( B ) CD86 and CD40 expression was quantified on WT ( blue dots ) and Irf3 - / - Irf7 - / - ( orange dots ) BMDCs plated at high density ( 2x105 cells / cm2 ) and stimulated with poly ( I : C ) or left unstimulated . Data are pooled from 2 independent experiments . ( C ) CD86 and CD40 expression on Irf3 - / - Irf7 - / - BMDCs plated at 2x105 cells / cm2 and stimulated with poly ( I : C ) ( dark blue dots ) , IFN - α ( light blue dots ) or a combination of both ( green dots ) . Data are representative of 2 independent experiments .

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

(B) CD86 and CD40 expression was quantified on WT (blue dots) and Irf3-/-Irf7-/- (orange dots) BMDCs plated at high density (2x105 cells/cm2) and stimulated with poly(I:C) or left unstimulated. Data are pooled from 2 independent experiments. (C) CD86 and CD40 expression on Irf3-/-Irf7-/- BMDCs plated at 2x105 cells/cm2 and stimulated with poly(I:C) (dark blue dots), IFN-α (light blue dots) or a combination of both (green dots). Data are representative of 2 independent experiments.


( A ) CD86 and CD40 expression was quantified on WT ( blue dots ) and Irf3 ‑ / ‑ Irf7 ‑ / ‑ ( orange dots ) DCs in non ‑ draining ( ndLN ) and draining ( dLN ) popliteal lymph nodes of WT and Irf3 ‑ / ‑ Irf7 ‑ / ‑ mice injected with poly ( I : C ) or PBS . Data are pooled data from 2 independent experiments .

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

(A) CD86 and CD40 expression was quantified on WT (blue dots) and Irf3‑/‑Irf7‑/‑ (orange dots) DCs in non‑draining (ndLN) and draining (dLN) popliteal lymph nodes of WT and Irf3‑/‑Irf7‑/‑ mice injected with poly(I:C) or PBS. Data are pooled data from 2 independent experiments.


( D ) Representative dot plots of CD86 and CD40 expression on WT ( blue dots ) and Irf3 ‑ / ‑ Irf7 ‑ / ‑ ( orange dots ) DCs of chimeras injected with PBS or poly ( I : C ) .

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

(D) Representative dot plots of CD86 and CD40 expression on WT (blue dots) and Irf3‑/‑Irf7‑/‑ (orange dots) DCs of chimeras injected with PBS or poly(I:C).


( B ) Representative dot plots showing CD86 and CD40 expression by low ‑ density BMDCs stimulated with poly ( I : C ) for different periods of IFN ‑ α exposure .

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

(B) Representative dot plots showing CD86 and CD40 expression by low‑density BMDCs stimulated with poly(I:C) for different periods of IFN‑α exposure.


Monocyte recruitment in the draining lymph node requires collective DC activation ( A ) Representative dot plots showing the percentage of monocytes ( CD11b + Ly6Chigh cells , black box ) present in popliteal dLN of WT : Irf3 ‑ / ‑ Irf7 ‑ / ‑ chimeric mice injected with poly ( I : C ) ( middle and right ) or with PBS ( left ) . CD11b + Ly6Clow / int cells corresponded to neutrophils and were not included in the analysis .

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

Monocyte recruitment in the draining lymph node requires collective DC activation (A) Representative dot plots showing the percentage of monocytes (CD11b+ Ly6Chigh cells, black box) present in popliteal dLN of WT:Irf3‑/‑Irf7‑/‑ chimeric mice injected with poly(I:C) (middle and right) or with PBS (left). CD11b+ Ly6Clow/int cells corresponded to neutrophils and were not included in the analysis.


Collective DC activation is essential for the generation of T cell responses . ( G ) Representative dot plots showing the percentage of CD44high tetramer H ‑ 2Kb ‑ OVA + CD8 + T cells in the draining lymph node of chimeric mice injected with PBS or a mixture of OVA + poly ( I : C ) .

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

Collective DC activation is essential for the generation of T cell responses. (G) Representative dot plots showing the percentage of CD44high tetramer H‑2Kb‑OVA+ CD8+ T cells in the draining lymph node of chimeric mice injected with PBS or a mixture of OVA+poly(I:C).


Collective DC activation is essential for the generation of T cell responses . ( H ) Flow cytometric quantification of CD44high H ‑ 2Kb ‑ OVA tetramer + CD8 + T cells in the draining lymph node of 90 : 10 and 5 : 95 ( WT : Irf3 ‑ / ‑ Irf7 ‑ / ‑ ) chimeric mice injected with PBS or OVA + poly ( I : C ) . Data are pooled from 2 independent experiments .

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

Collective DC activation is essential for the generation of T cell responses. (H) Flow cytometric quantification of CD44high H‑2Kb‑OVA tetramer+ CD8+ T cells in the draining lymph node of 90:10 and 5:95 (WT:Irf3‑/‑Irf7‑/‑) chimeric mice injected with PBS or OVA+poly(I:C). Data are pooled from 2 independent experiments.


B . Immunoblot of parental wild - type ( WT ) and UPF3B mutant cell lines showing levels of proteins on the right . In 3BΔ2BD , a smaller UPF3B protein with deletion of amino acids 20 - 155 ( UPF3BΔ20 - 155 ) is expressed . Relative Expression ( Rel Exp ) of this deletion protein as compared to the full - length UPF3B along with standard error of mean ( SEM ) are indicated below lane 2 . UPF3B antibody recognizes antigen outside the deleted region in 3BΔ2BD . HNRNPA1 is used as a loading control . ND = UPF3B levels not determined .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunoblot of parental wild-type (WT) and UPF3B mutant cell lines showing levels of proteins on the right. In 3BΔ2BD, a smaller UPF3B protein with deletion of amino acids 20-155 (UPF3BΔ20-155) is expressed. Relative Expression (Rel Exp) of this deletion protein as compared to the full-length UPF3B along with standard error of mean (SEM) are indicated below lane 2. UPF3B antibody recognizes antigen outside the deleted region in 3BΔ2BD. HNRNPA1 is used as a loading control. ND = UPF3B levels not determined.


C . Cumulative Distribution Function ( CDF ) plots of PTC + isoforms and PTC - isoforms from the same set of genes . X - axis represents fold change in 3BΔ2BD versus WT cells each with control knockdown ( siNC ) . Number of transcripts in each set ( n ) and p - value from Kolmogorov - Smirnov ( KS ) test comparing the two distributions are shown .

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

C. Cumulative Distribution Function (CDF) plots of PTC+ isoforms and PTC- isoforms from the same set of genes. X-axis represents fold change in 3BΔ2BD versus WT cells each with control knockdown (siNC). Number of transcripts in each set (n) and p-value from Kolmogorov-Smirnov (KS) test comparing the two distributions are shown.


D . Bar plots from isoform specific RT - qPCR analysis showing average fold change ( y - axis ) of PTC + and PTC - isoforms from genes indicated on the bottom in WT and the two UPF3B mutant cells identified in the legend on the top right . For each isoform , levels in mutant cells are compared to the levels in WT cells ( set to 1 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Bar plots from isoform specific RT-qPCR analysis showing average fold change (y-axis) of PTC+ and PTC- isoforms from genes indicated on the bottom in WT and the two UPF3B mutant cells identified in the legend on the top right. For each isoform, levels in mutant cells are compared to the levels in WT cells (set to 1).


E . Cumulative Distribution Function ( CDF ) plots of NMD + isoforms and NMD - isoforms . X - axis represents fold change in 3BΔ2BD versus WT cells each with control knockdown ( siNC ) . Number of transcripts in each set ( n ) and p - value from Kolmogorov - Smirnov ( KS ) test comparing the two distributions are shown . F . Cumulative Distribution Function ( CDF ) plots of PTC + isoforms and PTC - isoforms from same set of genes . X - axis represents fold change in UPF1 knockdown ( siUPF1 ) versus control knockdown ( siNC ) in 3BΔ2BD cells . Number of transcripts in each set ( n ) and p - value from Kolmogorov - Smirnov ( KS ) test comparing the two distributions are shown . G . Cumulative Distribution Function ( CDF ) plots of NMD + isoforms and NMD - isoforms . X - axis represents fold change in UPF1 knockdown ( siUPF1 ) versus control knockdown ( siNC ) in 3BΔ2BD cells . Number of transcripts in each set ( n ) and p - value from Kolmogorov - Smirnov ( KS ) test comparing the two distributions are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Cumulative Distribution Function (CDF) plots of NMD+ isoforms and NMD- isoforms. X-axis represents fold change in 3BΔ2BD versus WT cells each with control knockdown (siNC). Number of transcripts in each set (n) and p-value from Kolmogorov-Smirnov (KS) test comparing the two distributions are shown. F. Cumulative Distribution Function (CDF) plots of PTC+ isoforms and PTC- isoforms from same set of genes. X-axis represents fold change in UPF1 knockdown (siUPF1) versus control knockdown (siNC) in 3BΔ2BD cells. Number of transcripts in each set (n) and p-value from Kolmogorov-Smirnov (KS) test comparing the two distributions are shown. G. Cumulative Distribution Function (CDF) plots of NMD+ isoforms and NMD- isoforms. X-axis represents fold change in UPF1 knockdown (siUPF1) versus control knockdown (siNC) in 3BΔ2BD cells. Number of transcripts in each set (n) and p-value from Kolmogorov-Smirnov (KS) test comparing the two distributions are shown.


A . Immunoblots showing levels of proteins on the right in input or FLAG immunoprecipitates ( IP ) from WT and UPF3B mutant cells expressing endogenously FLAG - tagged UPF1 protein as indicated above each lane . The presence of RNase A during FLAG - IP is indicated above each lane .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunoblots showing levels of proteins on the right in input or FLAG immunoprecipitates (IP) from WT and UPF3B mutant cells expressing endogenously FLAG-tagged UPF1 protein as indicated above each lane. The presence of RNase A during FLAG-IP is indicated above each lane.


B . Immunoblots showing levels of proteins ( right ) in input and IP with normal rabbit IgG ( IgG - IP ) or antibody targeting EIF4A3 ( EIF4A3 - IP ) from WT and UPF3B mutant cells .

O O O O O O O O O O O O O O O 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. Immunoblots showing levels of proteins (right) in input and IP with normal rabbit IgG (IgG-IP) or antibody targeting EIF4A3 (EIF4A3-IP) from WT and UPF3B mutant cells.


C , D . CDF plots of ( C ) PTC + and PTC - isoforms , ( D ) NMD + and NMD - isoforms . X - axis represents fold change upon UPF3A knockdown ( siUPF3A ) versus negative control knockdown ( siNC ) in WT cells . Number of transcripts in each set ( n ) and p - value from KS test comparing the two distributions are shown on each plot . E , F . CDF plots of ( E ) PTC + and PTC - isoforms , ( F ) NMD + and NMD - isoforms . X - axis represents fold change upon UPF3A knockdown ( siUPF3A ) versus negative control knockdown ( siNC ) in 3BΔ2BD cells . Number of transcripts in each set ( n ) and p - value from KS test comparing the two distributions are shown on each plot . G , H . CDF plots of UPF3B - dependent ( G ) and - independent ( H ) PTC + isoforms and their respective PTC - isoforms . X - axis represents fold change upon UPF3A knockdown ( siUPF3A ) versus negative control knockdown ( siNC ) in 3BΔ2BD cells . Number of transcripts in each set ( n ) and p - value from KS test comparing the two distributions are shown on each plot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D. CDF plots of (C) PTC+ and PTC-isoforms, (D) NMD+ and NMD- isoforms. X-axis represents fold change upon UPF3A knockdown (siUPF3A) versus negative control knockdown (siNC) in WT cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot. E, F. CDF plots of (E) PTC+ and PTC-isoforms, (F) NMD+ and NMD- isoforms. X-axis represents fold change upon UPF3A knockdown (siUPF3A) versus negative control knockdown (siNC) in 3BΔ2BD cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot. G, H. CDF plots of UPF3B-dependent (G) and -independent (H) PTC+ isoforms and their respective PTC- isoforms. X-axis represents fold change upon UPF3A knockdown (siUPF3A) versus negative control knockdown (siNC) in 3BΔ2BD cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.


I . CDF plots of UPF3B - dependent and UPF3B - independent NMD + isoforms as compared to NMD - isoforms . X - axis represents fold change upon UPF3A knockdown ( siUPF3A ) versus negative control knockdown ( siNC ) in 3BΔ2BD cells . Number of transcripts in each set ( n ) and p - value from KS test comparing the two distributions are shown on each plot .

I. CDF plots of UPF3B-dependent and UPF3B-independent NMD+ isoforms as compared to NMD- isoforms. X-axis represents fold change upon UPF3A knockdown (siUPF3A) versus negative control knockdown (siNC) in 3BΔ2BD cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.


J . Bar plot showing average fold change as measured by isoform specific RT - qPCR of PTC + and PTC - isoform from genes indicated on the bottom in WT and two independent clones of 3AKO , 3BΔ2BD , 3BKO , and 3DKO cells . Relative levels from each replicate are shown by white circles . Error bars indicate standard errors of means .

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

J. Bar plot showing average fold change as measured by isoform specific RT-qPCR of PTC+ and PTC- isoform from genes indicated on the bottom in WT and two independent clones of 3AKO, 3BΔ2BD, 3BKO, and 3DKO cells. Relative levels from each replicate are shown by white circles. Error bars indicate standard errors of means.


B . Immunoblot showing levels of EJC and UPF proteins ( on the right ) in input or FLAG - IP samples from 3DKO # 2 cells stably expressing different FLAG - tagged proteins indicated above each lane . D . Immunoblot showing EJC / UPF proteins ( on the right ) in input or FLAG - IP samples from 3DKO # 2 cells stably expressing FLAG - tagged proteins given above each lane ( hUPF3B = human UPF3B ; hUPF3B - h3AC = human UPF3B protein with C - terminal domain from human UPF3A ; hUPF3B - m3AC = human UPF3B protein with C - terminal domain from mouse UPF3A ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunoblot showing levels of EJC and UPF proteins (on the right) in input or FLAG-IP samples from 3DKO#2 cells stably expressing different FLAG-tagged proteins indicated above each lane. D. Immunoblot showing EJC/UPF proteins (on the right) in input or FLAG-IP samples from 3DKO#2 cells stably expressing FLAG-tagged proteins given above each lane (hUPF3B = human UPF3B; hUPF3B-h3AC = human UPF3B protein with C-terminal domain from human UPF3A; hUPF3B-m3AC = human UPF3B protein with C-terminal domain from mouse UPF3A).


A . Northern blots showing levels of β - globin reporter mRNAs in wild - type HeLa Tet - off cells and UPF3B knockout HeLa Tet - off cells . β39 is a tetracycline ( Tet ) - inducible reporter with a PTC at codon 39 whose levels are shown at different timepoints after transcriptional shut - off ( chase ) as indicated above each lane . β - GAP is a stable , constitutively - expressed , longer β - globin mRNA used as transfection control . Proteins overexpressed ( OE ) in each condition are indicated on top and reporter mRNA half - lives ( t1 / 2 ) along with standard errors of means are on the bottom .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Northern blots showing levels of β-globin reporter mRNAs in wild-type HeLa Tet-off cells and UPF3B knockout HeLa Tet-off cells. β39 is a tetracycline (Tet)-inducible reporter with a PTC at codon 39 whose levels are shown at different timepoints after transcriptional shut-off (chase) as indicated above each lane. β-GAP is a stable, constitutively-expressed, longer β-globin mRNA used as transfection control. Proteins overexpressed (OE) in each condition are indicated on top and reporter mRNA half-lives (t1/2) along with standard errors of means are on the bottom.


C . Northern blot showing steady - state levels of β39 NMD reporter and β - GAP control in HeLa Tet - off UPF3B knockout cells upon transient overexpression of wild - type UPF3 proteins or different UPF3A chimeric proteins indicated above each lane . Below each lane , relative fold - change ( Rel FC ) indicates β39 reporter levels ( normalized to β - GAP control ) as compared to the normalized β39 reporter levels in UPF3B expressing cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Northern blot showing steady-state levels of β39 NMD reporter and β-GAP control in HeLa Tet-off UPF3B knockout cells upon transient overexpression of wild-type UPF3 proteins or different UPF3A chimeric proteins indicated above each lane. Below each lane, relative fold-change (Rel FC) indicates β39 reporter levels (normalized to β-GAP control) as compared to the normalized β39 reporter levels in UPF3B expressing cells.


D . Immunoblot showing levels of EJC proteins or HNRNPA1 in input or EIF4A3 - IP from 3DKO # 2 cells stably expressing different UPF3 proteins or EGFP as a control as indicated above each lane . Relative IP of FLAG - tagged proteins are quantified against EIF4A3 .

O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunoblot showing levels of EJC proteins or HNRNPA1 in input or EIF4A3-IP from 3DKO#2 cells stably expressing different UPF3 proteins or EGFP as a control as indicated above each lane. Relative IP of FLAG-tagged proteins are quantified against EIF4A3.


A , B . CDF plots of ( A ) PTC + and PTC - isoforms , ( B ) NMD + and NMD - isoforms . X - axis represents fold change upon UPF1 knockdown ( siUPF1 ) versus negative control knockdown ( siNC ) in 3DKO # 2 cells . Number of transcripts in each set ( n ) and p - value from KS test comparing the two distributions are shown on each plot .

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

A, B. CDF plots of (A) PTC+ and PTC-isoforms, (B) NMD+ and NMD- isoforms. X-axis represents fold change upon UPF1 knockdown (siUPF1) versus negative control knockdown (siNC) in 3DKO#2 cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.


D , E . CDF plots of UPF3A / 3B - independent ( UPF3A / 3B - indep . , D ) and - dependent ( UPF3A / 3B - dep . , E ) PTC + isoforms and their respective PTC - isoforms . X - axis represents fold change upon UPF1 knockdown ( siUPF1 ) versus negative control knockdown ( siNC ) in 3DKO # 2 cells . Number of transcripts in each set ( n ) and p - value from KS test comparing the two distributions are shown on each plot .

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

D, E. CDF plots of UPF3A/3B-independent (UPF3A/3B-indep., D) and -dependent (UPF3A/3B-dep., E) PTC+ isoforms and their respective PTC- isoforms. X-axis represents fold change upon UPF1 knockdown (siUPF1) versus negative control knockdown (siNC) in 3DKO#2 cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.


F . CDF plots of UPF3A / 3B - dependent ( UPF3A / 3B - dep . ) , UPF3A / 3B - independent ( UPF3A / 3B - indep . ) NMD + isoforms and NMD - isoforms . X - axis represents fold change upon UPF1 knockdown ( siUPF1 ) versus negative control knockdown ( siNC ) in 3DKO # 2 cells . Number of transcripts in each set ( n ) and p - value from KS test comparing the two distributions are shown on each plot .

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

F. CDF plots of UPF3A/3B-dependent (UPF3A/3B-dep.), UPF3A/3B-independent (UPF3A/3B-indep.) NMD+ isoforms and NMD- isoforms. X-axis represents fold change upon UPF1 knockdown (siUPF1) versus negative control knockdown (siNC) in 3DKO#2 cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.


A , B . Western blots showing levels of EJC proteins or HNRNPA1 ( control ) in input , IgG IP or EIF4A3 IP following overexpression ( OE ) of CASC3 wild - type ( WT ) and EJC binding deficient ( HDAA ) mutant proteins in ( A ) HeLa Tet - off cells , and ( B ) 3BKO # 3 HeLa Tet - off cells . Ramps above lanes indicate expression levels of the CASC3 proteins .

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

A, B. Western blots showing levels of EJC proteins or HNRNPA1 (control) in input, IgG IP or EIF4A3 IP following overexpression (OE) of CASC3 wild-type (WT) and EJC binding deficient (HDAA) mutant proteins in (A) HeLa Tet-off cells, and (B) 3BKO#3 HeLa Tet-off cells. Ramps above lanes indicate expression levels of the CASC3 proteins.


C . Western blots showing levels of EJC / UPF proteins and HNRNPA1 in input and FLAG followed by EIF4A3 tandem IP from HCT116 cells expressing the FLAG - tagged endogenous protein indicated above each lane . Quantifications of UPF3B and CASC3 protein enrichment from two replicates are shown at the bottom .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Western blots showing levels of EJC/UPF proteins and HNRNPA1 in input and FLAG followed by EIF4A3 tandem IP from HCT116 cells expressing the FLAG-tagged endogenous protein indicated above each lane. Quantifications of UPF3B and CASC3 protein enrichment from two replicates are shown at the bottom.


D . Meta - exon plot showing read distributions within the 100 nucleotide ( nt ) window from the exon 3 ′ end in RIPiT - Seq replicates of MAGOH : EIF4A3 , UPF3B : EIF4A3 , and CASC3 : EIF4A3 . The black vertical line indicates the - 24 nt position .

D. Meta-exon plot showing read distributions within the 100 nucleotide (nt) window from the exon 3′ end in RIPiT-Seq replicates of MAGOH:EIF4A3, UPF3B:EIF4A3, and CASC3:EIF4A3. The black vertical line indicates the -24 nt position.


F . Scatter plot comparing log2 - transformed fold change in occupancy of CASC3 : EIF4A3 EJC as compared to MAGOH : EIF4A3 EJC ( x - axis ) and UPF3B : EIF4A3 EJC compared to MAGOH : EIF4A3 EJC . Each dot represents a gene where gene - level occupancy of each EJC composition was quantified at the canonical position for EJC footprints . Pearson correlation coefficient is shown on the top left .

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F. Scatter plot comparing log2-transformed fold change in occupancy of CASC3:EIF4A3 EJC as compared to MAGOH:EIF4A3 EJC (x-axis) and UPF3B:EIF4A3 EJC compared to MAGOH:EIF4A3 EJC. Each dot represents a gene where gene-level occupancy of each EJC composition was quantified at the canonical position for EJC footprints. Pearson correlation coefficient is shown on the top left.


G . Box plots comparing occupancy of a specific EJC composition on mRNA ( indicated on y - axis ; normalized by gene expression from RNA - Seq ) between genes of different NMD efficiency . Up , no , and down stands for upregulation , no obvious change or downregulation of a representative PTC + isoform for each gene ( see material and methods ) that is used to reflect overall NMD efficiency for each gene . In the box plots , central band represents the median , boxes represent the interquartile range ( IQR ) , whiskers indicate maximum ( top ) and minimum ( bottom ) values ( 1 . 5 - times the highest or lowest IQR values , respectively ) , and circles represent the outliers . p - values are from Wilcoxon signed - rank test comparing the two indicated distributions .

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G. Box plots comparing occupancy of a specific EJC composition on mRNA (indicated on y-axis; normalized by gene expression from RNA-Seq) between genes of different NMD efficiency. Up, no, and down stands for upregulation, no obvious change or downregulation of a representative PTC+ isoform for each gene (see material and methods) that is used to reflect overall NMD efficiency for each gene. In the box plots, central band represents the median, boxes represent the interquartile range (IQR), whiskers indicate maximum (top) and minimum (bottom) values (1.5-times the highest or lowest IQR values, respectively), and circles represent the outliers. p-values are from Wilcoxon signed-rank test comparing the two indicated distributions.


H . Box plots as in G comparing specific EJC composition occupancy relative to EJC core occupancy ( normalized by MAGOH : EIF4A3 RIPiT - Seq ) between genes of different NMD efficiency . Up , no , down , central band , boxes , and whiskers are as in G .

H. Box plots as in G comparing specific EJC composition occupancy relative to EJC core occupancy (normalized by MAGOH:EIF4A3 RIPiT-Seq) between genes of different NMD efficiency. Up, no, down, central band, boxes, and whiskers are as in G.


I . Bar plots showing fold changes measured by isoform specific RT - qPCR of PTC + and PTC - isoform from genes indicated on the bottom in WT and CASC3 - KO HCT116 cells . Relative levels from each replicate are shown by white circles . Error bars indicate standard error of means .

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I. Bar plots showing fold changes measured by isoform specific RT-qPCR of PTC+ and PTC- isoform from genes indicated on the bottom in WT and CASC3-KO HCT116 cells. Relative levels from each replicate are shown by white circles. Error bars indicate standard error of means.


C ) Representative immunoblots of brain extracts of wild - type , Ttll1 - / - and Ttll7 - / - mice . Polyglutamylation of α - tubulin , detected with polyE and GT335 , is specifically lost in Ttll1 - / - brain . By contrast , β - tubulin glutamylation , which is specifically detected with β ‑ monoE , is absent from brains of Ttll7 - / - mice . D ) Representative immunoblots of extracts of wild - type , Ttll1 ‑ / - and Ttll7 - / - primary hippocampal neurons at DIV4 . As in brain ( C ) , α - tubulin polyglutamylation is specifically lost in Ttll1 - / - neurons , while β - tubulin glutamylation lacks in Ttll7 - / - neurons . E

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C) Representative immunoblots of brain extracts of wild-type, Ttll1-/- and Ttll7-/- mice. Polyglutamylation of α-tubulin, detected with polyE and GT335, is specifically lost in Ttll1-/- brain. By contrast, β-tubulin glutamylation, which is specifically detected with β‑monoE, is absent from brains of Ttll7-/- mice. D) Representative immunoblots of extracts of wild-type, Ttll1‑/- and Ttll7-/- primary hippocampal neurons at DIV4. As in brain (C), α-tubulin polyglutamylation is specifically lost in Ttll1-/- neurons, while β-tubulin glutamylation lacks in Ttll7-/- neurons. E


F , G ) Immunocytochemistry of DIV4 primary hippocampal neurons from wild type , Ttll1 - / - and Ttll7 ‑ / - mice . Microtubules in Ttll1 - / - cells have reduced levels of polyE signal , indicative of lower α - tubulin polyglutamylation ( F ) , Ttll7 ‑ / - neurons are specifically reduced in β ‑ monoE reactivity , indicating the lack of β ‑ tubulin glutamylation ( G ) .

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F,G) Immunocytochemistry of DIV4 primary hippocampal neurons from wild type, Ttll1-/- and Ttll7‑/- mice. Microtubules in Ttll1-/- cells have reduced levels of polyE signal, indicative of lower α-tubulin polyglutamylation (F), Ttll7‑/- neurons are specifically reduced in β‑monoE reactivity, indicating the lack of β‑tubulin glutamylation (G).


A ) Representative histology images of cerebella from 3 - months - old mice stained with anti - calbindin antibody , which specifically labels Purkinje cells ( Sequier et al , 1988 ) . Note the presence of an intact Purkinje - cell layer in cerebella of wild - type , Ttll1 - / - , Ttll7 - / - and Ccp1 - / - Ttll1 - / - mice , in contrast to the complete absence of these cells in Ccp1 - / - and Ccp1 - / - Ttll7 - / - mice .

A) Representative histology images of cerebella from 3-months-old mice stained with anti-calbindin antibody, which specifically labels Purkinje cells (Sequier et al, 1988). Note the presence of an intact Purkinje-cell layer in cerebella of wild-type, Ttll1-/-, Ttll7-/- and Ccp1-/-Ttll1-/- mice, in contrast to the complete absence of these cells in Ccp1-/- and Ccp1-/-Ttll7-/- mice.


B ) Histology of 25 - day old mice with partial degeneration of Purkinje Cells . Note that both , Ccp1 - / - and Ccp1 - / - Ttll7 - / - brains , show highly similar pattern of partial Purkinje - cell loss ( red arrow heads : gaps in the Purkinje - cell layer , indicating degenerated Purkinje cells ; orange arrow heads : swellings of Purkinje - cell axons , indicative of ongoing degeneration ) .

B) Histology of 25-day old mice with partial degeneration of Purkinje Cells. Note that both, Ccp1-/- and Ccp1-/-Ttll7-/- brains, show highly similar pattern of partial Purkinje-cell loss (red arrow heads: gaps in the Purkinje-cell layer, indicating degenerated Purkinje cells; orange arrow heads: swellings of Purkinje-cell axons, indicative of ongoing degeneration).


C ) Representative immunoblots of cerebellum extracts of wild - type , Ccp1 - / - , Ttll7 - / - , Ccp1 - / - Ttll7 - / - , Ttll1 - / - and Ccp1 - / - Ttll1 - / - mice . Loss of CCP1 leads to hyperglutamylation of α - and β - tubulin as revealed with polyE and GT335 antibodies . Concomitant deletion of TTLL7 lead to a loss of glutamylation from β - tubulin ( β ‑ monoE , GT335 ) , while hyperglutamylation of α - tubulin persists ( polyE , GT335 ) . Concomitant loss of CCP1 and TTLL1 eliminates hyperglutamylation from both , α - and β - tubulin ( polyE , GT335 ) , while β - tubulin glutamylation is unaltered ( β - monoE ) .

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C) Representative immunoblots of cerebellum extracts of wild-type, Ccp1-/-, Ttll7-/-, Ccp1-/-Ttll7-/-, Ttll1-/- and Ccp1-/-Ttll1-/- mice. Loss of CCP1 leads to hyperglutamylation of α- and β-tubulin as revealed with polyE and GT335 antibodies. Concomitant deletion of TTLL7 lead to a loss of glutamylation from β-tubulin (β‑monoE, GT335), while hyperglutamylation of α-tubulin persists (polyE, GT335). Concomitant loss of CCP1 and TTLL1 eliminates hyperglutamylation from both, α- and β-tubulin (polyE, GT335), while β-tubulin glutamylation is unaltered (β-monoE).


Semi - thin cross sections of femoral quadriceps nerves stained with alkaline methylene blue . Note the smaller size of Ccp1 - / - and Ccp1 - / - Ttll7 - / - nerves ( A ) .

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Semi-thin cross sections of femoral quadriceps nerves stained with alkaline methylene blue. Note the smaller size of Ccp1-/- and Ccp1-/-Ttll7-/- nerves (A).


Semi - thin cross sections of femoral quadriceps nerves stained with alkaline methylene blue . These sections were quantified for ( B ) number of myelinated axons , ( C ) nerve area , ( D ) nerve perimeter ( Fig . EV2 ) of femoral quadriceps nerves from wild - type , Ccp1 - / - , Ttll7 - / - , Ccp1 - / - Ttll7 - / - , Ttll1 - / - and Ccp1 - / - Ttll1 - / - mice . Scatter dot plots in which each point represents one nerve of an individual mouse are shown . The black lines indicate means ± SEM . Significance was tested with one - way ANOVA and Holm - Šídák ' s multiple comparisons test . Note that all three parameters are decreased for Ccp1 - / - and Ccp1 - / - Ttll7 ‑ / - mice , while Ccp1 - / - Ttll1 - / - mice showed no difference to the Ttll1 - / - control .

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Semi-thin cross sections of femoral quadriceps nerves stained with alkaline methylene blue. These sections were quantified for (B) number of myelinated axons, (C) nerve area, (D) nerve perimeter (Fig. EV2) of femoral quadriceps nerves from wild-type, Ccp1-/-, Ttll7-/-, Ccp1-/-Ttll7-/-, Ttll1-/- and Ccp1-/-Ttll1-/- mice. Scatter dot plots in which each point represents one nerve of an individual mouse are shown. The black lines indicate means ±SEM. Significance was tested with one-way ANOVA and Holm-Šídák's multiple comparisons test. Note that all three parameters are decreased for Ccp1-/- and Ccp1-/-Ttll7‑/- mice, while Ccp1-/-Ttll1-/- mice showed no difference to the Ttll1-/- control.


E , F ) Representative electron microscopy images of cross sections of femoral quadriceps nerves . ( E ) Nerves from Ttll1 - / - and Ccp1 - / - Ttll1 - / - mice show no abnormalities of myelinated axons ( green asterisks ) , indicating that loss of TTLL1 prevents axonal degeneration caused by the absence of CCP1 . ( F ) Ccp1 - / - and Ccp1 - / - Ttll7 - / - mice both show the presence of foamy macrophages ( magenta arrowheads ) , which are signs for myelin phagocytosis during axon degeneration . This indicates that loss of TTLL7 cannot prevent the CCP1 - induced degeneration of femoral quadriceps nerves .

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E,F) Representative electron microscopy images of cross sections of femoral quadriceps nerves. (E) Nerves from Ttll1-/- and Ccp1-/-Ttll1-/- mice show no abnormalities of myelinated axons (green asterisks), indicating that loss of TTLL1 prevents axonal degeneration caused by the absence of CCP1. (F) Ccp1-/- and Ccp1-/-Ttll7-/- mice both show the presence of foamy macrophages (magenta arrowheads), which are signs for myelin phagocytosis during axon degeneration. This indicates that loss of TTLL7 cannot prevent the CCP1-induced degeneration of femoral quadriceps nerves.


A ) Representative images of DIV4 primary hippocampal neurons from wild type , Ttll1 - / - and Ttll7 - / - mice Cells were incubated with MitoTracker ® ( black dots ) and mitochondria movements were recorded in the longest neurite ( orange lines ) .

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A) Representative images of DIV4 primary hippocampal neurons from wild type, Ttll1-/- and Ttll7-/- mice Cells were incubated with MitoTracker® (black dots) and mitochondria movements were recorded in the longest neurite (orange lines).


B ) Kymographs of one representative experiment representing mitochondrial movements C - F ) Analysis of one single experiment shown in ( B ) . Scatter dot plots of ( C ) run length and ( D ) speed distributions , where each point represents a single run extracted from the kymographs shown in ( B ) . The black lines indicate the median of the distribution , with whiskers at interquartile range . Numerical values of medians are indicated below each plot . ( E ) A bar graph showing the overall motility of mitochondria ( the fraction of time mitochondria spent in movement relative to the total number of mitochondria during the recording time of 60 s ) . ( F ) A bar graph showing mitochondrial density in analysed axons . Values of the overall motilities and mitochondrial densities are given below the bar plots .

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B) Kymographs of one representative experiment representing mitochondrial movements C - F) Analysis of one single experiment shown in (B). Scatter dot plots of (C) run length and (D) speed distributions, where each point represents a single run extracted from the kymographs shown in (B). The black lines indicate the median of the distribution, with whiskers at interquartile range. Numerical values of medians are indicated below each plot. (E) A bar graph showing the overall motility of mitochondria (the fraction of time mitochondria spent in movement relative to the total number of mitochondria during the recording time of 60 s). (F) A bar graph showing mitochondrial density in analysed axons. Values of the overall motilities and mitochondrial densities are given below the bar plots.


A . Typical fluorescence intensity trace collected over 10 seconds .

A. Typical fluorescence intensity trace collected over 10 seconds.


B . Typical normalized FCS curves of Smc3 - GFP at different cell cycle phases . Dotted lines indicate fitting of the one - component anomalous diffusion model to the respective FCS curve .

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B. Typical normalized FCS curves of Smc3-GFP at different cell cycle phases. Dotted lines indicate fitting of the one-component anomalous diffusion model to the respective FCS curve.


C - F . Diffusion times of Smc3 - GFP , Scc3 - GFP , and Scc1 - GFP at the G1 phase ( C ) , S phase ( D ) , G2 phase ( E ) , and mitosis ( F ) . Data shown as mean ± s . e . m . from at least three independent experiments . * : p < 0 . 01 , # : p < 10 - 13 with the Mann - Whitney test .

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C-F. Diffusion times of Smc3-GFP, Scc3-GFP, and Scc1-GFP at the G1 phase (C), S phase (D), G2 phase (E), and mitosis (F). Data shown as mean ± s.e.m. from at least three independent experiments. *: p<0.01, #: p<10-13 with the Mann-Whitney test.


Representative normalized FCS curve of Scc1 - GFP at different cell cycle phases . Dotted lines indicate fitting of one - component anomalous diffusion models to the respective FCS curve .

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Representative normalized FCS curve of Scc1-GFP at different cell cycle phases. Dotted lines indicate fitting of one-component anomalous diffusion models to the respective FCS curve.


The diffusion times of Scc1 - GFP at S phase ( B ) were measured in strains yIO664 ( wild - type ) and yEB005 ( WPL1 ∆ ) , and in auxin - depleted yEB002 ( PDS5 - AID ) , yAM085 ( SCC3 - AID ) and yAM946 ( ECO1 - AID ) cells . Data are shown as mean ± s . e . m . from at least three independent experiments . * : p < 10 - 5 , * * : p < 10 - 13 .

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The diffusion times of Scc1-GFP at S phase (B) were measured in strains yIO664 (wild-type) and yEB005 (WPL1∆), and in auxin-depleted yEB002 (PDS5-AID), yAM085 (SCC3-AID) and yAM946 (ECO1-AID) cells. Data are shown as mean ± s.e.m. from at least three independent experiments. *: p< 10-5, **: p< 10-13.


The diffusion times of Scc1 - GFP at G2 phase ( C ) and M phase ( D ) were measured in strains yIO664 ( wild - type ) and yEB005 ( WPL1 ∆ ) , and in auxin - depleted yEB002 ( PDS5 - AID ) , yAM085 ( SCC3 - AID ) and yAM946 ( ECO1 - AID ) cells . Data are shown as mean ± s . e . m . from at least three independent experiments . * : p < 10 - 5 , * * : p < 10 - 13 .

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

The diffusion times of Scc1-GFP at G2 phase (C) and M phase (D) were measured in strains yIO664 (wild-type) and yEB005 (WPL1∆), and in auxin-depleted yEB002 (PDS5-AID), yAM085 (SCC3-AID) and yAM946 (ECO1-AID) cells. Data are shown as mean ± s.e.m. from at least three independent experiments. *: p< 10-5, **: p< 10-13.


A . Typical fitted photon counting histograms of GFP and GFP - GFP in live yeast cells .

A. Typical fitted photon counting histograms of GFP and GFP-GFP in live yeast cells.


B . The molecular brightness of different GFP tagged constructs was obtained with GFP monomer and GFP - GFP dimer as the brightness standard . Cells of strain yIO664 ( SCC1 - GFP ) were analyzed throughout the cell cycle by PCH . To examine the effects of Wpl1 , Pds5 , and Eco1 on cohesin dimerization , the PCH analysis was repeated in strain yEB005 cells ( SCC1 - GFP WPL1 ∆ ) , strain yEB002 cells ( SCC1 - GFP PDS5 - AID ) , and yAM946 ( SCC1 - GFP ECO1 - AID ) in which Pds5 - AID / Eco1 - AID was depleted with auxin .

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B. The molecular brightness of different GFP tagged constructs was obtained with GFP monomer and GFP-GFP dimer as the brightness standard. Cells of strain yIO664 (SCC1-GFP) were analyzed throughout the cell cycle by PCH. To examine the effects of Wpl1, Pds5, and Eco1 on cohesin dimerization, the PCH analysis was repeated in strain yEB005 cells (SCC1-GFP WPL1∆), strain yEB002 cells (SCC1-GFP PDS5-AID), and yAM946 (SCC1-GFP ECO1-AID) in which Pds5-AID/Eco1-AID was depleted with auxin.


C . PCH analysis of strain yAM085 ( SMC3 - GFP ) throughout the cell cycle .

C. PCH analysis of strain yAM085 (SMC3-GFP) throughout the cell cycle.


D . PCH analysis of strains yAM953 ( SCC1 - GFP SMC3 - GFP ) and yAP941 ( SCC1 - GFP SCC3 - GFP ) containing two GFP tagged subunits . Data shown as boxplot ( + data overlap ) with values of minimum , 5 % percentile , 25 % percentile , median , 75 % percentile , maximum , and mean ( center square marks ) from at least three independent experiments .

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D. PCH analysis of strains yAM953 (SCC1-GFP SMC3-GFP) and yAP941 (SCC1-GFP SCC3-GFP) containing two GFP tagged subunits. Data shown as boxplot (+data overlap) with values of minimum, 5% percentile, 25% percentile, median, 75% percentile, maximum, and mean (center square marks) from at least three independent experiments.


Fluorescent and histological analysis of liver morphology in WT and yap - / - mutant larvae on a lf : CFP background at 5 dpf . The blue colour overlay represents the fluorescent liver tissue . Scale bar : 200μm ( bright field , BF ) and 50μm ( histology , H + E ) Quantitative analysis of fluorescent liver area in WT and lf : Yap larvae at 5 dpf . n > 30 , * * * * p < 0 . 0001 , two - sided Student ' s t - test ; values represent the mean ± standard error of the mean ( s . e . m . )

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Fluorescent and histological analysis of liver morphology in WT and yap-/- mutant larvae on a lf:CFP background at 5 dpf. The blue colour overlay represents the fluorescent liver tissue. Scale bar: 200μm (bright field, BF) and 50μm (histology, H+E) Quantitative analysis of fluorescent liver area in WT and lf:Yap larvae at 5 dpf. n>30, ****p<0.0001, two-sided Student's t-test; values represent the mean ± standard error of the mean (s.e.m.)


Whole - mount in situ hybridization ( WISH ) analysis of progenitor marker prox1 , hhex , and foxa3 expression in WT and yap - / - mutant embryos at 36 hpf . Arrow and bracket highlight the hepatic bud . Scale bar :

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Whole-mount in situ hybridization (WISH) analysis of progenitor marker prox1, hhex, and foxa3 expression in WT and yap-/- mutant embryos at 36 hpf. Arrow and bracket highlight the hepatic bud. Scale bar: 200μm


qPCR analysis of hhex and prox1 gene expression in WT and yap - / - mutant embryos at 48 hpf . n > 3 , * p < 0 . 05 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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qPCR analysis of hhex and prox1 gene expression in WT and yap-/- mutant embryos at 48 hpf. n>3, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m


WISH analysis of prox1 , hhex and foxa3 expression in WT , hs : dnYap and hs : Yap transgenic embryos heat shocked at 24 hpf and fixed at 36 hpf . Arrow and bracket highlight the hepatic bud . Scale bar :

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WISH analysis of prox1, hhex and foxa3 expression in WT, hs:dnYap and hs:Yap transgenic embryos heat shocked at 24 hpf and fixed at 36 hpf. Arrow and bracket highlight the hepatic bud. Scale bar: 200μm


Quantitative analysis of hepatic bud area ( prox1 staining ) in WT , hs : dnYap and hs : Yap transgenic embryos heat shocked at 24 hpf and fixed at 36 hpf . n > 10 , * p < 0 . 05 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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Quantitative analysis of hepatic bud area (prox1 staining) in WT, hs:dnYap and hs:Yap transgenic embryos heat shocked at 24 hpf and fixed at 36 hpf. n>10, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m


Fluorescent and histological analysis of liver morphology in dissected WT and yap - / - mutant adults on a lf : CFP background . The blue colour overlay represents the fluorescent liver tissue . Scale bar : 2mm ( brightfield , BF ) and 50μm ( histology , H + E )

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Fluorescent and histological analysis of liver morphology in dissected WT and yap-/- mutant adults on a lf:CFP background. The blue colour overlay represents the fluorescent liver tissue. Scale bar: 2mm (brightfield, BF) and 50μm (histology, H+E)


Quantitative determination of body mass in WT and yap - / - adults . n = 8 , * p < 0 . 05 , two - sided Student ' s t - test ; values represent the mean ± s . e . m Quantitative determination of liver size in WT and yap - / - adults as determined by measuring liver mass as a percentage of body mass . n = 12 , * p < 0 . 05 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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

Quantitative determination of body mass in WT and yap-/- adults. n=8, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m Quantitative determination of liver size in WT and yap-/- adults as determined by measuring liver mass as a percentage of body mass. n=12, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m


qPCR analysis of the hhex expression in adult liver tissue isolated from WT and yap - / - fish . n > 3 , * * p < 0 . 01 , two - sided Student ' s t - test ; values represent the mean ± s . e . m qPCR analysis of the prox1 expression in adult liver tissue isolated from WT and yap - / - mutant fish . n > 3 , * p < 0 . 05 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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

qPCR analysis of the hhex expression in adult liver tissue isolated from WT and yap-/- fish. n>3, **p<0.01, two-sided Student's t-test; values represent the mean ± s.e.m qPCR analysis of the prox1 expression in adult liver tissue isolated from WT and yap-/- mutant fish. n>3, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m


Heatmap of RNAseq analysis illustrating statistically significant differential gene expression between WT and yap - / - mutant larvae at 3 dpf . Blue indicates decreased expression , while red demonstrates increased

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Heatmap of RNAseq analysis illustrating statistically significant differential gene expression between WT and yap-/- mutant larvae at 3 dpf. Blue indicates decreased expression, while red demonstrates increased expression


Heatmap analysis of RNAseq data highlights the reduction of glucose transporters ( glut1 and glut2 ) in yap - / - mutant larvae at 3

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Heatmap analysis of RNAseq data highlights the reduction of glucose transporters (glut1 and glut2) in yap-/- mutant larvae at 3 dpf


qPCR analysis of glut1 and glut2 expression in WT and yap - / - mutant larvae at 3 dpf . n > 3 , * p < 0 . 05 , * * * p < 0 . 001 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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

qPCR analysis of glut1 and glut2 expression in WT and yap-/- mutant larvae at 3 dpf. n>3, *p<0.05, ***p<0.001, two-sided Student's t-test; values represent the mean ± s.e.m


qPCR analysis of glut1 and glut2 expression in WT , hs : dnYap and hs : Yap transgenic embryos heat shocked at 24 hpf and isolated at 30 hpf . n > 3 , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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

qPCR analysis of glut1 and glut2 expression in WT, hs:dnYap and hs:Yap transgenic embryos heat shocked at 24 hpf and isolated at 30 hpf. n>3, *p<0.05, **p<0.01, ****p<0.0001, two-sided Student's t-test; values represent the mean ± s.e.m


qPCR analysis of glut1 and glut2 expression in liver tissue dissected from WT , yap - / - mutant and lf : Yap transgenic adults . n > 3 , * p < 0 . 05 , * * p < 0 . 01 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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

qPCR analysis of glut1 and glut2 expression in liver tissue dissected from WT, yap-/- mutant and lf:Yap transgenic adults. n>3, *p<0.05, **p<0.01, two-sided Student's t-test; values represent the mean ± s.e.m


Glucose tolerance test in 3 - month old WT and yap - / - mutant zebrafish . Blood glucose was measured 0 , 30 , 60 , 120 , 180 and 240 min post glucose injection . n > 8 , * p < 0 . 05 , two - sided Student ' s t - test ; values represent the mean ± s . e . m Glucose tolerance test in 12 - month old WT and yap - / - mutant zebrafish . Blood glucose was measured 0 , 30 , 60 , 120 , 180 and 240 min post glucose injection . n > 8 , * p < 0 . 05 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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

Glucose tolerance test in 3-month old WT and yap-/- mutant zebrafish. Blood glucose was measured 0, 30, 60, 120, 180 and 240 min post glucose injection. n>8, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m Glucose tolerance test in 12-month old WT and yap-/- mutant zebrafish. Blood glucose was measured 0, 30, 60, 120, 180 and 240 min post glucose injection. n>8, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m


Clustergram analysis of polar metabolite abundance from WT and yap - / - mutant larvae at 3 dpf as determined by LC - MS / MS via selected reaction monitoring ( SRM ) analysis . n = 4 , p < 0 . 05 , two - sided Student ' s t -

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Clustergram analysis of polar metabolite abundance from WT and yap-/- mutant larvae at 3 dpf as determined by LC-MS/MS via selected reaction monitoring (SRM) analysis. n=4, p<0.05, two-sided Student's t-test


Steady state abundance of glycolytic intermediates in WT and yap - / - mutant larvae as determined by SRM analysis . n = 4 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , two - sided Student ' s t - test ; values represent the mean ± s . e . m Steady state abundance of nucleotides in WT and yap - / - mutant larvae as determined by SRM analysis . n = 4 , * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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

Steady state abundance of glycolytic intermediates in WT and yap-/- mutant larvae as determined by SRM analysis. n=4, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, two-sided Student's t-test; values represent the mean ± s.e.m Steady state abundance of nucleotides in WT and yap-/- mutant larvae as determined by SRM analysis. n=4, *p<0.05, ***p<0.001, ****p<0.0001, two-sided Student's t-test; values represent the mean ± s.e.m


Relative isotopic enrichment of 13C in glycolytic intermediates following incubation of WT and yap - / - mutant larvae in [ U13C6 ] - glucose as determined by SRM analysis . n = 3 , * p < 0 . 05 , two - sided Student ' s t - test ; values represent the mean ± s . e . m Relative isotopic enrichment of 13C in nucleotide precursors following incubation of WT and yap - / - larvae in [ U13C6 ] - glucose as determined by SRM analysis . n = 3 , * p < 0 . 05 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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

Relative isotopic enrichment of 13C in glycolytic intermediates following incubation of WT and yap-/- mutant larvae in [U13C6]-glucose as determined by SRM analysis. n=3, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m Relative isotopic enrichment of 13C in nucleotide precursors following incubation of WT and yap-/- larvae in [U13C6]-glucose as determined by SRM analysis. n=3, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m


Effect of nucleoside ( NS ) cocktail treatment from 1 - 3 dpf on the incidence of cardiac edema in WT and yap - / - mutant larvae . Each point represents the percentage of affected embryos in each independent clutch . n > 7 , * * p < 0 . 01 , two - sided Student ' s t - test ; values represent the mean s . e .

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

Effect of nucleoside (NS) cocktail treatment from 1-3 dpf on the incidence of cardiac edema in WT and yap-/- mutant larvae. Each point represents the percentage of affected embryos in each independent clutch. n>7, **p<0.01, two-sided Student's t-test; values represent the mean s.e.m


Effect of WZB - 117 ( WZB , 10 μM ) treatment from 3 - 5 dpf in WT and lf : Yap larvae on liver size as determined by fluorescence microscopy at 5 dpf in lf : GFP reporters . Scale bar :

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

Effect of WZB-117 (WZB, 10 μM) treatment from 3-5 dpf in WT and lf:Yap larvae on liver size as determined by fluorescence microscopy at 5 dpf in lf:GFP reporters. Scale bar: 200μm


Quantitative analysis of the effect of WZB treatment from 3 - 5 dpf on liver size as determined by fluorescence microscopy . n > 21 , * * * * p < 0 . 0001 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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Quantitative analysis of the effect of WZB treatment from 3-5 dpf on liver size as determined by fluorescence microscopy. n>21, ****p<0.0001, two-sided Student's t-test; values represent the mean ± s.e.m


Quantitative analysis of the effect of Trehalose ( 100 μM ) treatment from 3 - 5 dpf on liver size as determined by fluorescence microscopy . n > 70 , * * * * p < 0 . 0001 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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Quantitative analysis of the effect of Trehalose (100 μM) treatment from 3-5 dpf on liver size as determined by fluorescence microscopy. n>70, ****p<0.0001, two-sided Student's t-test; values represent the mean ± s.e.m


Dot plots showing the correlation between GLUT1 and YAP1 mRNA expression in the cancer cell line encyclopedia ( CCLE , 967 lines ) . Pearson coefficient is 0 . 46 Dot plots showing the correlation between GLUT1 and AMOTL2 mRNA expression in the cancer cell line encyclopedia ( CCLE , 967 lines ) . Pearson coefficient is 0 .

Dot plots showing the correlation between GLUT1 and YAP1 mRNA expression in the cancer cell line encyclopedia (CCLE, 967 lines). Pearson coefficient is 0.46 Dot plots showing the correlation between GLUT1 and AMOTL2 mRNA expression in the cancer cell line encyclopedia (CCLE, 967 lines). Pearson coefficient is 0.45


ChIPseq tracks at the GLUT1 locus highlighting YAP and TEAD enrichment overlaying H3K27Ac marks at an intragenic enhancer element in different contexts ( LATS2 mutant MSTO211H cancer cells and HA - YAP5SA overexpressing HuCCT1 liver cancer cells )

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ChIPseq tracks at the GLUT1 locus highlighting YAP and TEAD enrichment overlaying H3K27Ac marks at an intragenic enhancer element in different contexts (LATS2 mutant MSTO211H cancer cells and HA-YAP5SA overexpressing HuCCT1 liver cancer cells)


Gross morphology of the liver from WT and TetONYap transgenic mice , treated with doxycycline for 10 days . Scale bar : 1

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Gross morphology of the liver from WT and TetONYap transgenic mice, treated with doxycycline for 10 days. Scale bar: 1 cm


Immunohistochemical analysis of GLUT1 expression in liver tissue from WT and TetONYap transgenic mice , treated with doxycycline for 10 days . Scale bar :

Immunohistochemical analysis of GLUT1 expression in liver tissue from WT and TetONYap transgenic mice, treated with doxycycline for 10 days. Scale bar: 50μm


FDG PET imaging of coronal views from WT and TetONYap transgenic mice , treated with doxycycline for 10 days . Arrow is pointing to the

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FDG PET imaging of coronal views from WT and TetONYap transgenic mice, treated with doxycycline for 10 days. Arrow is pointing to the liver


Scintillation analysis of FDG uptake ( fraction of injected dose per gram of liver tissue , % ID / g ) in dissected liver tissue from WT and TetON Yap transgenic mice , treated with doxycycline for 10 days . n = 4 , * * * p < 0 . 001 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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

Scintillation analysis of FDG uptake (fraction of injected dose per gram of liver tissue, %ID/g) in dissected liver tissue from WT and TetON Yap transgenic mice, treated with doxycycline for 10 days. n=4, ***p<0.001, two-sided Student's t-test; values represent the mean ± s.e.m


A . Fura2 340 / 380 time traces from M - CSF - differentiated macrophages derived from conditionally - immortalised macrophage precursor cell lines ( M - MØP ) of Plcg2P522 mice ( P522 , blue ) and Plcg2R522 mice ( R522 , red ) . One set of cell lines , generated from male mice , is shown . Cells were exposed to 5µg / ml anti - FcγRII / III along with 20µM EGTA and 2µM Ionomycin as indicated . Data shows the mean ± SD of 3 independent experiments analysed by two - way ANOVA with Sidak post - hoc tests ( * * = p < 0 . 01 ; * * * * = p < 0 . 0001 ) . B . Fura2 340 / 380 time traces from primary microglia derived from the cortex of Plcg2R522 mice ( blue : P522 ) and Plcg2R522 mice ( red : R522 ) with or without pre - exposure for 2 hours with Edelfosine ( 10µM ) . Cells were exposed to 5µg / ml anti - FcγRII / III along with EGTA and 2µM Ionomycin . Data shows the mean ± SD of 3 independent experiments analysed by two - way ANOVA with Sidak post - hoc tests ( * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ) . C

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

A. Fura2 340/380 time traces from M-CSF-differentiated macrophages derived from conditionally-immortalised macrophage precursor cell lines (M-MØP) of Plcg2P522 mice (P522, blue) and Plcg2R522 mice (R522, red). One set of cell lines, generated from male mice, is shown. Cells were exposed to 5µg/ml anti-FcγRII/III along with 20µM EGTA and 2µM Ionomycin as indicated. Data shows the mean±SD of 3 independent experiments analysed by two-way ANOVA with Sidak post-hoc tests (** = p<0.01; **** = p< 0.0001). B. Fura2 340/380 time traces from primary microglia derived from the cortex of Plcg2R522 mice (blue: P522) and Plcg2R522 mice (red: R522) with or without pre-exposure for 2 hours with Edelfosine (10µM). Cells were exposed to 5µg/ml anti-FcγRII/III along with EGTA and 2µM Ionomycin. Data shows the mean±SD of 3 independent experiments analysed by two-way ANOVA with Sidak post-hoc tests (** = p<0.01; *** = p<0.001; **** = p< 0.0001). C


C - E . M - MOPS ( C ) , primary mouse microglia ( D ) and hIPSC ( E ) ( blue : P522 and red : R522 ) were loaded with Fluo - 8 Ca2 + indicator and examined for peak changes in fluoresce after exposure to DOPS - liposomes ( 25 μg / ml ) . Data information : Data shown as the mean ± SD of 3 independent experiments . The data were analysed by two - way ANOVA with Sidak ' s post - tests ( * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 ) .

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

C-E. M-MOPS (C), primary mouse microglia (D) and hIPSC (E) (blue: P522 and red: R522) were loaded with Fluo-8 Ca2+ indicator and examined for peak changes in fluoresce after exposure to DOPS-liposomes (25 μg/ml). Data information: Data shown as the mean±SD of 3 independent experiments. The data were analysed by two-way ANOVA with Sidak's post-tests (*=p<0.05, **=p<0.01, ***=p<0.001).


A - F . Phagocytotic activity of R522 and P522 M - MOP and microglia was assessed using pHrodo Red E . coli and zymosan BioParticles . Phagocytosis arbitrary units ( A . U ) describes the amount of bioparticle cellular fluorescence emission at each time point . E . coli uptake in M - MOP ( A ) , primary mouse microglia ( B ) , hIPSC - derived microglia ( C ) . Zymosan uptake in M - MOP ( D ) , primary mouse microglia ( E ) and hIPSC - derived microglia ( F ) . For hIPSC - derived microglia , 3 isogenic P522 and 3 isogenic R522 clones were examined with at least 6 wells in 3 independent experiments . All microglia and M - MOP data shows the mean ± SD of 3 independent experiments were analysed by two - way ANOVA using Sidak multiple comparison test . * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . ( blue : P522 ; red : R522 )

O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F. Phagocytotic activity of R522 and P522 M-MOP and microglia was assessed using pHrodo Red E. coli and zymosan BioParticles. Phagocytosis arbitrary units (A.U) describes the amount of bioparticle cellular fluorescence emission at each time point. E.coli uptake in M-MOP (A), primary mouse microglia (B), hIPSC-derived microglia (C). Zymosan uptake in M-MOP (D), primary mouse microglia (E) and hIPSC-derived microglia (F). For hIPSC-derived microglia, 3 isogenic P522 and 3 isogenic R522 clones were examined with at least 6 wells in 3 independent experiments. All microglia and M-MOP data shows the mean±SD of 3 independent experiments were analysed by two-way ANOVA using Sidak multiple comparison test. *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. (blue: P522; red: R522)


A - F . Endocytic activity of R522 and P522 M - MOP and microglia was assessed using FITC soluble Aβ1 - 42 oligomers and pHrodo Red Dexran ( 10 , 000 MW ) . Endocytosis arbitrary units ( A . U ) describes the amount of bioparticle cellular fluorescence emission at each time point . Aβ1 - 42 oligomer uptake in M - MOP ( A ) , primary microglia ( B ) , and hIPSC - derived microglia ( C ) . Dextran uptake in M - MOP ( D ) , primary microglia ( E ) and hIPSC - derived microglia ( F ) . For hIPSC - derived microglia , 3 isogenic P522 and 3 isogenic R522 clones were examined with at least 6 wells in 3 independent experiments . All microglia and M - MOP data shows the mean ± SD of 3 independent experiments were analysed by two - way ANOVA using Sidak ' s multiple comparison test . * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 . ( blue : P522 ; red : R522 )

O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F. Endocytic activity of R522 and P522 M-MOP and microglia was assessed using FITC soluble Aβ1-42 oligomers and pHrodo Red Dexran (10,000 MW). Endocytosis arbitrary units (A.U) describes the amount of bioparticle cellular fluorescence emission at each time point. Aβ1-42 oligomer uptake in M-MOP (A), primary microglia (B), and hIPSC-derived microglia (C). Dextran uptake in M-MOP (D), primary microglia (E) and hIPSC-derived microglia (F). For hIPSC-derived microglia, 3 isogenic P522 and 3 isogenic R522 clones were examined with at least 6 wells in 3 independent experiments. All microglia and M-MOP data shows the mean±SD of 3 independent experiments were analysed by two-way ANOVA using Sidak's multiple comparison test. *=p<0.05, **=p<0.01, ***=p<0.001. (blue: P522; red: R522)


level of PIP2 in M - MOPS was examined by measuring immunofluorescence from images at set time points after exposure after exposure to 5µg / ml anti - FcγRII / III ( 2 . 4G2 ) , 50 ng / ml LPS or oligomers of 40 µM Aβ1 - 42 Data shows the mean ± SD of 3 independent experiments were analysed by 2 way ANOVA with Sidak ' s multiple comparison * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . ( blue : P522 ; red : R522 ) .

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

level of PIP2 in M-MOPS was examined by measuring immunofluorescence from images at set time points after exposure after exposure to 5µg/ml anti-FcγRII/III (2.4G2), 50 ng/ml LPS or oligomers of 40 µM Aβ1-42 Data shows the mean±SD of 3 independent experiments were analysed by 2 way ANOVA with Sidak's multiple comparison *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. (blue: P522; red: R522).


A - C . A mass ELISA was used to detect specific PIP species after exposure to anti - FcγRII / III ( 2 . 4G2 ) with or without 3 - a - aminocholestane ( SHIP 1 inhibitor ) , SF1670 ( PTEN inhibitor ) and LY294002 ( PI - 3K inhibitor ) . In M - MOP cells ( left panel ) and primary microglia ( right panel ) PI ( 4 , 5 ) P2 ( A ) , PI ( 3 , 4 ) P2 ( B ) and PIP3 ( C ) were detected . All data shows the mean ± SD of 3 independent experiments were analysed by 2 way ANOVA ( log - transformed data was used in C ) with Sidak ' s multiple comparison tests performed * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . ( blue : P522 ; red : R522 )

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

A-C. A mass ELISA was used to detect specific PIP species after exposure to anti-FcγRII/III (2.4G2) with or without 3-a-aminocholestane (SHIP 1 inhibitor), SF1670 (PTEN inhibitor) and LY294002 (PI-3K inhibitor). In M-MOP cells (left panel) and primary microglia (right panel) PI(4,5)P2 (A), PI(3,4)P2 (B) and PIP3 (C) were detected. All data shows the mean±SD of 3 independent experiments were analysed by 2 way ANOVA (log-transformed data was used in C) with Sidak's multiple comparison tests performed *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. (blue: P522; red: R522)


A - C . Iba1 was used as a marker for microglia and PIP2 levels were detected by measuring the intensity of florescence per cell . The average fluorescence was calculated above background in the cortex and hippocampus at 2 ( A ) , 6 ( B ) and 9 ( C ) months . All data shows the mean ± SD of 3 independent experiments analysed using 2 way ANOVA with Sidak ' s multiple comparison tests performed * * = p < 0 . 01 , * * * * = p < 0 . 0001 .

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A-C. Iba1 was used as a marker for microglia and PIP2 levels were detected by measuring the intensity of florescence per cell. The average fluorescence was calculated above background in the cortex and hippocampus at 2 (A), 6 (B) and 9 (C) months. All data shows the mean±SD of 3 independent experiments analysed using 2 way ANOVA with Sidak's multiple comparison tests performed **=p<0.01, ****=p<0.0001.


Cell migration assays were performed using Transwell migration chambers . HCT116 cells were transfected with siRNAs targeting 11 candidate lincRNAs and cell migration activities were evaluated by Transwell migration assays ( n = 3 ) . CALIC is the lincRNA targeted by si - LINC00920 .

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Cell migration assays were performed using Transwell migration chambers. HCT116 cells were transfected with siRNAs targeting 11 candidate lincRNAs and cell migration activities were evaluated by Transwell migration assays (n = 3). CALIC is the lincRNA targeted by si-LINC00920.


Migration of HCT116 cells transfected with the indicated siRNAs or negative control . ( n = 4 )

Migration of HCT116 cells transfected with the indicated siRNAs or negative control. (n = 4)


Migration of colon cancer cells treated with an siRNA targeting CALIC . ( n = 3 )

Migration of colon cancer cells treated with an siRNA targeting CALIC. (n = 3)


Subcellular localization analysis of CALIC . RNAs were isolated from the nuclear and cytoplasmic fractions of HCT116 cells and quantified by qRT - PCR ( n = 3 ) . Nuclear controls : U6 , MALAT1 ; Cytoplasmic control : GAPDH .

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Subcellular localization analysis of CALIC. RNAs were isolated from the nuclear and cytoplasmic fractions of HCT116 cells and quantified by qRT-PCR (n = 3). Nuclear controls: U6, MALAT1; Cytoplasmic control: GAPDH.


CALIC expression levels in cancer tissues . N , normal mucosa ; T , tumor .

CALIC expression levels in cancer tissues. N, normal mucosa; T, tumor.


Expression of CALIC in primary skin tumors ( P ) and their metastases ( M ) . Expression of CALIC in primary skin tumors of patients without ( w / o ) or with ( w ) metastases .

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Expression of CALIC in primary skin tumors (P) and their metastases (M). Expression of CALIC in primary skin tumors of patients without (w/o) or with (w) metastases.


qRT - PCR analysis of CALIC expression in early ( I / II ) and late ( III / IV ) stage colon cancers . [ Normal ( N ) n = 8 , stage I / II n = 14 , stage III / IV n = 26 ]

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qRT-PCR analysis of CALIC expression in early (I/II) and late (III/IV) stage colon cancers. [Normal (N) n = 8, stage I/II n = 14, stage III/IV n = 26]


Kaplan - Meier curves for disease - free survival of colon cancer patients whose primary tumors expressed low ( blue , n = 113 ) or high ( red , n = 250 ) levels of CALIC .

Kaplan-Meier curves for disease-free survival of colon cancer patients whose primary tumors expressed low (blue, n = 113) or high (red, n = 250) levels of CALIC.


Biotinylated full - length CALIC or antisense CALIC ( negative control ) generated in vitro were incubated with lysates from DLD - 1 colon cancer cells and precipitated with streptavidin beads . Precipitated proteins were resolved by SDS - PAGE followed by silver staining . The protein band indicated by the arrow was excised and subjected to mass spectrometry .

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Biotinylated full-length CALIC or antisense CALIC (negative control) generated in vitro were incubated with lysates from DLD-1 colon cancer cells and precipitated with streptavidin beads. Precipitated proteins were resolved by SDS-PAGE followed by silver staining. The protein band indicated by the arrow was excised and subjected to mass spectrometry.


( Top ) schematic of the full - length and mutants of CALIC used for the precipitation of hnRNP - L from HCT116 cell lysates . The mutated elements are indicated by the cross marks . Se , sense transcript ; AS , antisense transcript . + , detectable binding activity ; - , no detectable activity . ( Bottom ) precipitated hnRNP - L and biotin - labeled fragments of CALIC are shown .

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(Top) schematic of the full-length and mutants of CALIC used for the precipitation of hnRNP-L from HCT116 cell lysates. The mutated elements are indicated by the cross marks. Se, sense transcript; AS, antisense transcript. +, detectable binding activity; -, no detectable activity. (Bottom) precipitated hnRNP-L and biotin-labeled fragments of CALIC are shown.


RIP analysis was performed using control rabbit IgG or anti - hnRNP - L antibody . HPRT1 and the lncRNA MALAT1 were used as negative controls .

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RIP analysis was performed using control rabbit IgG or anti-hnRNP-L antibody. HPRT1 and the lncRNA MALAT1 were used as negative controls.


Immunoblotting analysis of proteins precipitated by RIP assays using anti - hnRNP - L antibody are shown ( D ) .

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Immunoblotting analysis of proteins precipitated by RIP assays using anti-hnRNP-L antibody are shown (D).


ChIRP - immunoblotting analysis of hnRNP - L association with endogeneous CALIC in HCT116 cells .

ChIRP-immunoblotting analysis of hnRNP-L association with endogeneous CALIC in HCT116 cells.


RNA - Seq analysis of hnRNP - L - and CALIC - regulated genes in HCT116 cells . Venn diagram showing the overlap of genes differentially expressed by hnRNP - L ( blue ) or CALIC ( purple ) knockdown ( B , top ) . Expression levels of 59 genes regulated in common in both hnRNP - L and CALIC knockdown cells relative to control siRNA experiments ( B , bottom ) . Two biological replicates ( R1 and R2 ) were included for each siRNA treatment . The number of genes within each category is indicated .

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RNA-Seq analysis of hnRNP-L- and CALIC-regulated genes in HCT116 cells. Venn diagram showing the overlap of genes differentially expressed by hnRNP-L (blue) or CALIC (purple) knockdown (B, top). Expression levels of 59 genes regulated in common in both hnRNP-L and CALIC knockdown cells relative to control siRNA experiments (B, bottom). Two biological replicates (R1 and R2) were included for each siRNA treatment. The number of genes within each category is indicated.


Scatterplots showing correlations between CALIC - and hnRNP - L - regulated genes in ( B ) . Left , co - regulated 59 genes ; Right , all differentially regulated genes ( DEGs ) . The correlation was calculated using the data except for the gene indicated by the asterisk .

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Scatterplots showing correlations between CALIC- and hnRNP-L-regulated genes in (B). Left, co-regulated 59 genes; Right, all differentially regulated genes (DEGs). The correlation was calculated using the data except for the gene indicated by the asterisk.


Genes induced by CALIC are significantly enriched in those upregulated by hnRNP - L . GSEA comparing the genes repressed by CALIC and hnRNP - L knockdown . NES , normalized enrichment score .

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Genes induced by CALIC are significantly enriched in those upregulated by hnRNP-L. GSEA comparing the genes repressed by CALIC and hnRNP-L knockdown. NES, normalized enrichment score.


Heatmap showing the expression levels of genes positively regulated in common by both CALIC and hnRNP - L in colon and rectal cancers . Samples are ordered based on the expression levels of AXL .

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Heatmap showing the expression levels of genes positively regulated in common by both CALIC and hnRNP-L in colon and rectal cancers. Samples are ordered based on the expression levels of AXL.


qRT - PCR analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting

qRT-PCR analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting CALIC


immunoblotting ( B , D ) analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting

immunoblotting (B, D) analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting CALIC


qRT - PCR analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting hnRNP - L .

qRT-PCR analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting hnRNP-L.


immunoblotting ( B , D ) analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting hnRNP - L .

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immunoblotting (B, D) analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting hnRNP-L.


qRT - PCR analyses of AXL in HCT116 cells infected with a lentivirus expressing wild - type CALIC ( CALIC - full ) , CALIC - 296 - 913 or CALIC -

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qRT-PCR analyses of AXL in HCT116 cells infected with a lentivirus expressing wild-type CALIC (CALIC-full), CALIC-296-913 or CALIC-mut


HCT116 cells that had been treated with an siRNA targeting hnRNP - L or CALIC were transfected with an AXL reporter construct and subjected to luciferase assays .

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HCT116 cells that had been treated with an siRNA targeting hnRNP-L or CALIC were transfected with an AXL reporter construct and subjected to luciferase assays.


ChIP assays were performed using anti - hnRNP - L antibody or control mouse IgG . The promoter regions of GAPDH and Egr1 were used as negative and positive controls , respectively .

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ChIP assays were performed using anti-hnRNP-L antibody or control mouse IgG. The promoter regions of GAPDH and Egr1 were used as negative and positive controls, respectively.


Immunoblotting analysis of proteins precipitated by ChIP assays using anti - hnRNP - L

Immunoblotting analysis of proteins precipitated by ChIP assays using anti-hnRNP-L antibody


ChIP analysis of hnRNP - L association with the AXL promoter region in CALIC knockdown HCT116 cells .

ChIP analysis of hnRNP-L association with the AXL promoter region in CALIC knockdown HCT116 cells.


ChIP analysis of hnRNP - L association with the AXL promoter region in HCT116 cells expressing lentiviral CALIC - full .

ChIP analysis of hnRNP-L association with the AXL promoter region in HCT116 cells expressing lentiviral CALIC-full.


ChIRP analysis of CALIC binding to the AXL promoter . Binding of CALIC to the promoter regions of the indicated genes was compared with that of LacZ .

ChIRP analysis of CALIC binding to the AXL promoter. Binding of CALIC to the promoter regions of the indicated genes was compared with that of LacZ.


Migratory activity of AXL knockdown HCT116 cells .

Migratory activity of AXL knockdown HCT116 cells.


Lentiviral expression of AXL restores the migration activity of HCT116 cells transfected with an siRNA targeting CALIC . Immunoblotting and qRT - PCR analyses of HA - tagged AXL and CALIC expression , respectively ( left ) . α - Tubulin was used as a loading control .

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Lentiviral expression of AXL restores the migration activity of HCT116 cells transfected with an siRNA targeting CALIC. Immunoblotting and qRT-PCR analyses of HA-tagged AXL and CALIC expression, respectively (left). α-Tubulin was used as a loading control.


Representative images of gross specimens ( top ) and H & E stained sections ( bottom ) of lung metastatic lesions in mice injected with CALIC or AXL knockdown HCT116 cells . Tumors were marked with yellow arrows . The dashed lines depict the boundary between normal and tumor tissues . ( Right ) Percentage of mice with lung metastases .

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Representative images of gross specimens (top) and H&E stained sections (bottom) of lung metastatic lesions in mice injected with CALIC or AXL knockdown HCT116 cells. Tumors were marked with yellow arrows. The dashed lines depict the boundary between normal and tumor tissues. (Right) Percentage of mice with lung metastases.


Representative images of gross specimens of primary cecal tumors ( top ) and liver metastatic lesions ( bottom ) in mice orthotopically ( cecal ) injected with CALIC knockdown HCT116 cells . Tumors are marked with yellow arrows . ( Right ) Percentage of mice with primary tumors and liver metastases .

Representative images of gross specimens of primary cecal tumors (top) and liver metastatic lesions (bottom) in mice orthotopically (cecal) injected with CALIC knockdown HCT116 cells. Tumors are marked with yellow arrows. (Right) Percentage of mice with primary tumors and liver metastases.


Cloning and characterization of APG9 . A , Wild - type ( WT ; SEY6210 ) , apg9 ( THY154 ) , apg9Δ ( JKY007 ) , and the apg9Δ strain transformed with single copy ( CEN ; pAPG9 ( 414 ) ) or multicopy ( 2μ ; pAPG9 ( 424 ) ) plasmids encoding APG9 were grown to log phase in SMD . Protein extracts were prepared and analyzed by immunoblot using antiserum to API as described in Materials and Methods . The positions of precursor and mature API are indicated . The APG9 gene complements the prAPI accumulation phenotype of the apg9Δ mutant .

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

Cloning and characterization of APG9. A, Wild-type (WT; SEY6210), apg9 (THY154), apg9Δ (JKY007), and the apg9Δ strain transformed with single copy (CEN; pAPG9(414)) or multicopy (2μ; pAPG9(424)) plasmids encoding APG9 were grown to log phase in SMD. Protein extracts were prepared and analyzed by immunoblot using antiserum to API as described in Materials and Methods. The positions of precursor and mature API are indicated. The APG9 gene complements the prAPI accumulation phenotype of the apg9Δ mutant.


B , WT ( SEY6210 ) , apg9Δ ( JKY007 ) , and the apg9Δ strain transformed with pAPG9 ( 414 ) centromeric plasmid were grown in SMD and transferred to SD ( − N ) as described in Materials and Methods . Aliquots were removed at the indicated times and spread onto YPD plates in triplicate . Numbers of viable colonies were determined after two to three days . The APG9 gene complements the starvation - sensitive phenotype of the apg9Δ mutant .

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

B, WT (SEY6210), apg9Δ (JKY007), and the apg9Δ strain transformed with pAPG9(414) centromeric plasmid were grown in SMD and transferred to SD(−N) as described in Materials and Methods. Aliquots were removed at the indicated times and spread onto YPD plates in triplicate. Numbers of viable colonies were determined after two to three days. The APG9 gene complements the starvation-sensitive phenotype of the apg9Δ mutant.


Apg9p is an unglycosylated , integral membrane protein . A , Wild - type ( WT ; SEY6210 ) , apg9Δ ( JKY007 ) , and the apg9Δ strain transformed with single copy ( CEN ) or multicopy ( 2μ ) plasmids encoding APG9 were grown to log phase in SMD . Protein extracts were prepared and analyzed by immunoblot using antiserum to Apg9p . Apg9p is detected as an ∼ 125 - kD protein .

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Apg9p is an unglycosylated, integral membrane protein. A, Wild-type (WT; SEY6210), apg9Δ (JKY007), and the apg9Δ strain transformed with single copy (CEN) or multicopy (2μ) plasmids encoding APG9 were grown to log phase in SMD. Protein extracts were prepared and analyzed by immunoblot using antiserum to Apg9p. Apg9p is detected as an ∼125-kD protein.


. B , Hydrophobicity analysis of Apg9p based on the Kyte - Doolittle algorithm ( Kyte and Doolittle 1982 ) predicts six to eight transmembrane domains .

. B, Hydrophobicity analysis of Apg9p based on the Kyte-Doolittle algorithm (Kyte and Doolittle 1982) predicts six to eight transmembrane domains.


C , Cells of wild - type strain YW5 - 1B were grown in YPD , converted to spheroplasts , and osmotically lysed as described in Materials and Methods . After a preclearing step to remove cell debris , the lysates were incubated with or without 1 % Triton X - 100 on ice for 30 min , as indicated . Centrifugation at 10 , 000 g for 1 h separated the lysate into supernatant ( S ) and pellet ( P ) fractions , which were analyzed by immunoblots with antiserum to Apg9p . Apg9p is found in the low - speed pellet in the absence of Triton X - 100 . Detergent extraction completely solubilizes Apg9p and causes it to appear in the supernatant

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

C, Cells of wild-type strain YW5-1B were grown in YPD, converted to spheroplasts, and osmotically lysed as described in Materials and Methods. After a preclearing step to remove cell debris, the lysates were incubated with or without 1% Triton X-100 on ice for 30 min, as indicated. Centrifugation at 10,000 g for 1 h separated the lysate into supernatant (S) and pellet (P) fractions, which were analyzed by immunoblots with antiserum to Apg9p. Apg9p is found in the low-speed pellet in the absence of Triton X-100. Detergent extraction completely solubilizes Apg9p and causes it to appear in the supernatant fraction


D , Strain CTD1 harboring a multicopy 3 × HA APG9 plasmid was grown in YPD and converted to spheroplasts as described in Materials and Methods . A crude lysate was prepared and treated with or without endo H as indicated overnight at 37 ° C and analyzed by immunoblots with anti - HA antibodies or anti - CPY serum . Endo H treatment removes the N - linked glycosyl residues of CPY , causing a shift in its electrophoretic mobility . In contrast , Apg9p is not glycosylated and remains unchanged in the presence or absence of endo H treatment .

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D, Strain CTD1 harboring a multicopy 3×HA APG9 plasmid was grown in YPD and converted to spheroplasts as described in Materials and Methods. A crude lysate was prepared and treated with or without endo H as indicated overnight at 37°C and analyzed by immunoblots with anti-HA antibodies or anti-CPY serum. Endo H treatment removes the N-linked glycosyl residues of CPY, causing a shift in its electrophoretic mobility. In contrast, Apg9p is not glycosylated and remains unchanged in the presence or absence of endo H treatment.


Apg9p does not comigrate with typical endomembrane markers in sucrose density gradients . A and B , Strain STY1 ( pep4Δ ) was grown in YPD to log - phase , and osmotically lysed . Lysates were precleared , loaded on a sucrose density gradient ranging from 18 - 55 % ( wt / wt ) , and centrifuged for 2 . 5 h at 174 , 000 g as described in Materials and Methods . Fractions were collected from the top ( fraction number 1 ) to the bottom ( fraction number 16 ) . Fractions were subjected to SDS - PAGE and immunoblot with antiserum against Apg9p and : in A , Kex2p ( trans Golgi network ) , Pho8p ( vacuole ) , and Pma1p ( plasma membrane ) ; and in B , Sec12p ( ER ) and Pep12p ( endosome ) . The graphs in panels A and B are from the same gradient , but are presented separately for clarity .

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

Apg9p does not comigrate with typical endomembrane markers in sucrose density gradients. A and B, Strain STY1 (pep4Δ) was grown in YPD to log-phase, and osmotically lysed. Lysates were precleared, loaded on a sucrose density gradient ranging from 18-55% (wt/wt), and centrifuged for 2.5 h at 174,000 g as described in Materials and Methods. Fractions were collected from the top (fraction number 1) to the bottom (fraction number 16). Fractions were subjected to SDS-PAGE and immunoblot with antiserum against Apg9p and: in A, Kex2p (trans Golgi network), Pho8p (vacuole), and Pma1p (plasma membrane); and in B, Sec12p (ER) and Pep12p (endosome). The graphs in panels A and B are from the same gradient, but are presented separately for clarity.