counteracts the protective effect of miR-181a/b downregulation in cox7b-MO/miR-181a/b-MO embryos PD98059 treatment does not interfere with the modulation of the MLS phenotype mediated by miR-181a/b downregulation Scale bars are 100μm.


of with or without MLS phenotype in different conditions in hccs - MO . N ≥ 300 / conditions .

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

Percentage of embryos with or without MLS phenotype in the different conditions in hccs-MO embryos. N≥300 embryos/conditions.


of with or without MLS phenotype in different conditions in cox7b - MO ( N ) . N ≥ 300 / conditions .

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

Percentage of embryos with or without MLS phenotype in the different conditions in cox7b-MO (N) embryos. N≥300 embryos/conditions.


Immunofluorescence with anti - NeuN antibody in retina of miR - 181a / b - 1 + / + miR - 181a / b - 1 - / - mice intravitreally injected with Rotenone or DMSO . RGCs are preserved in miR - 181a / b - 1 - / - rotenone - injected mice with respect to controls at both one two after injection . Scale bars are 50μm .

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

Immunofluorescence analysis with anti-NeuN antibody in the retina of miR-181a/b-1+/+ and miR-181a/b-1-/- mice intravitreally injected with Rotenone or DMSO. RGCs are preserved in miR-181a/b-1-/- rotenone-injected mice with respect to controls at both one and two weeks after injection. Scale bars are 50μm.


Number of RGCs / mm ( indicated as % ) at one - two - post - injection . N = 10 .

O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O

Number of RGCs/mm (indicated as %) at one- and two-weeks post-injection. N=10.


NADH dehydrogenase histochemical reaction on retinal sections of miR - 181a / b - 1 + / + miR - 181a / b - 1 - / - mice intravitreally injected with Rotenone or DMSO . At one week post - injection , NADH dehydrogenase activity is lost in RGCs ( GCL , areas within dashed lines ) of miR - 181a / b - 1 + / + Rotenone - injected eyes , while it is preserved in those of miR - 181a / b - 1 - / - Rotenone - injected eyes . GCL , Ganglion Layer ONL ; INL , Inner Layer ; Outer Layer . Scale bars are 50μm .

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

NADH dehydrogenase histochemical reaction on retinal sections of miR-181a/b-1+/+ and miR-181a/b-1-/- mice intravitreally injected with Rotenone or DMSO. At one week post-injection, NADH dehydrogenase activity is lost in RGCs (GCL, areas within dashed lines) of miR-181a/b-1+/+ Rotenone-injected eyes, while it is preserved in those of miR-181a/b-1-/- Rotenone-injected eyes. GCL, Ganglion Cells Layer ONL; INL, Inner Nuclear Layer; Outer Nuclear Layer. Scale bars are 50μm.


Immunofluorescence , with an anti - CoxIV antibody , in retina of miR - 181a / b - 1 + / + miR - 181a / b - 1 - / - mice , injected with either Rotenone or DMSO , showed preserved mitochondria in miR - 181a / b - 1 - / - rotenone - injected eyes at one - week post - injection . Dashed boxes indicate of magnifications shown in right panels . Scale bars are 10μm .

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

Immunofluorescence analysis, with an anti-CoxIV antibody, in the retina of miR-181a/b-1+/+ and miR-181a/b-1-/- mice, injected with either Rotenone or DMSO, showed preserved mitochondria in miR-181a/b-1-/- rotenone-injected eyes at one-week post-injection. Dashed boxes indicate the area of magnifications shown in right panels. Scale bars are 10μm.


Graphical representation of results of optokinetic tracking assays reported as fold change of cycles / degree . Visual acuity is preserved in miR - 181a / b - 1 - / - rotenone - injected mice with respect to controls at both one two after injection . N = 10 .

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

Graphical representation of the results of the optokinetic tracking assays reported as fold change of cycles/degree. Visual acuity is preserved in miR-181a/b-1-/- rotenone-injected mice with respect to controls at both one and two weeks after injection. N=10.


Immunofluorescence with anti - NeuN antibody in retina of WT , Ndufs4 - / - , Ndufs4 - / - / miR - 181a / b - 1 - / - miR - 181a / b - 1 - / - mice . RGCs are preserved in Ndufs4 - / - / miR - 181a / b - 1 - / - with respect to Ndufs4 - / - mice . Scale bars are 50μm .

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

Immunofluorescence analysis with anti-NeuN antibody in the retina of WT, Ndufs4-/-, Ndufs4-/-/miR-181a/b-1-/- and miR-181a/b-1-/- mice. RGCs are preserved in Ndufs4-/-/miR-181a/b-1-/- with respect to Ndufs4-/- mice. Scale bars are 50μm.


Graphical representation of results of optokinetic tracking assays reported as fold change of cycles / degree . Visual acuity is preserved in Ndufs4 - / - / miR - 181a / b - 1 - / - with respect to Ndufs4 - / - mice . N ≥ 8 .

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

Graphical representation of the results of the optokinetic tracking assays reported as fold change of cycles/degree. Visual acuity is preserved in Ndufs4-/-/miR-181a/b-1-/- with respect to Ndufs4-/- mice. N≥8.


Electroretinographic reveals amelioration of b - wave patterns in Ndufs4 - / - / miR - 181a / b - 1 - / - with respect to Ndufs4 - / - mice . N ≥ 5 .

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

Electroretinographic analysis reveals amelioration of b-wave patterns in Ndufs4-/-/miR-181a/b-1-/- with respect to Ndufs4-/-mice. N≥5.


Electron microscopy shows amelioration of mitochondria increase of mitochondrial number in Ndufs4 - / - / miR - 181a / b - 1 - / - vs . Ndufs4 - / - mice . Scale bars are 1μm . quantitative increase of mitochondria is reported in C as number of mitochondria / RGC , at p30 p55 . N ≥ 2 / genotype .

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

Electron microscopy analysis shows amelioration of mitochondria morphology and increase of mitochondrial number in Ndufs4-/-/miR-181a/b-1-/- vs. Ndufs4-/- mice. Scale bars are 1μm. The quantitative increase of mitochondria is reported in C as number of mitochondria/RGC, at p30 and p55. N≥2 animals/genotype.


Ndufs4 - / - / miR - 181a / b - 1 - / - mice show increased mtDNA content vs . Ndufs4 - / - mice as measured by q - PCR . N ≥ 4 / genotype .

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

Ndufs4-/-/miR-181a/b-1-/- mice show increased mtDNA content vs. Ndufs4-/- mice as measured by q-PCR. N≥4 animals/genotype.


Biochemical activity of MRC complexes I , II IV normalized by of citrate synthase ( CS ) activity in Ndufs4 - / - / miR - 181a / b - 1 - / - vs . Ndufs4 - / - mice . N ≥ 3 / genotype . p ‐ values were calculated by two Student ' s t - ; Error bars are .

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

Biochemical activity of MRC complexes I, II and IV normalized by the percentage of citrate synthase (CS) activity in Ndufs4-/-/miR-181a/b-1-/- vs. Ndufs4-/- mice. N≥3 animals/genotype. p‐values were calculated by two tailed Student's t-test; Error bars are SEM.


Tap - 1 is required for secretion of TseL . Pellet ( P ) supernatant ( S ) of cultures were analyzed by SDS - PAGE . result of immunoblotting with antisera against TseL Hcp purified antibodies against DnaK is shown . asterisk marks an unspecific band , detected by antiserum against TseL , present in pre - immunized serum . One representative experiment of three independent experiments is shown .

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

Tap-1 is required for the secretion of TseL. Pellet (P) and supernatant (S) of bacterial cultures were analyzed by SDS-PAGE. The result of immunoblotting with antisera against TseL and Hcp and purified antibodies against DnaK is shown. The asterisk marks an unspecific band, detected by the antiserum against TseL, present in the pre-immunized serum. One representative experiment of three independent experiments is shown.


Tap - 1 is required for TseL - mediated killing . Wild - type C6706 or C6706 lacking tsiV1 were exposed to indicated V52 mutants in a killing . y - axis indicates number of surviving C6706 .

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

Tap-1 is required for TseL-mediated killing. Wild-type C6706 or C6706 lacking tsiV1 were exposed to the indicated V52 mutants in a killing assay. The y-axis indicates the number of surviving C6706.


Tap - 1 is required for TseL function . Killing assays in which V52Δtap - 1 was mixed with wild - type C6706 or mutants lacking tsiV1 , tsiV2 , or tsiV3 . y - axis indicates surviving C6706 .

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

Tap-1 is required for TseL function. Killing assays in which V52Δtap-1 was mixed with wild-type C6706 or mutants lacking tsiV1, tsiV2, or tsiV3. The y-axis indicates surviving C6706.


TseL secretion depends on VgrG - 1 . Western blot of pellet ( P ) supernatant ( S ) samples of indicated strains . Samples were immunoblotted with antisera against TseL Hcp purified antibodies against DnaK . unspecific band detected by rabbit serum is marked with an asterisk . One representative of three experiments is shown .

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

TseL secretion depends on VgrG-1. Western blot analysis of pellet (P) and supernatant (S) samples of the indicated strains. Samples were immunoblotted with antisera against TseL and Hcp and purified antibodies against DnaK. The unspecific band detected by rabbit serum is marked with an asterisk. One representative of three experiments is shown.


VgrG - 1 is required for TseL - mediated killing . Killing with wild - type V52 or indicated mutants wild - type C6706 or a mutant lacking immunity gene tsiV1 . y - axis indicates surviving C6706 .

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VgrG-1 is required for TseL-mediated killing. Killing assay with wild-type V52 or indicated mutants and wild-type C6706 or a mutant lacking the immunity gene tsiV1. The y-axis indicates surviving C6706.


VasX - VgrG - 3 - mediated killing but not TseL - mediated killing occurs in absence of VgrG - 1 . V52ΔvgrG - 1 was mixed in a killing with wild - type C6706 or mutants lacking immunity genes tsiV1 , tsiV2 , or tsiV3 ( specific for TseL , VasX , VgrG - 3 , respectively ) . y - axis indicates surviving C6706 .

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VasX- and VgrG-3-mediated killing but not TseL-mediated killing occurs in the absence of VgrG-1. V52ΔvgrG-1 was mixed in a killing assay with wild-type C6706 or mutants lacking immunity genes tsiV1, tsiV2, or tsiV3 (specific for TseL, VasX, and VgrG-3, respectively). The y-axis indicates surviving C6706.


A , B Interactions between Tap - 1 TseL ( A ) or VgrG - 1 ( B ) . Shown are immunoblots of lysates ( total ) immunoprecipitates with anti - FLAG affinity beads ( IP : FLAG ) of V52Δtap - 1 ( A ) or V52 vgrG - 1 : : myc ( B ) transformed with empty vector or a plasmid encoding His - tagged or FLAG - tagged Tap - 1 .

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A, B Interactions between Tap-1 and TseL (A) or VgrG-1 (B). Shown are immunoblots of lysates (total) and immunoprecipitates with anti-FLAG affinity beads (IP:FLAG) of V52Δtap-1 (A) or V52 vgrG-1::myc (B) transformed with empty vector or a plasmid encoding His-tagged or FLAG-tagged Tap-1.


C Interaction between TseL VgrG - 1 . Shown are immunoblots of lysates ( total ) immunoprecipitates with an anti - FLAG affinity beads ( IP : FLAG ) of V52 or V52Δtap - 1 transformed with empty vector or a plasmid encoding HIS - tagged or FLAG - tagged VgrG - 1 .

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C Interaction between TseL and VgrG-1. Shown are immunoblots of lysates (total) and immunoprecipitates with an anti-FLAG affinity beads (IP:FLAG) of V52 or V52Δtap-1 transformed with empty vector or a plasmid encoding HIS-tagged or FLAG-tagged VgrG-1.


Specificity of C - terminal Tap - 1 segment . Killing in which lack of tap - 1 in V52 is complemented by empty vector ( control ) or one of two different alleles of tap - 1 . ability to kill in a TseL - dependent manner is indicated by logarithm of surviving C6706ΔtsiV1 .

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Specificity of the C-terminal Tap-1 segment. Killing assay in which the lack of tap-1 in V52 is complemented by empty vector (control) or one of two different alleles of tap-1. The ability to kill in a TseL-dependent manner is indicated by the logarithm of surviving C6706ΔtsiV1.


Specificity of N - terminal segment of Tap - 1 . Killing in which lack of tap - 1 in 1587 is complemented by empty vector ( control ) or one of two different alleles of tap - 1 . ability to facilitate killing by effector A55 _ 1502 is indicated by logarithm of surviving 1587ΔA55 _ 1501 - 03 .

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Specificity of N-terminal segment of Tap-1. Killing assay in which the lack of tap-1 in 1587 is complemented by empty vector (control) or one of two different alleles of tap-1. The ability to facilitate killing by the effector A55_1502 is indicated by the logarithm of surviving 1587ΔA55_1501-03.


( A ) time courses of normalized FRET ( Mean ± ) of calcium biosensor before after shockwave stimulation in HBSS Ca2 + medium with ( yellow , n = 6 , N = 3 ) or without Piezo1 expression ( black , n = 6 , N = 3 ) . ( B ) representative FRET / ECFP of calcium biosensor in Piezo1 - expressed HEK before ( top left ) after ( top right ) LIS stimulation . Phase image with initiating point ( left ) image indicating Piezo1 expression ( right ) were also shown on bottom panels ( Scale bar , 40 μm ) . Color scale bars in figures are to show FRET / ECFP ratios , with cold hot colors representing low high ratios , respectively . ( C ) time course of normalized FRET ( Mean ± ) of calcium biosensor in Piezo1 - expressing HEK before after multiple shockwave stimulations in HBSS Ca2 + medium ( n = 6 , N = 3 ) .

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(A) The time courses of normalized FRET ratio (Mean ± SEM) of calcium biosensor before and after shockwave stimulation in HBSS Ca2+ medium with (yellow line, n = 6, N = 3) or without the Piezo1 expression (black line, n = 6, N = 3). (B) The representative FRET/ECFP ratio images of calcium biosensor in Piezo1-expressed HEK cells before (top left) and after (top right) LIS stimulation. Phase image with laser initiating point (left) and fluorescent image indicating Piezo1 expression (right) were also shown on the bottom panels (Scale bar, 40 μm). Color scale bars in the figures are to show the FRET/ECFP ratios, with cold and hot colors representing low and high ratios, respectively. (C) The time course of normalized FRET ratio (Mean ± SEM) of calcium biosensor in the Piezo1-expressing HEK cells before and after multiple shockwave stimulations in HBSS Ca2+ medium (n = 6, N = 3).