Quantitative RT - PCR analysis of auxin signaling gene expression in roots of wild type ( Col - 0 ) , uvr8 , myb73 myb77 , and myb73 myb77 uvr8 . ( D ) Results under white light and ( E ) under white plus UV - B light . Seedlings grown in continuous white light for 12 days were kept in white light or transferred to white light plus UV - B for 1 day with the addition of 0 . 4 μM NAA , and roots were collected to analyze auxin signaling gene expression . Error bars represent SD of three biological replicates .

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

Quantitative RT-PCR analysis of auxin signaling gene expression in roots of wild type (Col-0), uvr8, myb73 myb77, and myb73 myb77 uvr8. (D) Results under white light and (E) under white plus UV-B light. Seedlings grown in continuous white light for 12 days were kept in white light or transferred to white light plus UV-B for 1 day with the addition of 0.4 μM NAA, and roots were collected to analyze auxin signaling gene expression. Error bars represent SD of three biological replicates.


Electrophoretic mobility - shift assay ( EMSA ) showing that MYB73 / MYB77 binds to the MBSI motif in vitro . Cold probe was added as a competitor .

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Electrophoretic mobility-shift assay (EMSA) showing that MYB73/MYB77 binds to the MBSI motif in vitro. Cold probe was added as a competitor.


EMSA showing that MYB73 / MYB77 binds to the promoter of IAA19 . Cold IAA19p or IAA19mp ( MBSI motif mutated ) was added as a competitor .

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EMSA showing that MYB73/MYB77 binds to the promoter of IAA19. Cold IAA19p or IAA19mp (MBSI motif mutated) was added as a competitor.


EMSA results showing that monomeric UVR8 inhibits the DNA - binding activity of MYB73 ( C )

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EMSA results showing that monomeric UVR8 inhibits the DNA-binding activity of MYB73(C)


EMSA results showing that monomeric UVR8 inhibits the DNA - binding activity of MYB77 ( D ) .

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EMSA results showing that monomeric UVR8 inhibits the DNA-binding activity of MYB77(D).


ChIP - qPCR analysis showed that UV - B inhibits the DNA - binding activity of MYB73 . ChIP - qPCR assays were performed using transgenic seedlings expressing 35S : : MYB73 - TAP , treated with or without UV - B for 5 h before harvesting samples . Chromatin fragments ( ~ 500 bp ) were immunoprecipitated by anti - Myc antibody , and the precipitated DNA was analyzed by qPCR using the primer pairs indicated . The IP / Input ratios are shown with SDs ( n = 3 ) . ChIP - qPCR showing that UV - B inhibits the DNA - binding activity of MYB73 in a UVR8 - dependent manner . The IP / Input ratios are shown with SDs ( n = 3 ) .

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

ChIP-qPCR analysis showed that UV-B inhibits the DNA-binding activity of MYB73. ChIP-qPCR assays were performed using transgenic seedlings expressing 35S::MYB73-TAP, treated with or without UV-B for 5 h before harvesting samples. Chromatin fragments (~500 bp) were immunoprecipitated by anti-Myc antibody, and the precipitated DNA was analyzed by qPCR using the primer pairs indicated. The IP/Input ratios are shown with SDs (n=3). ChIP-qPCR showing that UV-B inhibits the DNA-binding activity of MYB73 in a UVR8-dependent manner. The IP/Input ratios are shown with SDs (n=3).


ChIP - qPCR showing that UV - B inhibits the DNA - binding activity of MYB73 in roots ( G ) in a UVR8 - dependent manner ( H ) . The IP / Input ratios are shown with SDs ( n = 3 ) .

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ChIP-qPCR showing that UV-B inhibits the DNA-binding activity of MYB73 in roots (G) in a UVR8-dependent manner (H). The IP/Input ratios are shown with SDs (n=3).


ChIP - qPCR showing that UV - B inhibits MYB77 from binding to the indicated promoters in a UVR8 - dependent manner . The IP / Input ratios are shown with SDs ( n = 3 ) .

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ChIP-qPCR showing that UV-B inhibits MYB77 from binding to the indicated promoters in a UVR8-dependent manner. The IP/Input ratios are shown with SDs (n=3).


Immunoblot with anti - HA Abs of lysates from parental ( Ctrl ) and tagged lines ( TgCRMPa - HA3 and TgCRMPb - HA3 ) together with inducible - knockdown lines ( TgCRMPa - HA3 _ iKD and TgCRMPb - HA3 _ iKD ) treated with ATc for 0 , 24 or 48h . TgROP5 was used as loading control . Two close bands around 300kDa were detected for TgCRMPa . A ~ 820kDa protein , corresponding to the predicted size for TgCRMPb , was observed together with a ~ 130kDa band .

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Immunoblot with anti-HA Abs of lysates from parental (Ctrl) and tagged lines (TgCRMPa-HA3 and TgCRMPb-HA3) together with inducible-knockdown lines (TgCRMPa-HA3_iKD and TgCRMPb-HA3_iKD) treated with ATc for 0, 24 or 48h. TgROP5 was used as loading control. Two close bands around 300kDa were detected for TgCRMPa. A ~820kDa protein, corresponding to the predicted size for TgCRMPb, was observed together with a ~130kDa band.


Quantification of microneme secretion in TgCRMPa - and TgCRMPb - depleted tachyzoites was measured by detecting the processed form ( arrowhead ) of TgMIC2 ( arrow ) in the media . Control , TgCRMPa _ iKD and TgCRMPb _ iKD parasites , ATc - treated ( + ) and untreated ( - ) , were stimulated with propranolol to release microneme contents . Blots were probed with anti - MIC2 ( secretion of micronemes ) and anti - GRA3 ( constitutive secretion of dense granules ) . P : Parasites pellet . Sup : Supernatant from untreated parasites . Sup + Prop : Supernatant from parasites treated with propranolol . The results are representative of two independent experiments .

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

Quantification of microneme secretion in TgCRMPa- and TgCRMPb-depleted tachyzoites was measured by detecting the processed form (arrowhead) of TgMIC2 (arrow) in the media. Control, TgCRMPa_iKD and TgCRMPb_iKD parasites, ATc-treated (+) and untreated (-), were stimulated with propranolol to release microneme contents. Blots were probed with anti-MIC2 (secretion of micronemes) and anti-GRA3 (constitutive secretion of dense granules). P: Parasites pellet. Sup: Supernatant from untreated parasites. Sup+Prop: Supernatant from parasites treated with propranolol. The results are representative of two independent experiments.


Co - immunoprecipitation of TgCRMPa and TgCRMPb . Lysates from parasites co - expressing TgCRMPa - FLAG3 and TgCRMPb - HA3 were split and incubated with either anti - FLAG ( left panels ) or anti - HA beads ( right panels ) . Eluates were subjected to SDS - PAGE and immunoblotted with anti - HA and anti - FLAG Abs . Untagged , TgCRMPa - FLAG3 and TgCRMPb - HA3 parasites were used as controls . TgCRMPs robustly associate with each other .

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Co-immunoprecipitation of TgCRMPa and TgCRMPb. Lysates from parasites co-expressing TgCRMPa-FLAG3 and TgCRMPb-HA3 were split and incubated with either anti-FLAG (left panels) or anti-HA beads (right panels). Eluates were subjected to SDS-PAGE and immunoblotted with anti-HA and anti-FLAG Abs. Untagged, TgCRMPa-FLAG3 and TgCRMPb-HA3 parasites were used as controls. TgCRMPs robustly associate with each other.


Quantification of microneme secretion in control and Tg277910 - depleted tachyzoites was measured as in Fig 2H . Blots were probed with anti - MIC2 ( secretion of micronemes ) and anti - GRA3 ( constitutive secretion of dense granules ) . P : Parasites pellet . Sup : Supernatant from untreated parasites . Sup + Prop : Supernatant from parasites treated with propranolol . The results are representative of two independent experiments .

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Quantification of microneme secretion in control and Tg277910-depleted tachyzoites was measured as in Fig 2H. Blots were probed with anti-MIC2 (secretion of micronemes) and anti-GRA3 (constitutive secretion of dense granules). P: Parasites pellet. Sup: Supernatant from untreated parasites. Sup+Prop: Supernatant from parasites treated with propranolol. The results are representative of two independent experiments.


Immunofluorescence images of extracellular tachyzoites of untagged , TgCRMPa - HA3 and TgCRMPb - HA3 parasites , incubated either with host cell monolayers for 2min , or with ionophore A23187 , to induce natural or artificial conoid extrusion , respectively . Parasites were immunostained with anti - HA Abs ; DNA was labeled by Hoechst . CRMPa and CRMPb consistently accumulate at the tip of extruded conoids ( arrows ) . The apexes of A23187 - treated parasites were magnified on the right , and increased in brightness and contrast to highlight the apical dots . DIC : differential interference contrast . Quantification of the dot pattern upon A23187 treatment shown in ( A ) . Values are expressed as percentage of parasites showing ( dot ) or lacking ( no dot ) the apical accumulation of TgCRMPa and TgCRMPb ; n = number of parasites analyzed per line .

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

Immunofluorescence images of extracellular tachyzoites of untagged, TgCRMPa-HA3 and TgCRMPb-HA3 parasites, incubated either with host cell monolayers for 2min, or with ionophore A23187, to induce natural or artificial conoid extrusion, respectively. Parasites were immunostained with anti-HA Abs; DNA was labeled by Hoechst. CRMPa and CRMPb consistently accumulate at the tip of extruded conoids (arrows). The apexes of A23187-treated parasites were magnified on the right, and increased in brightness and contrast to highlight the apical dots. DIC: differential interference contrast. Quantification of the dot pattern upon A23187 treatment shown in (A). Values are expressed as percentage of parasites showing (dot) or lacking (no dot) the apical accumulation of TgCRMPa and TgCRMPb; n= number of parasites analyzed per line.


Immunofluorescence images of extracellular TgCRMPb - HA3 tachyzoites in presence ( - ATc ) or absence ( + 48h ATc ) of TgCRMPa - FLAG3 ( iKD line ) . Parasites were stained with anti - HA Abs to visualize CRMPb . TgCRMPb localization at the tip of the extruded conoid ( arrow ) disappears upon TgCRMPa depletion , but it is still detected in the cytoplasm ( lower panel ) . DNA is labeled by Hoechst . Single focal planes are shown . DIC : differential interference contrast . Quantification of the dot pattern shown in ( C ) . The values are reported as in ( B ) .

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

Immunofluorescence images of extracellular TgCRMPb-HA3 tachyzoites in presence (-ATc) or absence (+48h ATc) of TgCRMPa-FLAG3 (iKD line). Parasites were stained with anti-HA Abs to visualize CRMPb. TgCRMPb localization at the tip of the extruded conoid (arrow) disappears upon TgCRMPa depletion, but it is still detected in the cytoplasm (lower panel). DNA is labeled by Hoechst. Single focal planes are shown. DIC: differential interference contrast.   Quantification of the dot pattern shown in (C). The values are reported as in (B).


Whole cell lysates from TIR1 - expressing parental line ( Ctrl ) , HA3 - miniAID - TgCRMPa _ iKD ( N - term ) and TgCRMPa - miniAID - HA3 _ iKD ( C - term ) lines were immunoblotted with anti - HA Abs to visualize tagged CRMPa in IAA - treated and untreated samples . CRMPa was undetectable upon 24h incubation with IAA when C - terminally , but not N - terminally , tagged with the miniAID - HA3 , suggesting that the C - terminus is the one exposed towards the cytosol . TgMIC2 was used as loading control and detected with anti - MIC2 Abs . MW : molecular weight standards .

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Whole cell lysates from TIR1-expressing parental line (Ctrl), HA3-miniAID-TgCRMPa_iKD (N-term) and TgCRMPa-miniAID-HA3_iKD (C-term) lines were immunoblotted with anti-HA Abs to visualize tagged CRMPa in IAA-treated and untreated samples. CRMPa was undetectable upon 24h incubation with IAA when C-terminally, but not N-terminally, tagged with the miniAID-HA3, suggesting that the C-terminus is the one exposed towards the cytosol. TgMIC2 was used as loading control and detected with anti-MIC2 Abs. MW: molecular weight standards.


Immunofluorescence images of extracellular A23187 - treated parasites expressing either N - or C - terminally HA3 - tagged TgCRMPa , and immunostained as in ( A ) . TgCRMPa accumulates at the tip of extruded conoids ( arrows ) in triton - permeabilized ( + ) or non - permeabilized ( - ) parasites . DIC : differential interference contrast . Single focal planes are shown . Quantification of the dot pattern upon natural ( + H F Fibroblasts ) or artificial ( + A23187 ) conoid extrusion in parasites expressing either N - or C - terminally HA3 - tagged TgCRMPa . Values are reported as in ( B ) .

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Immunofluorescence images of extracellular A23187-treated parasites expressing either N-or C-terminally HA3-tagged TgCRMPa, and immunostained as in (A). TgCRMPa accumulates at the tip of extruded conoids (arrows) in triton-permeabilized (+) or non-permeabilized (-) parasites. DIC: differential interference contrast. Single focal planes are shown.   Quantification of the dot pattern upon natural (+H F Fibroblasts) or artificial (+A23187) conoid extrusion in parasites expressing either N- or C-terminally HA3-tagged TgCRMPa. Values are reported as in (B).


Ultrastructure Expansion Microscopy of extracellular tachyzoites , either untagged or co - expressing TgCRMPa - HA3 / TgCRMPa - TY2 and TgCRMPb - HA3 together or in pairwise combination with TgNd6 - TY2 . Parasites were treated with A23187 prior to fixation and preparation for U - ExM , and stained with anti - HA , anti - TY and anti - α / β tubulin Abs to label CRMPs , Nd6 / CRMPa and microtubules , respectively . Shown are maximum intensity projections of z - stack confocal images . CRMPs overlap with Nd6 at the tip of the extruded conoid ( yellow selection ) . A magnified image of the apical tip of each parasite is shown on the right .

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

Ultrastructure Expansion Microscopy of extracellular tachyzoites, either untagged or co-expressing TgCRMPa-HA3/TgCRMPa-TY2 and TgCRMPb-HA3 together or in pairwise combination with TgNd6-TY2. Parasites were treated with A23187 prior to fixation and preparation for U-ExM, and stained with anti-HA, anti-TY and anti-α/β tubulin Abs to label CRMPs, Nd6/CRMPa and microtubules, respectively. Shown are maximum intensity projections of z-stack confocal images. CRMPs overlap with Nd6 at the tip of the extruded conoid (yellow selection). A magnified image of the apical tip of each parasite is shown on the right.


Extent of colocalization between TgCRMPa - TY2 and TgCRMPb - HA3 , and TgCRMPs - HA3 with TgNd6 - TY2 in the apical dot shown in B . Pearson ' s correlation coefficient was measured using the Fiji - JACoP plugin . Values are expressed as mean ± SD . Three - five parasites per line were analyzed . Quantification of the dot pattern for TgCRMPa - HA3 and TgCRMPb - HA3 in TgNd9 _ iKD lines . CRMPs accumulation at the apical tip of extracellular parasites was measured as in Fig EV4A . TgCRMPs were still found in the apical dot in absence of TgNd9 ( + ATc ) . Numbers are expressed as percentage of parasites showing ( dot ) or lacking ( no dot ) the tip accumulation of TgCRMPa and TgCRMPb . The number of parasites ( n ) analyzed for each line is reported at the top of the column .

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

Extent of colocalization between TgCRMPa-TY2 and TgCRMPb-HA3, and TgCRMPs-HA3 with TgNd6-TY2 in the apical dot shown in B. Pearson's correlation coefficient was measured using the Fiji-JACoP plugin. Values are expressed as mean ± SD. Three-five parasites per line were analyzed. Quantification of the dot pattern for TgCRMPa-HA3 and TgCRMPb-HA3 in TgNd9_iKD lines. CRMPs accumulation at the apical tip of extracellular parasites was measured as in Fig EV4A. TgCRMPs were still found in the apical dot in absence of TgNd9 (+ATc). Numbers are expressed as percentage of parasites showing (dot) or lacking (no dot) the tip accumulation of TgCRMPa and TgCRMPb. The number of parasites (n) analyzed for each line is reported at the top of the column.


( A - B ) Western - Blotting ( WB ) of p53 - negative lung cancer H1299 cells stably expressing ( A ) or transiently expressing ( B ) the indicated constructs . Post - translationally modified Δ133p53 and Δ160p53 isoforms are indicated with * .

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(A-B) Western-Blotting (WB) of p53-negative lung cancer H1299 cells stably expressing (A) or transiently expressing (B) the indicated constructs. Post-translationally modified Δ133p53 and Δ160p53 isoforms are indicated with *.


( C ) The same WB membrane containing lysates from R273Hp53 - expressing A431cells was incubated with rabbit polyclonal CM - 1 antibody and mouse monoclonal 1801 antibody against the N - terminus of p53 . Detection using anti - rabbit IRDye 680LT ( red ) and anti - mouse IRDye 800CW ( green ) secondary antibodies confirmed that the Δ160p53 band corresponds to a C - terminal isoform of p53 .

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(C) The same WB membrane containing lysates from R273Hp53-expressing A431cells was incubated with rabbit polyclonal CM-1 antibody and mouse monoclonal 1801 antibody against the N-terminus of p53. Detection using anti-rabbit IRDye 680LT (red) and anti-mouse IRDye 800CW (green) secondary antibodies confirmed that the Δ160p53 band corresponds to a C-terminal isoform of p53.


( D ) WB of H1299 cells transiently expressing the indicated constructs .

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(D) WB of H1299 cells transiently expressing the indicated constructs.


( E ) WB of cell lines expressing wild - type ( HCT116 , U2OS , A549 ) or mutant ( SW480 [ R273H / P309S ] , MDAMB231 [ R280K ] , A431 [ R273H ] , HT29 [ R273H ] and SKBR3 [ R175H ] ) p53 .

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(E) WB of cell lines expressing wild-type (HCT116, U2OS, A549) or mutant (SW480 [R273H/P309S], MDAMB231 [R280K], A431 [R273H], HT29 [R273H] and SKBR3 [R175H]) p53.


( F ) Cell lines harbouring endogenous wild - type ( A549 ) or R273H mutant ( A431 ) p53 were treated or not with control siRNA ( ctl ) or siRNA targeting both p53 transcripts ( ex6 and ex7 ) , full - length mRNA only ( ex2 / 3 ) or Δ133p53 mRNA only ( in4 ) before lysis and WB .

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(F) Cell lines harbouring endogenous wild-type (A549) or R273H mutant (A431) p53 were treated or not with control siRNA (ctl) or siRNA targeting both p53 transcripts (ex6 and ex7), full-length mRNA only (ex2/3) or Δ133p53 mRNA only (in4) before lysis and WB.


( G ) WB of H1299 cells stably expressing R273Hp53 and treated with DMSO , proteasome inhibitor MG132 or mRNA translation inhibitor cycloheximide ( CHX ) . Shown are representative data of three independent experiments . The numbers in parenthesis indicate the amounts of protein for the indicated bands according to WB quantifications and normalization against α - tubulin or GAPDH and relative to the value indicated in bold and set to 1 . 0 . In WBs for endogenous p53 cell lines the Δ160 lane is used as a marker lane showing Δ160p53 as transiently expressed in p53 - null H1299 cells .

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

(G) WB of H1299 cells stably expressing R273Hp53 and treated with DMSO, proteasome inhibitor MG132 or mRNA translation inhibitor cycloheximide (CHX). Shown are representative data of three independent experiments. The numbers in parenthesis indicate the amounts of protein for the indicated bands according to WB quantifications and normalization against α-tubulin or GAPDH and relative to the value indicated in bold and set to 1.0. In WBs for endogenous p53 cell lines the Δ160 lane is used as a marker lane showing Δ160p53 as transiently expressed in p53-null H1299 cells.


( A ) Cell lines endogenously expressing mutant R273Hp53 ( A431 and HT29 ) were treated with control siRNA ( ctl ) or siRNA targeting exon 7 of p53 , submitted to endoplasmic reticulum ( ER ) stress by exposure to thapsigargin ( Th ) and then analysed for apoptosis by incubation with propidium - iodide ( PI ) and FACS analysis . Data was normalized against si ctl condition , which was set to 100 .

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

(A) Cell lines endogenously expressing mutant R273Hp53 (A431 and HT29) were treated with control siRNA (ctl) or siRNA targeting exon 7 of p53, submitted to endoplasmic reticulum (ER) stress by exposure to thapsigargin (Th) and then analysed for apoptosis by incubation with propidium-iodide (PI) and FACS analysis. Data was normalized against si ctl condition, which was set to 100.


( B and C ) A549 ( B ) lung cancer cell lines stably expressing the indicated constructs were counted with Trypan blue for several days following stress stimuli ( over - confluency ( O - C ) for A549 ) . Data was normalized against 1st day values . Δ160p53 shows similar pro - proliferative capacities as mutant R273Hp53 . On the other hand R273Hp53 lost pro - proliferative functions when deficient for Δ160p53 expression ( M160AR273Hp53 ) . Also shown is the WB of A549 cells used in ( B ) and stably expressing the indicated constructs .

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

(B and C) A549 (B) lung cancer cell lines stably expressing the indicated constructs were counted with Trypan blue for several days following stress stimuli (over-confluency (O-C) for A549). Data was normalized against 1st day values. Δ160p53 shows similar pro-proliferative capacities as mutant R273Hp53. On the other hand R273Hp53 lost pro-proliferative functions when deficient for Δ160p53 expression (M160AR273Hp53). Also shown is the WB of A549 cells used in (B) and stably expressing the indicated constructs.


( B and C ) H1299 ( C ) lung cancer cell lines stably expressing the indicated constructs were counted with Trypan blue for several days following stress stimuli ( thapsigargin ( Th ) for H1299 ) . Data was normalized against 1st day values . Δ160p53 shows similar pro - proliferative capacities as mutant R273Hp53 . On the other hand R273Hp53 lost pro - proliferative functions when deficient for Δ160p53 expression ( M160AR273Hp53 ) .

O O O O O B-CELL_LINE O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O B-CELL_LINE O O O O O O O O O O O O O O O O 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 and C) H1299 (C) lung cancer cell lines stably expressing the indicated constructs were counted with Trypan blue for several days following stress stimuli (thapsigargin (Th) for H1299). Data was normalized against 1st day values. Δ160p53 shows similar pro-proliferative capacities as mutant R273Hp53. On the other hand R273Hp53 lost pro-proliferative functions when deficient for Δ160p53 expression (M160AR273Hp53).


( D to G ) H1299 cells transfected with the indicated constructs were submitted to ER stress ( E and G ) or cultured under regular conditions ( D ) . Data was normalized against empty vector ( E ) or p53 ( D ) condition , which were set to 100 . Cells were then analysed for apoptosis as in ( A ) . Shown are representative data or averages + s . d . of three independent experiments ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 005 and ^ P > 0 . 05 compared to control ( " Th + si ctl " or " - " ) or as indicated ) .

O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 to G) H1299 cells transfected with the indicated constructs were submitted to ER stress (E and G) or cultured under regular conditions (D). Data was normalized against empty vector (E) or p53 (D) condition, which were set to 100. Cells were then analysed for apoptosis as in (A). Shown are representative data or averages + s.d. of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.005 and ^P > 0.05 compared to control ("Th + si ctl" or "-" ) or as indicated).


( D to G ) H1299 cells transfected with the indicated constructs were submitted to ER stress ( E and G ) , DNA - damage by 36h 3 μM etoposide ( Eto ) treatment ( F ) . Data was normalized against empty vector ( E ) or p53 ( D ) condition , which were set to 100 . Cells were then analysed for apoptosis as in ( A ) . Shown are representative data or averages + s . d . of three independent experiments ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 005 and ^ P > 0 . 05 compared to control ( " Th + si ctl " or " - " ) or as indicated ) .

O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O

(D to G) H1299 cells transfected with the indicated constructs were submitted to ER stress (E and G), DNA-damage by 36h 3 μM etoposide (Eto) treatment (F). Data was normalized against empty vector (E) or p53 (D) condition, which were set to 100. Cells were then analysed for apoptosis as in (A). Shown are representative data or averages + s.d. of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.005 and ^P > 0.05 compared to control ("Th + si ctl" or "-" ) or as indicated).


( A ) Adhesion assay shows that MCF10A cells stably expressing Δ133p53 , Δ160p53 , R273HΔ160p53 or missense mutant R273Hp53 adhere more strongly than control cells . Excluding Δ160p53 expression from the mutant background ( M160A / R273Hp53 ) rescued control - cell phenotype . Scale bar represents 200 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O 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) Adhesion assay shows that MCF10A cells stably expressing Δ133p53, Δ160p53, R273HΔ160p53 or missense mutant R273Hp53 adhere more strongly than control cells. Excluding Δ160p53 expression from the mutant background (M160A/R273Hp53) rescued control-cell phenotype. Scale bar represents 200 μm.


( B ) WB of MCF10A cells used in ( A ) and stably expressing the indicated constructs . Shown are representative data of three independent experiments and quantifications .

O O O B-EXP_ASSAY O B-CELL_LINE 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) WB of MCF10A cells used in (A) and stably expressing the indicated constructs. Shown are representative data of three independent experiments and quantifications.


( C and D ) MCF10A human breast epithelial cells stably expressing the indicated constructs were cultured in matrigel . Control - and Δ133p53 - expressing cells formed regular three - dimensional ( 3D ) mammary acinar structures with hollow lumina but Δ160p53 - , R273HΔ160p53 and to a smaller extent mutant R273Hp53 - expressing cells formed acini with filled lumen and long invasive structures ( n > 30 acini per experiment per condition ) .

O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-SUBCELLULAR O O O O O O O O B-TISSUE O O O O O O

(C and D) MCF10A human breast epithelial cells stably expressing the indicated constructs were cultured in matrigel. Control- and Δ133p53-expressing cells formed regular three-dimensional (3D) mammary acinar structures with hollow lumina but Δ160p53-, R273HΔ160p53 and to a smaller extent mutant R273Hp53-expressing cells formed acini with filled lumen and long invasive structures (n > 30 acini per experiment per condition).


O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-TISSUE O O O O O O O O B-TISSUE O O O O O O


( E ) H1299 cells stably expressing empty vector ( control ) or the indicated constructs were assessed by confocal microscopy for invasion further than 30 μm through a Matrigel - fibronectin matrix towards EGF - supplemented growth media . The 30 μm plane is indicated and scale bars represent 100 μm . Quantifications are shown in the right panel .

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

(E) H1299 cells stably expressing empty vector (control) or the indicated constructs were assessed by confocal microscopy for invasion further than 30 μm through a Matrigel-fibronectin matrix towards EGF-supplemented growth media. The 30 μm plane is indicated and scale bars represent 100 μm. Quantifications are shown in the right panel.


( A ) WB of H1299 cells stably expressing Δ133p53 and treated with control morpholino oligos ( Ctl - 1 or Ctl - 2 ) or antisense morpholino oligo targeting Δ160p53 ' s translation initiation ( MO )

O O O B-EXP_ASSAY O B-CELL_LINE O O O O O O O O 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) WB of H1299 cells stably expressing Δ133p53 and treated with control morpholino oligos (Ctl-1 or Ctl-2) or antisense morpholino oligo targeting Δ160p53's translation initiation (MO)


( B ) A431 and HT29 cells expressing endogenous mutant R273Hp53 were treated with control morpholinos ( Ctl - 2 or Ctl - 1 ) or antisense morpholino oligo targeting Δ160p53 ' s translation initiation site ( MO ) before lysis and WB . wtp53 - expressing A549 cells were used as reference for endogenous wtp53 expression levels . Δ160 lane was used as a marker lane showing Δ160p53 as transiently expressed in p53 - null H1299 cells .

O O O B-CELL_LINE O B-CELL_LINE O O O O O O O O O O O O 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 B-CELL_LINE O 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-CELL_LINE O O

(B) A431 and HT29 cells expressing endogenous mutant R273Hp53 were treated with control morpholinos (Ctl-2 or Ctl-1) or antisense morpholino oligo targeting Δ160p53's translation initiation site (MO) before lysis and WB. wtp53-expressing A549 cells were used as reference for endogenous wtp53 expression levels. Δ160 lane was used as a marker lane showing Δ160p53 as transiently expressed in p53-null H1299 cells.


( C ) Shown are the average Δ160p53 protein levels expressed in R273Hp53 versus other mutant p53cell lines that were treated with control morpholino ( Ctl - 1 ) or morpholino targeting 160p53 ' s translation initiation site ( MO ) . MO efficiently targets 160p53 in endogenous R273Hp53 cell lines . See also figure EV3 for the raw data .

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

(C) Shown are the average Δ160p53 protein levels expressed in R273Hp53 versus other mutant p53cell lines that were treated with control morpholino (Ctl-1) or morpholino targeting 160p53's translation initiation site (MO). MO efficiently targets 160p53 in endogenous R273Hp53 cell lines. See also figure EV3 for the raw data.


( D - E ) A431 ( D ) and HT29 ( E ) cells endogenously expressing mutant R273Hp53 were treated with control siRNA ( si Ctl ) or control MO ( MO Ctl - 2 or MO Ctl - 1 ) or siRNA targeting exon 7 of p53 ( si ) or MO targeting Δ160p53 ' s translation initiation site ( MO ) , as indicated , submitted to endoplasmic reticulum stress by exposure to thapsigargin ( Th ) and then analysed for apoptosis by incubation with propidium - iodide ( PI ) and FACS analysis . wtp53 - expressing A549 cells were similarly control - treated and used as comparison . Data was normalized against A549 cell condition , which was set to 100 .

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

(D-E) A431 (D) and HT29 (E) cells endogenously expressing mutant R273Hp53 were treated with control siRNA (si Ctl) or control MO (MO Ctl-2 or MO Ctl-1) or siRNA targeting exon 7 of p53 (si) or MO targeting Δ160p53's translation initiation site (MO), as indicated, submitted to endoplasmic reticulum stress by exposure to thapsigargin (Th) and then analysed for apoptosis by incubation with propidium-iodide (PI) and FACS analysis. wtp53-expressing A549 cells were similarly control-treated and used as comparison. Data was normalized against A549 cell condition, which was set to 100.


( F ) A431 and HT29 cells endogenously expressing mutant R273Hp53 were treated with control morpholino Ctl - 1 or morpholino targeting Δ160p53 ' s translation initiation site ( MO ) and then assessed by confocal microscopy for invasion further than 30 μm through a Matrigel - fibronectin matrix towards EGF - supplemented growth media . The 30 μm plane is indicated and scale bars represent 100 μm . Quantifications are shown in the right panel .

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

(F) A431 and HT29 cells endogenously expressing mutant R273Hp53 were treated with control morpholino Ctl-1 or morpholino targeting Δ160p53's translation initiation site (MO) and then assessed by confocal microscopy for invasion further than 30 μm through a Matrigel-fibronectin matrix towards EGF-supplemented growth media. The 30 μm plane is indicated and scale bars represent 100 μm. Quantifications are shown in the right panel.


A . Western blot analysis of reporter HslO - FLAG protein produced with a custom - made PURExpress ® coupled in vitro transcription - translation system supplemented with the indicated EF - G proteins and incubated for the indicated times . Blot was developed using anti - FLAG antibodies . Shown is a representative from at least two independent experiments . Eco EF - G : E . coli EF - G .

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

A. Western blot analysis of reporter HslO-FLAG protein produced with a custom-made PURExpress® coupled in vitro transcription-translation system supplemented with the indicated EF-G proteins and incubated for the indicated times. Blot was developed using anti-FLAG antibodies. Shown is a representative from at least two independent experiments. Eco EF-G: E. coli EF-G.


B . Western blot analysis of reporter HslO - FLAG protein synthesized in vitro using a custom - made PURExpress ® system with EF - G1 , EF - G2 , and two engineered EF - G2 variants : one missing the 26 - amino acid insert , EF - G2 ( ∆ 514 - 539 ) and one missing the beta hairpin in the insert , EF - G2 ( ∆ 516 - 527 ) . Shown is a representative experiment from at least two independent experiments .

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

B. Western blot analysis of reporter HslO-FLAG protein synthesized in vitro using a custom-made PURExpress® system with EF-G1, EF-G2, and two engineered EF-G2 variants: one missing the 26-amino acid insert, EF-G2 (∆514-539) and one missing the beta hairpin in the insert, EF-G2 (∆516-527). Shown is a representative experiment from at least two independent experiments.


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 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O 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 O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O 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 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 .

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

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 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 .

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

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 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 .

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 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 O B-GENEPROD I-GENEPROD I-GENEPROD O O 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 O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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 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 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 .

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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 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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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 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 .

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(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 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 .

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(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 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 ) .

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(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 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 .

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(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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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

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

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

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

O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O B-CELL_LINE O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O 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 analysis of mTORC1 activity in C2C12 - Vector and ARF1 KO cells in CM , in response to nutrient starvation and AA stimulation .

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

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

O O O O O O O O O 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

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 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 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-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 analysis of mTORC1 activity in wild type ( WT ) and MYH10 KO MEF cells cultured in CM , or in response to starvation and AA stimulation .

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

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

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

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 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 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 .

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

O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O 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 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 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 ) .

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

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O 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 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 + cells in rostral and ventral domains of an E11 . 5 Rx - Dicer mutant embryo . NCx , neocortex ; OB , olfactory bulb ; Spt , septum .

O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O 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 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 .

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

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

O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-CELL_TYPE O B-GENEPROD O O O B-TISSUE I-TISSUE I-TISSUE 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 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 the rostral telencephalon of E17 . 5 control and Rx - Dicer mutant littermates showing the neocortex ( NCx ) and olfactory bulb ( OB ; dashed line ) .

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

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

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

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

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 B-GENEPROD O O B-EXP_ASSAY O O 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 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 and 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 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 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 .

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

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 complex proteins ( Par3 , β - Catenin ) , primary cilia ( Arl13b ) , apical mitoses ( PhVim ) and neurons ( Tuj1 ) .

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

O 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 O O O O B-EXP_ASSAY O B-GENEPROD O O B-CELL_TYPE O B-GENEPROD 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 O O O O O B-EXP_ASSAY O O B-TISSUE O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O 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 O B-GENEPROD O 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 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 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 the rostral telencephalon of an E14 . 5 Rx - Dicer mutant embryo displaying the three types of rosettes .

O 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

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 ) 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 .

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 O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O B-TISSUE O O O O O B-TISSUE O 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 the indicated ages ( C ) , and rostro - caudal distribution of total rosette abundance per age , independent of type ( D ) . n ≥ 2 brains per age .

O O O O O O O O O O O O O O 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 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 + 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 .

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

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