( A ) Representative blots of 4 and 12 months old hippocampal lysates from control , Miro1CKO and Miro2KO . B ) Quantification of protein levels in 4 month lysates and 12 month lysates ( n = 3 animals / genotype at 4 months and n = 4 animals / genotype at 12 months ; one - way ANOVA with post - hoc Dunnett ' s test ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 .

O O O O B-EXP_ASSAY O O O O O O B-TISSUE O O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative blots of 4 and 12 months old hippocampal lysates from control, Miro1CKO and Miro2KO. B) Quantification of protein levels in 4 month lysates and 12 month lysates (n=3 animals / genotype at 4 months and n=4 animals / genotype at 12 months; one-way ANOVA with post-hoc Dunnett's test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001.


C ) Representative images of hippocampal regions from control and Miro1CKO crossed with MitoDendra animals .

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C) Representative images of hippocampal regions from control and Miro1CKO crossed with MitoDendra animals.


( D ) Details from cortical regions of control , Miro1CKO and Miro2KO animals at 12 months of age showing large mitochondrial structures occurring only in the case of Miro1CKO animals .

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(D) Details from cortical regions of control, Miro1CKO and Miro2KO animals at 12 months of age showing large mitochondrial structures occurring only in the case of Miro1CKO animals.


( E ) Electron microcopy images of control and Miro1CKO brains showing the ultrastructure of the mitochondrial compartment . The mitochondrial units in Miro1CKO cells are enlarged and show altered cristae structure at 4 months of age . This process is progressive as mitochondrial structures appear larger and with reduced intramitochondrial complexity at 12 months of age .

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

(E) Electron microcopy images of control and Miro1CKO brains showing the ultrastructure of the mitochondrial compartment. The mitochondrial units in Miro1CKO cells are enlarged and show altered cristae structure at 4 months of age. This process is progressive as mitochondrial structures appear larger and with reduced intramitochondrial complexity at 12 months of age.


Representative images ( F ) and quantification ( G ) of Mfn1 and Mfn2 staining in cortical slices from 12 month WT and Miro1CKO mice ( n = 4 animals / genotype ; student ' s t - test ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 .

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

Representative images (F) and quantification (G) of Mfn1 and Mfn2 staining in cortical slices from 12 month WT and Miro1CKO mice (n=4 animals / genotype; student's t-test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001.


( A ) Representative western blot images of brain lysates from 12 - month WT , Miro1CKO and Miro2KO mice immunoblotted with the antibodies stated . ( B ) Quantification of eIF2α and P - eIF2α band intensity ( normalised to actin band intensity ) from 12 - month WT , Miro1CKO and Miro2KO brain lysates . Phosphorylation of eIF2α is defined as P - eIF2α / eIF2α ( n = 4 mice per genotype , unpaired t - test ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 .

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

(A) Representative western blot images of brain lysates from 12-month WT, Miro1CKO and Miro2KO mice immunoblotted with the antibodies stated. (B) Quantification of eIF2α and P-eIF2α band intensity (normalised to actin band intensity) from 12-month WT, Miro1CKO and Miro2KO brain lysates. Phosphorylation of eIF2α is defined as P-eIF2α/eIF2α (n=4 mice per genotype, unpaired t-test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001.


( C ) Representative confocal images of cortical regions of 12 - month mitoDendra - crossed WT and Miro1CKO mice stained with MAP2 and P - eIF2α . ( D ) Quantification of P - eIF2α signal intensity from cortical regions of 12 - month mitoDendra - crossed WT and Miro1CKO mice ( n = 4 mice per genotype ; unpaired t - test ) .

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

(C) Representative confocal images of cortical regions of 12-month mitoDendra-crossed WT and Miro1CKO mice stained with MAP2 and P-eIF2α. (D) Quantification of P-eIF2α signal intensity from cortical regions of 12-month mitoDendra-crossed WT and Miro1CKO mice (n=4 mice per genotype; unpaired t-test).


( E ) Representative confocal images ( 63x ) of cortical regions of 12 - month mitoDendra - crossed WT and Miro1CKO mice stained with MAP2 and P - eIF2α . Arrows indicate the presence of megamitochondria ( > 2 . 5 µm2 ) , stars indicate neuronal somas without megamitochondria .

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

(E) Representative confocal images (63x) of cortical regions of 12-month mitoDendra -crossed WT and Miro1CKO mice stained with MAP2 and P-eIF2α. Arrows indicate the presence of megamitochondria (>2.5 µm2), stars indicate neuronal somas without megamitochondria.


( F ) Example zoom images of neuronal somas with and without megamitochondria from cortical regions of 12 - month mitoDendra - crossed Miro1CKO mice stained with P - eIF2α . ( G ) Quantification P - eIF2α signal intensity ( a . u . ) from neuronal somas with and without megamitochondria from cortical regions of 12 - month mitoDendra - crossed Miro1CKO ( n = 16 sections from 4 mice ; paired t - test ) .

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

(F) Example zoom images of neuronal somas with and without megamitochondria from cortical regions of 12-month mitoDendra -crossed Miro1CKO mice stained with P-eIF2α. (G) Quantification P-eIF2α signal intensity (a.u.) from neuronal somas with and without megamitochondria from cortical regions of 12-month mitoDendra-crossed Miro1CKO (n=16 sections from 4 mice; paired t-test).


A Anti - SARS - CoV - 2 neutralizing antibodies in plasma of vaccine breakthrough cases ( n = 54 ) and close contacts ( n = 86 ) were measured by surrogate virus neutralization test ( sVNT ) . The percentage inhibition of ACE2 binding to RBD at plasma dilution of 1 : 800 is given . The wild type ( Wuhan ) strain or Delta ( B . 1 . 617 . 2 ) strain RBD was used respectively .

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

A Anti-SARS-CoV-2 neutralizing antibodies in plasma of vaccine breakthrough cases (n=54) and close contacts (n=86) were measured by surrogate virus neutralization test (sVNT). The percentage inhibition of ACE2 binding to RBD at plasma dilution of 1:800 is given. The wild type (Wuhan) strain or Delta (B.1.617.2) strain RBD was used respectively.


B Anti - SARS - CoV - 2 neutralizing antibodies in plasma of vaccine breakthrough cases ( n = 29 ) and close contacts ( n = 86 ) were measured by pseudovirus neutralization assay .

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B Anti-SARS-CoV-2 neutralizing antibodies in plasma of vaccine breakthrough cases (n=29) and close contacts (n=86) were measured by pseudovirus neutralization assay.


C Anti - SARS - CoV - 2 S protein antibodies in plasma of vaccine breakthrough cases ( n = 48 ) and close contacts ( n = 86 ) was determined by S protein flow cytometry - based assay ( SFB ) . The percentage of plasma antibody - bound cells out of the total population of cells transfected with membrane - anchored SARS - CoV - 2 S protein is given . In all graphs error bars denote median and interquartile range . Red solid circles indicate Delta strain vaccine breakthrough , red open circles indicate non - Delta or unknown strain .

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

C Anti-SARS-CoV-2 S protein antibodies in plasma of vaccine breakthrough cases (n=48) and close contacts (n=86) was determined by S protein flow cytometry-based assay (SFB). The percentage of plasma antibody-bound cells out of the total population of cells transfected with membrane-anchored SARS-CoV-2 S protein is given. In all graphs error bars denote median and interquartile range. Red solid circles indicate Delta strain vaccine breakthrough, red open circles indicate non-Delta or unknown strain.


B , C Levels of anti - SARS - CoV - 2 neutralizing antibodies inhibiting ACE2 binding to WT or Delta are determined by surrogate virus neutralization test ( sVNT ) , in supernatant from 5 - day culture of activated memory B cells and in plasma . The difference in sVNT % inhibition between WT and Delta is taken , and this difference is compared between plasma and memory B cell supernatant for vaccine breakthrough cases ( n = 48 pairs ) ( B ) , and for close contacts ( n = 86 pairs ) ( C ) .

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

B, C Levels of anti-SARS-CoV-2 neutralizing antibodies inhibiting ACE2 binding to WT or Delta are determined by surrogate virus neutralization test (sVNT), in supernatant from 5-day culture of activated memory B cells and in plasma. The difference in sVNT % inhibition between WT and Delta is taken, and this difference is compared between plasma and memory B cell supernatant for vaccine breakthrough cases (n=48 pairs) (B), and for close contacts (n=86 pairs) (C).


A - D PBMCs from vaccine breakthrough cases ( n = 15 ) and close contacts ( n = 26 ) were examined for T cell responses . PBMCs were left unstimulated ( Unstim ) , were stimulated with pooled SARS - CoV - 2 PepTivator ® S , S1 , M and N peptides for 6 hours ( SARS - CoV - 2 Peptide Stim ) , or non - specifically stimulated with phorbol 12 - myristate 13 - acetate ( PMA ) , then assessed by high - dimensional flow cytometry . CD4 + T cells positive for intracellular staining of IFNγ ( a ) , IL - 2 ( b ) , TNF ( c ) , or CD8 + T cells positive for intracellular staining of granzyme B ( d ) are shown . Error bars indicate median and interquartile range .

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

A-D PBMCs from vaccine breakthrough cases (n=15) and close contacts (n=26) were examined for T cell responses. PBMCs were left unstimulated (Unstim), were stimulated with pooled SARS-CoV-2 PepTivator® S, S1, M and N peptides for 6 hours (SARS-CoV-2 Peptide Stim), or non-specifically stimulated with phorbol 12-myristate 13-acetate (PMA), then assessed by high-dimensional flow cytometry. CD4+ T cells positive for intracellular staining of IFNγ (a), IL-2 (b), TNF (c), or CD8+ T cells positive for intracellular staining of granzyme B (d) are shown. Error bars indicate median and interquartile range.


F The differentiation status of SARS - CoV - 2 peptide - stimulated CD4 + T cells ( left ) and CD8 + T cells ( right ) were compared based on CD27 and CD45RA expression ( Naïve : CD27 + CD45RA + ; T central memory ( TCM ) : CD27 + CD45RA - ; T effector memory ( TEM ) : CD27 - CD45RA - ; TEMRA : CD27 - CD45RA + ) . The average of all vaccine breakthrough cases or close contacts are plotted respectively .

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

F The differentiation status of SARS-CoV-2 peptide-stimulated CD4+ T cells (left) and CD8+ T cells (right) were compared based on CD27 and CD45RA expression (Naïve: CD27+ CD45RA+; T central memory (TCM): CD27+ CD45RA-; T effector memory (TEM): CD27- CD45RA-; TEMRA: CD27- CD45RA+). The average of all vaccine breakthrough cases or close contacts are plotted respectively.


B Heatmap of selected immune mediator levels in plasma samples of healthy controls , vaccinated close contacts , COVID - 19 vaccine breakthrough cases , and unvaccinated COVID - 19 patients with primary infection . Each color represents the relative concentration of a particular analyte . Blue and red indicate low and high concentration , respectively .

O O O O O O O O B-TISSUE O O O O O O O O O B-DISEASE I-DISEASE I-DISEASE O O O O O O B-DISEASE I-DISEASE I-DISEASE B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O

B Heatmap of selected immune mediator levels in plasma samples of healthy controls, vaccinated close contacts, COVID-19 vaccine breakthrough cases, and unvaccinated COVID-19 patients with primary infection. Each color represents the relative concentration of a particular analyte. Blue and red indicate low and high concentration, respectively.


( B ) The upper panel showing qRT - PCR analysis of four identified LUAD - associated RBPs ( ADARB1 , CELF2 , QKI and ZFP36 ) mRNA levels in 61 LUAD tissues and matched paracarcinoma tissues . The lower panel showing relative mRNA expression ( T / N ) of the four RBPs in metastatic ( n = 28 ) and non - metastatic ( n = 33 ) LUAD tissues . LUAD tissues were classified into metastatic and non - metastatic tissues as described in Materials and Methods . T , LUAD tissues ; N , paracarcinoma tissues . Y axis represents the log10 transformed fold change of mRNA expression ( T / N ) of four RBPs .

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

(B) The upper panel showing qRT-PCR analysis of four identified LUAD-associated RBPs (ADARB1, CELF2, QKI and ZFP36) mRNA levels in 61 LUAD tissues and matched paracarcinoma tissues. The lower panel showing relative mRNA expression (T/N) of the four RBPs in metastatic (n = 28) and non-metastatic (n = 33) LUAD tissues. LUAD tissues were classified into metastatic and non-metastatic tissues as described in Materials and Methods. T, LUAD tissues; N, paracarcinoma tissues. Y axis represents the log10 transformed fold change of mRNA expression (T/N) of four RBPs.


( E - G ) Kaplan - Meier survival curves of LC , LUAD and LUSC patients ( n = 1540 , n = 865 and n = 675 , respectively ) with high or low expression levels of QKI . The log - rank test was used to analyze the difference between two groups .

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

(E-G) Kaplan-Meier survival curves of LC, LUAD and LUSC patients (n=1540, n=865 and n=675, respectively) with high or low expression levels of QKI. The log-rank test was used to analyze the difference between two groups.


( H ) qRT - PCR analysis of endogenous mRNA levels of QKI - 5 , QKI - 6 , and QKI - 7 in human bronchial epithelial cell line BEAS - 2B , LUAD cell lines A549 and H1299 .

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

(H) qRT-PCR analysis of endogenous mRNA levels of QKI-5, QKI-6, and QKI-7 in human bronchial epithelial cell line BEAS-2B, LUAD cell lines A549 and H1299.


( J ) In vitro RNA pull - down assays were performed using biotin - labeled TGFβR1 3 ' UTR segments in A549 and H1299 cells . After pull - down , endogenous QKI - 5 enrichments were detected by western blot analyses . β - actin was used as the protein control .

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

(J) In vitro RNA pull-down assays were performed using biotin-labeled TGFβR1 3' UTR segments in A549 and H1299 cells. After pull-down, endogenous QKI-5 enrichments were detected by western blot analyses. β-actin was used as the protein control.


( A and B ) qRT - PCR and western blot analyses of TGFβR1 mRNA and protein levels in QKI - 5 - overexpressing A549 and H1299 cells . ( C and D ) Expression of TGFβR1 mRNA and protein in QKI - 5 - silenced A549 and H1299 cells .

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

(A and B) qRT-PCR and western blot analyses of TGFβR1 mRNA and protein levels in QKI-5-overexpressing A549 and H1299 cells. (C and D) Expression of TGFβR1 mRNA and protein in QKI-5-silenced A549 and H1299 cells.


( K ) QKI - 5 - overexpressing A549 and H1299 cells were treated and subjected to qRT - PCR assays to detect the mRNA expression of downstream genes of the TGF - β / SMAD signaling , including PAI - 1 , Slug , Snail , E - cadherin , and / or N - cadherin .

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

(K) QKI-5-overexpressing A549 and H1299 cells were treated and subjected to qRT-PCR assays to detect the mRNA expression of downstream genes of the TGF-β/SMAD signaling, including PAI-1, Slug, Snail, E-cadherin, and/or N-cadherin.


( C - F ) Wound - healing migration assays were performed on QKI - 5 - overexpressing A549 and H1299 cells as well as control cells The wound healing was recorded ( C and E ) and quantitatively measured ( D and F ) at least six times .

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

(C-F) Wound-healing migration assays were performed on QKI-5-overexpressing A549 and H1299 cells as well as control cells The wound healing was recorded (C and E) and quantitatively measured (D and F) at least six times.


( G and H ) In the presence or absence of TGF - β1 , QKI - 5 - overexpressing A549 and H1299 cells were allowed to migrate through a polycarbonate membrane in Transwells . After 24 h , migrated cells were stained , photographed and counted in at least four random fields under a light microscope . Representative images ( left ) and the migrated cell numbers ( right ) were presented . ( I and J ) QKI - 5 - overexpressing A549 and H1299 cells were treated and allowed to invade through Matrigel - coated membrane in Transwells . Invasive cells were

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

(G and H) In the presence or absence of TGF-β1, QKI-5-overexpressing A549 and H1299 cells were allowed to migrate through a polycarbonate membrane in Transwells. After 24 h, migrated cells were stained, photographed and counted in at least four random fields under a light microscope. Representative images (left) and the migrated cell numbers (right) were presented. (I and J) QKI-5-overexpressing A549 and H1299 cells were treated and allowed to invade through Matrigel-coated membrane in Transwells. Invasive cells were determined


( E ) H & E staining was performed for the evaluation of lung micrometastases . Representative images showing micrometastases of lung tissues from a pair of mice Blue and red arrowheads indicate lung micrometastases of vector group and QKI - 5 - overexpressing group , respectively . ( F ) Dot plots showing the difference of the micrometastases counts in lung tissues between QKI - 5 - overexpressing group and vector control group ( n = 10 mice per group ) . ( G ) Representative microscopic photographs of H & E staining for liver micrometastases in a pair of mice Blue and red arrowheads indicate liver micrometastases . ( H ) Dot plots showing the difference of the micrometastases counts in liver tissues between QKI - 5 - overexpressing group and control vector group ( n = 10 mice per group ) .

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

(E) H&E staining was performed for the evaluation of lung micrometastases. Representative images showing micrometastases of lung tissues from a pair of mice Blue and red arrowheads indicate lung micrometastases of vector group and QKI-5-overexpressing group, respectively. (F) Dot plots showing the difference of the micrometastases counts in lung tissues between QKI-5-overexpressing group and vector control group (n = 10 mice per group). (G) Representative microscopic photographs of H&E staining for liver micrometastases in a pair of mice Blue and red arrowheads indicate liver micrometastases. (H) Dot plots showing the difference of the micrometastases counts in liver tissues between QKI-5-overexpressing group and control vector group (n = 10 mice per group).


( C - F ) In the presence or absence of TGF - β1 , the migratory and invasive capabilities of above - treated A549 and H1299 cells were evaluated by Transwell assays Migrated and invasive cells were stained and counted under a light microscope .

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

(C-F) In the presence or absence of TGF-β1, the migratory and invasive capabilities of above-treated A549 and H1299 cells were evaluated by Transwell assays Migrated and invasive cells were stained and counted under a light microscope.


( J ) Kaplan - Meier survival curves of LUAD patients ( n = 865 ) with high or low expression levels of TGFβR1 . The log - rank test was used to compare the difference between two groups .

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

(J) Kaplan-Meier survival curves of LUAD patients (n=865) with high or low expression levels of TGFβR1. The log-rank test was used to compare the difference between two groups.


qRT - PCR analyses of mRNA expression of QKI - 5 in A549 cells transfected with siRNAs of TFs , which have potential binding sites in QKI - 5 promoter .

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

qRT-PCR analyses of mRNA expression of QKI-5 in A549 cells transfected with siRNAs of TFs, which have potential binding sites in QKI-5 promoter.


( E ) Western blot analysis of QKI - 5 protein levels in KLF6 - silenced A549 cells .

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

(E) Western blot analysis of QKI-5 protein levels in KLF6-silenced A549 cells.


( F ) An overview of KLF6 - ChIP - seq data ( GSE96355 ) is illustrated using Integrative Genomics Viewer ( IGV ) software . An obvious peak was detected at position - 100 to + 100 in the QKI - 5 promoter .

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

(F) An overview of KLF6-ChIP-seq data (GSE96355) is illustrated using Integrative Genomics Viewer (IGV) software. An obvious peak was detected at position -100 to +100 in the QKI-5 promoter.


qRT - PCR analysis of KLF6 mRNA in 61 LUAD tissues and matched paracarcinoma tissues ( I ) T , LUAD tissues ; N , paracarcinoma tissues . Y axis represents the log10 transformed fold change of mRNA expression of KLF6 . Central dotted line of the violin plot represents median , and upper and lower dotted line represents quartile ( I ) .

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

qRT-PCR analysis of KLF6 mRNA in 61 LUAD tissues and matched paracarcinoma tissues (I) T, LUAD tissues; N, paracarcinoma tissues. Y axis represents the log10 transformed fold change of mRNA expression of KLF6. Central dotted line of the violin plot represents median, and upper and lower dotted line represents quartile (I).


( A ) Western blot analysis of supernatants containing long and short mouse and human DNGR - 1 ECD proteins under reducing and non - reducing conditions .

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

(A) Western blot analysis of supernatants containing long and short mouse and human DNGR-1 ECD proteins under reducing and non-reducing conditions.


( B ) Western blot analysis of supernatants after protein production of WT and C94S mutant long mouse DNGR - 1 ECD proteins under reducing and non - reducing conditions . C94S mutant - containing supernatant was 200x concentrated before analysis .

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

(B) Western blot analysis of supernatants after protein production of WT and C94S mutant long mouse DNGR-1 ECD proteins under reducing and non-reducing conditions. C94S mutant-containing supernatant was 200x concentrated before analysis.


( C ) Western blot analysis of supernatants containing the indicated DNGR - 1 ECD proteins after treatment with PNGase F overnight . HRP - conjugated anti - FLAG antibody was used for detection of all proteins . Numbers on the side of blots indicate positions of molecular weight markers .

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

(C) Western blot analysis of supernatants containing the indicated DNGR-1 ECD proteins after treatment with PNGase F overnight. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers.


( A ) 0 . 25 μg of purified long mouse DNGR - 1 ECD was diluted into 10 mM Tris buffer of the indicated pH ( left panel ) or into 10 mM Tris pH 7 . 4 buffer supplemented with increasing amounts of NaCl ( 1 - 250 mM ; right panel ) , and reduction sensitivity of DNGR - 1 was assessed by reducing SDS - PAGE and Western blot . ( B ) 0 . 25 μg of purified DNGR - 1 long mouse ECD was diluted into buffers of different pH and ionic strength , and its reduction sensitivity was assessed by reducing SDS - PAGE and Western blot . The intensity of bands corresponding to dimer and monomer was determined densitometrically , and the ratio was plotted as a function of buffer ionic strength and pH .

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

(A) 0.25 μg of purified long mouse DNGR-1 ECD was diluted into 10 mM Tris buffer of the indicated pH (left panel) or into 10 mM Tris pH 7.4 buffer supplemented with increasing amounts of NaCl (1 - 250 mM; right panel), and reduction sensitivity of DNGR-1 was assessed by reducing SDS-PAGE and Western blot. (B) 0.25 μg of purified DNGR-1 long mouse ECD was diluted into buffers of different pH and ionic strength, and its reduction sensitivity was assessed by reducing SDS-PAGE and Western blot. The intensity of bands corresponding to dimer and monomer was determined densitometrically, and the ratio was plotted as a function of buffer ionic strength and pH.


( C ) 0 . 25 μg of purified long mouse DNGR - 1 ECD was diluted into PBS or 10 mM MES pH 6 . 1 buffer , and its reduction sensitivity was tested under mildly ( Laemmli buffer ) or strongly denaturing ( 8 M urea ) conditions by reducing SDS - PAGE and Western blot .

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

(C) 0.25 μg of purified long mouse DNGR-1 ECD was diluted into PBS or 10 mM MES pH 6.1 buffer, and its reduction sensitivity was tested under mildly (Laemmli buffer) or strongly denaturing (8 M urea) conditions by reducing SDS-PAGE and Western blot.


( D ) 0 . 25 μg of purified long mouse DNGR - 1 ECD was diluted into PBS or 10 mM MES pH 6 . 1 buffers , and its reduction sensitivity after different lengths of heat - denaturation in Laemmli buffer was tested by reducing SDS - PAGE and Western blot . HRP - conjugated anti - FLAG antibody was used for detection of all proteins . Numbers on the side of blots indicate positions of molecular weight markers . Reduction - resistant dimers are indicated with an * and reduction - sensitive protein with a + .

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

(D) 0.25 μg of purified long mouse DNGR-1 ECD was diluted into PBS or 10 mM MES pH 6.1 buffers, and its reduction sensitivity after different lengths of heat-denaturation in Laemmli buffer was tested by reducing SDS-PAGE and Western blot. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction-sensitive protein with a +.


( A ) 1 μg of purified long mouse DNGR - 1 ECD was transferred into 10mM MES pH 6 . 1 and 0 . 5 μg into PBS . Half of the MES sample and the whole PBS sample were dialyzed against 2 l of PBS overnight at 4 ° C . After dialysis , all samples were prepared for reducing SDS - PAGE and Western blot .

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

(A) 1 μg of purified long mouse DNGR-1 ECD was transferred into 10mM MES pH 6.1 and 0.5 μg into PBS. Half of the MES sample and the whole PBS sample were dialyzed against 2 l of PBS overnight at 4°C. After dialysis, all samples were prepared for reducing SDS-PAGE and Western blot.


( B ) Supernatants after production of indicated proteins in 293F cells were harvested and diluted into indicated buffers at a 2 : 3 ratio and analyzed using reducing SDS - PAGE and Western blot . HRP - conjugated anti - FLAG antibody was used for detection of all proteins . Numbers on the side of blots indicate positions of molecular weight markers . Reduction - resistant dimers are indicated with an * and reduction - sensitive protein with a + .

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

(B) Supernatants after production of indicated proteins in 293F cells were harvested and diluted into indicated buffers at a 2:3 ratio and analyzed using reducing SDS-PAGE and Western blot. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction-sensitive protein with a +.


( B ) Supernatants containing ECD of long mouse DNGR - 1 , Dectin - 1 and the chimeric receptor were prepared for SDS - PAGE and Western blot under non - reducing conditions .

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

(B) Supernatants containing ECD of long mouse DNGR-1, Dectin-1 and the chimeric receptor were prepared for SDS-PAGE and Western blot under non-reducing conditions.


( C ) Supernatants containing indicated proteins were diluted into PBS or pH 6 . 1 MES buffer and analyzed using SDS - PAGE and Western blot under reducing conditions . HRP - conjugated anti - FLAG antibody was used for detection of all proteins . Numbers on the side of blots indicate positions of molecular weight markers . Reduction - resistant dimers are indicated with an * and reduction sensitive protein with a + .

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

(C) Supernatants containing indicated proteins were diluted into PBS or pH 6.1 MES buffer and analyzed using SDS-PAGE and Western blot under reducing conditions. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction sensitive protein with a +.


( A ) Long mouse and human DNGR - 1 ECD proteins were diluted in PBS or 10 mM MES pH 6 . 1 buffer and 20 independent far - UV spectra were acquired for each condition ( red and black lines depict the composite curve for each condition )

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

(A) Long mouse and human DNGR-1 ECD proteins were diluted in PBS or 10 mM MES pH 6.1 buffer and 20 independent far-UV spectra were acquired for each condition (red and black lines depict the composite curve for each condition)


( B ) Long mouse DNGR - 1 ECD was diluted in PBS or 10 mM MES pH 6 . 1 buffer and 20 independent near - UV spectra were acquired for each condition ( red and black lines depict the composite curve for each condition and the blue line shows the difference between the two ) .

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

(B) Long mouse DNGR-1 ECD was diluted in PBS or 10 mM MES pH 6.1 buffer and 20 independent near-UV spectra were acquired for each condition (red and black lines depict the composite curve for each condition and the blue line shows the difference between the two).


( B ) and ( C ) Supernatants containing the indicated ECD proteins ( mouse , long isoform ) were diluted 1 : 2 into the indicated buffers and analyzed by SDS - PAGE and Western blot under reducing or non reducing conditions . HRP - conjugated anti - FLAG antibody was used for detection of all proteins . Numbers on the side of blots indicate positions of molecular weight markers . Reduction - resistant dimers are indicated with an * and reduction - sensitive protein with a + .

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

(B) and (C) Supernatants containing the indicated ECD proteins (mouse, long isoform) were diluted 1:2 into the indicated buffers and analyzed by SDS-PAGE and Western blot under reducing or non reducing conditions. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction-sensitive protein with a +.


( D ) Phoenix cells expressing the indicated proteins were lysed in 1 % SDS in PBS or 10mM MES pH 6 . 1 buffers and the lysates were analyzed by SDS - PAGE and Western blot under reducing conditions . Anti - DNGR - 1 antibody ( clone 397 ) followed by HRP - conjugated secondary anti - rat antibody was used for detection . Numbers on the side of blots indicate positions of molecular weight markers . Reduction - resistant dimers are indicated with an * and reduction - sensitive protein with a + .

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

(D) Phoenix cells expressing the indicated proteins were lysed in 1% SDS in PBS or 10mM MES pH 6.1 buffers and the lysates were analyzed by SDS-PAGE and Western blot under reducing conditions. Anti-DNGR-1 antibody (clone 397) followed by HRP-conjugated secondary anti-rat antibody was used for detection. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction-sensitive protein with a +.


( A ) Decreasing amounts of F - actin were spotted onto a nitrocellulose membrane and binding of indicated DNGR - 1 ECD ( mouse , long isoform ) proteins was tested in PBS or MES pH 6 . 1 buffers . HRP - conjugated anti - FLAG antibody was used for detection . The signal was quantified by densitometry and quantitation of four independent experiments is shown .

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

(A) Decreasing amounts of F-actin were spotted onto a nitrocellulose membrane and binding of indicated DNGR-1 ECD (mouse, long isoform) proteins was tested in PBS or MES pH 6.1 buffers. HRP-conjugated anti-FLAG antibody was used for detection. The signal was quantified by densitometry and quantitation of four independent experiments is shown.


( B and C ) B3Z - Syk reporter cells expressing indicated mutant or WT DNGR - 1 receptors ( mouse , long isoform ) were incubated with UV - irradiated 293T cells ( ratio dead : reporter cells is indicated ) , or with plate - bound anti - DNGR - 1 antibody or medium alone at 37 ° C overnight . Activation of the NFAT reporter was measured at the end of the incubation period . Data are plotted as mean ± SD of experimental duplicates . One representative of three experiments is shown .

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

(B and C) B3Z-Syk reporter cells expressing indicated mutant or WT DNGR-1 receptors (mouse, long isoform) were incubated with UV-irradiated 293T cells (ratio dead:reporter cells is indicated), or with plate-bound anti-DNGR-1 antibody or medium alone at 37°C overnight. Activation of the NFAT reporter was measured at the end of the incubation period. Data are plotted as mean ± SD of experimental duplicates. One representative of three experiments is shown.


( D ) Phoenix cells expressing indicated mutant or WT DNGR - 1 proteins ( mouse , long isoform ) were treated with F - actin , F - actin buffer , anti - DNGR - 1 antibody or isotype - matched antibody of irrelevant specificity for 45 minutes , fixed in PFA , surface - stained for DNGR - 1 and analyzed by flow cytometry . One representative of three experiments is shown .

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

(D) Phoenix cells expressing indicated mutant or WT DNGR-1 proteins (mouse, long isoform) were treated with F-actin, F-actin buffer, anti-DNGR-1 antibody or isotype-matched antibody of irrelevant specificity for 45 minutes, fixed in PFA, surface-stained for DNGR-1 and analyzed by flow cytometry. One representative of three experiments is shown.


( A ) DNGR - 1 KO MuTu DCs were transduced to express indicated DNGR - 1 proteins , FACS - sorted and expanded . Surface ( left panel ) and total ( right panel ) DNGR - 1 expression was determined by flow cytometry .

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

(A) DNGR-1 KO MuTu DCs were transduced to express indicated DNGR-1 proteins, FACS-sorted and expanded. Surface (left panel) and total (right panel) DNGR-1 expression was determined by flow cytometry.


( B and C ) Ovalbumin - expressing mouse embryonic fibroblasts ( bm1 T OVA MEFs ) were UV - irradiated , left to undergo secondary necrosis and incubated overnight with MuTu cells transduced with indicated WT or mutant DNGR - 1 proteins or an empty - vector control ( ratio dead cells : MuTu cells is indicated ) and OVA - specific pre - activated OT - I CD8 + T - lymphocytes . The amount of IFNγ accumulating in the culture medium was assessed using ELISA . One representative of two ( B ) or four ( C ) experiments is shown .

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

(B and C) Ovalbumin-expressing mouse embryonic fibroblasts (bm1 T OVA MEFs) were UV-irradiated, left to undergo secondary necrosis and incubated overnight with MuTu cells transduced with indicated WT or mutant DNGR-1 proteins or an empty-vector control (ratio dead cells:MuTu cells is indicated) and OVA-specific pre-activated OT-I CD8+ T-lymphocytes. The amount of IFNγ accumulating in the culture medium was assessed using ELISA. One representative of two (B) or four (C) experiments is shown.


( B ) Immunoblot showing CENP - AEA protein level at the indicated time in RPE - 1 cells .

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

(B) Immunoblot showing CENP-AEA protein level at the indicated time in RPE-1 cells.


( C ) Representative immunofluorescence images showing CENP - A reloading at CENP - B - marked centromeres in DLD - 1 cells . White dashed circles contour nuclei . Scale bar , 5 µm .

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

(C) Representative immunofluorescence images showing CENP-A reloading at CENP-B-marked centromeres in DLD-1 cells. White dashed circles contour nuclei. Scale bar, 5 µm.


( D ) Quantification of the percentage of cells showing centromeric CENP - A 24 hr or 48 hr after IAA WO . Each dot represents one experiment ( ~ 30 - 50 cells per condition per experiment ) , error bars represent standard deviation ( SD ) of 5 independent experiments .

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

(D) Quantification of the percentage of cells showing centromeric CENP-A 24 hr or 48 hr after IAA WO. Each dot represents one experiment (~30-50 cells per condition per experiment), error bars represent standard deviation (SD) of 5 independent experiments.


( E ) Quantification of centromeric CENP - A levels normalized to non - treated level . Each dot represents one experiment , error bars represent SD . Unpaired t test : * * * p = 0 . 0005 .

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

(E) Quantification of centromeric CENP-A levels normalized to non-treated level. Each dot represents one experiment, error bars represent SD. Unpaired t test: ***p= 0.0005.


( F ) Stills of live cell imaging to follow de novo CENP - AEA reloading in RPE - 1 cells harboring endogenously tagged CENP - Bmcherry . Images were taken every 15 min . White dashed circles contour nuclei prior / after mitosis and cells during mitosis based on bright field images .

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

(F) Stills of live cell imaging to follow de novo CENP-AEA reloading in RPE-1 cells harboring endogenously tagged CENP-Bmcherry. Images were taken every 15 min. White dashed circles contour nuclei prior/after mitosis and cells during mitosis based on bright field images.


( G ) Dot plot showing the timing of CENP - AEA reloading after anaphase onset in the indicated cell lines . Each dot represents one cell , error bars represent standard deviation .

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(G) Dot plot showing the timing of CENP-AEA reloading after anaphase onset in the indicated cell lines. Each dot represents one cell, error bars represent standard deviation.


( B ) Full coverage plot of chromosome 17 centromeric array ( chr17 : 22 , 500 , 000 - 26 , 900 , 000 of the hg38 assembly ) showing enrichment of CENP - A by CUT & RUN - seq in the untreated ( NT ) and IAA wash out ( WO ) sample . CENP - A is reloaded to cen17 _ 1 ( D17Z1 ) . A shift of CENP - A occupancy to cen17 _ 2 ( D17Z1B ) is not detected .

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

(B) Full coverage plot of chromosome 17 centromeric array (chr17:22,500,000-26,900,000 of the hg38 assembly) showing enrichment of CENP-A by CUT&RUN-seq in the untreated (NT) and IAA wash out (WO) sample. CENP-A is reloaded to cen17_1 (D17Z1). A shift of CENP-A occupancy to cen17_2 (D17Z1B) is not detected.


( F ) Representative images showing ectopic CENP - A on a chromosome spread after induction of CENP - A overexpression in DLD - 1 cells . Centromere position is marked using immunofluorescence staining for CENP - B . Yellow dashed lines contour representative chromosomes . Scale bar , 2µm . Schematic to the right illustrate the observed CENP - AmRFP - DHFR localization pattern on a chromosome . Right panel : Comparison of CENP - ADHFR level per pixel at the centromere and on chromosome arms following IAA and DOX / TMP treatment . Each dot represents one centromere or a region on the chromosome arms .

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

(F) Representative images showing ectopic CENP-A on a chromosome spread after induction of CENP-A overexpression in DLD-1 cells. Centromere position is marked using immunofluorescence staining for CENP-B. Yellow dashed lines contour representative chromosomes. Scale bar, 2µm. Schematic to the right illustrate the observed CENP-AmRFP-DHFR localization pattern on a chromosome. Right panel: Comparison of CENP-ADHFR level per pixel at the centromere and on chromosome arms following IAA and DOX/TMP treatment. Each dot represents one centromere or a region on the chromosome arms.


( G ) Schematic illustration for the experiments shown in H , I . ( H ) Representative images of chromosome spreads after re - expression of endogenous CENP - AEA into a CENP - AmRFP - DHFR overexpression background and subsequent removal of the overexpression as illustrated in ( G ) . Antibody against CENP - C was used to score for the presence of the functional centromere . Scale bar , 5µm . ( I ) Quantification of the indicated events after one CENP - AOFF / ON cycle compared to treatment shown in ( G ) .

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

(G) Schematic illustration for the experiments shown in H, I. (H) Representative images of chromosome spreads after re-expression of endogenous CENP-AEA into a CENP-AmRFP-DHFR overexpression background and subsequent removal of the overexpression as illustrated in (G). Antibody against CENP-C was used to score for the presence of the functional centromere. Scale bar, 5µm. (I) Quantification of the indicated events after one CENP-AOFF/ON cycle compared to treatment shown in (G).


( A ) Schematic illustration of the CENP - AOFF / ON cycle performed in the experiments shown in B , C . ( B ) Representative images of de novo CENP - A reloading in CENP - B wild - type ( + / + ) and CENP - B knock - out ( - / - ) cells . Cells with centromeric CENP - A are marked with a red dashed contour line while a yellow contour lines mark cell without centromeric CENP - A . Scale bar , 5 µm . ( C ) Quantification of relative number of RPE - 1 cells with centromeric CENP - A in the indicated conditions . Each dot represents one experiment with at least 20 cells per condition . Error bars represent standard error of the mean ( SEM ) from 3 independent experiments .

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

(A) Schematic illustration of the CENP-AOFF/ON cycle performed in the experiments shown in B, C. (B) Representative images of de novo CENP-A reloading in CENP-B wild-type (+/+) and CENP-B knock-out (-/-) cells. Cells with centromeric CENP-A are marked with a red dashed contour line while a yellow contour lines mark cell without centromeric CENP-A. Scale bar, 5 µm. (C) Quantification of relative number of RPE-1 cells with centromeric CENP-A in the indicated conditions. Each dot represents one experiment with at least 20 cells per condition. Error bars represent standard error of the mean (SEM) from 3 independent experiments.


( E ) Representative images of DLD - 1 CENP - B ( + / + ) or ( - / - ) cells in late M phase following one CENP - AOFF / ON cycle . EdU staining was used to confirm successful wash - out of Palbociclib and cell progressing through S - phase . Yellow dashed lines contour nucleus of cells in late M phase . Scale bar 5 µm . ( F ) Quantification of indicated events observed in late M phase cells in the indicated cell lines . Error bars show SEM from 4 independent experiments .

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

(E) Representative images of DLD-1 CENP-B (+/+) or (-/-) cells in late M phase following one CENP-AOFF/ON cycle. EdU staining was used to confirm successful wash-out of Palbociclib and cell progressing through S-phase. Yellow dashed lines contour nucleus of cells in late M phase. Scale bar 5 µm. (F) Quantification of indicated events observed in late M phase cells in the indicated cell lines. Error bars show SEM from 4 independent experiments.


( C ) Representative IF - FISH images of mitotic spreads in non - treated cell ( NT ) and following a CENP - AOFF / ON cycle ( IAA WO ) . Chromosomes are contoured by a green ( X ) , magenta ( Y ) or white ( other autosomes ) dashed line . The centromeres of chromosome X / Y are highlighted by a green / magenta arrow . Scale bar , 2 µm .

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

(C) Representative IF-FISH images of mitotic spreads in non-treated cell (NT) and following a CENP-AOFF/ON cycle (IAA WO). Chromosomes are contoured by a green (X), magenta (Y) or white (other autosomes) dashed line. The centromeres of chromosome X/Y are highlighted by a green/magenta arrow. Scale bar, 2 µm.


( D ) Quantification of CENP - C ( as read - out of CENP - A ) presence at the X ( green ) or Y ( magenta ) centromere in the indicated cell lines . Each dot represents one experiment . Error bars represent SD from 3 independent experiments . Unpaired t test , * * p = 0 . 0042 .

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

(D) Quantification of CENP-C (as read-out of CENP-A) presence at the X (green) or Y (magenta) centromere in the indicated cell lines. Each dot represents one experiment. Error bars represent SD from 3 independent experiments. Unpaired t test, **p=0.0042.


( G ) Representative IF - FISH images showing CENP - C localization on the Y chromosome in cell growing under selective pressure ( G418 ) following a CENP - AOFF / ON cycle . Chromosomes are highlighted by a white dashed contour line . CENP - C was found at the native centromere position ( top panel , blue ) , the Y chromosome fused to another chromosome indicated by the increased size of the chromosome and the absence of Chr - Y painting probe staining ( second panel , orange ) or CENP - C was found on a different location on the Y chromosome and did not overlap with Cen - Y DNA ( lower panels , red ) . Scale bar , 2 µm . ( H ) Quantification of indicated events depicted in G . Each dot represents one experiment ( > 20 spreads for experiment ) . Error bars represent SEM from 3 independent experiments . Unpaired t - test , * p = 0 . 0137 and 0 . 039 .

O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative IF-FISH images showing CENP-C localization on the Y chromosome in cell growing under selective pressure (G418) following a CENP-AOFF/ON cycle. Chromosomes are highlighted by a white dashed contour line. CENP-C was found at the native centromere position (top panel, blue), the Y chromosome fused to another chromosome indicated by the increased size of the chromosome and the absence of Chr-Y painting probe staining (second panel, orange) or CENP-C was found on a different location on the Y chromosome and did not overlap with Cen-Y DNA (lower panels, red). Scale bar, 2 µm. (H) Quantification of indicated events depicted in G. Each dot represents one experiment (>20 spreads for experiment). Error bars represent SEM from 3 independent experiments. Unpaired t-test, *p=0.0137 and 0.039.


( B ) Representative immunofluorescence images showing CENP - C recruitment at the LacO array in the indicated conditions . LacO array is displayed in the insets .

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(B) Representative immunofluorescence images showing CENP-C recruitment at the LacO array in the indicated conditions. LacO array is displayed in the insets.


( C ) Bar plot showing frequency of CENP - C ( orange ) or CENP - A ( green ) recruitment to LacO array in the indicated conditions . Each dot represents one independent experiment ( > 20 cell analyzed for experiment ) .

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

(C) Bar plot showing frequency of CENP-C (orange) or CENP-A (green) recruitment to LacO array in the indicated conditions. Each dot represents one independent experiment (>20 cell analyzed for experiment).


( D ) Quantification of CENP - C ( orange ) or CENP - A ( green ) protein levels at the LacO array in the indicated conditions normalized to protein intensities in non - treated conditions at the mCherry - LacI - CENP - B - LacO array using CENP - A or CENP - C antibody . Each dot represents one independent experiment ( > 20 cell analyzed for experiment ) . Error bars represent SEM from 3 independent experiments .

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(D) Quantification of CENP-C (orange) or CENP-A (green) protein levels at the LacO array in the indicated conditions normalized to protein intensities in non-treated conditions at the mCherry-LacI-CENP-B-LacO array using CENP-A or CENP-C antibody. Each dot represents one independent experiment (>20 cell analyzed for experiment). Error bars represent SEM from 3 independent experiments.


( E ) Representative immunofluorescence images showing CENP - A recruitment to ectopic mCherry - LacI - CENP - B in the indicated conditions . LacO array is shown in the inset .

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

(E) Representative immunofluorescence images showing CENP-A recruitment to ectopic mCherry-LacI-CENP-B in the indicated conditions. LacO array is shown in the inset.


( F ) Quantification of the frequency of CENP - C recruitment to LacO arrays by different LacI constructs . Error bars represent SEM from 5 independent experiments .

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

(F) Quantification of the frequency of CENP-C recruitment to LacO arrays by different LacI constructs. Error bars represent SEM from 5 independent experiments.


( G ) CENP - B immunoblot following GST pulldown experiments using GST tagged CENP - C ( 1 - 727 ) as bait and with the indicated proteins as preys .

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(G) CENP-B immunoblot following GST pulldown experiments using GST tagged CENP-C (1-727) as bait and with the indicated proteins as preys.


( C ) Representative immunofluorescence images showing CENP - A and CENP - C reloading in the indicated conditions . Cells with centromeric CENP - A / CENP - C are marked with a red dashed contour line while a yellow contour lines mark cell without centromeric CENP - A . The centromere position is marked using immunofluorescence staining for CENP - B . White dashed lines contour DAPI stained nucleus of representative cells .

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(C) Representative immunofluorescence images showing CENP-A and CENP-C reloading in the indicated conditions. Cells with centromeric CENP-A/CENP-C are marked with a red dashed contour line while a yellow contour lines mark cell without centromeric CENP-A. The centromere position is marked using immunofluorescence staining for CENP-B. White dashed lines contour DAPI stained nucleus of representative cells.


( D ) Bar plot showing the percentage of cells with CENP - A and CENP - C co - localization after a CENP - A / COFF / ON cycle in the indicated conditions . Error bars show SD from 5 independent experiments .

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(D) Bar plot showing the percentage of cells with CENP-A and CENP-C co-localization after a CENP-A/COFF/ON cycle in the indicated conditions. Error bars show SD from 5 independent experiments.


( E ) Box plot of CUT & RUN qPCR for CENP - A ( green ) or CENP - C ( orange ) . Box plot shows median , 25th and 75th percentiles , whiskers show minimum and maximum values . Results from 3 independent experiments including combined data from qPCR reactions using alpha - satellite primers and primers binding at the centromere of chromosome 4 . Enrichment is measured relative to the IgG control and normalized to Alu repeats .

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

(E) Box plot of CUT&RUN qPCR for CENP-A (green) or CENP-C (orange). Box plot shows median, 25th and 75th percentiles, whiskers show minimum and maximum values. Results from 3 independent experiments including combined data from qPCR reactions using alpha-satellite primers and primers binding at the centromere of chromosome 4. Enrichment is measured relative to the IgG control and normalized to Alu repeats.


( C ) Representative immunofluorescence images showing exogenous CENP - AmRFP - DHFR reloading in the presence ( control ) , but not in the absence ( sample ) of endogenous CENP - A and CENP - C . Cells with centromeric CENP - A are marked with a red dashed contour line while a yellow contour line marks cell without centromeric CENP - A . ( D ) Quantification of relative number of cells showing centromeric eCENP - AmRFP - DHFR in the indicated conditions . Error bars represent SEM from 3 independent experiments .

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

(C) Representative immunofluorescence images showing exogenous CENP-AmRFP-DHFR reloading in the presence (control), but not in the absence (sample) of endogenous CENP-A and CENP-C. Cells with centromeric CENP-A are marked with a red dashed contour line while a yellow contour line marks cell without centromeric CENP-A. (D) Quantification of relative number of cells showing centromeric eCENP-AmRFP-DHFR in the indicated conditions. Error bars represent SEM from 3 independent experiments.


( E ) Bar plot of CUT & RUN qPCR quantification using CENP - A antibody and primers binding at the centromere of chromosome 4 . Enrichment is measured relative to the IgG control and normalized to Alu repeats .

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

(E) Bar plot of CUT&RUN qPCR quantification using CENP-A antibody and primers binding at the centromere of chromosome 4. Enrichment is measured relative to the IgG control and normalized to Alu repeats.


( F ) Representative immunofluorescence chromosome spreads showing reloading of exogenous eCENP - CmRFP - DHFR in the presence ( control ) and in the absence ( sample ) of endogenous CENP - A and CENP - C . Cells were arrested with Colcemid for 3 h prior to spread . Cells with centromeric CENP - C are marked with a red dashed contour line while a yellow contour line marks cell without centromeric CENP - C . Chromosomes in inset are highlighted using a white dashed line . ( G ) Quantification of relative number of cells with eCENP - CmRFP - DHFR in the indicated conditions . Each dot represents one independent experiment with at least 30 cells per condition .

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

(F) Representative immunofluorescence chromosome spreads showing reloading of exogenous eCENP-CmRFP-DHFR in the presence (control) and in the absence (sample) of endogenous CENP-A and CENP-C. Cells were arrested with Colcemid for 3 h prior to spread. Cells with centromeric CENP-C are marked with a red dashed contour line while a yellow contour line marks cell without centromeric CENP-C. Chromosomes in inset are highlighted using a white dashed line. (G) Quantification of relative number of cells with eCENP-CmRFP-DHFR in the indicated conditions. Each dot represents one independent experiment with at least 30 cells per condition.


( H ) Bar plot of CUT & RUN qPCR results using CENP - C antibody and primers binding at the centromere of chromosome 4 . Enrichment is measured relative to the IgG control and normalized to Alu repeats . Each dot represents one independent experiment .

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

(H) Bar plot of CUT&RUN qPCR results using CENP-C antibody and primers binding at the centromere of chromosome 4. Enrichment is measured relative to the IgG control and normalized to Alu repeats. Each dot represents one independent experiment.


( A ) Gating strategy for the sorting of freshly purified resting human CD4 + T cells based on CENP - A expression . Dead cells were excluded ( first plot ) using Fixable Viability Dye eFluor 780 , CENP - Ahigh and CENP - Alow cells ( both CENP - B + ) were sorted ( second plot ) . Post - sort analysis of each population is then shown ( third and fourth plots ) .

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

(A) Gating strategy for the sorting of freshly purified resting human CD4+ T cells based on CENP-A expression. Dead cells were excluded (first plot) using Fixable Viability Dye eFluor 780, CENP-Ahigh and CENP-Alow cells (both CENP-B+) were sorted (second plot). Post-sort analysis of each population is then shown (third and fourth plots).


( B ) Representative images of sorted CD4 + T cells with high or low CENP - A level . The nucleus is contoured by a white dashed line . Scale bar , 5 µm .

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

(B) Representative images of sorted CD4+ T cells with high or low CENP-A level. The nucleus is contoured by a white dashed line. Scale bar, 5 µm.


( D ) Tracking of cell division by CFSE dilution . The black line separates cells that have not divided ( nº of div = 0 , CFSE - high ) from those that have undergone at least one division ( nº of div ≥ 1 , CFSE - low ) . ( E ) CENP - A expression and CFSE dilution . Black lines were set based on CENP - A isotype control and CFSE maximal staining at day 0 ( cells in shaded quadrants are CFSE - high / CENP - A - low ) . One representative donor is depicted . ( F ) Absolute number of CFSE - high / CENP - A - low CD4 + T cells ( shaded gate in E ) . Symbols represent individual donors . Error bars represent SEM from 6 independent donors . One - way ANOVA , multiple comparisons , n = 6 . * p < 0 . 05 ; * * p < 0 . 01 . ( G ) Frequency of cells without centromeric CENP - A determined by immunofluorescence microscopy before and post activation . Symbols represent individual donors , n = 4 . Error bars represent SEM from 6 independent donors .

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

(D) Tracking of cell division by CFSE dilution. The black line separates cells that have not divided (nº of div=0, CFSE-high) from those that have undergone at least one division (nº of div≥1, CFSE-low). (E) CENP-A expression and CFSE dilution. Black lines were set based on CENP-A isotype control and CFSE maximal staining at day 0 (cells in shaded quadrants are CFSE-high/CENP-A-low). One representative donor is depicted. (F) Absolute number of CFSE-high/CENP-A-low CD4+ T cells (shaded gate in E). Symbols represent individual donors. Error bars represent SEM from 6 independent donors. One-way ANOVA, multiple comparisons, n=6. *p < 0.05; **p < 0.01. (G) Frequency of cells without centromeric CENP-A determined by immunofluorescence microscopy before and post activation. Symbols represent individual donors, n=4. Error bars represent SEM from 6 independent donors.


A A bright field image of a wild - type adult C . elegans . The position of the AIY interneurons in the head is indicated with green , and the gut is outlined with dashed lines . A ' A cartoon diagram of the C . elegans head modified from wormatlas with permission . The AIY processes and soma are indicated in grey , and the presynaptic sites are marked with green ( Altun et al . , 2002 - 2019 ) .

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

A A bright field image of a wild-type adult C. elegans. The position of the AIY interneurons in the head is indicated with green, and the gut is outlined with dashed lines. A' A cartoon diagram of the C. elegans head modified from wormatlas with permission. The AIY processes and soma are indicated in grey, and the presynaptic sites are marked with green (Altun et al., 2002-2019).


B - G ' Confocal images of the AIY labeled with synaptic vesicle marker GFP : : RAB - 3 ( B - G , green ) and active zone marker GFP : : SYD - 1 ( B ' - G ' , pseudo - red ) in wild - type ( B , B ' ) , cwn - 2 ( ok895 ) ( C , C ' ) , cfz - 2 ( ok1201 ) ( D , D ' ) , pop - 1 ( hu9 ) ( E , E ' ) , and sys - 1 RNAi ( G , G ' ) animals . H - H ' Quantification of the fluorescence intensity of AIY RAB - 3 ( H ) and SYD - 1 ( H ' ) for the indicated genotypes .

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

B-G' Confocal images of the AIY labeled with synaptic vesicle marker GFP::RAB-3 (B-G, green) and active zone marker GFP::SYD-1 (B'-G', pseudo-red) in wild-type (B, B'), cwn-2(ok895) (C, C'), cfz-2(ok1201) (D, D'), pop-1(hu9) (E, E'), and sys-1 RNAi (G, G') animals. H-H' Quantification of the fluorescence intensity of AIY RAB-3 (H) and SYD-1 (H') for the indicated genotypes.


I - M ' Confocal images of AIY labeled with mCherry : : RAB - 3 ( pseudo - GFP ) ( I - M ) , or GFP : : RAB - 3 ( I ' - M ' ) of tissue - specific RNAi treatment for control ( I - I ' ) , cwn - 2 ( J - J ' ) , cfz - 2 ( K - K ' ) , sys - 1 ( L - L ' ) and pop - 1 ( M - M ' ) . N - N ' Quantification of the fluorescence intensity of AIY mCherry : : RAB - 3 or GFP : : RAB - 3 for the indicated RNAi treatment .

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

I-M' Confocal images of AIY labeled with mCherry::RAB-3 (pseudo-GFP) (I-M), or GFP::RAB-3 (I'-M') of tissue-specific RNAi treatment for control (I-I'), cwn-2(J-J'), cfz-2(K-K'), sys-1(L-L') and pop-1(M-M'). N-N' Quantification of the fluorescence intensity of AIY mCherry::RAB-3 or GFP::RAB-3 for the indicated RNAi treatment.


A Quantification of the fluorescence intensity of AIY GFP : : RAB - 3 in wild - type , egl - 3 ( n589 ) and aex - 5 ( sa23 ) mutants .

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

A Quantification of the fluorescence intensity of AIY GFP::RAB-3 in wild-type , egl-3(n589) and aex-5(sa23) mutants.


C Quantification of the fluorescence intensity of AIY GFP : : RAB - 3 for the indicated RNAi treatment . Candidates are intestinal expressed nlp or flp .

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

C Quantification of the fluorescence intensity of AIY GFP::RAB-3 for the indicated RNAi treatment. Candidates are intestinal expressed nlp or flp.


Confocal images of AIY labeled with GFP : : RAB - 3 in wild - type ( E ) with a wild type nlp - 40 transgene .

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

Confocal images of AIY labeled with GFP::RAB-3 in wild-type (E) with a wild type nlp-40 transgene.


Confocal images of AIY labeled with nlp - 40 ( tm4085 ) ( F ) , nlp - 40 ( vj3 ) ( G ) , nlp - 40 ( tm4085 ) ( H ) or nlp - 40 ( vj3 ) ( I ) with a wild type nlp - 40 transgene . J Quantification of the fluorescence intensity of AIY GFP : : RAB - 3 for the indicated genotypes . Transgenic data are averaged from at least two independent lines .

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

Confocal images of AIY labeled with nlp-40(tm4085) (F), nlp-40(vj3) (G), nlp-40(tm4085) (H) or nlp-40(vj3) (I) with a wild type nlp-40 transgene. J Quantification of the fluorescence intensity of AIY GFP::RAB-3 for the indicated genotypes. Transgenic data are averaged from at least two independent lines.


K - L . Confocal images of AIY synaptic marker GFP : : RAB - 3 for the indicated intestinal - specific RNAi treatment . M Quantification for ( K ) and ( L ) .

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

K-L. Confocal images of AIY synaptic marker GFP::RAB-3 for the indicated intestinal-specific RNAi treatment. M Quantification for (K) and (L).


A - C Quantification of the fluorescence intensity of AIY presynaptic marker GFP : : RAB - 3 ( A , B ) and nlp - 40 mRNA level ( C ) for the indicated genotypes . The AIY synaptic region was quantified for the synaptic intensity analysis .

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

A-C Quantification of the fluorescence intensity of AIY presynaptic marker GFP::RAB-3 (A, B) and nlp-40 mRNA level (C) for the indicated genotypes. The AIY synaptic region was quantified for the synaptic intensity analysis.


D - I Confocal images of the transcriptional nlp - 40 reporter ( Pnlp - 40 : : GFP ) in wild - type ( D ) , cwn - 2 ( ok895 ) ( E ) , cfz - 2 ( ok1201 ) ( F ) , pop - 1 ( hu9 ) ( G ) , and control or sys - 1 RNAi ( H and I ) animals at adult Day 1 stage . J Quantification of the Pnlp - 40 : : GFP fluorescence intensity for the indicated genotypes .

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

D-I Confocal images of the transcriptional nlp-40 reporter (Pnlp-40::GFP) in wild-type (D), cwn-2(ok895) (E), cfz-2(ok1201) (F), pop-1(hu9) (G), and control or sys-1 RNAi (H and I) animals at adult Day 1 stage. J Quantification of the Pnlp-40::GFP fluorescence intensity for the indicated genotypes.


K - P Confocal images of an intestinal - specific reporter ERM - 1 : : GFP in wild type ( K ) , cwn - 2 ( ok895 ) ( L ) , cfz - 2 ( ok1201 ) ( M ) , pop - 1 ( hu9 ) ( N ) , and control or sys - 1 RNAi ( O and P ) animals at the adult Day1 stage . Q Quantification of the ERM - 1 : : GFP fluorescence intensity for the indicated genotype .

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

K-P Confocal images of an intestinal-specific reporter ERM-1::GFP in wild type (K), cwn-2(ok895) (L), cfz-2(ok1201) (M), pop-1(hu9) (N), and control or sys-1 RNAi (O and P) animals at the adult Day1 stage. Q Quantification of the ERM-1::GFP fluorescence intensity for the indicated genotype.


A Diagram of aex - 2 genomic structure . The boxes and lines represent exons and introns . Black and gray indicate coding sequence and UTRs . The white asterisk marks the location of the R232Q mutation site of sa3 allele . B - K Confocal images of AIY presynaptic site labeled with GFP : : RAB - 3 in wild type ( B ) , nlp - 40 ( tm4085 ) ( C ) , aex - 2 ( sa3 ) ( D ) , nlp - 40 ( tm4085 ) ; aex - 2 ( sa3 ) ( E ) over - expression NLP - 40 in aex - 2 ( sa3 ) mutants ( F ) and aex - 2 ( sa3 ) with a wild - type aex - 2 transgene driven by the endogenous promoter ( Paex - 2 ) ( G ) , neuron - specific promoter ( Prab - 3 ) ( H ) , AIY - specific promoter ( Pttx - 3 ) ( I ) intestinal - specific promoter ( Pges - 1 ) ( J ) or muscle - specific promoter ( Pmyo - 3 ) ( K ) . L - M Quantification of the fluorescence intensity of AIY GFP : : RAB - 3 for the indicated genotypes . Each dot represents one animal . Data were collected from at least three independent experiments . Transgenic data are averaged from at least two independent lines . tg : Paex - 2 : : aex - 2 , Pttx - 3 : : aex - 2 or Pges - 1 : : aex - 2 transgene , + : wild type ; - : mutant or without tg . The AIY synaptic region was quantified for the synaptic intensity analysis .

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

A Diagram of aex-2 genomic structure. The boxes and lines represent exons and introns. Black and gray indicate coding sequence and UTRs. The white asterisk marks the location of the R232Q mutation site of sa3 allele. B-K Confocal images of AIY presynaptic site labeled with GFP::RAB-3 in wild type(B), nlp-40(tm4085)(C), aex-2(sa3)(D), nlp-40(tm4085); aex-2(sa3) (E) over-expression NLP-40 in aex-2(sa3) mutants(F) and aex-2(sa3) with a wild-type aex-2 transgene driven by the endogenous promoter (Paex-2) (G), neuron-specific promoter (Prab-3) (H) , AIY-specific promoter (Pttx-3) (I) intestinal-specific promoter (Pges-1) (J) or muscle-specific promoter (Pmyo-3)(K). L-M Quantification of the fluorescence intensity of AIY GFP::RAB-3 for the indicated genotypes. Each dot represents one animal. Data were collected from at least three independent experiments. Transgenic data are averaged from at least two independent lines. tg: Paex-2::aex-2, Pttx-3::aex-2 or Pges-1::aex-2 transgene, +: wild type ; -: mutant or without tg. The AIY synaptic region was quantified for the synaptic intensity analysis.


N - Q ' Confocal images of Paex - 2 : : mNeonGreen of whole animal with bright field ( N ) , the head region labeled with Paex - 2 : : mNeonGreen and Pttx - 3 : : mCherry with green channel ( O ) , red channel ( P ) and the merged channels ( Q ) . ( O ' - Q ' ) are the cross sections corresponding to the dashed line sites .

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

N-Q' Confocal images of Paex-2::mNeonGreen of whole animal with bright field (N), the head region labeled with Paex-2::mNeonGreen and Pttx-3::mCherry with green channel (O), red channel (P) and the merged channels (Q). (O'-Q') are the cross sections corresponding to the dashed line sites.


A - A ' Representative confocal images showing inactive ( A ) and active ( A ' ) state of AIY : : GCaMP6s ( with mod - 1 promoter ) colabeled with mCherry ( with ttx - 3 promoter ) ( red ) . Dashed ovals mark the AIY zone 2 region where GCaMP was quantified .

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

A-A' Representative confocal images showing inactive (A) and active(A') state of AIY::GCaMP6s (with mod-1 promoter) colabeled with mCherry (with ttx-3 promoter) (red). Dashed ovals mark the AIY zone 2 region where GCaMP was quantified.


B Representative AIY calcium activity over 60 seconds for the indicated genotypes . C - D Quantification of the peak frequency and amplitude of AIY : : GCaMP6s in for the indicated genotypes .

O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O

B Representative AIY calcium activity over 60 seconds for the indicated genotypes. C-D Quantification of the peak frequency and amplitude of AIY::GCaMP6s in for the indicated genotypes.


B - G Confocal images of Prab - 3 : : GFP : : RAB - 3 in the nerve region corresponding to the dashed box in ( A ) of wild type ( B ) , cwn - 2 ( ok895 ) ( C ) , cfz - 2 ( ok1201 ) ( D ) , pop - 1 ( hu9 ) ( E ) , nlp - 40 ( tm4085 ) ( F ) and aex - 2 ( sa3 ) ( G ) mutants . H - I Quantification of the GFP : : RAB - 3 intensity in the nerve ring synaptic region for the indicated genotypes . Each dot represents one animal . Data were collected from at least three independent experiments . Transgenic data are averaged from at least two independent lines .

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

B-G Confocal images of Prab-3::GFP::RAB-3 in the nerve region corresponding to the dashed box in (A) of wild type (B), cwn-2(ok895) (C), cfz-2(ok1201) (D), pop-1(hu9) (E), nlp-40(tm4085) (F) and aex-2(sa3) (G) mutants. H-I Quantification of the GFP::RAB-3 intensity in the nerve ring synaptic region for the indicated genotypes. Each dot represents one animal. Data were collected from at least three independent experiments. Transgenic data are averaged from at least two independent lines.


A APB Northern blot using a tRNAHis probe . m5C38 levels of tRNAAspGUC were measured by 454 bisulfite sequencing at the indicated time points . Both queuosinylation and methylation levels could be restored by the addition of queuine . The slower migration of tRNAHis is eliminated by oxidizing the ribose with periodate , producing a single faster - migrating band ( ox ) . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , Q ( queuosine ) , G ( guanine ) .

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

A APB Northern blot using a tRNAHis probe. m5C38 levels of tRNAAspGUC were measured by 454 bisulfite sequencing at the indicated time points. Both queuosinylation and methylation levels could be restored by the addition of queuine. The slower migration of tRNAHis is eliminated by oxidizing the ribose with periodate, producing a single faster-migrating band (ox). Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).


B The addition of queuine to HCT116 cells cultured in serum free medium resulted in an increase of both queuosinylation and m5C38 levels . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , Q ( queuosine ) , G ( guanine ) .

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

B The addition of queuine to HCT116 cells cultured in serum free medium resulted in an increase of both queuosinylation and m5C38 levels. Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).


C The addition of 20 nM queuine to the SF medium is sufficient to restore tRNAHisQUG in HeLa , whereas 200 nM of q is necessary for queuosinylation of tRNAHisGUG in HCT116 cells . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , Q ( queuosine ) , G ( guanine ) .

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

C The addition of 20 nM queuine to the SF medium is sufficient to restore tRNAHisQUG in HeLa, whereas 200 nM of q is necessary for queuosinylation of tRNAHisGUG in HCT116 cells. Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).


B Corresponding queuosinylation and methylation levels , as determined by Northern blot using a tRNAHisGUG probe and bisulfite sequencing of tRNAAspGUC . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , Q ( queuosine ) , G ( guanine ) .

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

B Corresponding queuosinylation and methylation levels, as determined by Northern blot using a tRNAHisGUG probe and bisulfite sequencing of tRNAAspGUC. Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).


C Representative metaplot of SF cells . 25 - 32 mer ribosome footprint reads were summed across all annotated reading frames with the AUG staring codon at position zero . In - frame 27 - 30 mer ribosome footprints showing the correct periodicity .

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

C Representative metaplot of SF cells. 25-32 mer ribosome footprint reads were summed across all annotated reading frames with the AUG staring codon at position zero. In-frame 27-30 mer ribosome footprints showing the correct periodicity.


D Representative metagene plot of 27 - 30 mer ribosome footprints in S and SF culture conditions for two biological replicates ( rep1 and rep2 ) . Data information : SF ( serum free medium ) , q ( queuine )

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

D Representative metagene plot of 27-30 mer ribosome footprints in S and SF culture conditions for two biological replicates (rep1 and rep2). Data information: SF (serum free medium), q (queuine)