( H ) Chi - G4S - DR5 stable 4T1 tumors ( after reaching ~ 100mm3 ) were treated with 6 doses of indicated treatment . Isolated tumors were imaged together ( n = 4 - 5 ) . ROCK1i indicates GSK269962A was administered to via intra - injections ( 2mg / kg ) .

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

(H) Chi-G4S-DR5 stable 4T1 tumors (after reaching ~100mm3) were treated with 6 doses of indicated treatment. Isolated tumors were imaged together (n=4-5). ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections (2mg/kg).


( H ) Chi - G4S - DR5 stable 4T1 tumors ( after reaching ~ 100mm3 ) were treated with 6 doses of indicated treatment . ROCK1i indicates GSK269962A was administered to via intra - injections ( 2mg / kg ) . average is shown ( n = 4 ) .

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

(H) Chi-G4S-DR5 stable 4T1 tumors (after reaching ~100mm3) were treated with 6 doses of indicated treatment. ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections (2mg/kg). average tumor weight is shown (n=4).


( J - K ) Confirmation of selective CD8 + T population depletion in mice spleen grafted tumors after injecting with either anti - PD - L1 avelumab alone or avelumab + anti - CD8a antibody . CD4 + T - remained unchanged .

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

(J-K) Confirmation of selective CD8+ T cell population depletion in mice spleen and grafted tumors after injecting animals with either anti-PD-L1 avelumab alone or avelumab+anti-CD8a antibody. CD4+ T-cells remained unchanged.


( L ) Average of weights ( harvested at same time ) from mice treated ( i . p . ) with IgG1 , lexatumumab , ROCK1i , lexatumumab + ROCK1i anti - CD8 + lexatumumab + ROCK1i ( n = 4 - 6 , 50μg Lexatumumab , 2mg / kg ROCK1i , 6 doses ) . ROCK1i indicates GSK269962A was administered to via intra - injections . Various treatments were started when tumors were ~ 100mm3 .

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

(L) Average of tumor weights (harvested at same time) from mice treated (i.p.) with IgG1, lexatumumab, ROCK1i, lexatumumab + ROCK1i and anti-CD8+ lexatumumab + ROCK1i (n=4-6, 50μg Lexatumumab, 2mg/kg ROCK1i, 6 doses). ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections. Various treatments were started when tumors were ~100mm3 size.


( M ) Average of weights ( harvested at same time ) from mice treated ( i . p . ) with IgG1 , lexatumumab , avelumab , lexatumumab + avelumab anti - CD8 + lexatumumab + avelumab ( n = 4 - 6 ) .

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(M) Average of tumor weights (harvested at same time) from mice treated (i.p.) with IgG1, lexatumumab, avelumab, lexatumumab +avelumab and anti-CD8+ lexatumumab +avelumab (n=4-6).


( N ) 6 - 8 old C57BL / 6 bearing Chi - G4S - DR5 expressing MC38 tumors were intraperitoneally ( i . p . ) injected with 50μg of indicated antibody every third day ( n = 4 - 6 ) . Various treatments were started when tumors were ~ 100mm3 . volumes were quantified at indicated days by caliper measurements .

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

(N) 6-8 weeks old C57BL/6 bearing Chi-G4S-DR5 expressing MC38 tumors were intraperitoneally (i.p.) injected with 50μg of indicated antibody every third day (n=4-6). Various treatments were started when tumors were ~100mm3 size. Tumor volumes were quantified at indicated days by caliper measurements.


Chi - G4S - DR5 stable 4T1 tumors ( after reaching ~ 100mm3 ) were treated with 6 doses of indicated treatment . Isolated tumors were imaged together ( n = 4 - 5 ) . ROCK1i indicates GSK269962A was administered to via intra - injections ( 2mg / kg ) . ( O ) KMTR2 ( 8 doses ) antibody used as DR5 agonist for experiment . ( P ) Average of weights of data shown in ( O ) .

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

Chi-G4S-DR5 stable 4T1 tumors (after reaching ~100mm3) were treated with 6 doses of indicated treatment. Isolated tumors were imaged together (n=4-5). ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections (2mg/kg). (O) KMTR2 (8 doses) antibody used as the DR5 agonist for the experiment. (P) Average of tumor weights of data shown in (O).


( Q ) Kaplan - Meier plot depicting survival of syngeneic Chi - G4S - DR5 4T1 bearing injected i . p . with 100μg of indicated antibodies such as IgG1 , KMTR2 avelumab . were injected with GSK269962A 2mg / kg ( in PBS ) directly into tumors wherever ROCK1i is indicated .

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

(Q) Kaplan-Meier plot depicting the survival of syngeneic Chi-G4S-DR5 4T1 tumor bearing animals injected i.p. with 100μg of indicated antibodies such as IgG1, KMTR2 and avelumab. Animals were injected with GSK269962A 2mg/kg (in PBS) directly into tumors wherever ROCK1i is indicated.


( A ) Chi - G4S - DR5 stable 4T1 tumors harboring mice were treated lexatumumab , lexatumumab + ROCK1i , avelumab + lexatumumab other controls as indicated . Antibodies were treated i . p at 100μg dose ( 6 total ) , ROCK1i ( in PBS ) was injected directly into tumors at 2mg / kg dose ( 6 total ) . Various treatments were started when tumors were ~ 400mm3 . Harvested tumors were grouped together sized matched ( 3 independent sets : n = 2 - 6 tumors in each set ) followed by TIL isolation CD8 / CD45 CD4 / CD45 expressing were measured by flow cytometry .

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

(A) Chi-G4S-DR5 stable 4T1 tumors harboring mice were treated lexatumumab, lexatumumab +ROCK1i, and avelumab + lexatumumab and other controls as indicated. Antibodies were treated i.p at 100μg dose (6 total), ROCK1i (in PBS) was injected directly into tumors at 2mg/kg dose (6 total). Various treatments were started when tumors were ~400mm3 size. Harvested tumors were grouped together and sized matched (3 independent sets: n=2-6 tumors in each set) followed by TIL isolation CD8/CD45 and CD4/CD45 expressing cells were measured by flow cytometry.


( B ) Plots showing % of total double positive TILs ( CD8 + CD45 + + CD4 + CD45 + ) in right upper quadrant after combining 3 independent experiments . ( C ) of CD8 + CD45 + / CD4 + CD45 + isolated TILs from tumors in each indicated treatment ( n = 3 ) .

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

(B) Plots showing % of total double positive TILs (CD8+CD45+ + CD4+CD45+) in right upper quadrant after combining 3 independent experiments. (C) Ratio of CD8+CD45+/CD4+CD45+ isolated TILs from tumors in each indicated treatment (n=3).


Chi - G4S - DR5 stable MC38 tumors harboring mice were treated ( 6 total doses ) lexatumumab , avelumab , ROCK1i , lexatumumab + ROCKi , avelumab + lexatumumab IgG1 control as indicated . Harvested tumors homogenized followed by quantitation . lysates were run on SDS - Page followed by immunoblotting using indicated CD8 , CD4 , Foxp3 , caspase - 3 granzyme - b antibody . GAPDH is loading control . Red arrows indicated caspase - 3 p19 p17 fragments .

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

Chi-G4S-DR5 stable MC38 tumors harboring mice were treated (6 total doses) lexatumumab, avelumab, ROCK1i, lexatumumab +ROCKi, and avelumab + lexatumumab and IgG1 control as indicated. Harvested tumors homogenized followed by quantitation. Protein lysates were run on SDS-Page followed by immunoblotting using indicated CD8, CD4, Foxp3, caspase-3 and granzyme-b antibody. GAPDH is loading control. Red arrows indicated cleaved caspase-3 p19 and p17 fragments.


( H ) killing of 4T1 treated with murine DR5 agonist MD5 - 1 bispecific avelu - MD5 antibody .

(H) Cell killing assay of 4T1 cells treated with murine DR5 agonist MD5-1 and bispecific avelu-MD5 antibody.


( I ) Balb / c mice harboring luciferase stable 4T1 tumors were i . p . injected with indicated antibodies . 50μg dose mice were imaged after 5 doses .

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

(I) Balb/c mice harboring luciferase stable 4T1 tumors were i.p. injected with indicated antibodies. 50μg dose and mice were imaged after 5 doses.


( J ) 6 - 8 old C57BL / 6 mice bearing MC38 tumors were intraperitoneally ( i . p . ) injected with 50μg of indicated antibody every third day ( n = 4 - 6 ) . Indicated treatments were started when tumors were ~ 100mm3 . volumes were quantified at indicated days by caliper measurements .

O O O O O O O O O O B-ORGANISM O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O

(J) 6-8 weeks old C57BL/6 mice bearing MC38 tumors were intraperitoneally (i.p.) injected with 50μg of indicated antibody every third day (n=4-6). Indicated treatments were started when tumors were ~100mm3 size. Tumor volumes were quantified at indicated days by caliper measurements.


( K ) ~ 150 - 200mm3 MC38 bearing C57LB / 6 mice were treated with indicated MD5 - 1 , avelumab bispecific antibodies along with control IgG1 , 6 total doses . Harvested tumors were homogenized followed by quantitation . lysates were run on SDS - Page followed by immunoblotting using indicated CD8 , CD4 , caspase - 3 granzyme - b antibody . GAPDH is loading control . Red arrows indicated caspase - 3 p19 p17 fragments .

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

(K) ~150-200mm3 size MC38 tumor bearing C57LB/6 mice were treated with indicated MD5-1, avelumab and bispecific antibodies along with control IgG1, 6 total doses. Harvested tumors were homogenized followed by quantitation. Protein lysates were run on SDS-Page followed by immunoblotting using indicated CD8, CD4, caspase-3 and granzyme-b antibody. GAPDH is loading control. Red arrows indicated cleaved caspase-3 p19 and p17 fragments.


( L ) 6 - 8 old C57BL / 6 mice were injected with MC38 . When tumors reached ~ 150 - 200mm3 , were intraperitoneally ( i . p . ) injected with 50μg of indicated antibody every third day . On day 18 , tumors were harvested , sized matched pooled by treatment group , exposed to collagenase / DNase were single suspensions enriched for CD8 + . Enriched CD8 + T - from various treatments were restimulated with anti - CD3 ( OKT3 ) antibody for 4 additional hours . CD8 gated were next analyzed for IFN - γ intracellular expression using flow cytometry .

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

(L) 6-8 weeks old C57BL/6 mice were injected with MC38 cells. When tumors reached ~150-200mm3, animals were intraperitoneally (i.p.) injected with 50μg of indicated antibody every third day. On day 18, tumors were harvested, sized matched and pooled by treatment group, exposed to collagenase/DNase and were single cell suspensions enriched for CD8+ cells. Enriched CD8+ T-cells from various treatments were restimulated with anti-CD3 (OKT3) antibody for 4 additional hours. CD8 gated cells were next analyzed for IFN-γ intracellular expression using flow cytometry.


( M ) of IFN - γ + CD8 + double positive from 3 independent experiments .

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

(M) Percentage of IFN-γ+CD8+ double positive cells from 3 independent experiments.


Median CSF abundance distribution as calculated from MS - intensities of quantified peptides of each . top ten most abundant hemoglobin are highlighted .

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

Median CSF protein abundance distribution as calculated from MS-intensities of quantified peptides of each protein. The top ten most abundant proteins and hemoglobin proteins are highlighted.


A - C AD / non - AD fold changes plotted vs statistical significance for Sweden ( A ) , Magdeburg / Kiel ( B ) , Berlin ( C ) cohort . associated with GO annotation neuron projection labelled in orange . above dashed green are statistically significant ( p < 0 . 05 ) those above black curves have a q - value below 0 . 05

O O O B-DISEASE O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Protein AD/non-AD fold changes plotted vs statistical significance for Sweden (A), Magdeburg/Kiel (B), and Berlin (C) cohort. Proteins associated with the GO annotation neuron projection labelled in orange. Proteins above the dashed green line are statistically significant (p < 0.05) and those above the black curves have a q-value below 0.05


A - C AD / non - AD fold changes plotted vs statistical significance for Sweden ( A ) , Magdeburg / Kiel ( B ) , Berlin ( C ) cohort . associated with GO annotation neuron projection labelled in orange . above dashed green are statistically significant ( p < 0 . 05 ) those above black curves have a q - value below 0 .

O O O B-DISEASE O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O


Correlation of AD / non - AD fold changes in pairwise combinations of two cohorts each . Combinations are Sweden vs Magdeburg / Kiel included differ significantly ( p < 0 . 05 ) consistently in abundance by AD status in both cohorts each .

O O B-DISEASE O O O B-DISEASE O O O O O O O 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-DISEASE O O O O O O

Correlation of protein AD/non-AD fold changes in pairwise combinations of two cohorts each. Combinations are Sweden vs Magdeburg/Kiel Proteins included differ significantly (p < 0.05) and consistently in abundance by AD status in both cohorts each.


Correlation of AD / non - AD fold changes in pairwise combinations of two cohorts each . Combinations are Sweden vs Berlin ( D ) , Magdeburg / Kiel vs Berlin ( E ) . included differ significantly ( p < 0 . 05 ) consistently in abundance by AD status in both cohorts each .

O O B-DISEASE O O O B-DISEASE O O O O O O O O O O O O O O O O O O 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-DISEASE O O O O O O

Correlation of protein AD/non-AD fold changes in pairwise combinations of two cohorts each. Combinations are Sweden vs Berlin (D), and Magdeburg/Kiel vs Berlin (E). Proteins included differ significantly (p < 0.05) and consistently in abundance by AD status in both cohorts each.


that differ significantly ( p - value < 0 . 05 in E ; q - value < 0 . 05 in F ) in abundance by AD status across three cohorts . Z - scored abundances of in AD non - AD groups of cohorts shown by heat map Hierarchical clustering separates AD from non - AD groups . Pyruvat kinase PKM ( PKM ) was quantified in two isoforms UniProt IDs are given in parenthesis . Black frames highlight with consistent AD / non - AD fold changes across cohorts .

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

Proteins that differ significantly (p-value < 0.05 in E; q-value < 0.05 in F) in abundance by AD status across all three cohorts. Z-scored abundances of proteins in the AD and non-AD groups of all cohorts shown by the heat map Hierarchical clustering separates AD from non-AD groups. Pyruvat kinase PKM (PKM) was quantified in two isoforms and UniProt IDs are given in parenthesis. Black frames highlight proteins with consistent AD/non-AD fold changes across cohorts.


A - C ) Correlation of with ELISA - measured t - tau across samples within Sweden ( A ) , Magdeburg ( B ) Berlin ( C ) cohorts . with a q - value below 0 . 05 are labeled in yellow . of 40 signature are colored in red for those with higher abundance in AD CSF in blue for those with higher abundance in non - AD CSF .

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

A-C) Correlation of proteins with ELISA-measured t-tau concentration across samples within the Sweden (A), Magdeburg (B) and Berlin (C) cohorts. Proteins with a q-value below 0.05 are labeled in yellow. Proteins of the 40 protein signature are colored in red for those with higher abundance in AD CSF and in blue for those with higher abundance in non-AD CSF.


D ) Three cohort summary of significantly correlating with ELISA - measured t - tau . names given for 29 out of 40 signature with significant ( p < 0 . 05 ) correlation in each of three cohorts . Pyruvat kinase PKM ( PKM ) was quantified in two isoforms UniProt IDs are given in parenthesis .

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

D) Three cohort summary of proteins significantly correlating with ELISA-measured t-tau. Protein names given for the 29 proteins out of the 40 protein signature with significant (p < 0.05) correlation in each of the three cohorts. Pyruvat kinase PKM (PKM) was quantified in two isoforms and UniProt IDs are given in parenthesis.


abundance distribution of CSF showing abundances of AD - modulated CSF . of our 40 signature are highlighted in red ( elevated abundance in AD ) blue ( elevated abundance in non - AD ) . linked to glucose metabolism are highlighted in purple labelled .

O O O B-TISSUE O O O B-DISEASE O O B-TISSUE O O O O O O O O O O O O O B-DISEASE O O O O O O O O B-DISEASE O O O O B-SMALL_MOLECULE O O O O O O O

Protein abundance distribution of CSF showing the abundances of AD-modulated CSF proteins. Proteins of our 40 protein signature are highlighted in red (elevated abundance in AD) and blue (elevated abundance in non-AD). Proteins linked to glucose metabolism are highlighted in purple and labelled.


abundance distribution of brain showing abundances of AD - modulated CSF . of our 40 signature are highlighted in red ( elevated abundance in AD ) blue ( elevated abundance in non - AD ) . linked to glucose metabolism are highlighted in purple labelled .

Protein abundance distribution of brain showing the abundances of AD-modulated CSF proteins. Proteins of our 40 protein signature are highlighted in red (elevated abundance in AD) and blue (elevated abundance in non-AD). Proteins linked to glucose metabolism are highlighted in purple and labelled.


Circular representation of methylation for mothers ( outer circle ) children ( inner circle ) . height of each bar indicates methylation change between smoking non ‐ smoking group ( hue : hypermethylation , hue : hypomethylation ) .

O O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Circular representation of DNA methylation levels for mothers (outer circle) and children (inner circle). The height of each bar indicates the methylation change between the smoking and non‐smoking group (dark hue: hypermethylation, light hue: hypomethylation).


( A ) Endogenous FAM134B forms oligomers . FAM134B knockout ( KO ) or wild type ( WT ) 293T were treated with 1μM of Thapsigargin ( Tg ) for different time lysates were prepared analyzed by Western - blot .

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

(A) Endogenous FAM134B forms oligomers. FAM134B knockout (KO) or wild type (WT) 293T cells were treated with 1μM of Thapsigargin (Tg) for different time and the cell lysates were prepared and analyzed by Western-blot.


( B ) oligomer of recombinant FAM134B WT using Native PAGE .

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

(B) Analysis the oligomer size of recombinant FAM134B WT using Native PAGE.


( C ) In vitro liposome fragmentation . After injection of recombinant ( 100μg / 100μl ) into chamber ( 500μl ) , morphological changes of liposomes were monitored by live imaging for 20 min . at different time points as indicated are presented . Scale bars , 10 μm . " ns " means no significance , one - way ANOVA , error bars indicate ( n = 3 ) .

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

(C) In vitro liposome fragmentation assay. After the injection of recombinant proteins (100μg/100μl) into the chamber (500μl), the morphological changes of liposomes were monitored by live imaging for 20 min. The images at different time points as indicated are presented. Scale bars, 10 μm. "ns" means no significance, one-way ANOVA, error bars indicate SEM (n=3).


( D ) of time between addition liposome fragmentation . * * * P < 0 . 001 , one - way ANOVA , error bars indicate ( n = 3 ) .

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

(D) Quantification of the time between protein addition and liposome fragmentation. ***P<0.001, one-way ANOVA, error bars indicate SEM (n=3).


( E , F ) Measurement of intracellular ER - scission activity . U2OS transiently expressing EGFP - FAM134B ( WT ) , EGFP - FAM134B ( 84 - 233 ) or EGFP - FAM134B ( Δ84 - 233 ) at same , lysosomal degradation of EGFP - FAM134B was blocked by Bafilomycin A1 ( Baf A1 ) or DMSO . GFP - positive were quantified for each in ( F ) . For Each group , at least 30 were counted . Scale bars , 10 μm . * * * P < 0 . 001 , " ns " means no significance , one - way ANOVA , error bars indicate .

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

(E, F) Measurement of the intracellular ER-scission activity. U2OS cells transiently expressing EGFP-FAM134B (WT), EGFP-FAM134B (84-233) or EGFP-FAM134B (Δ84-233) at same levels, lysosomal degradation of EGFP-FAM134B was blocked by Bafilomycin A1 (Baf A1) or DMSO. GFP-positive puncta were quantified for each cell in (F). For Each group, at least 30 cells were counted. Scale bars, 10 μm. ***P<0.001, "ns" means no significance, one-way ANOVA, error bars indicate SEM.


( G , H ) Measurement of ER - phagy activity . U2OS transiently expressing mCherry - EGFP - FAM134B ( WT ) , mCherry - EGFP - FAM134B ( 84 - 233 ) or mCherry - EGFP - FAM134B ( Δ84 - 233 ) at same . ER - phagy is indicated by mCherry - positive but EGFP - negative were quantified for each in ( H ) . For mCherry - EGFP - FAM134B ( WT ) , 50 were counted ( n = 50 ) ; for mCherry - EGFP - FAM134B ( 84 - 233 ) , n = 56 ; for mCherry - EGFP - FAM134B ( Δ84 - 233 ) , n = 66 . Scale bars , 10 μm . scale bars in magnification boxes are 2μm . * * * P < 0 . 001 , one - way ANOVA , error bars indicate .

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

(G, H) Measurement of the ER-phagy activity. U2OS cells transiently expressing mCherry-EGFP-FAM134B (WT), mCherry-EGFP-FAM134B (84-233) or mCherry-EGFP-FAM134B (Δ84-233) at same levels. ER-phagy is indicated by mCherry-positive but EGFP-negative puncta were quantified for each cell in (H). For mCherry-EGFP-FAM134B (WT), 50 cells were counted (n=50); for mCherry-EGFP-FAM134B (84-233), n=56; for mCherry-EGFP-FAM134B (Δ84-233), n=66. Scale bars, 10 μm. The scale bars in the magnification boxes are 2μm. ***P<0.001, one-way ANOVA, error bars indicate SEM.


( I ) Lysosomal cleavage of GFP was analyzed by Western blot for co - expressing GFP - SEC61B FAM134B WT - Flag , FAM134B ( 84 - 233 ) - Flag or FAM134B ( Δ84 - 233 ) - Flag .

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(I) Lysosomal cleavage of GFP was analyzed by Western blot for the cells co-expressing GFP-SEC61B and FAM134B WT-Flag, FAM134B (84-233)-Flag or FAM134B (Δ84-233)-Flag.


( A ) Mass spectrometry uncovered that FAM134B was phosphorylated at serine 151 .

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(A) Mass spectrometry analysis uncovered that FAM134B was phosphorylated at serine 151.


( B ) Comparison of self - interaction of FAM134B mono - phosphorylation mutants using co - IP .

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(B) Comparison of the self-interaction of FAM134B mono-phosphorylation mutants using co-IP.


( C ) Comparison of self - interaction of FAM134 tri - phosphorylation mutants using co - IP .

(C) Comparison of the self-interaction of FAM134 tri-phosphorylation mutants using co-IP.


( D ) Comparison of scission activity using in vitro liposome fragmentation . Scale bars , 10 μm . * * * P < 0 . 001 , one - way ANOVA , error bars indicate ( n ≧ 3 ) .

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

(D) Comparison of membrane scission activity using in vitro liposome fragmentation assay. Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM (n≧3).


( E ) Lysosomal cleavage of GFP was analyzed by Western blot for co - expressing GFP - SEC61B FAM134B ( WT ) - Flag , FAM134B ( SA ) - Flag or FAM134B ( SD ) - Flag .

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(E) Lysosomal cleavage of GFP was analyzed by Western blot for the cells co-expressing GFP-SEC61B and FAM134B (WT)-Flag, FAM134B (SA)-Flag or FAM134B (SD)-Flag.


( F , G ) Measurement of intracellular ER - scission activity . FAM134B knockout ( KO ) U2OS was engineered to express EGFP - FAM134B ( WT ) , EGFP - FAM134B ( SA ) or EGFP - FAM134B ( SD ) at endogenous , lysosomal degradation of EGFP - FAM134B was blocked by Bafilomycin A1 ( Baf A1 ) . GFP - positive were quantified for each in ( G ) . For control , 25 were counted ( n = 25 ) ; For EGFP - FAM134B ( WT ) , n = 27 ; for EGFP - FAM134B ( SA ) , n = 29 ; for EGFP - FAM134B ( SD ) , n = 25 . Scale bars , 10 μm . * * * P < 0 . 001 , one - way ANOVA , error bars indicate .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O 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, G) Measurement of the intracellular ER-scission activity. FAM134B knockout (KO) U2OS cells was engineered to express EGFP-FAM134B (WT), EGFP-FAM134B (SA) or EGFP-FAM134B (SD) at endogenous levels, lysosomal degradation of EGFP-FAM134B was blocked by Bafilomycin A1 (Baf A1). GFP-positive puncta were quantified for each cell in (G). For control, 25 cells were counted (n=25); For EGFP-FAM134B (WT), n=27; for EGFP-FAM134B (SA), n=29; for EGFP-FAM134B (SD), n=25. Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM.


( H , I ) Measurement of ER - phagy activity . FAM134B knockout ( KO ) U2OS was engineered to express mCherry - EGFP - FAM134B ( WT ) , mCherry - EGFP - FAM134B ( S151A ) or mCherry - EGFP - FAM134B ( S151D ) at endogenous . Lysosomal mCherry - positive but GFP - negative were quantified for each in ( I ) . For mCherry - EGFP - FAM134B ( WT ) , 25 were counted ( n = 25 ) ; for mCherry - EGFP - FAM134B ( SA ) , n = 23 ; for mCherry - EGFP - FAM134B ( SD ) , n = 21 . Scale bars , 10 μm . scale bars in magnification boxes are 2 μm . * * * P < 0 . 001 , one - way ANOVA , error bars indicate .

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

(H, I) Measurement of the ER-phagy activity. FAM134B knockout (KO) U2OS cells was engineered to express mCherry-EGFP-FAM134B (WT), mCherry-EGFP-FAM134B (S151A) or mCherry-EGFP-FAM134B (S151D) at endogenous levels. Lysosomal mCherry-positive but GFP-negative puncta were quantified for each cell in (I). For mCherry-EGFP-FAM134B (WT), 25 cells were counted (n=25); for mCherry-EGFP-FAM134B (SA), n=23; for mCherry-EGFP-FAM134B (SD), n=21. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM.


( A , B ) Endogenous CAMK2B redistribution to ER structures labeled by mCherry - FAM134B BAP31 upon Tg treatment . Scale bars , 10 μm . scale bars in magnification boxes are 2 μm . colocalization was analyzed by Pearson ' s correlation coefficient ( PCC ) in ( B ) . For Ctrl , 16 were counted ( n = 16 ) ; for Tg treatment , 18 were counted ( n = 18 ) . * * * P < 0 . 001 , one - way ANOVA , error bars indicate .

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

(A, B) Endogenous CAMK2B redistribution to ER membrane structures labeled by mCherry-FAM134B and BAP31 upon Tg treatment. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. The colocalization was analyzed by Pearson's correlation coefficient (PCC) in (B). For Ctrl, 16 cells were counted(n=16); for Tg treatment, 18 cells were counted (n=18). ***P<0.001, one-way ANOVA, error bars indicate SEM.


( C ) In vitro FAM134B S151 phosphorylation by CAMK2B was detected by Western blot . Recombinant for FAM134BWT FAM134BS151A were purified from E . coil were incubated with purified CAMK2B by IP in kinase buffer . FAM134B phosphorylation was analyzed by a specific antibody recognizing phos - Serine151 of human FAM134B .

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(C) In vitro FAM134B S151 phosphorylation by CAMK2B was detected by Western blot. Recombinant proteins for FAM134BWT and FAM134BS151A were purified from E. coil were incubated with purified CAMK2B by IP in kinase buffer. FAM134B phosphorylation was analyzed by a specific antibody recognizing phos-Serine151 of human FAM134B.


( D ) FAM134B S151 phosphorylation in treated with CAMK2 activator ( 100nM EB1089 for 1h ) or / inhibitor ( 10μM KN93 for 2h ) . 293T or SKN - SH ( a derived from neuroblastoma ) were treated with drugs as indicated , whole lysates were analyzed by phospho - FAM134B ( S151 ) antibody .

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(D) FAM134B S151 phosphorylation in cells treated with CAMK2 activator (100nM EB1089 for 1h) or/and inhibitor (10μM KN93 for 2h). 293T or SKN-SH (a cell line derived from neuroblastoma) cells were treated with drugs as indicated, whole cell lysates were analyzed by phospho-FAM134B (S151) antibody.


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(E) FAM134B S151 phosphorylation in CAMK2B knockdown 293T cells.


( F ) In vitro reconstitution of CAMK2B - FAM134B - mediated fragmentation using liposome . Purified recombinant FAM134B CAMK2B were preincubated in kinase buffer with ADP or ATP at 30 ° C for 10min , resultant mixtures were transferred to chamber coated with liposomes . Liposome fragmentation was monitored by live imaging . Scale bars , 10 μm . * * * P < 0 . 001 , one - way ANOVA , error bars indicate ( n = 3 ) .

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

(F) In vitro reconstitution of CAMK2B-FAM134B-mediated membrane fragmentation using liposome assay. Purified recombinant FAM134B and CAMK2B were preincubated in kinase buffer with ADP or ATP at 30°C for 10min, the resultant protein mixtures were transferred to chamber coated with liposomes. Liposome fragmentation was monitored by live imaging. Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM (n=3).


( G , H ) Measurement of ER - phagy activity . U2OS transiently expressing mCherry - EGFP - FAM134B ( WT ) were treated with compounds as indicated . Lysosomal mCherry - positive but GFP - negative were quantified for each in ( H ) . For vehicle control , 53 were counted ( n = 53 ) , for Ionomycin , n = 56 ; for EB1089 , n = 54 ; for KN - 93 , n = 58 . Scale bars , 10 μm . scale bars in magnification boxes are 2 μm . * * * P < 0 . 001 , one - way ANOVA , error bars indicate .

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

(G, H) Measurement of the ER-phagy activity. U2OS cells transiently expressing mCherry-EGFP-FAM134B (WT) were treated with compounds as indicated. Lysosomal mCherry-positive but GFP-negative puncta were quantified for each cell in (H). For vehicle control, 53 cells were counted (n=53), for Ionomycin, n=56; for EB1089, n=54; for KN-93, n=58. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM.


( I ) FAM134B WT or knockout ( KO ) U2OS were treated with CAMK2 activator Ionomycin ( Iono ) , EB1089 or inhibitor KN93 for 24 hours , Western - blot was performed to analyze as indicated .

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(I) FAM134B WT or knockout (KO) U2OS cells were treated with CAMK2 activator Ionomycin (Iono), EB1089 or inhibitor KN93 for 24 hours, Western-blot was performed to analyze the proteins as indicated.


( J ) CAMK2B WT or knockdown ( KD ) U2OS were treated with 1 μM Thapsigargin ( Tg ) to induce ER stress for different time as indicated . were collected analyzed for as indicated by Western blot .

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(J) CAMK2B WT or knockdown (KD) U2OS cells were treated with 1 μM Thapsigargin (Tg) to induce ER stress for different time as indicated. Cells were collected and analyzed for proteins as indicated by Western blot.


( K ) GFP - cleavage . CAMK2B WT or knockdown ( KD ) U2OS were co - transfected with FAM134B - Flag GFP - SEC61B , 24 hours post transfection , were treated with drugs as indicated analyzed for GFP - cleavage by Western blot . Note : of Flag - FAM134B , CAMK2B , GFP - SEC61B Tubulin were analyzed by Western blot using different membranes .

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(K) GFP-cleavage assay. CAMK2B WT or knockdown (KD) U2OS cells were co-transfected with FAM134B-Flag and GFP-SEC61B, 24 hours post transfection, cells were treated with drugs as indicated and analyzed for GFP-cleavage by Western blot. Note: the levels of Flag-FAM134B, CAMK2B, GFP-SEC61B and Tubulin were analyzed by Western blot using different membranes.


( A ) Comparison of self - interaction of FAM134B WT with mutants as indicated using co - IP . SA - G216R was constructed by simultaneously mutating S149 , S151 S153 to alanine ( SA ) on basis of FAM134B - G216R .

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

(A) Comparison of the self-interaction of FAM134B WT with mutants as indicated using co-IP. SA-G216R was constructed by simultaneously mutating S149, S151 and S153 to alanine (SA) on the basis of FAM134B-G216R.


( B ) In vitro liposome fragmentation . Scale bars , 10 μm . * * * P < 0 . 001 , one - way ANOVA , error bars indicate ( n ≧ 3 ) .

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

(B) In vitro liposome fragmentation assay. Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM (n≧3).


( C , D ) Measurement of intracellular ER - scission activity of FAM134B WT , G216R , S151A - G216R SA - G216R . GFP - tagged FAM134B were expressed in U2OS at same . autolyosomal degradation of ER fragments labelled by GFP - FAM134B were blocked by Baf A1 or DMSO . GFP - positive were quantified for each in ( D ) . For of groups , at least 30 were included for ; Scale bars , 10 μm . * * * P < 0 . 001 , one - way ANOVA , error bars indicate .

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

(C, D) Measurement of the intracellular ER-scission activity of FAM134B WT, G216R, S151A-G216R and SA-G216R. GFP-tagged FAM134B proteins were expressed in U2OS cells at same levels. The autolyosomal degradation of ER fragments labelled by GFP-FAM134B were blocked by Baf A1 or DMSO. GFP-positive puncta were quantified for each cell in (D). For all of the groups, at least 30 cells were included for quantification; Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM.


( E , F ) Measurement of ER - phagy activity of FAM134BG216R . U2OS transiently expressing mCherry - EGFP - FAM134B ( WT ) , mCherry - EGFP - FAM134B ( G216R ) , mCherry - EGFP - FAM134B ( S151A - G216R ) or mCherry - EGFP - FAM134B ( SA - G216R ) at same . Lysosomal mCherry - positive but GFP - negative were quantified for each in ( F ) . For WT , 50 were counted ( n = 50 ) ; for G216R , n = 55 ; for S151A - G216R , n = 51 ; for SA - G216R , n = 52 . Scale bars , 10 μm . scale bars in magnification boxes are 2 μm . * P < 0 . 05 , * * * P < 0 . 001 , one - way ANOVA , error bars indicate .

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(E, F) Measurement of the ER-phagy activity of FAM134BG216R. U2OS cells transiently expressing mCherry-EGFP-FAM134B (WT), mCherry-EGFP-FAM134B (G216R), mCherry-EGFP-FAM134B (S151A-G216R) or mCherry-EGFP-FAM134B (SA-G216R) at same levels. Lysosomal mCherry-positive but GFP-negative puncta were quantified for each cell in (F). For WT, 50 cells were counted (n=50); for G216R, n=55; for S151A-G216R, n=51; for SA-G216R, n=52. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. *P<0.05, ***P<0.001, one-way ANOVA, error bars indicate SEM.


( A , A ' ) Specific expression of FAM134B mRNA ( green ) in sensory neurons ( red ) of P4d vGlut2Cre ; R26lsl - tdTommouse . Scale bars , 100μm . dotted indicates dorsal root ganglia ( DRG ) where sensory neurons are genetically labeled with Tomato . Insets of ( a ) ( a ' ) are high magnifications of sensory neurons showing co - localization of FAM134B mRNA Tomato . Scale bars , 10 μm .

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(A,A') Specific expression of FAM134B mRNA (green) in sensory neurons (red) of P4d vGlut2Cre;R26lsl-tdTommouse. Scale bars, 100μm. The white dotted line indicates the dorsal root ganglia (DRG) where sensory neurons are genetically labeled with Tomato. Insets of (a) and (a') are high magnifications of sensory neurons showing the co-localization of FAM134B mRNA and Tomato. Scale bars, 10 μm.


Cultured sensory neurons from DRGs of E14 . 5 vGlut2Cre ; R26lsl - tdTom were infected with same titers of recombinant lentivirus expressing GFP - FAM134B WT ( B ) , G216R ( C ) , S151A - G216R ( D ) , SA - G216R ( E ) , G216R - ΔLIR ( F ) respectively . Infected sensory neurons ( GFP + / Tom + ) are indicated with arrowheads . nuclei of are shown with DAPI . Scale bars , 20 μm .

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

Cultured sensory neurons from DRGs of E14.5 vGlut2Cre;R26lsl-tdTom embryos were infected with same titers of recombinant lentivirus expressing GFP-FAM134B WT (B), G216R(C), S151A-G216R(D), SA-G216R(E), G216R-ΔLIR (F) respectively. Infected sensory neurons (GFP+/Tom+) are indicated with arrowheads. The nuclei of all cells are shown with DAPI staining. Scale bars, 20 μm.


Cultured sensory neurons from DRGs of E14 . 5 vGlut2Cre ; R26lsl - tdTom were infected with same titers of recombinant lentivirus expressing G216R supplemented with 5μM KN93 ( G ) Infected sensory neurons ( GFP + / Tom + ) are indicated with arrowheads . nuclei of are shown with DAPI . Scale bars , 20 μm .

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

Cultured sensory neurons from DRGs of E14.5 vGlut2Cre;R26lsl-tdTom embryos were infected with same titers of recombinant lentivirus expressing G216R supplemented with 5μM KN93 (G) Infected sensory neurons (GFP+/Tom+) are indicated with arrowheads. The nuclei of all cells are shown with DAPI staining. Scale bars, 20 μm.


( H ) of viability of sensory neuron after 48h , 72h 96h of infection . Sensory neuron viability was calculated as of transfected sensory neuron ( GFP + / Tom + ) in total sensory neurons ( Tom + ) . For each time point , four biological replicates were included for statistical . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , two - way ANOVA , error bars indicate . " ns " is abbreviation for " not significant " . For of groups , at least 10 fields of slides were included .

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

(H) Quantification of the viability of sensory neuron after 48h, 72h and 96h of infection. Sensory neuron viability was calculated as the percentage of transfected sensory neuron (GFP+/Tom+) in total sensory neurons (Tom+). For each time point, four biological replicates were included for statistical analysis. *P<0.05, **P<0.01, ***P<0.001, two-way ANOVA, error bars indicate SEM. "ns" is the abbreviation for "not significant". For all of the groups, at least 10 fields of the slides were included.


Entry of MLV pseudotyped with SARS - CoV - 2 S , SARS - CoV - 2 Sfur / mut SARS - CoV S in VeroE6 .

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

Entry of MLV pseudotyped with SARS-CoV-2 S, SARS-CoV-2 Sfur/mut and SARS-CoV S in VeroE6 cells.


Entry of MLV pseudotyped with SARS - CoV - 2 S or SARS - CoV - 2 Sfur / mut in BHK transiently transfected with hACE2 . experiments were carried out in triplicate with two independent pseudovirus preparations a representative experiment is shown .

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

Entry of MLV pseudotyped with SARS-CoV-2 S or SARS-CoV-2 Sfur/mut in BHK cells transiently transfected with hACE2. The experiments were carried out in triplicate with two independent pseudovirus preparations and a representative experiment is shown.


. Western blot of SARS - CoV - 2 S - MLV , SARS - CoV - 2 Sfur / mut - MLV SARS - CoV S - MLV pseudovirions using an anti - SARS - CoV S2 antibody .

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

. Western blot analysis of SARS-CoV-2 S-MLV, SARS-CoV-2 Sfur/mut-MLV and SARS-CoV S-MLV pseudovirions using an anti-SARS-CoV S2 antibody.


Biolayer interferometry binding of hACE2 ectodomain to immobilized SARS - CoV - 2 SB ( B ) or SARS - CoV

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

Biolayer interferometry binding analysis of the hACE2 ectodomain to immobilized SARS-CoV-2 SB (B) or SARS-CoV SB


Two orthogonal views of closed SARS - CoV - 2 S trimer cryoEM map . C . Atomic model of closed SARS - CoV - 2 S trimer in same orientation as in panel A .

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

Two orthogonal views of the closed SARS-CoV-2 S trimer cryoEM map. C. Atomic model of the closed SARS-CoV-2 S trimer in the same orientation as in panel A.


D - E . Two orthogonal views of partially open SARS - CoV - 2 S trimer cryoEM map ( one SB domain is open ) . F . Atomic model of closed SARS - CoV - 2 S trimer in same orientation as in panel D . glycans were omitted for clarity .

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

D-E. Two orthogonal views of the partially open SARS-CoV-2 S trimer cryoEM map (one SB domain is open). F. Atomic model of the closed SARS-CoV-2 S trimer in the same orientation as in panel D. The glycans were omitted for clarity.


Entry of SARS - CoV - 2 SARS - CoV S pseudotyped is potently inhibited by four SARS - CoV S mouse polyclonal immune plasma .

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

Entry of SARS-CoV-2 and SARS-CoV S pseudotyped viruses is potently inhibited by four SARS-CoV S mouse polyclonal immune plasma.


( A ) Western blot showing expression of ASCL1 / or NEUROD1 in NCI - H69 , NCI - H82 , NCI - H146 , NCI - H510A , NCI - H526 , SHP - 77 DMS - 53 SCLC ( lanes 4 - 10 ) ; A549 NCI - H460 NSCLC a IMR - 90 HFL lines ( lanes 1 - 3 ) . Tubulin ( α - Tub ) is shown as loading control . Each blot is representative from at least three independent experiments .

(A) Western blot showing the protein expression of ASCL1 and/or NEUROD1 in NCI-H69, NCI-H82, NCI-H146, NCI-H510A, NCI-H526, SHP-77 and DMS-53 SCLC cells (lanes 4-10); A549 and NCI-H460 NSCLC cells and a IMR-90 HFL cell lines (lanes 1-3). Tubulin (α-Tub) is shown as loading control. Each blot is representative from at least three independent experiments.


( A - E ) ChIP - seq experiments indicates localization of ( A ) ASCL1 , ( B ) NEUROD1 ( E ) RNAPII . ATAC - seq ( C ) ( D ) presence of H3K27Ac mark indicate accessibility around TSS at t = 0 ( blue ) t = 4h ( green ) after lurbinectedin treatment ; - 2 . 0 + 2 . 0 kb to TSS coordinates of hg19 genes .

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

(A-E) ChIP-seq experiments indicates the localization of (A) ASCL1, (B) NEUROD1 and (E) RNAPII. ATAC-seq (C) and (D) the presence of H3K27Ac mark indicate the chromatin accessibility around the TSS at t=0 (blue) and t=4h (green) after lurbinectedin treatment; -2.0 and +2.0 kb to TSS coordinates of all hg19 genes.


( A - D ) Chip - seq genome track of ASCL1 / NEUROD1 , RNAPII , H3K27Ac on ASCL1 , BCL2 ; INSM1 MYB ( hg19 ) , in untreated ( blue ) in lurbinectedin treated ( green ) conditions respectively ; to be noticed location of CGG rich motifs ( lower part of each panel ) which parallel presence of lurbinectedin ( red ) .

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

(A-D) Chip-seq genome track of ASCL1/NEUROD1, RNAPII, and H3K27Ac on ASCL1, BCL2; INSM1 and MYB (hg19), in untreated (dark blue) and in lurbinectedin treated (green) conditions respectively; to be noticed the location of CGG rich motifs (lower part of each panel) which parallel the presence of lurbinectedin (red).


( a ) Western blot showing RNAPII across panel of ; A549 NCI - H460 NSCLC ; IMR - 90 HFL ; NCI - H146 , NCI - H82 , DMS - 53 , NCI - H510A , NCI - H526 SHP - 77 SCLC before ( - ) after ( + ) treatment with lurbinectedin . α - Tubulin ( α - Tub ) is shown as loading control ; lower panel , Histogram showing average of RNAPII before ( blue ) after ( Blue ) lurbinectedin treatment in arbitrary units ( a . u . ) normalized against α - Tub . Data is presented as Mean ± as determined by Two Way RM ANOVA Šídák ' s multiple comparisons ( n = 3 biological replicas ) . * * * * P ≤ 0 . 0001 .

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

(a) Western blot showing RNAPII across the panel of; A549 and NCI-H460 NSCLC cells; IMR-90 HFL cells; NCI-H146, NCI-H82, DMS-53, NCI-H510A, NCI-H526 and SHP-77 SCLC cells before (-) and after (+) treatment with lurbinectedin. α-Tubulin (α-Tub) is shown as loading control; lower panel, Histogram showing the average of RNAPII before (dark blue) and after (light Blue) lurbinectedin treatment in arbitrary units (a.u.) normalized against α-Tub. Data is presented as Mean ± SEM as determined by Two Way RM ANOVA and Šídák's multiple comparisons test (n=3 biological replicas). ****P ≤0.0001.


( B Immunofluorescence of RNAPII ( red ) before ( Ctrl ) after lurbinectedin treatment . DAPI ( blue ) was used to stain nuclei ; scale bar is 65 μm .

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

(B Immunofluorescence of RNAPII (red) before (Ctrl) and after lurbinectedin treatment. DAPI (blue) was used to stain nuclei; scale bar is 65 μm.


( D ) Affinity purification of Bio - lur fractions containing elongating RNAPII Ser2 ( lane 4 ) . Input ( lanes 1 - 2 ) represents 10 % of starting material . Respective molecular weights are shown on right ( kDa ) .

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

(D) Affinity purification of Bio-lur fractions containing elongating RNAPII Ser2 (lane 4). Input (lanes 1-2) represents 10% of the starting material. Respective molecular weights are shown on the right (kDa).


( E ) Upper panel : GST - pulldown of ubiquitinated showing hypo - ( IIA ) , - hyper ( II0 ) phosphorylated RNAPII ( lanes 4 - 5 upper panel ) Ser2 Phosphorylated RNAPII ( Ser2P ) ( lanes 4 - 5 lower panel ) after lurbinectedin treatment . Empty beads ( Mock ) ( lane 3 ) as a negative control ; Lower Panel : GST - pulldown of ubiquitinated Ser2P ) ( lanes 4 - 5 ) . Representative smear of successful ubiquitinated pulldown ( Lanes 4 5 ) as compared to input ( lanes 1 2 ) . Empty beads ( Mock ) ( lane 3 ) as a negative control . Each Western blot is representative of three independent experiments .

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

(E) Upper panel: GST-pulldown of ubiquitinated proteins showing hypo- (IIA), -hyper (II0) phosphorylated RNAPII (lanes 4-5 upper panel) and Ser2 Phosphorylated RNAPII (Ser2P) (lanes 4-5 lower panel) after lurbinectedin treatment. Empty beads (Mock) (lane 3) as a negative control; Lower Panel: GST-pulldown of ubiquitinated proteins Ser2P) (lanes 4-5). Representative smear of successful ubiquitinated proteins pulldown (Lanes 4 and 5) as compared to input (lanes 1 and 2). Empty beads (Mock) (lane 3) as a negative control. Each Western blot is representative of three independent experiments.


A ) GFP tagged Syb expressed in Hh - receiving co - localizes with endogenous immuno - labelled Ptc along apico - basal axis ( 3D reconstruction ) . Vesicles containing Syb Ptc are visualized at lateral basal planes ( arrows ) , can be detected along cytonemes marked with Syb - GFP ( arrowhead ) .

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

A) GFP tagged Syb protein expressed in Hh-receiving cells co-localizes with endogenous immuno-labelled Ptc along the apico-basal axis (3D reconstruction). Vesicles containing Syb and Ptc are visualized at lateral and basal planes (arrows), and can be detected along cytonemes marked with Syb-GFP (arrowhead).


B ) Expression of GFP tagged Syt1 also co - localizes with inmmuno - labelled endogenous Ptc at vesicles ( lateral plane , arrows point at vesicles with co - localization ) decorates cytoneme membranes ( basal plane , arrowheads indicate cytonemes ) .

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

B) Expression of the GFP tagged Syt1 also co-localizes with inmmuno-labelled endogenous Ptc at vesicles (lateral plane, arrows point at vesicles with co-localization) and decorates the cytoneme membranes (basal plane, arrowheads indicate cytonemes).


C ) GRASP experiment shows exocytic events in Hh - receiving at basal contacts . Hh - receiving express GFP barrel 1 - 10 tagged to Syb Hh - producing express complementary GFP barrel 11 tagged to CD4 . Note discrete ( arrows ) along Hh producing cytonemes ( stabilized with Ihog - RFP ) crossing reception .

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

C) GRASP experiment shows exocytic events in Hh-receiving cells at basal membrane contacts. Hh-receiving cells express the GFP barrel 1-10 tagged to Syb and Hh-producing cells express the complementary GFP barrel 11 tagged to the membrane protein CD4. Note the fluorescent discrete punctae (arrows) along Hh producing cells cytonemes (stabilized with Ihog-RFP) crossing the reception area.


D ) Expression in receiving of Syb functional partner Syx1A tagged to GFP . Note a strong localization in clusters close to basal in 3D reconstruction ( left panel ) in confocal lateral basal planes , including cytonemes ( right panels , arrows arrowhead indicate vesicle - like structures along cytoenemes ) .

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

D) Expression in receiving cells of the Syb functional partner Syx1A tagged to GFP. Note a strong localization in clusters close to the basal membrane in the 3D reconstruction (left panel) and in confocal lateral and basal planes, including cytonemes (right panels, arrows and arrowhead indicate vesicle-like structures along cytoenemes).


A ) Ptc distribution in a wing disc ( 3D reconstitution ) under down - regulation of SNARE Syb Syt1 in dorsal compartment ( D ) , keeping ventral compartment ( V ) as a WT internal control . Note basal accumulation of endogenous Ptc ( arrowheads ) when knocking down Syb or Syt1 .

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

A) Ptc protein distribution in a wing disc (3D reconstitution) under down-regulation of the SNARE proteins Syb and Syt1 in the dorsal compartment (D), keeping the ventral compartment (V) as a WT internal control. Note the basal accumulation of endogenous Ptc (arrowheads) when knocking down Syb or Syt1.


B ) Confocal of immuno - labelled endogenous Ptc cytonemes stabilized with Ihog - RFP protruding from Hh producing in wild type receiving ( left panel ) , or B ' ) when either blocking exocytosis by down - regulating Syb ( middle panel ) or B ' ' ) blocking endocytosis by expressing a dominant negative form of Drosophila Dynamin , Shibire ( right panel ) in Hh - receiving . Endogenous Ptc in wild type conditions cannot be visualized due to its rapid internalization processing after Hh reception ; while blocking exocytosis causes an accumulation of basal Ptc in intracellular ( arrowheads ) , endocytosis inhibition leads to Ptc accumulation at plasma ( arrows ) .

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

B) Confocal images of immuno-labelled endogenous Ptc and cytonemes stabilized with Ihog-RFP protruding from Hh producing cells in wild type receiving cells (left panel), or B') when either blocking exocytosis by down-regulating Syb (middle panel) or B'') blocking endocytosis by expressing a dominant negative form of the Drosophila Dynamin, Shibire (right panel) in the Hh-receiving cells. Endogenous Ptc in wild type conditions cannot be visualized due to its rapid internalization and processing after Hh reception; while blocking exocytosis causes an accumulation of basal Ptc in intracellular punctae (arrowheads), and endocytosis inhibition leads to Ptc accumulation at the plasma membrane (arrows).


C ) Confocal apical ( left ) basal ( right ) of a shits mutant wing disc expressing Syb RNAi dorsally ( D ) to also block exocytosis , keeping ventral compartment ( V ) as an internal control where just endocytosis is inhibited . After dorsal Syb RNAi induction , endocytosis was inhibited in whole shits disc by incubation at restrictive . Note dorsal reduction of Ptc at plasma after blocking exocytosis .

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

C) Confocal apical (left) and basal (right) images of a shits mutant wing disc expressing Syb RNAi dorsally (D) to also block exocytosis, keeping the ventral compartment (V) as an internal control where just endocytosis is inhibited. After dorsal Syb RNAi induction, endocytosis was inhibited in the whole shits disc by incubation at restrictive temperature. Note the dorsal reduction of Ptc at plasma membrane after blocking exocytosis.


D ) of Ptc - GFP ( Yac construct ) along wing disc apico - basal axis , integrating both signal ( Integrated Density ) . graph on left shows values for wild type situations in purple ( average in blue ) , while Syb RNAi treatment values are shown in green ( average in orange ) . a clear shift of higher values towards basal side of discs after inhibition of Syb function . graph to right shows correlation coefficients between maximum Ptc value from basal side ( - 1 = basal , + 1 = apical ) of discs after RNAi expression for different SNARE wild type . Central horizontal lines show median values of N = 7 - 16 , box shows lower upper quartiles whiskers show maximum minimum excluding outliers . A basal association is particularly noticeable for Syb , Syx1A α - Snap down - regulation . Right panel is a scheme illustrating wild type RNAi - treated patterns of quantified Ptc - GFP distribution along wing disc apico - basal axis .

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

D) Quantification of Ptc-GFP levels (Yac construct) along the wing disc apico-basal axis, integrating both fluorescence intensity and signal area (Integrated Density). The graph on the left shows values for wild type situations in purple (average in blue), while Syb RNAi treatment values are shown in green (average in orange). a clear shift of higher values towards the basal side of the discs after inhibition of Syb function. The graph to the right shows correlation coefficients between the maximum Ptc value and distance from the basal side (-1=basal, +1=apical) of discs after RNAi expression for different SNARE proteins and the wild type. Central horizontal lines show median values of N=7-16, box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers. A basal association is particularly noticeable for Syb, Syx1A and α-Snap down-regulation. Right panel is a scheme illustrating the wild type and RNAi-treated patterns of quantified Ptc-GFP levels distribution along the wing disc apico-basal axis.


A ) Electron Microscopy imaging of wing discs expressing UAS - Ptc - GFP construct for 24 hours in receiving using Ptc - Gal4 ; TubGal80ts . Left panel shows areas with anti - GFP immuno - labelling ( marked in blue ) throughout apico / basal axis of wing disc epithelium a ) anti - GFP gold - labelling corresponding to Ptc is located on apical early endosomes towards MVB formation . b ) Ptc immuno - gold labelling is present in multivesicular bodies ( MVBs ) subapically ( top right panels ) . b1 ) subapical MVBs appear as less dense with greater Ptc labelling on MVB outer membranes . c ) Ptc label is also present in MVBs close to basal ( lower right panels ) , c1 ) basolateral Ptc MVBs are significantly denser richer in Ptc positive intraluminal vesicles ( ILV ) .

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

A) Electron Microscopy imaging of wing discs expressing the UAS-Ptc-GFP construct for 24 hours in receiving cells using Ptc-Gal4; TubGal80ts. Left panel shows areas with anti-GFP immuno-labelling (marked in blue) throughout the apico/basal axis of the wing disc epithelium a) anti-GFP gold-labelling corresponding to Ptc is located on the apical membrane and early endosomes towards MVB formation. b) Ptc immuno-gold labelling is present in multivesicular bodies (MVBs) subapically (top right panels). b1) subapical MVBs appear as less dense with greater Ptc labelling on the MVB outer membranes. c) Ptc label is also present in MVBs close to the basal membrane (lower right panels), c1) basolateral Ptc MVBs are significantly denser and richer in Ptc positive intraluminal vesicles (ILV).


B ) Endogenous Ptc subcellular localization after endocytosis inhibition by dorsal expression of a dominant negative form of Rab5 . top panel is a schematic representation of localized expression . left panel is a digital 3D reconstruction right panel shows an apical basal confocal sections . that accumulation of Ptc occurs at both apical basal sides of dorsal part of disc , compared to wild type control on ventral side .

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B) Endogenous Ptc subcellular localization after endocytosis inhibition by the dorsal expression of a dominant negative form of Rab5. The top panel is a schematic representation of the localized expression. The left panel is a digital 3D reconstruction and the right panel shows an apical and basal confocal sections. that accumulation of Ptc occurs at both the apical and basal sides of the dorsal part of the disc, compared to the wild type control on the ventral side.


accumulation of Ptc occurs at both apical basal sides of dorsal part of disc , compared to wild type control on ventral side . C ) Digital 3D reconstruction ( left ) apical basal confocal sections ( right ) of endogenous Ptc localization after endocytosis inhibition by dorsal expression of a dominant negative form of shibire .

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accumulation of Ptc occurs at both the apical and basal sides of the dorsal part of the disc, compared to the wild type control on the ventral side. C) Digital 3D reconstruction (left) and apical and basal confocal sections (right) of endogenous Ptc localization after endocytosis inhibition by the dorsal expression of a dominant negative form of shibire.


A ) 3D reconstructions of immuno - labelled endogenous Ptc of wing discs in wild type condition after RNAi expression for different ESCRT components . RNAis ' expression result in Ptc accumulation in large structures , located apically under Hrs ( ESCRT - 0 component ) inhibition , basally under inhibition of ESCRT components I ( Tsg101 ) , II ( Vps22 ) III ( Shrub ) . Graphs depicting signal values from apical to basal show a shift of higher curves either towards acute apical or basal accumulation .

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

A) 3D reconstructions of immuno-labelled endogenous Ptc of wing discs in wild type condition and after RNAi expression for different ESCRT complex components. All RNAis' expression result in Ptc accumulation in large structures, located apically under Hrs (ESCRT-0 component) inhibition, and basally under inhibition of the ESCRT components I (Tsg101), II (Vps22) and III (Shrub). Graphs depicting signal values from apical to basal show a shift of higher intensity curves either towards acute apical or basal accumulation.


B ) Detailed confocal imaging of immuno - labelled endogenous Ptc localization after clonal Tsg101 down - regulation . image shows abnormal accumulation of Ptc clusters in basal cytonemes emanating from receiving ( arrows ) , since in wild type conditions Ptc visualization at basal membranes is not possible due to its rapid internalization processing after Hh

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B) Detailed confocal imaging of immuno-labelled endogenous Ptc localization after clonal Tsg101 down-regulation. The image shows abnormal accumulation of Ptc clusters in basal cytonemes emanating from receiving cells (arrows), since in wild type conditions Ptc visualization at basal membranes is not possible due to its rapid internalization and processing after Hh reception


C ) Ptc accumulation in these punctated structures ( arrows ) is also visualized on receiving cytonemes stabilized with co - receptor Ihog - RFP after Tsg101 inhibition .

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C) Ptc accumulation in these punctated structures (arrows) is also visualized on receiving cytonemes stabilized with the co-receptor Ihog-RFP and after Tsg101 inhibition.


D ) 3D reconstruction of Hh producing cytoneme network stabilized by Ihog - RFP shows that Ptc accumulation after Tsg101 treatment localizes above signal sending cytoneme extensions ( arrow ) .

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D) 3D reconstruction of Hh producing cytoneme network stabilized by Ihog-RFP shows that Ptc accumulation after Tsg101 treatment localizes above the signal sending cytoneme extensions (arrow).


A ) Confocal image of wing disc expressing human tetraspanin CD63 - GFP expressed in Hh receiving colocalizing with Ptc ( Bac . Ptc - cherry ) in vesicle - like structures ( yellow arrows ) along basal cytonemes .

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A) Confocal image of wing disc expressing the human tetraspanin CD63-GFP expressed in the Hh receiving cells colocalizing with Ptc (Bac.Ptc-cherry) in vesicle-like structures (yellow arrows) along basal cytonemes.


B ) In vivo confocal frames of abdominal histoblasts showing colocalization of CD63 - Cherry Ptc ( Yac . Ptc - GFP ) at moving in anterograde direction along cytonemes ( arrowheads show trajectory in time - lapse frames ) .

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B) In vivo confocal frames of abdominal histoblasts showing colocalization of CD63-Cherry and Ptc (Yac.Ptc-GFP) at punctae moving in anterograde direction along cytonemes (white arrowheads show trajectory in time-lapse frames).


C ) Lateral confocal plane of wing disc showing endogenous Ptc colocalizing with Drosophila tetraspanin Tsp96F - RFP ( orthologous to mammalian tetraspanin CD9 / CD81 ) ( yellow arrows indicate co - localization in punctate structures ) .

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C) Lateral confocal plane of wing disc showing endogenous Ptc colocalizing with the Drosophila tetraspanin Tsp96F-RFP (orthologous to the mammalian tetraspanin CD9/CD81) (yellow arrows indicate co-localization in punctate structures).


D ) Basolateral in vivo imaging of abdominal histoblasts expressing Tsp96F - RFP in Hh receiving shows colocalization with Ptc - GFP at mobile ( yellow arrows ) .

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D) Basolateral in vivo imaging of abdominal histoblasts expressing Tsp96F-RFP in the Hh receiving cells shows colocalization with Ptc-GFP at mobile punctae (yellow arrows).


E ) Tsp96F - RFP expressed in wing disc Hh - receiving also shows localization in basal cytonemes stabilized by Ihog - CFP expression ( yellow arrows indicate Tsp96F - RFP - positive punctate structures ) .

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E) Tsp96F-RFP expressed in wing disc Hh-receiving cells also shows localization in basal cytonemes stabilized by Ihog-CFP expression (yellow arrows indicate Tsp96F-RFP-positive punctate structures).


F ) Frames showing in vivo localization of Tsp96 - RFP to cytonemes stabilized with Ihog - CFP expressed in abdominal histoblasts Hh - receiving , moving in an anterograde fashion towards P compartment ( arrowheads show trajectory in time - lapse frames ) .

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F) Frames showing in vivo localization of Tsp96-RFP punctae to cytonemes stabilized with Ihog-CFP expressed in the abdominal histoblasts Hh-receiving cells, moving in an anterograde fashion towards the P compartment (white arrowheads show trajectory in time-lapse frames).


G ) Confocal imaging of receiving expressing HA tagged Tsp96F construct also shows co - localization with endogenous Ptc at cytonemes ( yellow arrows indicate Tsp96F - HA Ptc co - localizing punctate structures ) .

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G) Confocal imaging of receiving cells expressing the HA tagged Tsp96F construct also shows co-localization with endogenous Ptc at cytonemes (yellow arrows indicate Tsp96F-HA and Ptc co-localizing punctate structures).


H ) Western blot against HA - tag of Immuno - Precipitated sample ( IP ) , Depleted Sample ( Dep ) , High Speed Sample ( HSS ) Whole Sample Control sample from a co - immunoprecipitation experiment isolating GFP of Tsp96F - HA Ptc - GFP expressing larvae or GFP Tsp96F - HA expressing larvae as negative control . Arrowhead indicates positive band corresponding to Tsp96F - HA expected molecular ( 30 - 40 kDa ) , present in samples including immuno - precipitated one not present in negative control sample .

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H) Western blot against HA-tag of Immuno-Precipitated sample (IP), Depleted Sample (Dep), High Speed Sample (HSS) Whole Sample and Control sample from a co-immunoprecipitation experiment isolating GFP of Tsp96F-HA and Ptc-GFP expressing larvae or GFP and Tsp96F-HA expressing larvae as negative control. Arrowhead indicates the positive band corresponding to Tsp96F-HA expected molecular weight (30-40 kDa), present in all samples including the immuno-precipitated one and not present in the negative control sample.


A ) 3D reconstruction of wing disc immuno - labelled for endogenous Ptc dorsally over - expressing Tsp96F - HA construct , keeping ventral side of disc in wild type condition as internal control . dorsal accumulation of Ptc in very large across apico - basal axis , but mainly at basal side of epithelium . On right , graphs represent values from apical to basal , comparing dorsal ventral sides showing a shift of highest values towards basal side after Tsp96 - HA expression .

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A) 3D reconstruction of wing disc immuno-labelled for endogenous Ptc and dorsally over-expressing the Tsp96F-HA construct, keeping the ventral side of the disc in wild type condition as internal control. the dorsal accumulation of Ptc in very large punctae across the apico-basal axis, but mainly at the basal side of the epithelium. On the right, graphs represent fluorescence values from apical to basal, comparing dorsal and ventral sides and showing a shift of the highest values towards the basal side after Tsp96-HA expression.


B ) Confocal image of a wing disc with a clone expressing Tsp96F - HA immuno - labelled with anti - Ptc anti - HA antibodies , which co - localize in large basolateral ( yellow arrows ) .

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B) Confocal image of a wing disc with a clone expressing Tsp96F-HA and immuno-labelled with anti-Ptc and anti-HA antibodies, which co-localize in the large basolateral punctae (yellow arrows).


C ) Electron microscopy image from immunolabelled ( anti - GFP ) Hh - receiving of a wing disc expressing Ptc - GFP Tsp96F - HA . detailed image shows large sac structures that accumulate several dense MVBs positive labelled for Ptc .

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C) Electron microscopy image from immunolabelled (anti-GFP) Hh-receiving cells of a wing disc expressing Ptc-GFP and Tsp96F-HA. The detailed image shows large sac structures that accumulate several dense MVBs positive labelled for Ptc.


A ) Ptc is mainly secreted in small EVs ( P100K ) from Cl8 .

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A) Ptc is mainly secreted in small EVs (P100K) from Cl8 cells.


B ) Large EVs ( lEVs ) , small EVs ( sEVs ) very small EVs ( vsEVs ) were analysed in triplicate by Western blotting . sEVs are enriched in Ptc in EV - associated Syntaxin1 ( Synt1A ) , Late bloomer ( Lbm ) Hsp70 .

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B) Large EVs (lEVs), small EVs (sEVs) and very small EVs (vsEVs) were analysed in triplicate by Western blotting. sEVs are enriched in Ptc and in the EV-associated proteins Syntaxin1 (Synt1A), Late bloomer (Lbm) and Hsp70.


C ) Fractionation of sEVs by Sucrose Density Gradient : Ptc partially co - fractioned with Synt1A , Lbm Hsp70 . Note that while unprocessed form of co - receptor Ihog does not co - fraction with Ptc , processed form does in same subpopulation .

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C) Fractionation of sEVs by Sucrose Density Gradient: Ptc partially co-fractioned with Synt1A, Lbm and Hsp70. Note that while unprocessed form of co-receptor Ihog does not co-fraction with Ptc, the processed form does in the same subpopulation.


D ) Fractionation of sEVs by Exclusion Chromatography : Ptc perfectly co - fractioned with Synt1A , Lbm Hsp70 . In agreement with density gradient , unprocessed form of co - receptor Ihog does not co - fraction with Ptc in same subpopulation of sEVs .

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D) Fractionation of sEVs by Size Exclusion Chromatography: Ptc perfectly co-fractioned with Synt1A, Lbm and Hsp70. In agreement with the density gradient analysis, unprocessed form of co-receptor Ihog does not co-fraction with Ptc in the same subpopulation of sEVs.


E ) Effect on EV - associated Ptc secretion after gene silencing on Cl8 by dsRNAs against SNARE Syb α - Snap , tetraspanin Tsp96F ESCRT components Hrs , Tsg101 , Shrub Vps4 . Hsp70 in EVs is detected by different exposition time .

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E) Effect on EV-associated Ptc secretion after gene silencing on Cl8 cells by dsRNAs against the SNARE proteins Syb and α-Snap, the tetraspanin Tsp96F and the ESCRT components Hrs, Tsg101, Shrub and Vps4. Hsp70 in cells and EVs is detected by different exposition time.


A ) Projection of three confocal sections ( 1 μm ) at basal side of wing discs expressing Bac constructs for Hh - GFP Ptc - cherry , used to analyse co - localization between ligand receptor in wild type conditions after inhibition of vesicle fusion ( Syb RNAi ) , vesicle trafficking ( Hrs RNAi ) or Ptc degradation ( Dor RNAi ) . Boxplot shows co - localization rates using Manders coefficient for proportion of red covered by green in projected for wing discs temporally expressing RNAi against ESCRT Hrs Vps4 , SNAREs Syb α - Snap , Dor as control for effects after Ptc degradation - blocking , as well as temporally over - expressing Tsp96F - HA . Central horizontal lines show median values of N = 8 - 12 box shows lower upper quartiles whiskers show maximum minimum excluding . Note significant reduction in co - localization rates ( 1 = total coverage of red by green ) after RNAi expression for both ESCRT SNARE as well as after Tsp96F - HA over - expression , while RNAi expression for Dor presents greater co - localization .

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A) Projection images of three confocal sections (1 μm) at the basal side of wing discs expressing the Bac constructs for Hh-GFP and Ptc-cherry, used to analyse co-localization between the ligand and the receptor in wild type conditions and after inhibition of vesicle fusion (Syb RNAi), vesicle trafficking (Hrs RNAi) or Ptc protein degradation (Dor RNAi). Boxplot shows co-localization rates using the Manders coefficient for proportion of red covered by green in projected images for wing discs temporally expressing RNAi against ESCRT proteins Hrs and Vps4, SNAREs Syb and α-Snap, and Dor as control for effects after Ptc degradation-blocking, as well as temporally over-expressing the Tsp96F-HA. Central horizontal lines show median values of N=8-12 box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding. Note the significant reduction in co-localization rates (1= total coverage of red by green) after RNAi expression for both ESCRT and SNARE proteins as well as after Tsp96F-HA over-expression, while RNAi expression for Dor presents greater co-localization.


B ) Projection of three confocal sections ( 1 μm ) from apical side of wing discs . of co - localization rates using apical projection ; central horizontal lines show median values of N = 8 - 12 , box shows lower upper quartiles whiskers show maximum minimum excluding outliers . Note that at apical side no clear significant reduction in co - localization rates ( 1 = total coverage of red by green ) is observed after RNAi expression or in Tsp96F - HA over - expression .

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

B) Projection images of three confocal sections (1 μm) from the apical side of the wing discs. Quantification of co-localization rates using apical projection images; central horizontal lines show median values of N=8-12 , box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers. Note that at the apical side no clear significant reduction in co-localization rates (1= total coverage of red by green) is observed after RNAi expression or in Tsp96F-HA over-expression.


C ) Correlation coefficients between maximum value of quantified integrated density ( integrating both signal ) for Ptc - GFP values ( Yac construct ) from basal side ( - 1 = basal , + 1 = apical ) . Box plot shows data of discs after RNAi expression for vacuole sorting Dor ( necessary for Ptc degradation ) , ESCRT Hrs , SNARE Syb as well as after over - expression of Tsp96F - HA construct wild type condition . Central horizontal lines show median values of N = 8 - 12 , box shows lower upper quartiles whiskers show maximum minimum excluding outliers . Note apical localization of Ptc - GFP highest values after degradation impairment ( Dor RNAi ) in contrast to basal shift resulting from exocytosis deficiency ( Syb RNAi ) .

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

C) Correlation coefficients between the maximum value of quantified integrated density (integrating both fluorescence intensity and signal area) for Ptc-GFP values (Yac construct) and distance from the basal side (-1=basal, +1=apical). Box plot shows quantification data of discs after RNAi expression for the vacuole sorting protein Dor (necessary for Ptc degradation), the ESCRT Hrs, the SNARE Syb as well as after over-expression of the Tsp96F-HA construct and wild type condition. Central horizontal lines show median values of N=8-12, box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers. Note the apical localization of Ptc-GFP highest values after degradation impairment (Dor RNAi) in contrast to the basal shift resulting from exocytosis deficiency (Syb RNAi).


A ) Confocal at lateral plane showing immunostaining of medium threshold Hh target Collier ( Col ) of wild type wing discs discs after inhibition of vesicle fusion ( α - Snap RNAi ) , vesicle trafficking ( Hrs RNAi ) or in over - expression of Tsp96F - HA . of gradient lengths ( μm ) using Collier signal normalized to wing pouch , showing proportion of wing pouch covered by signal ( 1 = total coverage ) . was performed for wild type condition as well as in temporally regulated RNAi expression for ESCRT Hrs , Tsg101 , Shrub Vps4 for SNARE Syb , Snap24 , α - Snap , as well as in transient over - expression of Tsp96F - HA . Central horizontal lines show median values of N = 7 - 22 , box shows lower upper quartiles whiskers show maximum minimum excluding outliers . Note a slight but significant increase in of Col signal for treatment compared to wild type condition .

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

A) Confocal images at the lateral plane showing the immunostaining of the medium threshold Hh target Collier (Col) of wild type wing discs and discs after inhibition of vesicle fusion (α-Snap RNAi), vesicle trafficking (Hrs RNAi) or in over-expression of the Tsp96F-HA. Quantification of gradient lengths (μm) using Collier signal and normalized to the wing pouch size, showing the proportion of wing pouch covered by signal (1=total coverage). Quantification was performed for wild type condition as well as in temporally regulated RNAi expression for the ESCRT complex proteins Hrs, Tsg101, Shrub and Vps4 and for the SNARE proteins Syb, Snap24, α-Snap, as well as in transient over-expression of the Tsp96F-HA. Central horizontal lines show median values of N=7-22 , box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers. Note a slight but significant increase in the length of Col signal for all treatment compared to the wild type condition.


B ) Lateral confocal of extension of immuno - labeled Engrailed as high threshold Hh target . shows number of diameters covered by signal from posterior / anterior border marked by Hh - GFP ( Bac construct ) ; central horizontal lines show median values of N = 6 - 10 box shows lower upper quartiles whiskers show maximum minimum excluding outliers . Note a significant reduction after RNAi expression , suggesting Hh reception impairment flattening of signal gradient .

O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O 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) Lateral confocal images and quantification of the extension of immuno-labeled Engrailed as the high threshold Hh target. Quantification shows number of cell diameters covered by signal from the posterior/anterior border marked by Hh-GFP (Bac construct); central horizontal lines show median values of N=6-10 box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers. Note a significant reduction after RNAi expression, suggesting Hh reception impairment and flattening of the signal gradient.