A . SDS - PAGE analysis of the D391N DIS3L2 - RNA complexes . RNAs were radioactively labeled at the 5 ' end and DIS3L2 - bound RNAs were detected by autoradiography . The negative control was performed with HEK293T - Rex cells ( Control )

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A. SDS-PAGE analysis of the D391N DIS3L2-RNA complexes. RNAs were radioactively labeled at the 5' end and DIS3L2-bound RNAs were detected by autoradiography. The negative control was performed with HEK293T-Rex cells (Control)


B . DIS3L2 CLIP reads possess oligo ( U ) 3 ' termini . Histograms of reads with 3 ' - terminal stretches of identical nucleotides . Only oligo ( U ) tails longer than 4 nucleotides are observed .

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B. DIS3L2 CLIP reads possess oligo(U) 3' termini. Histograms of reads with 3'-terminal stretches of identical nucleotides. Only oligo(U) tails longer than 4 nucleotides are observed.


C . The untemplated uridylation is mostly found in the proximity of mature 3 ' RNA ends . The heat map shows the coverage of various RNA types by uridylated reads . RNA class and number of uridylated genes / transcripts for each category are indicated on the left . The length of each mature RNA was scaled to 100 % .

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C. The untemplated uridylation is mostly found in the proximity of mature 3' RNA ends. The heat map shows the coverage of various RNA types by uridylated reads. RNA class and number of uridylated genes/transcripts for each category are indicated on the left. The length of each mature RNA was scaled to 100%.


D . List of DIS3L2 - bound uridylated miRNAs with the number of uridylated reads identified in all three replicate CLIPs . Members of the let - 7 family are shown in bold .

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D. List of DIS3L2-bound uridylated miRNAs with the number of uridylated reads identified in all three replicate CLIPs. Members of the let-7 family are shown in bold.


F . Secondary structure of pre - hsa - let - 7i , with both 5p and 3p mature products highlighted in bold . The intramolecular ligation position is indicated in dash line between nt 28 and 61 . It corresponds to cleavage positions of RNaseT1 , which leaves 3 ' - terminal guanosine residues .

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F. Secondary structure of pre-hsa-let-7i, with both 5p and 3p mature products highlighted in bold. The intramolecular ligation position is indicated in dash line between nt 28 and 61. It corresponds to cleavage positions of RNaseT1, which leaves 3'-terminal guanosine residues.


G . D391N DIS3L2 overexpression does not cause abundance changes of CLIPed uridylated RNAs . Scatter plot of transcript abundances estimated from RNA - seq from the cell line overexpressing D391N DIS3L2 and control HEK293T - Rex cells . CLIPed transcripts are marked in white .

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G. D391N DIS3L2 overexpression does not cause abundance changes of CLIPed uridylated RNAs. Scatter plot of transcript abundances estimated from RNA-seq from the cell line overexpressing D391N DIS3L2 and control HEK293T-Rex cells. CLIPed transcripts are marked in white.


A . Types of uridylated ncRNAs identified by DIS3L2 CLIP - seq .

A. Types of uridylated ncRNAs identified by DIS3L2 CLIP-seq.


B . U + read coverage in downstream regions of mature U12 snRNA , Vault1 and 7SL ncRNAs . The full dark box is representing mature ncRNA , the thin line indicates the region downstream of the mature 3 ' end ( extension ) .

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B. U+ read coverage in downstream regions of mature U12 snRNA, Vault1 and 7SL ncRNAs. The full dark box is representing mature ncRNA, the thin line indicates the region downstream of the mature 3' end (extension).


C . Quantification of DIS3L2 D391N RIP analysis of U12 and U5D snRNAs showing number of snRNA clones with mature ( Mat ) , extended ( ext ) or trimmed ( Trim ) 3 ' end . + U is number of clones with untemplated 3 ' oligo ( U ) ends . For more details see Figure S2 . ( D ) An example of hybrid reads ( produced by intra - strand ligation of RNaseT1 products during CLIP protocol ) for RNU5D snRNA .

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C. Quantification of DIS3L2 D391N RIP analysis of U12 and U5D snRNAs showing number of snRNA clones with mature (Mat), extended (ext) or trimmed (Trim) 3' end. +U is number of clones with untemplated 3' oligo(U) ends. For more details see Figure S2. (D) An example of hybrid reads (produced by intra-strand ligation of RNaseT1 products during CLIP protocol) for RNU5D snRNA.


E . U + reads mapped to the rDNA locus by using the artificial chromosome U13369 . 1 .

E. U+ reads mapped to the rDNA locus by using the artificial chromosome U13369.1.


B . Uridylated tRNA reads overlap mainly with 3 ' trailers , T - loop and anticodon loop regions . Heat map showing the coverage of uridylated reads on pre - tRNAs . The pre - tRNA sub - regions ( as indicated in ( A ) ) are in small letters on the x - axis .

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B. Uridylated tRNA reads overlap mainly with 3' trailers, T-loop and anticodon loop regions. Heat map showing the coverage of uridylated reads on pre-tRNAs. The pre-tRNA sub-regions (as indicated in (A)) are in small letters on the x-axis.


C . Northern blot analysis of RNAs immunoprecipitated with Flag - tagged WT ( DIS3L2 ) and D391N ( MUT ) DIS3L2 , AGO2 and DIS3 , respectively . The blots were probed with radioactively labeled probes for 5 ' and 3 ' ends of mature tRNAVal , respectively .

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C. Northern blot analysis of RNAs immunoprecipitated with Flag-tagged WT (DIS3L2) and D391N (MUT) DIS3L2, AGO2 and DIS3, respectively. The blots were probed with radioactively labeled probes for 5' and 3' ends of mature tRNAVal, respectively.


D . Sequencing of MUT DIS3L2 - bound tRNAVal and tRNALeu confirmed the uridylation status of the tRNA fragments identified by CLIP - seq . Genomic sequence for each tRNA is indicated on top with the 3 ' trailer region separated by a vertical line . Non - templated uridine residues are in red .

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D. Sequencing of MUT DIS3L2-bound tRNAVal and tRNALeu confirmed the uridylation status of the tRNA fragments identified by CLIP-seq. Genomic sequence for each tRNA is indicated on top with the 3' trailer region separated by a vertical line. Non-templated uridine residues are in red.


E . Identification of hybrid reads ( produced by intra - strand ligation of RNaseT1 products during CLIP protocol ) mapping to tRNAPhe ( GAA ) and tRNAAsn ( GTT ) genes . To the right : the schematic representation of selected hybrid reads with corresponding color - code .

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E. Identification of hybrid reads (produced by intra-strand ligation of RNaseT1 products during CLIP protocol) mapping to tRNAPhe(GAA) and tRNAAsn(GTT) genes. To the right: the schematic representation of selected hybrid reads with corresponding color-code.


A . DIS3L2 - bound uridylated mRNA reads map to the 5 ' and 3 ' UTRs .

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A. DIS3L2-bound uridylated mRNA reads map to the 5' and 3' UTRs.


B . Most 3 ' UTR U + reads match the stem - loop structure of histone mRNAs . Heat map representation of the uridylation position .

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B. Most 3' UTR U+ reads match the stem-loop structure of histone mRNAs. Heat map representation of the uridylation position.


C . The uridylated aberrant RNAs are stabilized in cells with nonfunctional DIS3L2 . Small RNAs were modified with 3 ' end linker , and linker - specific primer was used for cDNA synthesis . Uridylated RNAs were PCR amplified in a semi - quantitative way with the same amount of input RNA used for RT reaction . We used a combination of the gene - and linker - specific primers , - RT ctrl is RT - PCR control , where no reverse transcriptase was added ; PCR control is a reaction with no cDNA added . The graph on the right summarizes the percentage of uridylated and nonuridylated 5 ' fragments of OAT mRNA in HEK293T - Rex cells ( Ctrl ) and cells overexpressing mutant DIS3L2 ( D391N ) .

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C. The uridylated aberrant RNAs are stabilized in cells with nonfunctional DIS3L2. Small RNAs were modified with 3' end linker, and linker-specific primer was used for cDNA synthesis. Uridylated RNAs were PCR amplified in a semi-quantitative way with the same amount of input RNA used for RT reaction. We used a combination of the gene- and linker-specific primers, -RT ctrl is RT-PCR control, where no reverse transcriptase was added; PCR control is a reaction with no cDNA added. The graph on the right summarizes the percentage of uridylated and nonuridylated 5' fragments of OAT mRNA in HEK293T-Rex cells (Ctrl) and cells overexpressing mutant DIS3L2 (D391N).


D . DIS3L2 - bound uridylated RNAs are degraded by DIS3L2 in vivo . RT - PCR amplification of 5 ' mRfs of OAT and RPL12 genes and 3 ' extended forms of U12 snRNA ( RNU12 ext ) from HEK293T cell lines with modified expression of DIS3L2 ( as marked on top ) was performed as described in C . Regions of mature 5S rRNA and 7SL RNA were used as loading controls . DIS3L2 KO is a DIS3L2 knock - out cell line . DIS3L2 rescue is DIS3L2 KO cells with stably integrated mutant ( D391N ) and wild type ( WT ) DIS3L2 expressing constructs , respectively . Bands marked with * are unspecific primer - dimer PCR products .

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D. DIS3L2-bound uridylated RNAs are degraded by DIS3L2 in vivo. RT-PCR amplification of 5'mRfs of OAT and RPL12 genes and 3' extended forms of U12 snRNA (RNU12 ext) from HEK293T cell lines with modified expression of DIS3L2 (as marked on top) was performed as described in C. Regions of mature 5S rRNA and 7SL RNA were used as loading controls. DIS3L2 KO is a DIS3L2 knock-out cell line. DIS3L2 rescue is DIS3L2 KO cells with stably integrated mutant (D391N) and wild type (WT) DIS3L2 expressing constructs, respectively. Bands marked with * are unspecific primer-dimer PCR products.


E . Metagene analysis of the position of DIS3L2 D391N clipped U + reads peak maximum around mRNA transcription start sites ( TSS ) and of the ChIP - seq data for RNAP II .

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E. Metagene analysis of the position of DIS3L2 D391N clipped U+ reads peak maximum around mRNA transcription start sites (TSS) and of the ChIP-seq data for RNAP II.


F . U + 5 ' mRfs and aberrant forms of U12 snRNA are highly enriched in the cytoplasm . RT - PCR detection of 5 ' mRfs of the OAT and RPL12 and aberrant form of U12 snRNA was performed as described in C with RNA isolated from nuclear and cytoplasmic fractions , respectively . The RT - PCR amplification of the body of 5S rRNA was used as a positive control for RNA isolation and RT reaction in both fractions . The identity and uridylation status of cytoplasmic OAT U + 5 ' mRfs was confirmed by sequencing ( shown on the bottom ) . In red are untemplated nucleotides , in green region corresponding to the first intron of OAT pre - mRNA . The efficiency of the subcellular fractionation was monitored by western blot ( right panel , WB ) . DDX5 is nucleoplasmic , ACO2 is mitochondrial , and DIS3L2 and TUBa are cytoplasmic markers .

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F. U+5'mRfs and aberrant forms of U12 snRNA are highly enriched in the cytoplasm. RT-PCR detection of 5'mRfs of the OAT and RPL12 and aberrant form of U12 snRNA was performed as described in C with RNA isolated from nuclear and cytoplasmic fractions, respectively. The RT-PCR amplification of the body of 5S rRNA was used as a positive control for RNA isolation and RT reaction in both fractions. The identity and uridylation status of cytoplasmic OAT U+5'mRfs was confirmed by sequencing (shown on the bottom). In red are untemplated nucleotides, in green region corresponding to the first intron of OAT pre-mRNA. The efficiency of the subcellular fractionation was monitored by western blot (right panel, WB). DDX5 is nucleoplasmic, ACO2 is mitochondrial, and DIS3L2 and TUBa are cytoplasmic markers.


( G ) Immunostaining of pS6 ( Ser240 / 244 ) in skin lesions from control and LL37 - induced mice . Bottom right panels , magnified images of boxed areas . DAPI staining ( blue ) indicates nuclear localization . Scale bar : 50 μm . Right panel , the quantification of relative fluorescence intensity for pS6 ( Ser240 / 244 ) in control and LL37 group ( n = 5 for each group ) . Data represent the mean ± SEM . * * P < 0 . 01 . two - tailed unpaired Student ' s t - test was used .

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(G) Immunostaining of pS6 (Ser240/244) in skin lesions from control and LL37-induced mice. Bottom right panels, magnified images of boxed areas. DAPI staining (blue) indicates nuclear localization. Scale bar: 50 μm. Right panel, the quantification of relative fluorescence intensity for pS6 (Ser240/244) in control and LL37 group (n=5 for each group). Data represent the mean ± SEM. **P < 0.01. two-tailed unpaired Student's t-test was used.


( D ) HE staining of lesional skin sections from WT and Raptor cKO mice injected with LL37 or control vehicle ( n = 6 for each group ) . Scale bar : 50 μm . ( E ) Dermal infiltrating cells were quantified ( n = 6 for each group ) for each high power field ( HPF ) .

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(D) HE staining of lesional skin sections from WT and Raptor cKO mice injected with LL37 or control vehicle (n=6 for each group). Scale bar: 50 μm. (E) Dermal infiltrating cells were quantified (n = 6 for each group) for each high power field (HPF).


( F ) The mRNA expression levels of Tnf - α , Il6 , Mmp9 and Vegf in skin lesions ( n = 6 for each group ) .

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(F) The mRNA expression levels of Tnf-α, Il6, Mmp9 and Vegf in skin lesions (n = 6 for each group).


( I ) HE staining of lesional skin sections from LL37 or control mice topically applied with RAPA or vehicle . Scale bar : 50 μm . ( J ) Dermal infiltrating cells were quantified ( n = 6 for each group ) . Data represent the mean ± SEM . * * P < 0 . 01 . 1 - way ANOVA with Bonferroni ' s post hoc test was used .

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(I) HE staining of lesional skin sections from LL37 or control mice topically applied with RAPA or vehicle. Scale bar: 50 μm. (J) Dermal infiltrating cells were quantified (n = 6 for each group). Data represent the mean ± SEM. **P < 0.01. 1-way ANOVA with Bonferroni's post hoc test was used.


( A ) The back skins of WT ( Tsc2 + / + ) and Tsc2 + / - mice were intradermally injected with LL37 or control vehicle ( n = 5 for each group ) . Pictures were taken on day 0 . 5 , 2 , 5 , 8 , 12 after the first LL37 injection . The mouse experiments were repeated for three times , and 5 mice were included in each group for each time . The results of a representative mouse experiment were displayed . Scale bar : 2 mm .

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(A) The back skins of WT (Tsc2+/+) and Tsc2+/- mice were intradermally injected with LL37 or control vehicle (n=5 for each group). Pictures were taken on day 0.5, 2, 5, 8, 12 after the first LL37 injection. The mouse experiments were repeated for three times, and 5 mice were included in each group for each time. The results of a representative mouse experiment were displayed. Scale bar: 2 mm.


( A ) The mRNA expression level of human cathelicidin ( CAMP ) in skin lesions from healthy individuals ( n = 10 ) and patients with rosacea ( n = 15 ) .

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(A) The mRNA expression level of human cathelicidin (CAMP) in skin lesions from healthy individuals (n = 10) and patients with rosacea (n = 15). Data


( G ) Immunostaining of cathelicidin in HaCaT keratinocytes treated with LL37 ( 4 μM ) ± RAPA for 24 h . DAPI staining ( blue ) indicates nuclear localization . Scale bar : 50 μm . All results are representative of at least 3 independent experiments . Data represent the mean ± SEM .

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(G) Immunostaining of cathelicidin in HaCaT keratinocytes treated with LL37 (4 μM) ± RAPA for 24 h. DAPI staining (blue) indicates nuclear localization. Scale bar: 50 μm. All results are representative of at least 3 independent experiments. Data represent the mean ± SEM.


( E ) Primary human keratinocytes expressing TLR2 shRNAs or scramble shRNA were treated with FITC - labeled LL37 or sLL37 ± PKH26 for 30 min followed by PBS washed , then live cells were analyzed by fluorescent microscope . Scale bar : 50 μm .

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(E) Primary human keratinocytes expressing TLR2 shRNAs or scramble shRNA were treated with FITC-labeled LL37 or sLL37 ± PKH26 for 30 min followed by PBS washed, then live cells were analyzed by fluorescent microscope. Scale bar: 50 μm.


( F , G ) Primary human keratinocytes were incubated with sLL37 - flag or LL37 - flag for 1 h . Cell lysates were immunoprecipitated with anti - flag or anti - TLR2 antibodies , showing an interaction between LL37 and TLR2 . Medium , the medium of primary human keratinocytes treated with sLL37 - flag or LL37 - flag . All results are representative of at least 3 independent experiments .

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(F, G) Primary human keratinocytes were incubated with sLL37-flag or LL37-flag for 1 h. Cell lysates were immunoprecipitated with anti-flag or anti-TLR2 antibodies, showing an interaction between LL37 and TLR2. Medium, the medium of primary human keratinocytes treated with sLL37-flag or LL37-flag. All results are representative of at least 3 independent experiments.


( E ) Immunostaining of phospho - p65 ( p - p65 ) in skin sections . Bottom right panels , magnified images of dotted line areas . Epi , epidermis . Der , dermis . Scale bar : 50 μm . ( F ) Quantification of relative fluorescence intensity for p - p65 in epidermis ( n = 6 ) .

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(E) Immunostaining of phospho-p65 (p-p65) in skin sections. Bottom right panels, magnified images of dotted line areas. Epi, epidermis. Der, dermis. Scale bar: 50 μm. (F) Quantification of relative fluorescence intensity for p-p65 in epidermis (n=6).


( G ) The mRNA expression levels of NF - κB family of transcription factors ( Nfκb1 , Nfκb1 , Rela and Relb ) in mice skin lesions ( n = 6 for each group ) .

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(G) The mRNA expression levels of NF-κB family of transcription factors (Nfκb1, Nfκb1, Rela and Relb) in mice skin lesions (n=6 for each group).


( I ) IHC of p65 on skin sections from HS and rosacea . Scale bar , 50 μm . ( J ) Percentage of nuclear p65 positive cells in the epidermis from HS ( n = 8 ) and rosacea ( n = 13 ) . All results are representative of at least 3 independent experiments . Data represent the mean ± SEM . * * P < 0 . 01 . 1 - way ANOVA with Bonferroni ' s post hoc test ( F and G ) or two - tailed unpaired Student ' s t - test ( J ) was used .

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(I) IHC of p65 on skin sections from HS and rosacea. Scale bar, 50 μm. (J) Percentage of nuclear p65 positive cells in the epidermis from HS (n = 8) and rosacea (n = 13). All results are representative of at least 3 independent experiments. Data represent the mean ± SEM. **P < 0.01. 1-way ANOVA with Bonferroni's post hoc test (F and G) or two-tailed unpaired Student's t-test (J) was used.


( B ) The mRNA expression levels of mouse chemokines ( Cxcl11 , Cxcl12 , Ccl2 and Ccl3 ) in skin lesions ( n = 6 for each group ) .

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(B) The mRNA expression levels of mouse chemokines (Cxcl11, Cxcl12, Ccl2 and Ccl3) in skin lesions (n=6 for each group).


( E ) Heatmap of upregulated chemokines and cytokines in rosacea skin lesions determined by RNA - sequencing . All results are representative of at least 3 independent experiments . Data represent the mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 . 1 - way ANOVA with Bonferroni ' s post hoc test was used .

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(E) Heatmap of upregulated chemokines and cytokines in rosacea skin lesions determined by RNA-sequencing. All results are representative of at least 3 independent experiments. Data represent the mean ± SEM. *P < 0.05, **P < 0.01. 1-way ANOVA with Bonferroni's post hoc test was used.


Topical rapamycin treatment twice daily for 4 weeks showed an obvious therapeutic effect in rosacea patients . ( C ) The change of patients ' CEA score showed in scatter histogram at 0 week and 4th week . ( D ) The change of patients ' IGA score showed in scatter histogram at 0 week and 4th week . " The Change value < 0 " indicates that skin lesions show positive improvement after treatment ; " The Change value > 0 " indicates skin lesions show exacerbation after treatment ; " The Change value = 0 " indicates that skin lesions show no change after treatment ; Data represent the mean ± SEM , and two - tailed unpaired Student ' s t - test was used ( C , D ) .

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Topical rapamycin treatment twice daily for 4 weeks showed an obvious therapeutic effect in rosacea patients. (C) The change of patients' CEA score showed in scatter histogram at 0 week and 4th week. (D) The change of patients' IGA score showed in scatter histogram at 0 week and 4th week. "The Change value < 0" indicates that skin lesions show positive improvement after treatment; "The Change value > 0" indicates skin lesions show exacerbation after treatment; "The Change value = 0" indicates that skin lesions show no change after treatment; Data represent the mean ± SEM, and two-tailed unpaired Student's t-test was used (C, D).


RAW 264 . 7 mouse macrophages were treated with either oxLDL ( 50 µg / ml ) or TG ( 300 nM ) for 6 hours . Protein lysates were treated with λ Phosphatase ( PPase ) for 30 minutes and analyzed by western blotting using specific antibodies for pFMRP , FMRP , pIRE1 , IRE1 and β - Actin . pFMRP / FMRP fold induction is depicted above the blots ( n = 6 biological replicates ) .

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RAW 264.7 mouse macrophages were treated with either oxLDL (50 µg/ml) or TG (300 nM) for 6 hours. Protein lysates were treated with λ Phosphatase (PPase) for 30 minutes and analyzed by western blotting using specific antibodies for pFMRP, FMRP, pIRE1, IRE1 and β-Actin. pFMRP/FMRP fold induction is depicted above the blots (n=6 biological replicates).


Control - or IRE1 - siRNA transfected HEK293T cells were stimulated by either PA ( 500 µM ) or TG ( 600 nM ) for 4 hours . Protein lysates were analyzed by western blotting using specific antibodies for pFMRP , FMRP , pIRE1 , IRE1 and β - Actin . pFMRP / FMRP fold induction is depicted above the blots ( n = 4 biological replicates ) .

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Control- or IRE1-siRNA transfected HEK293T cells were stimulated by either PA (500 µM) or TG (600 nM) for 4 hours. Protein lysates were analyzed by western blotting using specific antibodies for pFMRP, FMRP, pIRE1, IRE1 and β-Actin. pFMRP/FMRP fold induction is depicted above the blots (n=4 biological replicates).


MEF cells were transfected with either empty vector , EGFP - FMRP or 3xFLAG - IRE1 plasmids then pre - treated either with vehicle ( dimethyl sulfoxide , DMSO ) or AMG - 18 ( 25 µM ; 1 hour ) followed by TG ( 600 nM ) stimulation for 4 hours . Protein lysates were analyzed by western blotting using specific antibodies for pFMRP , FMRP , pIRE1 , IRE1 and β - Actin . pFMRP / FMRP fold induction is depicted above the blots ( n = 4 biological replicates ) .

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

MEF cells were transfected with either empty vector, EGFP-FMRP or 3xFLAG-IRE1 plasmids then pre-treated either with vehicle (dimethyl sulfoxide, DMSO) or AMG-18 (25 µM; 1 hour) followed by TG (600 nM) stimulation for 4 hours. Protein lysates were analyzed by western blotting using specific antibodies for pFMRP, FMRP, pIRE1, IRE1 and β-Actin. pFMRP/FMRP fold induction is depicted above the blots (n=4 biological replicates).


Purified FMRP and IRE1 kinase ( activated ) proteins were subjected to kinase assay and analyzed by western blotting using specific antibodies for ThioP , IRE1 and FMRP ( n = 3 biological replicates ) and with LC - MS / MS . Identified IRE1 kinase - mediated FMRP phosphorylation sites ( bottom ) .

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Purified FMRP and IRE1 kinase (activated) proteins were subjected to kinase assay and analyzed by western blotting using specific antibodies for ThioP, IRE1 and FMRP (n=3 biological replicates) and with LC-MS/MS. Identified IRE1 kinase-mediated FMRP phosphorylation sites (bottom).


Fmr1 + / + and Fmr1 - / - mice were injected with AAV _ PCSK9 and fed with 16 weeks of WD . Residential PM were stained with Oil Red O ( ORO ) and imaged ( n = 7 mice per group ; Scale bar = 50 µm ) . Apoe - / - mice were fed with WD ( 12 weeks ) and injected with vehicle ( DMSO ) or AMG - 18 ( 30 mg / kg / day ) in the last 4 weeks of WD . Residential PM were stained with ORO and imaged ( n = 5 mice per group ; Scale bar = 50 µm ) .

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Fmr1+/+ and Fmr1-/- mice were injected with AAV_PCSK9 and fed with 16 weeks of WD. Residential PM were stained with Oil Red O (ORO) and imaged (n=7 mice per group; Scale bar = 50 µm).    Apoe-/- mice were fed with WD (12 weeks) and injected with vehicle (DMSO) or AMG-18 (30 mg/kg/day) in the last 4 weeks of WD. Residential PM were stained with ORO and imaged (n=5 mice per group; Scale bar = 50 µm).


RCT experiment ( I ) plasma cholesterol levels after 24 and 48 hours , ( J ) liver cholesterol levels after 48 hours , and ( K ) feces cholesterol levels after 48 hours ( n = 12 mice per group ) .

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RCT experiment (I) plasma cholesterol levels after 24 and 48 hours, (J) liver cholesterol levels after 48 hours, and (K) feces cholesterol levels after 48 hours (n=12 mice per group).


A - E In vitro and in vivo efferocytosis experiments , where percentage of macrophages F4 / 80 + ( red ) that ingested apoptotic cells ( AC ) labeled with carboxyfluorescein succinimidyl ester ( CFSE ) + ( green ) were reported as % efferocytosis . ( A ) BMDMs were transfected with Fmr1 - or control - siRNA and incubated CFSE - labeled AC for the indicated hours ( n = 4 biological replicates ) . ( B ) Fmr1 + / + and Fmr1 - / - BMDMs were treated with PA ( 500 µM ) for 6 hours and then incubated with CFSE - labeled ACs for 4 hours ( n = 4 biological replicates ) . ( C ) Fmr1 + / + and Fmr1 - / - mice were fed WD ( 16 weeks ) and injected intraperitoneally with CFSE - labeled AC ( 1 . 5 hours ) , followed by PM elicitation ( n = 4 - 5 mice per group ) . ( D ) BMDM were pre - treated either with vehicle ( DMSO ) or AMG - 18 ( 5 µM ) for 1 hour then incubated with CFSE - labeled ACs for 4 hours ( n = 3 biological replicates ) . ( E ) C57BL / 6 mice were injected with AMG - 18 ( 30 mg / kg ) or vehicle ( DMSO ) for 8 hours , followed by intraperitoneal injection with CFSE - labeled ACs for 1 . 5 hours and PM elicitation ( n = 4 mice per group ) .

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

A-E In vitro and in vivo efferocytosis experiments, where percentage of macrophages F4/80+ (red) that ingested apoptotic cells (AC) labeled with carboxyfluorescein succinimidyl ester (CFSE)+ (green) were reported as % efferocytosis. (A) BMDMs were transfected with Fmr1- or control-siRNA and incubated CFSE-labeled AC for the indicated hours (n=4 biological replicates). (B) Fmr1+/+ and Fmr1-/- BMDMs were treated with PA (500 µM) for 6 hours and then incubated with CFSE-labeled ACs for 4 hours (n=4 biological replicates). (C) Fmr1+/+ and Fmr1-/- mice were fed WD (16 weeks) and injected intraperitoneally with CFSE-labeled AC (1.5 hours), followed by PM elicitation (n=4-5 mice per group). (D) BMDM were pre-treated either with vehicle (DMSO) or AMG-18 (5 µM) for 1 hour then incubated with CFSE-labeled ACs for 4 hours (n=3 biological replicates). (E) C57BL/6 mice were injected with AMG-18 (30 mg/kg) or vehicle (DMSO) for 8 hours, followed by intraperitoneal injection with CFSE-labeled ACs for 1.5 hours and PM elicitation (n=4 mice per group).


F - G In vitro continuous efferocytosis experiments , where macrophages were stained for F4 / 80 + ( red ) , AC were labeled with CFSE ( AC - 1 ; green ) or Violet ( AC2 ; violet ) . % continuous efferocytosis was determined by the ratio of F4 / 80 + , CFSE + and Violet + ( triple positive ) cells to total F4 / 80 + and CFSE + ( double positive ) cells . ( F ) Fmr1 + / + and Fmr1 - / - BMDM were incubated with AC - 1 for 2 hours , and after 2 hours interval , incubated with AC - 2 for 2 more hours ( n = 4 - 3 biological replicates ) . ( G ) BMDM were pre - treated either with vehicle ( DMSO ) or AMG - 18 ( 5 µM ) for 1 hour , incubated with CFSE - labeled AC - 1 for 2 hours , followed by incubation with Violet - labeled AC - 2 for 2 hours and PM collection ( n = 4 biological replicates ) .

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F-G In vitro continuous efferocytosis experiments, where macrophages were stained for F4/80+ (red), AC were labeled with CFSE (AC-1; green) or Violet (AC2; violet). % continuous efferocytosis was determined by the ratio of F4/80+, CFSE+ and Violet+ (triple positive) cells to total F4/80+ and CFSE+ (double positive) cells. (F) Fmr1+/+ and Fmr1-/- BMDM were incubated with AC-1 for 2 hours, and after 2 hours interval, incubated with AC-2 for 2 more hours (n=4-3 biological replicates). (G) BMDM were pre-treated either with vehicle (DMSO) or AMG-18 (5 µM) for 1 hour, incubated with CFSE-labeled AC-1 for 2 hours, followed by incubation with Violet-labeled AC-2 for 2 hours and PM collection (n=4 biological replicates).


RNA lysates from Fmr1 + / + and Fmr1 - / - BMDM that were treated with PA ( 500 µM ; 6 hours ) were fractionated using a 10 % - 50 % sucrose gradient and separated to polysome , monosome / NTR fractions . The absorbance ( 260 nm ) of RNA was measured and plotted as a function of time ( n = 3 biological replicates ) . ( B ) The ratio of the Abca1 , Abcg1 , Mertk , Lrp1 , Cd36 , Cd47 and Rac1 mRNA in polysome to NTR fraction ( n = 3 biological replicates ) .

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

RNA lysates from Fmr1+/+ and Fmr1-/- BMDM that were treated with PA (500 µM; 6 hours) were fractionated using a 10%-50% sucrose gradient and separated to polysome, monosome/NTR fractions. The absorbance (260 nm) of RNA was measured and plotted as a function of time (n=3 biological replicates). (B) The ratio of the Abca1, Abcg1, Mertk, Lrp1, Cd36, Cd47 and Rac1 mRNA in polysome to NTR fraction (n=3 biological replicates).


D Fmr1 - / - MEF cells were transfected with EV , WT - FMRP or STSA - FMRP plasmids followed by PA treatment ( 500 µM ; 6 hours ) . Protein lysates were analyzed by western blotting using specific antibodies for ABCA1 , MerTK , LRP1 , pFMRP , FMRP and β - Actin and fold inductions relative to β - Actin are depicted above the blots ( n = 5 biological replicates ) .

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D Fmr1-/- MEF cells were transfected with EV, WT-FMRP or STSA-FMRP plasmids followed by PA treatment (500 µM; 6 hours). Protein lysates were analyzed by western blotting using specific antibodies for ABCA1, MerTK, LRP1, pFMRP, FMRP and β-Actin and fold inductions relative to β-Actin are depicted above the blots (n=5 biological replicates).


Lesion area calculated from en face aorta , stained with ORO ( n = 12 - 13 mice per group ; Scale bar = 5 mm ) .

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Lesion area calculated from en face aorta, stained with ORO (n=12-13 mice per group; Scale bar = 5 mm).


Lesion area calculated from en face aorta , stained with ORO ( n = 9 mice per group ; Scale bar = 5 mm ) .

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Lesion area calculated from en face aorta, stained with ORO (n=9 mice per group; Scale bar = 5 mm).


Lesion area calculated from en face aorta , stained with ORO ( n = 5 mice per group ; Scale bar = 5 mm ) .

Lesion area calculated from en face aorta, stained with ORO (n=5 mice per group; Scale bar = 5 mm).


Lesion area calculated from en face aorta , stained with ORO ( n = 6 mice per group ; Scale bar = 5 mm ) .

Lesion area calculated from en face aorta, stained with ORO (n=6 mice per group; Scale bar = 5 mm).


Coronal and axial brain MRI of the patient with compound heterozygote mutations in ECE2 shows nodules of heterotopic neurons lining the lateral ventricles .

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Coronal and axial brain MRI of the patient with compound heterozygote mutations in ECE2 shows nodules of heterotopic neurons lining the lateral ventricles.


ECE2 expression on RNA level . In situ hybridisation for ECE2 RNA in 50d old cerebral organoids ( COs ) shows higher signal in the cortical plate - like zone ( CP ' ) ( Scalebar = 100 µm ) . Ventricle - like lumen in COs is marked as V ' .

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ECE2 expression on RNA level. In situ hybridisation for ECE2 RNA in 50d old cerebral organoids (COs) shows higher signal in the cortical plate-like zone (CP') (Scalebar=100 µm). Ventricle-like lumen in COs is marked as V'.


ECE2 expression on protein level . Immunohistochemistry ( IHC ) for ECE2 in 50d old COs shows accumulation in the CP ' and at the apical surface ( Scalebar = 50µm ) . Ventricle - like lumen in COs is marked as V ' .

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ECE2 expression on protein level. Immunohistochemistry (IHC) for ECE2 in 50d old COs shows accumulation in the CP' and at the apical surface (Scalebar=50µm). Ventricle-like lumen in COs is marked as V'.


COs transfected with micro - RNAs targeting ECE2 ( KD ) or scrambled negative control ( CTRL ) and GFP and analysed 7 days later reveal an increase in ectopic neurons upon ECE2 KD ( Transfected cells are shown in green , NEUN + neuronal nuclei in magenta . d = day . Scalebar = 25 µm ) . Ventricle - like lumen in COs is marked as V ' .

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COs transfected with micro-RNAs targeting ECE2 (KD) or scrambled negative control (CTRL) and GFP and analysed 7 days later reveal an increase in ectopic neurons upon ECE2 KD (Transfected cells are shown in green, NEUN+ neuronal nuclei in magenta. d=day. Scalebar=25 µm). Ventricle-like lumen in COs is marked as V'.


Graph depicting the number of ectopically located NEUN + cells in CTRL and KD electroporated COs using two different micro - RNAs targeting ECE2 . Data shown as box plot ( mean = red line , median = black line , box represents 25th and 75th percentiles , whiskers extend to 10th and 90th percentiles , all outliers are shown ; n = number of ventricles analysed ; * * * P < 0 . 001 in Kruskal - Wallis One Way ANOVA on Ranks and Dunn ' s Pairwise Multiple Comparison ) .

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Graph depicting the number of ectopically located NEUN+ cells in CTRL and KD electroporated COs using two different micro-RNAs targeting ECE2. Data shown as box plot (mean = red line, median = black line, box represents 25th and 75th percentiles, whiskers extend to 10th and 90th percentiles, all outliers are shown; n=number of ventricles analysed; ***P < 0.001 in Kruskal-Wallis One Way ANOVA on Ranks and Dunn's Pairwise Multiple Comparison).


Scheme depicting the experimental setup of neuronal differentiation and timelapse imaging under treatment with the ECE2 - inhibitor PHOS [ 42 ] .

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Scheme depicting the experimental setup of neuronal differentiation and timelapse imaging under treatment with the ECE2-inhibitor PHOS [42].


Graphs showing different parameters of neuronal migration dynamics , tracked via live imaging . Upon ECE2 inhibition , significant decrease in velocity , increase in resting TP and increase in tortuosity are observed . Data shown as z - scores ( CTRL = DMSO control ; PHOS = Phosphoramidon ; Resting TP = Resting timepoints ; N = number of individual neurons analysed from 2 batches ; Resting TP : * P = 0 . 024 ; tortuosiy : * P = 0 . 035 , * * * P < 0 . 001 in 2 - tailed Chi - Sqare test ) .

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Graphs showing different parameters of neuronal migration dynamics, tracked via live imaging. Upon ECE2 inhibition, significant decrease in velocity, increase in resting TP and increase in tortuosity are observed. Data shown as z-scores (CTRL = DMSO control; PHOS = Phosphoramidon; Resting TP = Resting timepoints; N=number of individual neurons analysed from 2 batches; Resting TP: *P=0.024; tortuosiy: *P=0.035, ***P < 0.001 in 2-tailed Chi-Sqare test).


Quantification of distribution of GFP + transfected cells 1dpe upon CTRL and Ece2 KD by IUE ( n = 3 CTRL and 4 Ece2 KD brains ; * P = 0 . 044 in One - way ANOVA and Tukey ' s Multiple Pairwise Comparison ; data shown as mean ± SEM ) .

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Quantification of distribution of GFP+ transfected cells 1dpe upon CTRL and Ece2 KD by IUE (n=3 CTRL and 4 Ece2 KD brains; *P=0.044 in One-way ANOVA and Tukey's Multiple Pairwise Comparison; data shown as mean±SEM).


IHC staining of GFP ( transfected cells ) and Pax6 1 dpe showing ectopic Pax6 + progenitors ( red circle ) and a reduction in their apical localisation ( yellow circle ) .

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IHC staining of GFP (transfected cells) and Pax6 1 dpe showing ectopic Pax6+ progenitors (red circle) and a reduction in their apical localisation (yellow circle).


High magnification images of GFP and Pax6 showing that ectopic progenitors are mostly not transfected . D - J , E13 - 16 Ece2 KD .

High magnification images of GFP and Pax6 showing that ectopic progenitors are mostly not transfected. D-J, E13-16 Ece2 KD.


GFP - Pax6 + progenitors delaminate upon Ece2 KD ( red arrows ) , leaving behind a region free of Pax6 + cells in the VZ ( yellow circle ) and forming ectopic rosettes ( yellow box ) .

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GFP-Pax6+ progenitors delaminate upon Ece2 KD (red arrows), leaving behind a region free of Pax6+ cells in the VZ (yellow circle) and forming ectopic rosettes (yellow box).


Reduction in apical Arl13b in the electroporation site ( white arrowheads ) indicates loss of apical belt integrity and disruption of apico - basal polarity upon Ece2 KD .

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Reduction in apical Arl13b in the electroporation site (white arrowheads) indicates loss of apical belt integrity and disruption of apico-basal polarity upon Ece2 KD.


Inside the nodule , which is surrounded by thickened GFP + processes ( pink arrows ) , Arl13b staining shows the formation of an ectopic apical

Inside the nodule, which is surrounded by thickened GFP+ processes (pink arrows), Arl13b staining shows the formation of an ectopic apical surface


Reduction in β - catenin in the electroporation site ( white arrowheads ) indicates loss of apical belt integrity and disruption of apico - basal polarity upon Ece2 KD .

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Reduction in β-catenin in the electroporation site (white arrowheads) indicates loss of apical belt integrity and disruption of apico-basal polarity upon Ece2 KD.


, Deep layer and I , upper layer neurons localise next to ectopic rosettes , forming nodules of ectopic neurons ( blue arrowheads for single ectopic neurons and blue circle for large nodule ) .

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, Deep layer and I, upper layer neurons localise next to ectopic rosettes, forming nodules of ectopic neurons (blue arrowheads for single ectopic neurons and blue circle for large nodule).


Quantification at 3 dpe , counting the proportion of brains with reduced density of Pax6 in the VZ , ectopic progenitors or neurons , and changes in apico - basal polarity ( including ectopic apical surface formation and reduction in apically localised apical markers ; n = 6 brains per condition ; Exact binomial test : * * * * P < 0 . 0001 ) . Scalebars in A , B , D , E , H , I = 100 µm ; in C , F , G Scalebar = 50 µm . V marks the position of the ventricle .

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

Quantification at 3 dpe, counting the proportion of brains with reduced density of Pax6 in the VZ, ectopic progenitors or neurons, and changes in apico-basal polarity (including ectopic apical surface formation and reduction in apically localised apical markers; n=6 brains per condition; Exact binomial test: ****P < 0.0001). Scalebars in A, B, D, E, H, I=100 µm; in C, F, G Scalebar = 50 µm. V marks the position of the ventricle.


Brightfield images of 60d old CTRL and ECE2 KO COs show normal formation of neuroepithelial structures .

Brightfield images of 60d old CTRL and ECE2 KO COs show normal formation of neuroepithelial structures.


ISH for ECE2 confirms the absence of ECE2 mRNA in 60d old ECE2 KO COs ( pink dotted line indicates position of the CP ' ) .

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ISH for ECE2 confirms the absence of ECE2 mRNA in 60d old ECE2 KO COs (pink dotted line indicates position of the CP').


, FACS analysis of 60d old ECE2 KO COs reveals an increase in progenitors and decrease in neurons ( n = 7 - 8 samples of 3 pooled organoids each from 3 different batches ; * * P < 0 . 01 ; * * * P < 0 . 001 in One Way ANOVA and Tukey Pairwise Multiple Comparison ) .

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, FACS analysis of 60d old ECE2 KO COs reveals an increase in progenitors and decrease in neurons (n=7-8 samples of 3 pooled organoids each from 3 different batches; **P < 0.01; ***P < 0.001 in One Way ANOVA and Tukey Pairwise Multiple Comparison).


Example images of IHC for PH3 + cells in 60d old CTRL and ECE2 KO COs . Quantification of PH3 + cycling cells in 60d old COs , normalised to length of the apical surface of ventricle - like structures , shows an increase upon ECE2 KO ( Data shown as box plots with median as black line , mean as red line ; n = number of analysed ventricles from 2 batches of COs ; One Way ANOVA : * * P = 0 . 004 ) .

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Example images of IHC for PH3+ cells in 60d old CTRL and ECE2 KO COs. Quantification of PH3+ cycling cells in 60d old COs, normalised to length of the apical surface of ventricle-like structures, shows an increase upon ECE2 KO (Data shown as box plots with median as black line, mean as red line; n=number of analysed ventricles from 2 batches of COs; One Way ANOVA: **P = 0.004).


In ECE2 KO COs , more ventricles are disorganised in terms of neuronal localisation as revealed from MAP2 + cell bodies in the progenitor zone ( n = number of ventricles from 2 independent batches ; two - tailed Mann Whitney U ( U = 458 ) and chi - square test : χ2 ( 2 ) = 25 . 42 , * * * * P < 0 . 0001 ) .

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In ECE2 KO COs, more ventricles are disorganised in terms of neuronal localisation as revealed from MAP2+ cell bodies in the progenitor zone (n=number of ventricles from 2 independent batches; two-tailed Mann Whitney U (U = 458) and chi-square test: χ2(2)=25.42, ****P < 0.0001).


Quantification of actin levels from Westernblot of whole CO lysate reveals a reduction in ECE2 KO COs ( n = 4 independent batches of CTRL and ECE2 KO COs with 3 pooled COs each sample ; * P = 0 . 021 in Kruskal - Wallis One Way Analysis of Variance on Ranks and Dunn ' s Pairwise Comparison ) .

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Quantification of actin levels from Westernblot of whole CO lysate reveals a reduction in ECE2 KO COs (n=4 independent batches of CTRL and ECE2 KO COs with 3 pooled COs each sample; *P = 0.021 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison).


Fractionation of G - and F - actin from CTRL and ECE2 KO COs and analysis via Westernblot additionally reveals an increase in G - at the expense F - actin upon ECE2 KO , suggesting a reduction in F - actin belt integrity ( n = 4 independent batches of CTRL and ECE2 KO COs with 3 pooled COs each ; * P = 0 . 021 in Kruskal - Wallis One Way Analysis of Variance on Ranks and Dunn ' s Pairwise Comparison ) .

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Fractionation of G- and F-actin from CTRL and ECE2 KO COs and analysis via Westernblot additionally reveals an increase in G- at the expense F-actin upon ECE2 KO, suggesting a reduction in F-actin belt integrity (n=4 independent batches of CTRL and ECE2 KO COs with 3 pooled COs each; *P = 0.021 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison).


The thickness of the apical F - actin belt is increased in ECE2 KO COs . C , Example images of apical F - actin belt in 60d old CTRL and ECE2 KO COs with F - actin labeled by AlexaFluor594 - conjugated Phalloidin ( Scalebar = 100 µm ) . D , Quantification of the thickness of apical F - actin belt in Fiji [ 86 ] by measuring the area of F - actin and dividing by the length of apical surface reveals significant increase in ECE2 KO COs ( Box plots : mean = red line , median = black line , box represents 25th and 75th percentiles , whiskers extend to 10th and 90th percentiles , all outliers are shown ; n = number of analysed ventricles in 2 batches ; Kruskal - Wallis One Way ANOVA on Ranks and Dunn ' s Pariwise Multiple Comparison : * * * P < 0 . 001 ) .

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

The thickness of the apical F-actin belt is increased in ECE2 KO COs. C, Example images of apical F-actin belt in 60d old CTRL and ECE2 KO COs with F-actin labeled by AlexaFluor594-conjugated Phalloidin (Scalebar=100 µm). D, Quantification of the thickness of apical F-actin belt in Fiji [86] by measuring the area of F-actin and dividing by the length of apical surface reveals significant increase in ECE2 KO COs (Box plots: mean=red line, median=black line, box represents 25th and 75th percentiles, whiskers extend to 10th and 90th percentiles, all outliers are shown; n=number of analysed ventricles in 2 batches; Kruskal-Wallis One Way ANOVA on Ranks and Dunn's Pariwise Multiple Comparison: ***P < 0.001).


IHC for ARL13B and F - ACTIN in 60d old CTRL and ECE2 KO COs shows disruption of honeycomb - like structure of the apical adherens belt and of apico - basal polarity in terms of apically localised primary cilium in ECE2 KO COs ( Scalebar = 10µm ) .

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

IHC for ARL13B and F-ACTIN in 60d old CTRL and ECE2 KO COs shows disruption of honeycomb-like structure of the apical adherens belt and of apico-basal polarity in terms of apically localised primary cilium in ECE2 KO COs (Scalebar = 10µm).


The microtubule cytoskeleton is changed in COs upon ECE2 KO . Example images of ac - tub IHC in CTRL vs . ECE2 KO COs ( Scalebar = 100 µm ) .

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The microtubule cytoskeleton is changed in COs upon ECE2 KO. Example images of ac-tub IHC in CTRL vs. ECE2 KO COs (Scalebar = 100 µm).


The microtubule cytoskeleton is changed in COs upon ECE2 KO . Quantification of ac - tub in ventricles of ECE2 KO COs by measurement of the mean grey value in Fiji shows significant reduction upon ECE2 KO ( n = number of analysed ventricles ; * P = 0 . 019 in One Way ANOVA and Tukey Pairwise Multiple Comparison ) .

The microtubule cytoskeleton is changed in COs upon ECE2 KO. Quantification of ac-tub in ventricles of ECE2 KO COs by measurement of the mean grey value in Fiji shows significant reduction upon ECE2 KO (n=number of analysed ventricles; *P = 0.019 in One Way ANOVA and Tukey Pairwise Multiple Comparison).


Apico - basal polarity is impaired upon ECE2 inhibition and KO as visible in example images of ARL13B IHC in CTRL vs . KO COs ( H ; Scalebar = 100 µm ) . Quantification of germinal zones for normally high ( " 1 " ) vs . reduced ( " 0 " ) apical ARL13B reveals a reduction in the proportion of normal ventricles in the absence of ECE2 ( n = number of analysed ventricles from 2 batches ; * * * * P < 0 . 0001 in exact binomial test ) .

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

Apico-basal polarity is impaired upon ECE2 inhibition and KO as visible in example images of ARL13B IHC in CTRL vs. KO COs (H; Scalebar = 100 µm). Quantification of germinal zones for normally high ("1") vs. reduced ("0") apical ARL13B reveals a reduction in the proportion of normal ventricles in the absence of ECE2 (n=number of analysed ventricles from 2 batches; ****P < 0.0001 in exact binomial test).


Analysis of CTRL and ECE2 KO COs 1 dpe with GAP43 - GFP reveals RG that delaminated ( white arrowheads ) and / or lost their bipolar morphology ( red arrowheads ) in the KO COs ( Scalebar = 25 µm ) .

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Analysis of CTRL and ECE2 KO COs 1 dpe with GAP43-GFP reveals RG that delaminated (white arrowheads) and/or lost their bipolar morphology (red arrowheads) in the KO COs (Scalebar = 25 µm).


Binning analysis 1 dpe after forced exression of ECE2 in the developing mouse cortex reveals ectopic positioning of Pax6 + GFP + neural progenitors with significantly reduced localisation to bin2 ( A ) and increased localisation of Ctip2 + deep layer neurons to bin2 with reduction in bin5 ( B ) ( for binning strategy see Figure EV2B ; n = 3 CTRL and 5 ECE2 OX brains E13 - 14 ; data shown as bars with mean ± SEM ; * P < 0 . 05 and * * P < 0 . 01 in Student ' s t - test ) .

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Binning analysis 1 dpe after forced exression of ECE2 in the developing mouse cortex reveals ectopic positioning of Pax6+GFP+ neural progenitors with significantly reduced localisation to bin2 (A) and increased localisation of Ctip2+ deep layer neurons to bin2 with reduction in bin5 (B) (for binning strategy see Figure EV2B; n=3 CTRL and 5 ECE2 OX brains E13-14; data shown as bars with mean±SEM; *P < 0.05 and **P < 0.01 in Student's t-test).


Example images E13 - 14 of IHC for Pax6 and Ctip2 upon CTRL and ECE2 OX conditions ( Scalebar = 100 µm ; electroporated cells shown in green ) . Ventricle marked by V .

Example images E13-14 of IHC for Pax6 and Ctip2 upon CTRL and ECE2 OX conditions (Scalebar = 100 µm; electroporated cells shown in green). Ventricle marked by V.


E13 - 16 images and analysis of ECE2 OX in the developing mouse cortex reveal ectopic positioning of Sox2 + ( D ) progenitors and Ctip2 + deep - ( D ) and Satb2 + upper layer neurons ( E ) ( Scalebars = 100 µm ; electroporated cells shown in green ) . F , Quantification at 3dpe , counting the proportion of brains with normal vs . abnormal positioning of Sox2 + neural progenitors and Ctip2 + and Satb2 + neurons and that of brains with intact vs . disrupted basal membrane ( n = 5 CTRL and 8 ECE2 OX brains ; exact binomial test : * * * * P < 0 . 0001 ) . Ventricle marked by V .

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E13-16 images and analysis of ECE2 OX in the developing mouse cortex reveal ectopic positioning of Sox2+ (D) progenitors and Ctip2+ deep- (D) and Satb2+ upper layer neurons (E) (Scalebars = 100 µm; electroporated cells shown in green). F, Quantification at 3dpe, counting the proportion of brains with normal vs. abnormal positioning of Sox2+ neural progenitors and Ctip2+ and Satb2+ neurons and that of brains with intact vs. disrupted basal membrane (n=5 CTRL and 8 ECE2 OX brains; exact binomial test: ****P < 0.0001). Ventricle marked by V.


ECE2 OX in COs ( electroporated cells shown in green ; Scalebars = 50 µm ; n = number of CO ventricles analysed from 2 batches ) . G , Example images of COs 7 days after CTRL or ECE2 OX electroporation and IHC for PH3 + mitotic cells and NEUN + neuronal nuclei . H , Quantification of PH3 + cells relative to electroporated apical surface length reveals a reduction upon ECE2 OX in CTRL CO background ( * P = 0 . 046 in Kruskal - Wallis One Way Analysis of Variance on Ranks and Dunn ' s Pairwise Comparison ) . I , Quantification of GFP + neurons as proportion of all GFP + cells reveals an increase upon ECE2 OX in COs ( * P = 0 . 021 in Kruskal - Wallis One Way Analysis of Variance on Ranks and Dunn ' s Pairwise Comparison ) . J , Quantification of ectopic neurons localising to the VZ ' relative to electroporated radial units ( GFP + cells in the VZ ' ) reveals a significant increase upon ECE2 OX ( * * * P < 0 . 001 in Kruskal - Wallis One Way Analysis of Variance on Ranks and Dunn ' s Pairwise Comparison ) . Ventricle - like lumen marked by V ' .

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

ECE2 OX in COs (electroporated cells shown in green; Scalebars = 50 µm; n = number of CO ventricles analysed from 2 batches). G, Example images of COs 7 days after CTRL or ECE2 OX electroporation and IHC for PH3+ mitotic cells and NEUN+ neuronal nuclei. H, Quantification of PH3+ cells relative to electroporated apical surface length reveals a reduction upon ECE2 OX in CTRL CO background (*P = 0.046 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison). I, Quantification of GFP+ neurons as proportion of all GFP+ cells reveals an increase upon ECE2 OX in COs (*P = 0.021 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison). J, Quantification of ectopic neurons localising to the VZ' relative to electroporated radial units (GFP+ cells in the VZ') reveals a significant increase upon ECE2 OX (***P < 0.001 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison). Ventricle-like lumen marked by V'.


, Forced expression of ECE2 in ECE2 KO COs rescues neuronal positioning . K , Example images of CTRL and ECE2 electroporated ECE2 KO COs 7 dpe . L , Quantification of ectopic neurons relative to electroporated radial units reveals a decrease upon ECE2 OX in ECE2 KO COs , thus rescueing the neuronal positioning phenotype in the absence of ECE2 OX ( * * P = 0 . 002 in Kruskal - Wallis One Way Analysis of Variance on Ranks and Dunn ' s Pairwise Comparison ) . Ventricle - like lumen marked by V ' .

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

, Forced expression of ECE2 in ECE2 KO COs rescues neuronal positioning. K, Example images of CTRL and ECE2 electroporated ECE2 KO COs 7 dpe. L, Quantification of ectopic neurons relative to electroporated radial units reveals a decrease upon ECE2 OX in ECE2 KO COs, thus rescueing the neuronal positioning phenotype in the absence of ECE2 OX (**P = 0.002 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison). Ventricle-like lumen marked by V'.


Volcano plot visualizes the proteomic analysis from 60d old CO lysates showing proteins with significantly lower and higher expression upon ECE2 KO ( n = 3 batches of 60d old CTRL and ECE2 KO COs ; FDR 0 . 05 ; s0 1 ; dashed lines indicating the cut - off ; names of example proteins are shown ) .

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Volcano plot visualizes the proteomic analysis from 60d old CO lysates showing proteins with significantly lower and higher expression upon ECE2 KO (n=3 batches of 60d old CTRL and ECE2 KO COs; FDR 0.05; s0 1; dashed lines indicating the cut-off; names of example proteins are shown).


Volcano plot of proteomic analysis of cell culture supernatant from 55d old CTRL and ECE2 KO COs shows two significantly downregulated proteins ( red ) in the secretome ( n = 3 batches of CTRL and ECE2 KO COs ; FDR 0 . 05 ; s0 1 ; dashed lines indicating the cut - off ) . Non - significantly changed proteins of interest are highlighted in green below the cut - off curves .

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

Volcano plot of proteomic analysis of cell culture supernatant from 55d old CTRL and ECE2 KO COs shows two significantly downregulated proteins (red) in the secretome (n=3 batches of CTRL and ECE2 KO COs; FDR 0.05; s0 1; dashed lines indicating the cut-off). Non-significantly changed proteins of interest are highlighted in green below the cut-off curves.


( A ) Immunoblots of mitochondrial fractions from siRNA ‐ transfected HEK293T cells ± CCCP .

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(A) Immunoblots of mitochondrial fractions from siRNA‐transfected HEK293T cells±CCCP.


( B ) Immunoblots against indicated proteases in siRNA ‐ transfected cells .

(B) Immunoblots against indicated proteases in siRNA‐transfected cells.


( C ) Quantification of full ‐ length PINK1 ( n = 4 ) and knockdown efficiency ( n = 2 ) compared with non ‐ targeting ( NT ) siRNA from experiments performed as in B . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 001 . CCCP , carbonyl cyanide m ‐ chlorophenyl hydrazone ; MPP , mitochondrial processing peptidase ; PARL , presenilin ‐ associated rhomboid ‐ like protease ; PINK1 , phosphatase and tensin homologue ‐ induced kinase 1 ; siRNA , short interfering RNA .

(C) Quantification of full‐length PINK1 (n=4) and knockdown efficiency (n=2) compared with non‐targeting (NT) siRNA from experiments performed as in B. *P0.05; **P0.01; ***P0.001. CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; MPP, mitochondrial processing peptidase; PARL, presenilin‐associated rhomboid‐like protease; PINK1, phosphatase and tensin homologue‐induced kinase 1; siRNA, short interfering RNA.


( A ) Live confocal micrographs of siRNA ‐ transfected HEK293T cells ± CCCP stained with MitoTracker ( MTR ) Green and Deep Red ( DR ) ; Scale bar , 5 μm .

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

(A) Live confocal micrographs of siRNA‐transfected HEK293T cells±CCCP stained with MitoTracker (MTR) Green and Deep Red (DR); Scale bar, 5 μm.


( B ) MTR DR / Green ratio from images as in A . N = 3 ; * P0 . 05 compared with NT siRNA .

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(B) MTR DR/Green ratio from images as in A. N=3; *P0.05 compared with NT siRNA.


( C ) Mitochondrial interconnectivity from images as in A , N = 3 .

(C) Mitochondrial interconnectivity from images as in A, N=3.


( D ) Oxygen consumption in state 3 respiration in mitochondrial fractions from siRNA ‐ transfected cells . Antimycin A with NT siRNA was used as a control . A . U . , arbitrary unit ; CCCP , carbonyl cyanide m ‐ chlorophenyl hydrazone ; MPP , mitochondrial processing peptidase ; NT , non ‐ targeting ; PARL , presenilin ‐ associated rhomboid ‐ like protease ; siRNA , short interfering RNA .

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(D) Oxygen consumption in state 3 respiration in mitochondrial fractions from siRNA‐transfected cells. Antimycin A with NT siRNA was used as a control. A.U., arbitrary unit; CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; MPP, mitochondrial processing peptidase; NT, non‐targeting; PARL, presenilin‐associated rhomboid‐like protease; siRNA, short interfering RNA.


( A ) Immunoblots of mitochondrial ( M ) and cytosolic ( C ) fractions from siRNA ‐ transfected HEK293T cells ± CCCP for 3 . 5 h , ± MG132 for the final hour .

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

(A) Immunoblots of mitochondrial (M) and cytosolic (C) fractions from siRNA‐transfected HEK293T cells±CCCP for 3.5 h, ±MG132 for the final hour.


( B ) Quantification of 52 ‐ kDa PINK1 from A ( with MG132 ) , n = 3 ; * P0 . 05 ; * * P0 . 001 compared with NT siRNA .

O O O O O O O O O O O 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) Quantification of 52‐kDa PINK1 from A (with MG132), n=3; *P0.05; **P0.001 compared with NT siRNA.


( C ) Mitochondria from siRNA ‐ transfected cells treated with 10 μM CCCP or dimethylsulphoxide were incubated with or without respiration substrate ( Resp . mix ) for 30 min at 37 ° C . al hour .

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(C) Mitochondria from siRNA‐transfected cells treated with 10 μM CCCP or dimethylsulphoxide were incubated with or without respiration substrate (Resp. mix) for 30 min at 37°C.al hour.


( A ) Mitochondria from CCCP ‐ treated or siRNA ‐ transfected HEK293T cells were incubated with increasing concentrations of proteinase K ( PK ) .

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

(A) Mitochondria from CCCP‐treated or siRNA‐transfected HEK293T cells were incubated with increasing concentrations of proteinase K (PK).


( B ) Mitochondria from CCCP ‐ treated or MPPβ knockdown cells were incubated ± 0 . 1 M Na2CO3 before supernatant ( S ) and pellet ( P ) were separated by centrifugation . CCCP , carbonyl cyanide m ‐ chlorophenyl hydrazone ; MPP , mitochondrial processing peptidase ; NT , non ‐ targeting ; PARL , presenilin ‐ associated rhomboid ‐ like protease ; PINK1 , phosphatase and tensin homologue ‐ induced kinase 1 ; siRNA , short interfering RNA ; WB , western blot .

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

(B) Mitochondria from CCCP‐treated or MPPβ knockdown cells were incubated ±0.1 M Na2CO3 before supernatant (S) and pellet (P) were separated by centrifugation. CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; MPP, mitochondrial processing peptidase; NT, non‐targeting; PARL, presenilin‐associated rhomboid‐like protease; PINK1, phosphatase and tensin homologue‐induced kinase 1; siRNA, short interfering RNA; WB, western blot.


( A ) Live confocal micrographs of siRNA ‐ , GFP ‐ Parkin ‐ and OCT ‐ DsRed ‐ transfected HEK293T cells ± CCCP ; Scale bars , 20 μm ( low magnification ) and 5 μm ( high magnification ) .

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(A) Live confocal micrographs of siRNA‐, GFP‐Parkin‐ and OCT‐DsRed‐transfected HEK293T cells ±CCCP; Scale bars, 20 μm (low magnification) and 5 μm (high magnification).


( B ) Percentage of transfected cells displaying a punctate mitochondrial pattern of GFP ‐ Parkin distribution , n = 3 ; * P0 . 05 ; * * P0 . 01 compared with NT siRNA .

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(B) Percentage of transfected cells displaying a punctate mitochondrial pattern of GFP‐Parkin distribution, n=3; *P0.05; **P0.01 compared with NT siRNA.


( C ) Immunoblots of endogenous Parkin in mitochondrial fractions from siRNA ‐ transfected cells ± CCCP .

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(C) Immunoblots of endogenous Parkin in mitochondrial fractions from siRNA‐transfected cells ±CCCP.


( D ) Mitochondrial mass measured by flow cytometry of MTR Green fluorescence in siRNA ‐ transfected cells treated ± CCCP for 24 h , n = 3 . A . U . , arbitrary unit ; CCCP , carbonyl cyanide m ‐ chlorophenyl hydrazone ; GFP , green fluorescent protein ; MPP , mitochondrial processing peptidase ; MTR , MitoTracker ; NT , non ‐ targeting ; PARL , presenilin ‐ associated rhomboid ‐ like protease ; PINK1 , phosphatase and tensin homologue ‐ induced kinase 1 ; siRNA , short interfering RNA .

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(D) Mitochondrial mass measured by flow cytometry of MTR Green fluorescence in siRNA‐transfected cells treated ±CCCP for 24 h, n=3. A.U., arbitrary unit; CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; GFP, green fluorescent protein; MPP, mitochondrial processing peptidase; MTR, MitoTracker; NT, non‐targeting; PARL, presenilin‐associated rhomboid‐like protease; PINK1, phosphatase and tensin homologue‐induced kinase 1; siRNA, short interfering RNA.


( A - B ) Expression patterns ( blue stain ) of GUS reporter genes driven by the SKM1 ( A ) or SKM2 ( B ) promoter in roots of 5 - day - old Col - 0seedlings .

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(A-B) Expression patterns (blue stain) of GUS reporter genes driven by the SKM1(A) or SKM2(B) promoter in roots of 5-day-old Col-0seedlings.


( C ) Non - complementation of CLE45 insensitivity of the bam3mutant by BAM3expressed under control of the SKM1promoter ( 7 - day - old seedlings ; 2 independent transgenic lines per construct are shown ) .

(C) Non-complementation of CLE45 insensitivity of the bam3mutant by BAM3expressed under control of the SKM1promoter (7-day-old seedlings; 2 independent transgenic lines per construct are shown).


( D ) Full CLE45 sensitivity of skm1or skm2 single and double mutant roots ( 7 - day - old seedlings ) .

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(D) Full CLE45 sensitivity of skm1or skm2 single and double mutant roots (7-day-old seedlings).


( E ) Failure of SKM1 or SKM2 expressed under control of the BAM3 promoter to complement CLE45 insensitivity of the bam3 mutant ( 7 - day - old seedlings ; 3 independent transgenic lines per construct are shown ) .

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(E) Failure of SKM1 or SKM2 expressed under control of the BAM3 promoter to complement CLE45 insensitivity of the bam3 mutant (7-day-old seedlings; 3 independent transgenic lines per construct are shown).


( A ) Root phenotypes of 7 - day - old seedlings of indicated genotypes . ( B ) Primary root length of 7 - day - old seedlings . ( C ) Root meristem size in 5 - day - old seedlings .

O O O O O O O O O O O O O O O O O O O O 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) Root phenotypes of 7-day-old seedlings of indicated genotypes. (B) Primary root length of 7-day-old seedlings. (C) Root meristem size in 5-day-old seedlings.


( D ) Root meristem phenotype of 5 - day - old seedlings , confocal microscopy , propidium iodide - stained ( red ) .

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(D) Root meristem phenotype of 5-day-old seedlings, confocal microscopy, propidium iodide-stained (red).


( E ) Auxin activity as visualized by the constitutively expressed inverse DII - NLS - VENUS marker ( green ) in root meristems of 5 - day - old seedlings .

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(E) Auxin activity as visualized by the constitutively expressed inverse DII-NLS-VENUS marker (green) in root meristems of 5-day-old seedlings.


( F ) Gap cell frequency in brxsingle or brxmakr5 double mutants ( note : gap cells were absent from Col - 0 wild type or makr5single mutants ) . ( G ) Illustration of gap cells ( arrowheads ) in developing protophloem sieve element strands ( asterisks ) of brxsingle or brxmakr5 double mutants .

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(F) Gap cell frequency in brxsingle or brxmakr5 double mutants (note: gap cells were absent from Col-0 wild type or makr5single mutants). (G) Illustration of gap cells (arrowheads) in developing protophloem sieve element strands (asterisks) of brxsingle or brxmakr5 double mutants.


( A ) Primary root length of 7 - day - old seedlings of indicated genotypes in response to continuous CLE45 treatments .

(A) Primary root length of 7-day-old seedlings of indicated genotypes in response to continuous CLE45 treatments.


( B ) Root meristem phenotypes of ( CLE45 - treated ) 5 - day - old seedlings ( asterisks : protophloem sieve element strands ) . Note absence of protophloem in CLE45 - treated Col - 0 wild type roots .

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(B) Root meristem phenotypes of (CLE45-treated) 5-day-old seedlings (asterisks: protophloem sieve element strands). Note absence of protophloem in CLE45-treated Col-0 wild type roots.


( C ) Transverse sections of ( CLE45 - treated ) 5 - day - old roots , just before the onset of protoxylem ( asterisks ) differentiation . Note failure of protophloem sieve elements ( arrowheads ) to differentiate in CLE45 - treated Col - 0 ( indicated by dense cytoplasm ) .

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(C) Transverse sections of (CLE45-treated) 5-day-old roots, just before the onset of protoxylem (asterisks) differentiation. Note failure of protophloem sieve elements (arrowheads) to differentiate in CLE45-treated Col-0 (indicated by dense cytoplasm).


( D ) Expression pattern of GUS reporter gene driven by the MAKR5 promoter in roots of 5 - day - old Col - 0seedlings . ( E ) Corresponding transverse sections from different positions along the root meristem .

(D) Expression pattern of GUS reporter gene driven by the MAKR5 promoter in roots of 5-day-old Col-0seedlings. (E) Corresponding transverse sections from different positions along the root meristem.


( F ) Expression of a MAKR5 - GFP fusion protein under control of the MAKR5 promoter in roots of 5 - day - old seedlings .

O O O 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) Expression of a MAKR5-GFP fusion protein under control of the MAKR5 promoter in roots of 5-day-old seedlings.


( G ) Corresponding optical sections illustrating MAKR5 - GFP expression in developing protophloem sieve elements ( asterisk ) and adjacent cell files .

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) Corresponding optical sections illustrating MAKR5-GFP expression in developing protophloem sieve elements (asterisk) and adjacent cell files.


( H ) Complementation of makr5CLE45 insensitivity by MAKR5expression under control of a promoter specific for developing sieve elements ( CVP2 ) , but not by expression under control of a companion cell - specific promoter ( SUC2 ) ( 7 - day - old seedlings ; 3 independent transgenic lines per construct are shown ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Complementation of makr5CLE45 insensitivity by MAKR5expression under control of a promoter specific for developing sieve elements (CVP2), but not by expression under control of a companion cell-specific promoter (SUC2) (7-day-old seedlings; 3 independent transgenic lines per construct are shown).


( I ) Subcellular localization of MAKR5 - GFP ( meristematic zone ) . Note plasma membrane association ( white , green arrowheads ) and dissociation from the cell wall ( red arrowhead ) upon osmosis .

(I) Subcellular localization of MAKR5-GFP (meristematic zone). Note plasma membrane association (white, green arrowheads) and dissociation from the cell wall (red arrowhead) upon osmosis.


( B ) Expression of truncated MAKR5 fusion proteins under control of the MAKR5 promoter in 5 - day - old roots ( asterisks : protophloem sieve element strands ) .

O O O O O O O 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 of truncated MAKR5 fusion proteins under control of the MAKR5 promoter in 5-day-old roots (asterisks: protophloem sieve element strands).


( C ) Close up of protophloem sieve element strands expressing truncated MAKR5 - CITRINE fusion proteins under control of the UBQ10 promoter .

(C) Close up of protophloem sieve element strands expressing truncated MAKR5-CITRINE fusion proteins under control of the UBQ10 promoter.


( D ) Failure of truncated MAKR5 fusion proteins to complement CLE45 insensitivity of the makr5 mutant ( 7 - day - old seedlings ; 1 - 2 independent transgenic lines per construct are shown ) .

(D) Failure of truncated MAKR5 fusion proteins to complement CLE45 insensitivity of the makr5 mutant (7-day-old seedlings; 1-2 independent transgenic lines per construct are shown).


( E ) Sensitivity of makr5 mutant roots to continuous brassinolide ( BL ) treatment ( 7 - day - old seedlings ) .

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

(E) Sensitivity of makr5 mutant roots to continuous brassinolide (BL) treatment (7-day-old seedlings).


( F ) Expression of MAKR5 - BKI1 fusion proteins under control of the MAKR5 promoter in roots of 5 - day - old makr5 seedlings .

(F) Expression of MAKR5-BKI1 fusion proteins under control of the MAKR5 promoter in roots of 5-day-old makr5 seedlings.


( G ) Complementation of CLE45 insensitivity of makr5 roots by BKI1 - MAKR5 , but not MAKR5 - BKI1 fusion protein ( 7 - day - old seedlings ; 3 independent transgenic lines per construct are shown ) .

(G) Complementation of CLE45 insensitivity of makr5 roots by BKI1-MAKR5, but not MAKR5-BKI1 fusion protein (7-day-old seedlings; 3 independent transgenic lines per construct are shown).


( A ) Increased expression of BAM3 - CITRINE fusion protein under control of the BAM3 promoter in developing protophloem sieve elements ( asterisks ) of 5 - day - old bam3 roots upon CLE45 treatment .

O O O O O O O O O O O O O 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) Increased expression of BAM3-CITRINE fusion protein under control of the BAM3 promoter in developing protophloem sieve elements (asterisks) of 5-day-old bam3 roots upon CLE45 treatment.


( B ) Expression of BAM3 - CITRINE fusion protein under control of the BAM3 promoter in 5 - day - old Col - 0 roots or makr5 roots .

O O O O O O 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 of BAM3-CITRINE fusion protein under control of the BAM3 promoter in 5-day-old Col-0 roots or makr5 roots.


( C ) Expression of MAKR5 - GFP fusion protein under control of the MAKR5 promoter in 5 - day - old Col - 0 or bam3 roots .

O O O O O 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) Expression of MAKR5-GFP fusion protein under control of the MAKR5 promoter in 5-day-old Col-0 or bam3 roots.


( D ) GUS reporter gene expression driven by the MAKR5 promoter in roots of 5 - day - old Col - 0seedlings upon CLE45 treatment .

(D) GUS reporter gene expression driven by the MAKR5 promoter in roots of 5-day-old Col-0seedlings upon CLE45 treatment.


( E ) Expression of MAKR5 - GFP fusion protein under control of the MAKR5 promoter in 5 - day - old roots upon CLE45 treatment ( 3 h ) . Note accumulation of MAKR5 - GFP in protophloem sieve elements ( magnified ) and its dependence on functional BAM3 and CLE45 . The frequency of representative observations per total observations is indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Expression of MAKR5-GFP fusion protein under control of the MAKR5 promoter in 5-day-old roots upon CLE45 treatment (3 h). Note accumulation of MAKR5-GFP in protophloem sieve elements (magnified) and its dependence on functional BAM3 and CLE45. The frequency of representative observations per total observations is indicated.


( F ) Close up view of MAKR5 - GFP accumulation in the protophloem cell file upon CLE45 treatment .

(F) Close up view of MAKR5-GFP accumulation in the protophloem cell file upon CLE45 treatment.


( G - H ) Accumulation of MAKR5 - BKI1 fusion proteins in protophloem cells upon CLE45 treatment . The frequency of representative observations per total observations is indicated . ( I - J ) Quantification of fusion protein abundance in developing protophloem cells upon expression under control of the MAKR5 promoter , showing mean total fluorescence ( I ) and plasma membrane to cytosol signal ratio ( J ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Accumulation of MAKR5-BKI1 fusion proteins in protophloem cells upon CLE45 treatment. The frequency of representative observations per total observations is indicated. (I-J) Quantification of fusion protein abundance in developing protophloem cells upon expression under control of the MAKR5 promoter, showing mean total fluorescence (I) and plasma membrane to cytosol signal ratio (J).


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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


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

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


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

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


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

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


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

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

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


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

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


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

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


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

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

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


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

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

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


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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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