Figure 5Principal component analysis ( PCA ) of the DIA - MS phosphoproteomic data for the different stimuli separated by time points . Hierarchical clustering analysis of z - scored intensity values of significantly regulated ( ANOVA , permutation - based FDR < 0 . 05 ) phosphopeptides of the different time points . Column clustering hierarchy is indicated at the top . Row clusters for term enrichment analysis are color coded and indicated on the right .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Principal component analysis (PCA) of the DIA-MS phosphoproteomic data for the different stimuli separated by time points. Hierarchical clustering analysis of z-scored intensity values of significantly regulated (ANOVA, permutation-based FDR <0.05) phosphopeptides of the different time points. Column clustering hierarchy is indicated at the top. Row clusters for term enrichment analysis are color coded and indicated on the right.


Figure 1B - D ' ' ) Representative images showing that Hh accumulates on the surface of LDs in glial cells of the CB ( yellow arrows ) Glial cells are marked by repo - GAL4 > GFP and CB is circled in ( B ) . E - G ' ' ) In the posterior compartment of the developing wing disc ( WD ) pouch region where Hh is expressed , LDs and Hh are not tightly associated ( white arrows ) , H - J ) Hh - LD association is quantified by plotting the pixel intensities of both Hh ( cyan ) and LDs ( red ) along a line across LDs . Y axis represents gray intensity values , and X axis represents relative LD position . K - L ' ' ) Hh - LD associations are observed in the cortex glia ( yellow arrows , NP2222 - GAL4 > mGFP ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B-D'') Representative images showing that Hh accumulates on the surface of LDs in glial cells of the CB (yellow arrows) Glial cells are marked by repo-GAL4>GFP and CB is circled in (B).E-G'') In the posterior compartment of the developing wing disc (WD) pouch region where Hh is expressed, LDs and Hh are not tightly associated (white arrows),H-J) Hh-LD association is quantified by plotting the pixel intensities of both Hh (cyan) and LDs (red) along a line across LDs. Y axis represents gray intensity values, and X axis represents relative LD position.K-L'') Hh-LD associations are observed in the cortex glia (yellow arrows, NP2222-GAL4>mGFP).


Figure 2A - D ) Representative images showing that upon knockdown of Hh in cortex glial cells ( NP2222 - GAL4 > mGFP with UAS - dcr2 ) , cortex glial membrane and overall Repo + glial cell number are significantly reduced , quantified in ( C ) ( n = 7 , 8 brain lobes ) and ( D ) ( n = 7 , 8 brain lobes ) , respectively . E - G ) Hh knockdown in glia ( repo - GAL4 > GFP ) results in niche disruption and clustering of NBs ( circled with yellow dashed line ) , as well as an increase in the percentage of NBs in M phase ( pH3 + ) , quantified in ( E ) ( n = 12 , 10 brain lobes ) . H - K ) Representative images showing that Hh overexpression using pan - glial ( repo - GAL4 ) and cortex - glial ( NP2222 - GAL4 ) drivers both result in a decrease in NB EdU index , quantified in ( J ) ( n = 16 , 20 brain lobes ) and ( K ) ( n = 11 , 14 brain lobes ) , respectively . L - M ' ' ) Representative images showing ci - lacZ is expressed in NBs ( yellow arrows ) . ( M - M ' ' ) are zoomed in images of ( L ) . N - P ) Overexpression of ciACT in NBs ( dnab - GAL4 ) reduces EdU index , quantified in ( P ) ( n = 12 , 10 brain lobes ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A-D) Representative images showing that upon knockdown of Hh in cortex glial cells (NP2222-GAL4>mGFP with UAS-dcr2), cortex glial membrane and overall Repo+ glial cell number are significantly reduced, quantified in (C) (n=7, 8 brain lobes) and (D) (n=7, 8 brain lobes), respectively. E-G) Hh knockdown in glia (repo-GAL4>GFP) results in niche disruption and clustering of NBs (circled with yellow dashed line), as well as an increase in the percentage of NBs in M phase (pH3+), quantified in (E) (n=12, 10 brain lobes). H-K) Representative images showing that Hh overexpression using pan-glial (repo-GAL4) and cortex-glial (NP2222-GAL4) drivers both result in a decrease in NB EdU index, quantified in (J) (n=16, 20 brain lobes) and (K) (n=11, 14 brain lobes), respectively.L-M'') Representative images showing ci-lacZ is expressed in NBs (yellow arrows). (M-M'') are zoomed in images of (L).N-P) Overexpression of ciACT in NBs (dnab-GAL4) reduces EdU index, quantified in (P) (n=12, 10 brain lobes).


Figure 3A - B ' ' ) Representative images showing that Lsd2YFP is localised to the surface of LDs ( yellow arrows ) . C - D ' ' ) Lsd2YFP co - localises with Hh antibody staining in the cortex glia ( yellow arrows ) but not surface glial cells ( white arrow ) . E - G ) Lsd - 2 knockdown in cortex glial cells ( NP2222 - GAL4 ) where hh is overexpressed effectively reduces LD number in CB ( outlined in yellow dashed lines ) . H - K ) Representative images showing that NB EdU index is rescued upon Lsd - 2 knockdown in cortex glial cells ( NP2222 - GAL4 ) where hh is overexpressed , quantified in ( K ) ( n = 11 , 14 ; 16 , 14 ; 6 , 14 brain lobes ) . The NP2222 - GAL4 > w1118 vs hhOE columns depict the same data as Fig 2K . EdU + NBs are circled with yellow , dashed lines .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A-B'') Representative images showing that Lsd2YFP is localised to the surface of LDs (yellow arrows).C-D'') Lsd2YFP co-localises with Hh antibody staining in the cortex glia (yellow arrows) but not surface glial cells (white arrow).E-G) Lsd-2 knockdown in cortex glial cells (NP2222-GAL4) where hh is overexpressed effectively reduces LD number in CB (outlined in yellow dashed lines). H-K) Representative images showing that NB EdU index is rescued upon Lsd-2 knockdown in cortex glial cells (NP2222-GAL4) where hh is overexpressed, quantified in (K) (n=11, 14; 16, 14; 6, 14 brain lobes). The NP2222-GAL4>w1118 vs hhOE columns depict the same data as Fig 2K. EdU+ NBs are circled with yellow, dashed lines.


Figure 4A - D ' ) Representative images showing that pan - glial overexpression of htlACT , but not InRwt or EgfrACT causes an expansion of cortex glia that enwraps NBs . Glial cells are marked with repo - GAL4 > GFP , and NBs are marked with Mira . ( A ' , B ' , C ' and D ' ) are zoomed in images of ( A , B , C and D ) , respectively . Representative images showing that cortex glial overexpression of htlACT but not InRwt or EgfrACT causes an increase in cortex glial membrane size ( H ) ( n = 10 , 10 brain lobes ) NP2222 - GAL4 > mGFP is used to mark cortex glial membrane in ( E - G ) Representative images showing that cortex glial overexpression of EgfrACT to mark cortex glial membrane wrapper - GAL4 > is used in ( I , J ) . cortex glial overexpression of htlACT but not InRwt or EgfrACT causes an increase in glial cell ( Repo + ) numbers quantified in ( K ) ( n = 10 , 10 , 10 ; 7 , 12 brain lobes ) L ) Glial ( repo - GAL4 > ) overexpression of InRwt but not htlACT or EgfrACT significantly reduces the number of CB NBs ( n = 10 , 10 , 8 , 11 brain lobes ) . M ) Glial ( repo - GAL4 > ) overexpression of htlACT but not InRwt or EgfrACT significantly reduces the pH3 index of CB

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A-D') Representative images showing that pan-glial overexpression of htlACT, but not InRwt or EgfrACT causes an expansion of cortex glia that enwraps NBs. Glial cells are marked with repo-GAL4 >GFP, and NBs are marked with Mira. (A', B', C' and D') are zoomed in images of (A, B, C and D), respectively.Representative images showing that cortex glial overexpression of htlACT but not InRwt or EgfrACT causes an increase in cortex glial membrane size (H) (n=10, 10 brain lobes) NP2222-GAL4>mGFP is used to mark cortex glial membrane in (E-G)Representative images showing that cortex glial overexpression of EgfrACT to mark cortex glial membrane wrapper-GAL4> is used in (I, J).cortex glial overexpression of htlACT but not InRwt or EgfrACT causes an increase in glial cell (Repo+) numbers quantified in (K) (n=10, 10, 10; 7, 12 brain lobes)L) Glial (repo-GAL4>) overexpression of InRwt but not htlACT or EgfrACT significantly reduces the number of CB NBs (n= 10, 10, 8, 11 brain lobes).M) Glial (repo-GAL4>) overexpression of htlACT but not InRwt or EgfrACT significantly reduces the pH3 index of CB NBs


Figure 5A - E ) Representative images showing that both pan - glial ( repo - GAL4 > ) and cortex glial ( NP2222 - GAL4 > ) htlACT overexpression significantly reduce NB EdU index , quantified in ( E ) ( n = 15 , 14 ; 10 , 16 brain lobes ) . ( A , B , C , D ) are single sections with Mira and EdU staining , and ( A ' , B ' C ' , D ' ) are Z - projection of the EdU staining . pan - glial ( repo - GAL4 ) htlACT overexpression lengthens NB cell cycle , quantified in ( F ) The cell cycle length is measured as the length between consecutive divisions . Representative still images from ex vivo CNS live imaging at 72ALH showing that pan - glial ( repo - GAL4 ) htlACT overexpression lengthens NB cell cycle , ( n = 25 , 6 NBs imaged from 3 brains per genotype ) . NBs ( Dpn : : GFP , red ; Histone RFP , grey ) are circled with blue dashed lines . J - L ) Representative images showing that the percentage of NBs in G1 - S transition and G2 / M phase are both significantly increased with significantly less cells remaining in G1 phase , quantified in ( L ) ( n = 9 , 10 brain lobes ) . NBs in G1 phase ( Mira + , GFP + ) are circled by green dashed lines ; NBs in G1 - S transition ( Mira + , GFP - RFP - ) are circled by grey dashed lines ; NBs in S phase ( Mira + , RFP + ) are circled by red dashed lines ; and NBs in G2 / M phase ( Mira + , GFP + RFP + ) are circled by yellow dashed lines . M - O ) Representative images showing that the number of EdU + neurons generated per NB is significantly reduced upon pan - glial overexpression of FGF ( repo - LexA > LexAop - htlACT ; yellow arrows ) , quantified in ( O ) n = 94 , 127 NB lineages imaged from 5 and 7 brain lobes , respectively ) . NB lineages are marked with dnab - gal4 > GFP .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A-E) Representative images showing that both pan-glial (repo-GAL4>) and cortex glial (NP2222-GAL4>) htlACT overexpression significantly reduce NB EdU index, quantified in (E) (n=15 ,14; 10, 16 brain lobes). (A, B, C, D) are single sections with Mira and EdU staining, and (A', B' C', D') are Z-projection of the EdU staining.pan-glial (repo-GAL4) htlACT overexpression lengthens NB cell cycle, quantified in (F) The cell cycle length is measured as the length between consecutive divisions.Representative still images from ex vivo CNS live imaging at 72ALH showing that pan-glial (repo-GAL4) htlACT overexpression lengthens NB cell cycle, (n= 25, 6 NBs imaged from 3 brains per genotype). NBs (Dpn::GFP, red; Histone RFP, grey) are circled with blue dashed lines.J-L) Representative images showing that the percentage of NBs in G1-S transition and G2/M phase are both significantly increased with significantly less cells remaining in G1 phase, quantified in (L) (n= 9, 10 brain lobes). NBs in G1 phase (Mira+, GFP+) are circled by green dashed lines; NBs in G1-S transition (Mira+, GFP-RFP-) are circled by grey dashed lines; NBs in S phase (Mira+, RFP+) are circled by red dashed lines; and NBs in G2/M phase (Mira+, GFP+RFP+) are circled by yellow dashed lines.M-O) Representative images showing that the number of EdU+ neurons generated per NB is significantly reduced upon pan-glial overexpression of FGF (repo-LexA > LexAop-htlACT; yellow arrows), quantified in (O) n= 94, 127 NB lineages imaged from 5 and 7 brain lobes, respectively). NB lineages are marked with dnab-gal4 > GFP.


Figure 6A ) Pan - glial ( repo - GAL4 ) htlACT overexpression causes upregulation of hh , fasn1 and lsd2 transcripts ( n = 3 biological replicates pooled from 20 brains for each genotype ; for each biological replicate , we ran 3 technical replicates for each PCR reaction ) . The lipogenesis genes ( acc , lipin ) , and lipolysis gene bmm transcripts are not significantly altered . We utilised rpl32 as a reference gene in these experiments , as it is not altered by htlACT overexpression . The data are represented by log2 fold change relative to the control ( repo - GAL4 > w1118 ) . C - G ) Representative images showing that Hh staining normally localised to a ring like structure ( yellow arrows ) , becomes delocalises to the glial cytoplasm upon htlACT overexpression , quantified in ( E ) ( n = 4 , 6 brain lobes ) . Glial cells are marked with repo - GAL4 > GFP . ( E - F ' ) are zoomed in images of ( C - D ' ) . H ) Cortex glial ( NP2222 - GAL4 > ) overexpression of two independent hh RNAis significantly rescue EdU incorporation defects caused by htlACT overexpression ( n = 10 , 16 ; 7 , 12 ; 14 , 11 brain lobes ) . I ) Knockdown of NB Hh signalling pathway ( dnab - GAL4 > UAS - ciRNAi ) rescues NB EdU incorporation defects induced by glial htlACT overexpression ( repo - LexA > LexAop - htlACT ) . Induction of ciRNAi in NBs alone increases NB EdU incorporation ( n = 8 , 13 ; 10 , 10 ; 30 , 26 ; 12 , 7 brain lobes ) . J ) The NB EdU incorporation defects due to cortex glial ( NP2222 - GAL4 > ) overexpression of htlACT , is significantly rescued by overexpression of RNAis against fasn1 and lsd2 , compared to corresponding control RNAis ( n = 10 , 16 ; 25 , 17 ; 14 , 8 brain lobes ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A) Pan-glial (repo-GAL4) htlACT overexpression causes upregulation of hh, fasn1 and lsd2 transcripts (n = 3 biological replicates pooled from 20 brains for each genotype; for each biological replicate, we ran 3 technical replicates for each PCR reaction). The lipogenesis genes (acc, lipin), and lipolysis gene bmm transcripts are not significantly altered. We utilised rpl32 as a reference gene in these experiments, as it is not altered by htlACT overexpression. The data are represented by log2 fold change relative to the control (repo-GAL4> w1118).C-G) Representative images showing that Hh staining normally localised to a ring like structure (yellow arrows), becomes delocalises to the glial cytoplasm upon htlACT overexpression, quantified in (E) (n=4, 6 brain lobes). Glial cells are marked with repo-GAL4>GFP. (E-F') are zoomed in images of (C-D').H) Cortex glial (NP2222-GAL4>) overexpression of two independent hh RNAis significantly rescue EdU incorporation defects caused by htlACT overexpression (n=10, 16; 7, 12; 14, 11 brain lobes).I) Knockdown of NB Hh signalling pathway (dnab-GAL4> UAS-ciRNAi) rescues NB EdU incorporation defects induced by glial htlACT overexpression (repo-LexA> LexAop-htlACT). Induction of ciRNAi in NBs alone increases NB EdU incorporation (n=8, 13; 10, 10; 30, 26; 12, 7 brain lobes).J) The NB EdU incorporation defects due to cortex glial (NP2222-GAL4>) overexpression of htlACT, is significantly rescued by overexpression of RNAis against fasn1 and lsd2, compared to corresponding control RNAis (n=10, 16; 25, 17; 14, 8 brain lobes).


Figure 7B ) Inhibition of palmitoylation ( via two independent rasp RNAis ) rescues NB EdU incorporation defects induced by cortex glial ( NP2222 - GAL4 > ) htlACT overexpression , while knockdown of Rasp in cortex glial cells alone does not alter NB EdU index ( n = 10 , 15 ; 10 , 16 ; 18 , 21 ; 26 , 29 brain lobes ) . C - C ' ) Representative images showing that Hh . N . EGFP ( which cannot undergo cholesterol modification ) are found as puncta on the surface of NBs ( yellow arrows ) when overexpressed in neighbouring cortex glial cells ( NP2222 - GAL4 > ) . Representative images showing that knockdown of Fasn1 in glial cells ( repo - GAL4 > ) , where Hh . N . EGFP is overexpressed , significantly reduces the number of Hh . N . EGFP puncta on the surface of NBs ( yellow arrows ) n = 38 , 38 , 57 NBs from 4 , 4 , 8 brain lobes , respectively ) . knockdown of Fasn1 in glial cells ( repo - GAL4 > ) , where Hh . N . EGFP is overexpressed , significantly reduces the number of Hh . N . EGFP puncta on the surface of NBs phenocopying the effect of Rasp knockdown , quantified in ( G ) H ) Cortex glial ( NP2222 - GAL4 > ) overexpression of Hh . N . EGFP significantly reduces NB EdU incorporation ( n = 13 , 12 brain lobes ) . I ) Knockdown of Fasn1 in glial cells rescues NB EdU incorporation defects , caused by glial Hh . N . EGFP overexpression , quantified in ( I ) ( n = 15 , 13 brain lobes ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7B) Inhibition of palmitoylation (via two independent rasp RNAis) rescues NB EdU incorporation defects induced by cortex glial (NP2222-GAL4>) htlACT overexpression, while knockdown of Rasp in cortex glial cells alone does not alter NB EdU index (n=10, 15; 10, 16; 18, 21; 26, 29 brain lobes).C-C') Representative images showing that Hh.N.EGFP (which cannot undergo cholesterol modification) are found as puncta on the surface of NBs (yellow arrows) when overexpressed in neighbouring cortex glial cells (NP2222-GAL4>).Representative images showing that knockdown of Fasn1 in glial cells (repo-GAL4>), where Hh.N.EGFP is overexpressed, significantly reduces the number of Hh.N.EGFP puncta on the surface of NBs (yellow arrows) n=38, 38, 57 NBs from 4, 4, 8 brain lobes, respectively).knockdown of Fasn1 in glial cells (repo-GAL4>), where Hh.N.EGFP is overexpressed, significantly reduces the number of Hh.N.EGFP puncta on the surface of NBs phenocopying the effect of Rasp knockdown, quantified in (G)H) Cortex glial (NP2222-GAL4>) overexpression of Hh.N.EGFP significantly reduces NB EdU incorporation (n= 13,12 brain lobes).I) Knockdown of Fasn1 in glial cells rescues NB EdU incorporation defects, caused by glial Hh.N.EGFP overexpression, quantified in (I) (n=15, 13 brain lobes).


Figure 1A ) Ventral nests expansion at 0 min ( onset ) ; 245 min , when ventral and spiracle nests fuse ; 515 min , when intersegment nests fuse ; and 750 min , when most LECs are gone . We employed for these analyses the following stock : w ; If / CyO ; ZCL2207 : : hh - DsRed / MKRS . The green signal corresponds to ZCL2207 ( Septate junction GFP marker ) outlining all apical cell membranes and the red signal to nuclei from the posterior compartment expressing RFP under the control of the hedgehog promoter . Arrows point to the spiracular and ventral nests on the top panel and to the junction between these two nests in the second panel . Images of confocal sections have been rotated to maintain consistent alignment of the dorso - ventral axis ( dorsal is up ) . The outlines of the confocal images are marked with a yellow dotted line . Scale bar = 40 µm . B ) Comparison of manual vs . PIV surface area kinetics [ evolution of nests ( red - manual , black - PIV ) and LECs ( cyan - manual , blue - PIV ) ] C ) Nests rate of growth from a representative single nest . Note the similarity in behavior ( red - manual , black - PIV ) in three sequential phases : Phase 1 ( Ph 1 ) , accelerated growth ; Phase 2 ( Ph 2 ) , sustained growth ; and Phase 3 ( Ph 3 ) decelerated growth ) . D ) Progression of the elongation ratio ( red - manual , black - PIV ) of nests Note the initial fast decay on the elongated shape of the nests . E ) Nest orientation evolution ( red - manual , black - PIV ) . Anterior is π ( 3 . 1415 rads , dorso ventral is π / 2 ( 1 . 57 rads ) and posterior is 0 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A) Ventral nests expansion at 0 min (onset); 245 min, when ventral and spiracle nests fuse; 515 min, when intersegment nests fuse; and 750 min, when most LECs are gone. We employed for these analyses the following stock: w; If/CyO; ZCL2207::hh-DsRed/MKRS. The green signal corresponds to ZCL2207 (Septate junction GFP marker) outlining all apical cell membranes and the red signal to nuclei from the posterior compartment expressing RFP under the control of the hedgehog promoter. Arrows point to the spiracular and ventral nests on the top panel and to the junction between these two nests in the second panel. Images of confocal sections have been rotated to maintain consistent alignment of the dorso-ventral axis (dorsal is up). The outlines of the confocal images are marked with a yellow dotted line. Scale bar = 40 µm.B) Comparison of manual vs. PIV surface area kinetics [evolution of nests (red - manual, black - PIV) and LECs (cyan - manual, blue -PIV)]C) Nests rate of growth from a representative single nest. Note the similarity in behavior (red - manual, black - PIV) in three sequential phases: Phase 1 (Ph 1), accelerated growth; Phase 2 (Ph 2), sustained growth; and Phase 3 (Ph 3) decelerated growth).D) Progression of the elongation ratio (red - manual, black - PIV) of nests Note the initial fast decay on the elongated shape of the nests.E) Nest orientation evolution (red - manual, black - PIV). Anterior is π (3.1415 rads, dorso ventral is π/2 (1.57 rads) and posterior is 0.


Figure 2A ) Snapshots of the divergence of the velocity field from a representative movie . Note the areas of positive divergence in the nests , areas of positive and negative divergence in the LECs , and an increase of magnitude on the differences between them over time . Scale bar = 40 µm . B ) Frequency distribution of the divergence magnitude in the nests and LECs in the different growth phases . Nests are always shown in red and LECs in blue . C ) Color coded radial kymographs of divergence for a representative movie . Low divergences are blue , high are red ( see scales ) . Note the overall positive divergence in the nests and negative in the LECs .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A) Snapshots of the divergence of the velocity field from a representative movie. Note the areas of positive divergence in the nests, areas of positive and negative divergence in the LECs, and an increase of magnitude on the differences between them over time. Scale bar = 40 µm.B) Frequency distribution of the divergence magnitude in the nests and LECs in the different growth phases. Nests are always shown in red and LECs in blue.C) Color coded radial kymographs of divergence for a representative movie. Low divergences are blue, high are red (see scales). Note the overall positive divergence in the nests and negative in the LECs.


Figure 3A ) Cell numbers in nests ( blue - left scale ) and LECs territories ( red - right scale ) ( profiles are averaged from three segmented movies ) . B ) Mean cell area evolution of histoblasts ( blue - left scale ) and LECs ( red - right scale ) . Note the progressive increase in mean histoblast area ( profiles are average from three segmented movies ) . C ) Net change rate in cell number variation for nests ( red line ) versus nests growth ( black line ) . Note the temporal correlation in phases , but the reduced magnitude in cell numbers ( profiles are collected from single nests and normalized ; control black lines are retrieved from Figure 1 ) . D ) Net change rate in cell number variation for LECs ( blue line ) versus tissue growth ( black line ) . Note the full pattern correlation and the oscillations in LECs net rate numbers ( profiles are collected from single nests and normalized ; control black lines are retrieved from Figure 1 ) . E ) Evolution of the net change rate of histoblasts MCA ( red line ) versus net change rate of nest growth ( black line ) . Note the delay in reaching the sustained growth phase and the reduced magnitude of the MCA rate ( profiles are collected from single nests and normalized ; control black lines are retrieved from Figure 1 ) . F ) Evolution of the net change rate of LECs MCA ( blue line ) versus net change rate of LECs tissue growth ( black line ) . Note the oscillatory MCA rate around 0 and the tendency toward negative values ( MCA decrease ) ( profiles are collected from single nests and normalized ; control black lines are retrieved from Figure 1 ) .

O B-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A) Cell numbers in nests (blue - left scale) and LECs territories (red - right scale) (profiles are averaged from three segmented movies).B) Mean cell area evolution of histoblasts (blue - left scale) and LECs (red - right scale). Note the progressive increase in mean histoblast area (profiles are average from three segmented movies).C) Net change rate in cell number variation for nests (red line) versus nests growth (black line). Note the temporal correlation in phases, but the reduced magnitude in cell numbers (profiles are collected from single nests and normalized; control black lines are retrieved from Figure 1).D) Net change rate in cell number variation for LECs (blue line) versus tissue growth (black line). Note the full pattern correlation and the oscillations in LECs net rate numbers (profiles are collected from single nests and normalized; control black lines are retrieved from Figure 1).E) Evolution of the net change rate of histoblasts MCA (red line) versus net change rate of nest growth (black line). Note the delay in reaching the sustained growth phase and the reduced magnitude of the MCA rate (profiles are collected from single nests and normalized; control black lines are retrieved from Figure 1).F) Evolution of the net change rate of LECs MCA (blue line) versus net change rate of LECs tissue growth (black line). Note the oscillatory MCA rate around 0 and the tendency toward negative values (MCA decrease) (profiles are collected from single nests and normalized; control black lines are retrieved from Figure 1).


Figure 4A ) Histoblasts Mean Cell Area ( MCA ) increases over time for all ( black - the whole nest ) , central ( blue ) and peripheral histoblasts ( red ) . B ) Snapshots at specific time points of segmented movies depicting histoblasts with color - coded cell areas . Color scale on the right . Scale bar = 40 µm . Note the progressive increase of the histoblasts size , specially the peripheral ones . Data information : In ( B ) the top panel represents the central histoblasts and the bottom panel the peripheral . We employed an arbitrary annotation and consider peripheral those cells present in the 2 outermost rows of cells all throughout the perimeter of the nest at the earliest recorded time point . C ) Histoblasts Elongation Ratio decreases over time for all ( black - the whole nest ) , central ( blue ) and peripheral histoblasts ( red ) . D ) Color coded snapshots of cell elongation at different time points . Color scale on the right . Scale bar = 40 µm . Data information : In ( D ) , the top panel represents the central histoblasts and the bottom panel the peripheral . We employed an arbitrary annotation and consider peripheral those cells present in the 2 outermost rows of cells all throughout the perimeter of the nest at the earliest recorded time point .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A) Histoblasts Mean Cell Area (MCA) increases over time for all (black - the whole nest), central (blue) and peripheral histoblasts (red). B) Snapshots at specific time points of segmented movies depicting histoblasts with color-coded cell areas. Color scale on the right. Scale bar = 40 µm. Note the progressive increase of the histoblasts size, specially the peripheral ones. Data information: In (B) the top panel represents the central histoblasts and the bottom panel the peripheral. We employed an arbitrary annotation and consider peripheral those cells present in the 2 outermost rows of cells all throughout the perimeter of the nest at the earliest recorded time point.C) Histoblasts Elongation Ratio decreases over time for all (black - the whole nest), central (blue) and peripheral histoblasts (red). D) Color coded snapshots of cell elongation at different time points. Color scale on the right. Scale bar = 40 µm. Data information: In (D), the top panel represents the central histoblasts and the bottom panel the peripheral. We employed an arbitrary annotation and consider peripheral those cells present in the 2 outermost rows of cells all throughout the perimeter of the nest at the earliest recorded time point.


Figure 6A ) Density Power radial kymograph of a representative time - lapse . Each horizontal line displays for each time point ( left scale ) the mean of the power value at every distance ( bottom scale ) to the nest - LECs border ( vertical black line ) . Each phase is represented in a colored background with three different densities of purple . Values scale is on the right . Graph representing the mean ( line ) and standard deviation ( shadow ) of the power density radial kymographs ( left scale ) at different distances ( bottom scale ) by phases ( in coded densities of purple ) for all samples . Surface Stress radial kymograph of a representative time - lapse Phases are represented in different densities of brown . Graph representing the mean and standard deviation of the surface stress radial kymographs Values color coded in different densities of brown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A) Density Power radial kymograph of a representative time-lapse. Each horizontal line displays for each time point (left scale) the mean of the power value at every distance (bottom scale) to the nest-LECs border (vertical black line). Each phase is represented in a colored background with three different densities of purple. Values scale is on the right. Graph representing the mean (line) and standard deviation (shadow) of the power density radial kymographs (left scale) at different distances (bottom scale) by phases (in coded densities of purple) for all samples.Surface Stress radial kymograph of a representative time-lapse Phases are represented in different densities of brown. Graph representing the mean and standard deviation of the surface stress radial kymographs Values color coded in different densities of brown.


Figure 7A ) Density power radial kymograph of a representative time - lapse movie of the nests expansion process after blocking histoblasts proliferation by overexpressing Dacapo . Each horizontal line displays at each time point ( left scale ) the mean of the power value at every distance ( bottom scale ) to the nest - LECs border ( vertical black line ) . Each phase is represented in a colored background with three different densities of purple . Power values color scale is on the right . B ) Comparison of the means and standard deviations of the density power radial kymographs in phase 3 for Dacapo overexpressing nests ( green ) and wild type ( purple ) . C ) Surface Stress radial kymograph of a representative time - lapse movie Phases are represented in different densities of brown . Stress values color scale is on the right . D ) Comparison of the means and standard deviations of the surface stress radial kymographs in phase 3 for Dacapo overexpressing nests ( green ) and wild type ( brown ) . E ) Density power radial kymograph of a representative time - lapse movie after blocking LECs delamination by overexpressing P35 . Power values color scale is on the right . F ) Comparison of the means and standard deviations of the density power radial kymographs in phase 3 for P35 overexpressing LECs ( green ) and wild type ( purple ) . G ) Surface Stress radial kymograph of a representative time - lapse movie Phases are represented in different densities of brown . Power values color scale is on the right . H ) Comparison of the means and standard deviations of the surface stress radial kymograph in phase 3 for P35 overexpressing LECs ( green ) and wild type ( brown ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A) Density power radial kymograph of a representative time-lapse movie of the nests expansion process after blocking histoblasts proliferation by overexpressing Dacapo. Each horizontal line displays at each time point (left scale) the mean of the power value at every distance (bottom scale) to the nest-LECs border (vertical black line). Each phase is represented in a colored background with three different densities of purple. Power values color scale is on the right.B) Comparison of the means and standard deviations of the density power radial kymographs in phase 3 for Dacapo overexpressing nests (green) and wild type (purple).C) Surface Stress radial kymograph of a representative time-lapse movie Phases are represented in different densities of brown. Stress values color scale is on the right.D) Comparison of the means and standard deviations of the surface stress radial kymographs in phase 3 for Dacapo overexpressing nests (green) and wild type (brown).E) Density power radial kymograph of a representative time-lapse movie after blocking LECs delamination by overexpressing P35. Power values color scale is on the right.F) Comparison of the means and standard deviations of the density power radial kymographs in phase 3 for P35 overexpressing LECs (green) and wild type (purple).G) Surface Stress radial kymograph of a representative time-lapse movie Phases are represented in different densities of brown. Power values color scale is on the right.H) Comparison of the means and standard deviations of the surface stress radial kymograph in phase 3 for P35 overexpressing LECs (green) and wild type (brown).


Figure 8A ) Laser microsurgery was performed at the different sides of the ventral nests during phase 2 ( 300 minutes ) in parallel cuts [ in the LEC tissue at 40 µm of the nests / LEC border ( cyan ) and within the nests at 10 µm ( red ) and 40 µm ( green ) of the edge ] . Myo - GFP transgenic pupae were used to visualize the cortex displacements . This example illustrates at the anterior edge the laser cuts positions . Scale bar = 15 µm . B ) Cortex recoil velocity ( pixels per second ) at different time points after laser cutting ( + 10 sec ) for a single experiment . Note the exponentially decaying speed . D ) Snapshots of a laser cut in the nest area . PIV arrows are shown in red . Scale bar = 5 µm . E ) Comparison of the mean laser recoil velocities in phase 3 for Dacapo overexpressing nests ( green ) with wild type ( blue ) at different distances ( - 40 - n = 16 , - 10 - n = 22 , + 40 - n = 14 ) Error bars represent Standard Deviations . F ) Comparison of the mean laser recoil velocities in phase 3 for P35 overexpressing nests ( green ) with wild type ( blue ) at different distances ( - 40 - n = 17 , - 10 - n = 20 , + 40 - n = 15 ) Error bars represent Standard Deviations .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A) Laser microsurgery was performed at the different sides of the ventral nests during phase 2 (300 minutes) in parallel cuts [in the LEC tissue at 40 µm of the nests/LEC border (cyan) and within the nests at 10 µm (red) and 40 µm (green) of the edge]. Myo-GFP transgenic pupae were used to visualize the cortex displacements. This example illustrates at the anterior edge the laser cuts positions. Scale bar = 15 µm.B) Cortex recoil velocity (pixels per second) at different time points after laser cutting (+ 10 sec) for a single experiment. Note the exponentially decaying speed.D) Snapshots of a laser cut in the nest area. PIV arrows are shown in red. Scale bar = 5 µm.E) Comparison of the mean laser recoil velocities in phase 3 for Dacapo overexpressing nests (green) with wild type (blue) at different distances (-40 - n = 16, -10 - n =22, +40 - n = 14) Error bars represent Standard Deviations.F) Comparison of the mean laser recoil velocities in phase 3 for P35 overexpressing nests (green) with wild type (blue) at different distances (-40 - n = 17, -10 - n = 20, +40 - n = 15) Error bars represent Standard Deviations.


Figure 1A CD4 + T cells from wild - type ( WT ) , CBLOST and CBLBOSTmice were left unstimulated ( - ) or stimulated for 2 min with anti - CD3 and anti - CD4 antibodies ( + ) . Equal amounts of cell lysates were subjected to affinity purification on Strep - Tactin - Sepharose beads , followed by elution of proteins with D - biotin . Eluted proteins were analyzed by immunoblot with antibodies specific for CBLB and CBL . Equal amounts of proteins from whole cell lysates ( WCL ) were also probed for CBLB and CBL . Note that in the case of CBLB two bands are detected and likely corresponded to the presence of two isoforms differing at their N - terminus ( http : / / www . uniprot . org / uniprot / Q3TTA7 ) . Also shown are loading control corresponding to the same immunoblot probed with anti - CD5 , and molecular masses ( kDa ) . Data are representative of at least three experiments . B CD4 + CD8 + thymocytes and mature CD4 + T cells isolated from mice with the specified genotypes were permeabilized and stained with saturating amounts of Strep - Tactin - APC and analyzed by flow cytometry . Background staining was deduced using cells from WT mouse . Data are representative of 2 experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A CD4+T cells from wild-type (WT), CBLOST and CBLBOSTmice were left unstimulated (-) or stimulated for 2 min with anti-CD3 and anti-CD4 antibodies (+). Equal amounts of cell lysates were subjected to affinity purification on Strep-Tactin-Sepharose beads, followed by elution of proteins with D-biotin. Eluted proteins were analyzed by immunoblot with antibodies specific for CBLB and CBL. Equal amounts of proteins from whole cell lysates (WCL) were also probed for CBLB and CBL. Note that in the case of CBLB two bands are detected and likely corresponded to the presence of two isoforms differing at their N-terminus (http://www.uniprot.org/uniprot/Q3TTA7). Also shown are loading control corresponding to the same immunoblot probed with anti-CD5, and molecular masses (kDa). Data are representative of at least three experiments.B CD4+CD8+thymocytes and mature CD4+T cells isolated from mice with the specified genotypes were permeabilized and stained with saturating amounts of Strep-Tactin-APC and analyzed by flow cytometry. Background staining was deduced using cells from WT mouse. Data are representative of 2 experiments.


Figure 2CD4 + T cells from wild - type ( WT ) , CBLOST and CBLBOSTmice were left unstimulated ( unstim ) or stimulated for 30 , 120 , 300 or 600 seconds with anti - CD3 and anti - CD4 antibodies . Equal amounts of cell lysates were subjected to affinity purification using Strep - Tactin - Sepharose beads and subjected to MS analysis as described in Material and Methods . A Histogram comparing the normalized CBL protein intensities resulting from AP - MS analysis of CD4 + T cells from WT and CBLOSTmice prior to and after the specified stimulation times . For each time point , the distributions of the normalized intensities of the 9 data points ( 3 biological replicates x 3 technical replicates ) corresponding to CD4 + T from wild - type ( WT ) and CBLOSTmice were compared using the adjusted p - value from a one - way ANOVA test ( denoted as p ( t ) ) and the value r ( t ) corresponding to the enrichment observed in the CBLOST samples as compared to WT samples . Different colors represent distinct biological replicates . CD4 + T cells from wild - type ( WT ) , CBLOST and CBLBOSTmice were left unstimulated ( unstim ) or stimulated for 30 , 120 , 300 or 600 seconds with anti - CD3 and anti - CD4 antibodies . Equal amounts of cell lysates were subjected to affinity purification using Strep - Tactin - Sepharose beads and subjected to MS analysis as described in Material and Methods . B Proteins are classified as CBL interactors according to their position in a volcano plot in which the mean p - valueis plotted against the corresponding mean enrichment for CBL ( t ) > ( t ) > OST versus WT samples . The time point shown corresponds to the unstimulated condition ( t = 0s ) . Mean was calculated using bootstrap resampling ( see Results and Material and Methods ) . Proteins that displayed an enrichment r ( t ) greater than 2 - fold and a corresponding p - value p ( t ) lower than a set threshold ( see Material and Methods ) in more than 90 % of the bootstrap iterations were selected as specific partners ( indicated in red ) . Names of four of the most significantly enriched proteins are indicated . Dashed lines represent thresholds on the p - value p * = 10 - 3 and the enrichment r * = 2 used to identify specific interactors of CBL . CD4 + T cells from wild - type ( WT ) , CBLOST and CBLBOSTmice were left unstimulated ( unstim ) or stimulated for 30 , 120 , 300 or 600 seconds with anti - CD3 and anti - CD4 antibodies . Equal amounts of cell lysates were subjected to affinity purification using Strep - Tactin - Sepharose beads and subjected to MS analysis as described in Material and Methods . C , D List of specific interacting partners of CBLB ( C ) and CBL ( D ) ranked according to their maximum mean enrichment across all time points . Heat maps show , for each time point , the fraction of bootstrap iterations for which the corresponding proteins were detected as specific partners ( denoted as " P ( Detection ) " ) . Red dots indicate the proteins that have been previously reported as CBLB ( C ) interactors in the BioGRID database . Ubiquitin ( UB ) is highlighted with a black arrow . CD4 + T cells from wild - type ( WT ) , CBLOST and CBLBOSTmice were left unstimulated ( unstim ) or stimulated for 30 , 120 , 300 or 600 seconds with anti - CD3 and anti - CD4 antibodies . Equal amounts of cell lysates were subjected to affinity purification using Strep - Tactin - Sepharose beads and subjected to MS analysis as described in Material and Methods . C , D List of specific interacting partners of CBLB ( C ) and CBL ( D ) ranked according to their maximum mean enrichment across all time points . Heat maps show , for each time point , the fraction of bootstrap iterations for which the corresponding proteins were detected as specific partners ( denoted as " P ( Detection ) " ) . Red dots indicate the proteins that have been previously reported as CBL ( D ) interactors in the BioGRID database . Ubiquitin ( UB ) is highlighted with a black arrow . CD4 + T cells from wild - type ( WT ) , CBLOST and CBLBOSTmice were left unstimulated ( unstim ) or stimulated for 30 , 120 , 300 or 600 seconds with anti - CD3 and anti - CD4 antibodies . Equal amounts of cell lysates were subjected to affinity purification using Strep - Tactin - Sepharose beads and subjected to MS analysis as described in Material and Methods . E Histograms showing the numbers of specific interactors binding to the CBL - OST or CBLB - OST proteins prior to and after TCR stimulation for different times and for which P ( Detection ) > 0 . 9 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2CD4+T cells from wild-type (WT), CBLOST and CBLBOSTmice were left unstimulated (unstim) or stimulated for 30, 120, 300 or 600 seconds with anti-CD3 and anti-CD4 antibodies. Equal amounts of cell lysates were subjected to affinity purification using Strep-Tactin-Sepharose beads and subjected to MS analysis as described in Material and Methods.A Histogram comparing the normalized CBL protein intensities resulting from AP-MS analysis of CD4+T cells from WT and CBLOSTmice prior to and after the specified stimulation times. For each time point, the distributions of the normalized intensities of the 9 data points (3 biological replicates x 3 technical replicates) corresponding to CD4+ T from wild-type (WT) and CBLOSTmice were compared using the adjusted p-value from a one-way ANOVA test (denoted as p(t)) and the value r(t) corresponding to the enrichment observed in the CBLOST samples as compared to WT samples. Different colors represent distinct biological replicates.CD4+T cells from wild-type (WT), CBLOST and CBLBOSTmice were left unstimulated (unstim) or stimulated for 30, 120, 300 or 600 seconds with anti-CD3 and anti-CD4 antibodies. Equal amounts of cell lysates were subjected to affinity purification using Strep-Tactin-Sepharose beads and subjected to MS analysis as described in Material and Methods.B Proteins are classified as CBL interactors according to their position in a volcano plot in which the mean p-valueis plotted against the corresponding mean enrichment for CBL(t)>(t)>OST versus WT samples. The time point shown corresponds to the unstimulated condition (t=0s). Mean was calculated using bootstrap resampling (see Results and Material and Methods). Proteins that displayed an enrichment r(t) greater than 2-fold and a corresponding p-value p(t) lower than a set threshold (see Material and Methods) in more than 90% of the bootstrap iterations were selected as specific partners (indicated in red). Names of four of the most significantly enriched proteins are indicated. Dashed lines represent thresholds on the p-value p*=10-3 and the enrichment r*=2 used to identify specific interactors of CBL.CD4+T cells from wild-type (WT), CBLOST and CBLBOSTmice were left unstimulated (unstim) or stimulated for 30, 120, 300 or 600 seconds with anti-CD3 and anti-CD4 antibodies. Equal amounts of cell lysates were subjected to affinity purification using Strep-Tactin-Sepharose beads and subjected to MS analysis as described in Material and Methods.C, D List of specific interacting partners of CBLB (C) and CBL (D) ranked according to their maximum mean enrichment across all time points. Heat maps show, for each time point, the fraction of bootstrap iterations for which the corresponding proteins were detected as specific partners (denoted as "P(Detection)"). Red dots indicate the proteins that have been previously reported as CBLB (C) interactors in the BioGRID database. Ubiquitin (UB) is highlighted with a black arrow.CD4+T cells from wild-type (WT), CBLOST and CBLBOSTmice were left unstimulated (unstim) or stimulated for 30, 120, 300 or 600 seconds with anti-CD3 and anti-CD4 antibodies. Equal amounts of cell lysates were subjected to affinity purification using Strep-Tactin-Sepharose beads and subjected to MS analysis as described in Material and Methods.C, D List of specific interacting partners of CBLB (C) and CBL (D) ranked according to their maximum mean enrichment across all time points. Heat maps show, for each time point, the fraction of bootstrap iterations for which the corresponding proteins were detected as specific partners (denoted as "P(Detection)"). Red dots indicate the proteins that have been previously reported as CBL (D) interactors in the BioGRID database. Ubiquitin (UB) is highlighted with a black arrow.CD4+T cells from wild-type (WT), CBLOST and CBLBOSTmice were left unstimulated (unstim) or stimulated for 30, 120, 300 or 600 seconds with anti-CD3 and anti-CD4 antibodies. Equal amounts of cell lysates were subjected to affinity purification using Strep-Tactin-Sepharose beads and subjected to MS analysis as described in Material and Methods.E Histograms showing the numbers of specific interactors binding to the CBL-OST or CBLB-OST proteins prior to and after TCR stimulation for different times and for which P(Detection) >0.9.


Figure 5D , E , F , G , H Experimental validation of the PPIs predicted on the basis of the high confidence CN shown in C . CD4 + T cells were left unstimulated ( - ) or stimulated ( + ) with anti - CD3 and anti - CD4 antibodies and subsequently lysed . Equal amounts of cell lysates were incubated with isotype control ( Iso Ctrl ) or the specified antibodies and the resulting immunoprecipitates ( IP ) analyzed by immunoblot with the antibodies specified in the left margin . The validated PPIs predicted in C correspond to CSK - CBLB and CSK - CD5 ( D ) , CRKL - PI3Kp85β and CSK - PI3Kp85β ( E ) . Molecular masses are shown ( kDa ) . Data are representative of at least 2 experiments . D , E , F , G , H Experimental validation of the PPIs predicted on the basis of the high confidence CN shown in C . CD4 + T cells were left unstimulated ( - ) or stimulated ( + ) with anti - CD3 and anti - CD4 antibodies and subsequently lysed . Equal amounts of cell lysates were incubated with isotype control ( Iso Ctrl ) or the specified antibodies and the resulting immunoprecipitates ( IP ) analyzed by immunoblot with the antibodies specified in the left margin . The validated PPIs predicted in C correspond to PI3Kp85β - PI3Kp110α , CSK - PI3Kp110α ( F ) CRKL - PI3Kp110α ( G ) . Molecular masses are shown ( kDa ) . Data are representative of at least 2 experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5D, E, F, G, H Experimental validation of the PPIs predicted on the basis of the high confidence CN shown in C. CD4+T cells were left unstimulated (-) or stimulated (+) with anti-CD3 and anti-CD4 antibodies and subsequently lysed. Equal amounts of cell lysates were incubated with isotype control (Iso Ctrl) or the specified antibodies and the resulting immunoprecipitates (IP) analyzed by immunoblot with the antibodies specified in the left margin. The validated PPIs predicted in C correspond to CSK-CBLB and CSK-CD5 (D), CRKL-PI3Kp85β and CSK- PI3Kp85β (E). Molecular masses are shown (kDa). Data are representative of at least 2 experiments.D, E, F, G, H Experimental validation of the PPIs predicted on the basis of the high confidence CN shown in C. CD4+T cells were left unstimulated (-) or stimulated (+) with anti-CD3 and anti-CD4 antibodies and subsequently lysed. Equal amounts of cell lysates were incubated with isotype control (Iso Ctrl) or the specified antibodies and the resulting immunoprecipitates (IP) analyzed by immunoblot with the antibodies specified in the left margin. The validated PPIs predicted in C correspond to PI3Kp85β-PI3Kp110α, CSK-PI3Kp110α (F) CRKL-PI3Kp110α (G). Molecular masses are shown (kDa). Data are representative of at least 2 experiments.


Figure 6A CD4 + T cells from wild - type ( WT ) , CBLOST and CBLBOSTmice were left unstimulated ( - ) or stimulated for 1 min with anti - CD3 and anti - CD4 antibodies ( + ) . Equal amounts of cell lysates were subjected to affinity purification on Strep - Tactin - Sepharose beads and the resulting protein eluates were analyzed by immunoblotting with anti - ubiquitin ( UB ) , anti - CBLB or anti - CBL antibodies . Also shown are a loading control corresponding to equal amounts of proteins from whole cell lysates ( WCL ) probed with anti - CD5 , and molecular masses ( kDa ) . TCR stimulation resulted in some CBLB degradation and in the detection of a higher molecular weight CBLB form , likely due to its ubiquitylation ( marked with an asterisk ) . In contrast , no degradation of CBL was detectable upon TCR stimulation and its molecular weight remained unchanged . Molecular masses are shown ( kDa ) . Data are representative of at least 2 experiments . C Immunoprecipitation of CBL from cell lysates of CD4 + T cells isolated from WT or CBLB - deficient mouse ( Cblb - / - ) and kept unstimulated ( - ) or stimulated as in ( A ) . Immunoprecipitates and whole cell lystates ( WCLs ) were analyzed by immunoblot with antibodies specific for the proteins specified in the left margin . Molecular masses are shown ( kDa ) . Data are representative of at least 2 experiments . D Immunoprecipitation of CBLB from cell lysates of CD4 + T cells isolated from WT or CBL - deficient mouse ( Cbl - / - ) and kept unstimulated ( - ) or stimulated as in ( A ) . Immunoprecipitates and whole cell lystates ( WCLs ) were analyzed by immunoblot with antibodies specific for the proteins specified in the left margin . Molecular masses are shown ( kDa ) . Data are representative of at least 3 experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A CD4+T cells from wild-type (WT), CBLOST and CBLBOSTmice were left unstimulated (-) or stimulated for 1 min with anti-CD3 and anti-CD4 antibodies (+). Equal amounts of cell lysates were subjected to affinity purification on Strep-Tactin-Sepharose beads and the resulting protein eluates were analyzed by immunoblotting with anti-ubiquitin (UB), anti-CBLB or anti-CBL antibodies. Also shown are a loading control corresponding to equal amounts of proteins from whole cell lysates (WCL) probed with anti-CD5, and molecular masses (kDa). TCR stimulation resulted in some CBLB degradation and in the detection of a higher molecular weight CBLB form, likely due to its ubiquitylation (marked with an asterisk). In contrast, no degradation of CBL was detectable upon TCR stimulation and its molecular weight remained unchanged. Molecular masses are shown (kDa). Data are representative of at least 2 experiments.C Immunoprecipitation of CBL from cell lysates of CD4+T cells isolated from WT or CBLB-deficient mouse (Cblb-/-) and kept unstimulated (-) or stimulated as in (A). Immunoprecipitates and whole cell lystates (WCLs) were analyzed by immunoblot with antibodies specific for the proteins specified in the left margin. Molecular masses are shown (kDa). Data are representative of at least 2 experiments.D Immunoprecipitation of CBLB from cell lysates of CD4+T cells isolated from WT or CBL-deficient mouse (Cbl-/-) and kept unstimulated (-) or stimulated as in (A). Immunoprecipitates and whole cell lystates (WCLs) were analyzed by immunoblot with antibodies specific for the proteins specified in the left margin. Molecular masses are shown (kDa). Data are representative of at least 3 experiments.


Figure 7A , B CD4 + T cells from wild - type , Cblb - / - and Cd5 - / - mice were stimulated for 1 min with anti - CD3 and anti - CD4 antibodies ( + ) . CBL ( A ) were immunoprecipitated from equal amounts of protein lysates . Level of ubiquitylation and amount of CD5 associated proteins were evaluated by immunoblot with antibodies specific for the proteins specified in the left margin . Whole cell lystates ( WCLs ) were also analyzed with the specified antibodies . Molecular masses are shown ( kDa ) . Data are representative of at least 2 experiments . A , B CD4 + T cells from wild - type , Cblb - / - and Cd5 - / - mice were stimulated for 1 min with anti - CD3 and anti - CD4 antibodies ( + ) . PI3KR1 ( PI3Kp85α ) ( B ) were immunoprecipitated from equal amounts of protein lysates . Level of ubiquitylation and amount of CD5 associated proteins were evaluated by immunoblot with antibodies specific for the proteins specified in the left margin . Whole cell lystates ( WCLs ) were also analyzed with the specified antibodies . Molecular masses are shown ( kDa ) . Data are representative of at least 2 experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A, B CD4+T cells from wild-type, Cblb-/- and Cd5-/- mice were stimulated for 1 min with anti-CD3 and anti-CD4 antibodies (+). CBL (A) were immunoprecipitated from equal amounts of protein lysates. Level of ubiquitylation and amount of CD5 associated proteins were evaluated by immunoblot with antibodies specific for the proteins specified in the left margin. Whole cell lystates (WCLs) were also analyzed with the specified antibodies. Molecular masses are shown (kDa). Data are representative of at least 2 experiments.A, B CD4+T cells from wild-type, Cblb-/- and Cd5-/- mice were stimulated for 1 min with anti-CD3 and anti-CD4 antibodies (+). PI3KR1 (PI3Kp85α) (B) were immunoprecipitated from equal amounts of protein lysates. Level of ubiquitylation and amount of CD5 associated proteins were evaluated by immunoblot with antibodies specific for the proteins specified in the left margin. Whole cell lystates (WCLs) were also analyzed with the specified antibodies. Molecular masses are shown (kDa). Data are representative of at least 2 experiments.


Figure 1D . The model proteins consist of a fusion between Ura3p and GFP ( URA3sol , left ) and the amyloid - β - peptide of 42 residues ( Aβ ) ( URA3agg , right ) . After 18 hours of expression Ura3psol remains homogeneously distributed whereas Ura3pagg is accumulated into intracellular foci .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1D. The model proteins consist of a fusion between Ura3p and GFP (URA3sol, left) and the amyloid-β-peptide of 42 residues (Aβ) (URA3agg, right). After 18 hours of expression Ura3psol remains homogeneously distributed whereas Ura3pagg is accumulated into intracellular foci.


Figure 2S values measured in different environments . In all cases the standard error is below 5 % . Color scale : purple in absence of uracil ( - Ura ) , white in the presence of uracil ( + Ura ) and orange in the presence of 5FOA ( + 5FOA ) . At 37 ° C , the heat stress together with the inability to fold Ura3p have such a strong fitness effect that the strains barely grew in absence of uracil , impeding the measurement of S . A bar of purple - white diagonal lines indicates a presumed value of S at 37 ° C without uracil .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2S values measured in different environments. In all cases the standard error is below 5%. Color scale: purple in absence of uracil (-Ura), white in the presence of uracil (+Ura) and orange in the presence of 5FOA (+5FOA). At 37°C, the heat stress together with the inability to fold Ura3p have such a strong fitness effect that the strains barely grew in absence of uracil, impeding the measurement of S. A bar of purple-white diagonal lines indicates a presumed value of S at 37°C without uracil.


Figure 3B . Plots showing the distribution and relationship between the fractions of deposited or free Ura3p , its magnitude of effect on fitness ( α / β / γ ) and the selection coefficient ( S ) in different environments . C . Average of the selection coefficient ( left ) and average of the cytosolic fluorescence ( equivalent to the fractional abundance of the free protein in URA3agg , right ) .

O B-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B. Plots showing the distribution and relationship between the fractions of deposited or free Ura3p, its magnitude of effect on fitness (α/β/γ) and the selection coefficient (S) in different environments.C. Average of the selection coefficient (left) and average of the cytosolic fluorescence (equivalent to the fractional abundance of the free protein in URA3agg, right).


Figure 5Panels from A to D show that the variability in foci formation contributes to variability in free protein abundance . A . Confocal microscopy images of URA3agg and URA3sol incubated for 18 hours at 30 ° C . B . Coefficient of variation and p - values ( Wilcoxon test ) of the total fluorescence measured from populations of URA3agg and URA3sol . C . Boxplot showing the distribution of foci fluorescence ( left ) or cytoplasmic fluorescence ( right ) , from the foci containing subset of a population of URA3agg grown at 30ºC . Coefficient of Variation and p - values of each boxplot are shown below . D . Schematic showing how the formation of protein deposit can affect the abundance of free protein in a cell . Pie charts showing the average fraction of deposited and free protein of URA3agg in different media composition and 30ºC . E . Confocal microscopy image of URA3agg incubated in the presence of 5FOA after 18 hours at 30 ° C . F . Schematic showing that in our system , the phase separation - promoting region can modulate Ura3p activity and thus its phenotypic effect in a cell . G . Schematic highlighting that higher / lower level of free protein inside the cell results in higher / lower Ura3p activity leading to the manifestation of a stronger / weaker phenotype . H . Boxplots of the cytoplasmic fluorescence normalized by the cell area ( FCYTOagg / cell area ) for individual cells of the URA3agg population in 5FOA . Light green box , cells with foci containing at least 70 % of the total fluorescence ( big foci ) . Dark green box , cells with foci containing less than 70 % of the total fluorescence ( small foci ) . I - J . Cell cycle arrest phenotype . Density plots showing the fluorescence distribution of individual cells from the URA3agg population stained with propidium iodide to measure their DNA content . The cells were incubated for 18 hours at 30 ° C without ( I ) and with 5FOA ( J ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Panels from A to D show that the variability in foci formation contributes to variability in free protein abundance. A. Confocal microscopy images of URA3agg and URA3sol incubated for 18 hours at 30°C. B. Coefficient of variation and p-values (Wilcoxon test) of the total fluorescence measured from populations of URA3agg and URA3sol. C. Boxplot showing the distribution of foci fluorescence (left) or cytoplasmic fluorescence (right), from the foci containing subset of a population of URA3agg grown at 30ºC. Coefficient of Variation and p-values of each boxplot are shown below. D. Schematic showing how the formation of protein deposit can affect the abundance of free protein in a cell. Pie charts showing the average fraction of deposited and free protein of URA3agg in different media composition and 30ºC. E. Confocal microscopy image of URA3agg incubated in the presence of 5FOA after 18 hours at 30°C. F. Schematic showing that in our system, the phase separation-promoting region can modulate Ura3p activity and thus its phenotypic effect in a cell. G. Schematic highlighting that higher/lower level of free protein inside the cell results in higher/lower Ura3p activity leading to the manifestation of a stronger/weaker phenotype. H. Boxplots of the cytoplasmic fluorescence normalized by the cell area (FCYTOagg/cell area) for individual cells of the URA3agg population in 5FOA. Light green box, cells with foci containing at least 70% of the total fluorescence (big foci). Dark green box, cells with foci containing less than 70% of the total fluorescence (small foci). I-J. Cell cycle arrest phenotype. Density plots showing the fluorescence distribution of individual cells from the URA3agg population stained with propidium iodide to measure their DNA content. The cells were incubated for 18 hours at 30°C without (I) and with 5FOA (J).


Figure 1 ( D , E ) Kaplan - Meier curve analysis was conducted to evaluate the relapse - free survival or overall survival probabilities of colon cancer ( n = 1342 ) or lung cancer ( n = 2166 ) patients based on low or high levels of FBL expression . Data were sourced from an online tool website ( https : / / kmplot . com / analysis / ) . The lower quartile - based partitioning method was used to select upper and lower thresholds to subset tumor samples into low and high groups . A cut - off value of the 25th percentile was defined as the low level of FBL in colon or lung cancer patients . ( F ) Representative images of immunohistochemical analysis of FBL expression levels in human lung or colon tumors and their respective normal tissues ( n = 40 ) . Scale bar , 25 μm . ( H , I ) Enzymatic activity of FBL is not required for cell proliferation . Colony formation assays were conducted to investigate the role of enzymatic activity in cell proliferation . FBL knockdown HCT116 or A549 cells with ectopic expression of FBL or the catalytically inactive mutant ( FBL - E191A / D236A ) were used , with a bar graph showing the relative number of colonies per well ( mean ± SD ) . All cells were allowed to form colonies for 10 days and representative images of colonies in plates stained with Giemsa were displayed . The bar graphs present data as mean ± SD from three independent experiments . The statistical significance was determined using one - way ANOVA . ns denotes no statistical significance , * indicates P < 0 . 05 , * * * indicates P < 0 . 001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(D, E) Kaplan-Meier curve analysis was conducted to evaluate the relapse-free survival or overall survival probabilities of colon cancer (n=1342) or lung cancer (n=2166) patients based on low or high levels of FBL expression. Data were sourced from an online tool website (https://kmplot.com/analysis/). The lower quartile-based partitioning method was used to select upper and lower thresholds to subset tumor samples into low and high groups. A cut-off value of the 25th percentile was defined as the low level of FBL in colon or lung cancer patients.(F) Representative images of immunohistochemical analysis of FBL expression levels in human lung or colon tumors and their respective normal tissues (n=40). Scale bar, 25 μm.(H, I) Enzymatic activity of FBL is not required for cell proliferation. Colony formation assays were conducted to investigate the role of enzymatic activity in cell proliferation. FBL knockdown HCT116 or A549 cells with ectopic expression of FBL or the catalytically inactive mutant (FBL-E191A/ D236A) were used, with a bar graph showing the relative number of colonies per well (mean ± SD). All cells were allowed to form colonies for 10 days and representative images of colonies in plates stained with Giemsa were displayed. The bar graphs present data as mean ± SD from three independent experiments. The statistical significance was determined using one-way ANOVA. ns denotes no statistical significance, * indicates P < 0.05, *** indicates P < 0.001.


Figure 2 ( A , B ) Knockdown of FBL results in increased cellular sensitivity to DNA damage treatment . Cell viability analysis was performed on control ( shNC ) and shFBL - HCT116 / shFBL - A549 cells treated with indicated concentrations of 5 μM MMC using CCK - 8 assays . Each data point represents the mean ± SD from three replicates . The statistical significance was determined using Student ' s t - test . * * * p < 0 . 001 . ( D - F ) Immunofluorescence staining showed the formation of γH2AX and BRCA1 foci in shFBL and shNC HCT116 cells treated with 5 μM MMC or mock treatment . The scale bar represents 10 μm . The ( E ) and ( F ) panel displays quantification of the ( D ) panel : indicating the number of γH2AX or BRCA1 foci per cell ( n ≥ 100 cells ) . Data are shown as the mean ± SD . The statistical significance was determined using one - way ANOVA , with ns indicating no statistical significance , * * * indicating P < 0 . 001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A, B) Knockdown of FBL results in increased cellular sensitivity to DNA damage treatment. Cell viability analysis was performed on control (shNC) and shFBL-HCT116/shFBL-A549 cells treated with indicated concentrations of 5 μM MMC using CCK-8 assays. Each data point represents the mean ± SD from three replicates. The statistical significance was determined using Student's t-test. ***p < 0.001.(D-F) Immunofluorescence staining showed the formation of γH2AX and BRCA1 foci in shFBL and shNC HCT116 cells treated with 5 μM MMC or mock treatment. The scale bar represents 10 μm. The (E) and (F) panel displays quantification of the (D) panel: indicating the number of γH2AX or BRCA1 foci per cell (n≥100 cells). Data are shown as the mean ± SD. The statistical significance was determined using one-way ANOVA, with ns indicating no statistical significance, *** indicating P < 0.001.


Figure 3 ( B ) . Co - IP analyses of GFP - tagged FBL and SFB - tagged YBX1 in HCT116 cells using indicated antibodies . HCT116 cells were transfected with vectors encoding GFP - FBL and SFB - YBX1 . Cells were lyzed with NETN300 buffer . Cell extracts were examined by IP and western blot with indicated antibodies . ( C ) . Co - IP analyses of endogenous FBL and YBX1 in HCT116 cells using indicated antibodies . Cells were lyzed with NETN300 buffer , and lysates were examined by IP and western blot with indicated antibodies . GAPDH was used as the protein - loading control . ( E ) The N - terminal GAR domain is essential for the interaction between FBL and YBX1 . Upper panel , a schematic representation of wild - type FBL with the GAR domain and methyltransferase domain ( MTase ) . Lower panel , Co - IP analyses of GFP - tagged FBL deletion mutants and SFB - tagged YBX1 in HCT116 cells . ( F ) The B / A repeat domain of YBX1 is essential for the interaction between YBX1 and FBL . Upper panel , a schematic representation of wild - type YBX1 with the Alanine / proline rich domain , Cold shock domain and B / A repeat domain . Lower panel , co - IP analyses of GFP - tagged YBX1 deletion mutants and SFB - tagged FBL in HCT116 cells .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(B). Co-IP analyses of GFP- tagged FBL and SFB-tagged YBX1 in HCT116 cells using indicated antibodies. HCT116 cells were transfected with vectors encoding GFP-FBL and SFB-YBX1. Cells were lyzed with NETN300 buffer. Cell extracts were examined by IP and western blot with indicated antibodies.(C). Co-IP analyses of endogenous FBL and YBX1 in HCT116 cells using indicated antibodies. Cells were lyzed with NETN300 buffer, and lysates were examined by IP and western blot with indicated antibodies. GAPDH was used as the protein-loading control.(E) The N-terminal GAR domain is essential for the interaction between FBL and YBX1. Upper panel, a schematic representation of wild-type FBL with the GAR domain and methyltransferase domain (MTase). Lower panel, Co-IP analyses of GFP-tagged FBL deletion mutants and SFB-tagged YBX1 in HCT116 cells.(F) The B/A repeat domain of YBX1 is essential for the interaction between YBX1 and FBL. Upper panel, a schematic representation of wild-type YBX1 with the Alanine/proline rich domain, Cold shock domain and B/A repeat domain. Lower panel, co-IP analyses of GFP-tagged YBX1 deletion mutants and SFB-tagged FBL in HCT116 cells.


Figure 4 ( B ) HCT116 cells were treated with or without 5 μM MMC overnight or 25 μM cisplatin for 2 hours , cells were then harvested and separated into nuclear and cytoplasmic fractions . The protein level of YBX1 in each fraction was detected by Western blotting assays . GAPDH and Histone 3 ( H3 ) were used as loading controls for cytoplasmic and nuclear fractions , respectively . ( C ) Subcellular localization of YBX1 / FBL was confirmed by immunofluorescence staining following MMC ( 5 μM , overnight ) or cisplatin ( 25 μM , 2 hours ) treatment in shFBL and shNC cells . Scale bar , 10 μm . ( D ) The endogenous association between FBL and YBX1 in HCT116 cells was examined by co - immunoprecipitation assays with 5 μM MMC treatment . Cells were lyzed with NETN300 buffer , and nuclear lysates were examined by IP and western blot with indicated antibodies . GAPDH was used as the protein - loading control . ( E ) The distribution of YBX1 proteins in the nuclear and cytoplasmic fractions of control or FBL KD HCT116 cells after 5 μM MMC incubation overnight were determined by western blotting assays . H3 was used as a nuclear control . GAPDH was used as a cytoplasmic control . ( F ) The protein contents of YBX1 in the nuclear and cytoplasmic fractions in FBL KD HCT116 cells reconstituted with wild - type FBL or deletion mutants of FBL with 5 μM MMC treatment overnight were detected by western blotting assays with indicated antibodies .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(B) HCT116 cells were treated with or without 5 μM MMC overnight or 25 μM cisplatin for 2 hours, cells were then harvested and separated into nuclear and cytoplasmic fractions. The protein level of YBX1 in each fraction was detected by Western blotting assays. GAPDH and Histone 3 (H3) were used as loading controls for cytoplasmic and nuclear fractions, respectively.(C) Subcellular localization of YBX1/FBL was confirmed by immunofluorescence staining following MMC (5 μM, overnight) or cisplatin (25 μM, 2 hours) treatment in shFBL and shNC cells. Scale bar, 10 μm.(D) The endogenous association between FBL and YBX1 in HCT116 cells was examined by co-immunoprecipitation assays with 5 μM MMC treatment. Cells were lyzed with NETN300 buffer, and nuclear lysates were examined by IP and western blot with indicated antibodies. GAPDH was used as the protein-loading control.(E) The distribution of YBX1 proteins in the nuclear and cytoplasmic fractions of control or FBL KD HCT116 cells after 5 μM MMC incubation overnight were determined by western blotting assays. H3 was used as a nuclear control. GAPDH was used as a cytoplasmic control.(F) The protein contents of YBX1 in the nuclear and cytoplasmic fractions in FBL KD HCT116 cells reconstituted with wild-type FBL or deletion mutants of FBL with 5 μM MMC treatment overnight were detected by western blotting assays with indicated antibodies.


Figure 5 ( A ) A Volcano plot of differentially expressed gene analysis results for RNA - seq data comparing FBL KD HCT116 cells ( n = 3 ) to control cells ( n = 3 ) . The dots for significant upregulated or downregulated genes were colored in red or blue , respectively . ( E ) The mRNA and protein levels of BRCA1 and RAD51 were validated by RT - qPCR and western blot assays using FBL KD HCT116 cells or control cells . Ct values were normalized to GAPDH . The bar graphs present data as mean ± SD from three independent experiments . The statistical significance was determined using Student ' s t - test . * * * P < 0 . 001 . ( F ) The mRNA and protein levels of BRCA1 were validated by RT - qPCR and Western blotting assays using YBX1 - silenced HCT116 cells or control cells . Ct values were normalized to GAPDH . The bar graphs present data as mean ± SD from three independent experiments . The statistical significance was determined using Student ' s t - test . * * * P < 0 . 001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) A Volcano plot of differentially expressed gene analysis results for RNA-seq data comparing FBL KD HCT116 cells (n = 3) to control cells (n = 3). The dots for significant upregulated or downregulated genes were colored in red or blue, respectively.(E) The mRNA and protein levels of BRCA1 and RAD51 were validated by RT-qPCR and western blot assays using FBL KD HCT116 cells or control cells. Ct values were normalized to GAPDH. The bar graphs present data as mean ± SD from three independent experiments. The statistical significance was determined using Student's t-test. ***P < 0.001.(F) The mRNA and protein levels of BRCA1 were validated by RT-qPCR and Western blotting assays using YBX1-silenced HCT116 cells or control cells. Ct values were normalized to GAPDH. The bar graphs present data as mean ± SD from three independent experiments. The statistical significance was determined using Student's t-test. ***P < 0.001.


Figure 6 ( C ) HE staining of tumor tissues . Scale bar , 50 µm . ( D Representative images of immunohistochemical staining showing BRCA1 and γH2AX expression levels in MMC - treated and untreated xenograft tumors driven by control or FBL KD HCT116 cells . panel displays quantification of the ( D ) panel : showing BRCA1 and γH2AX expression levels . Scale bar , 25 µm . Graphs represent the mean ± SD ( n = 3 mice in each group ) . The statistical significance was determined using one - way ANOVA . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . ( F , G ) Immunofluorescence for TUNEL staining of tumor tissues . Quantification of the percentage of apoptotic cells is shown on the ( G ) panel . Scale bar , 50 µm . Graphs represent the mean ± SD ( n = 3 mice in each group ) . The statistical significance was determined using one - way ANOVA . * P < 0 . 05 , * * * P < 0 . 001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(C) HE staining of tumor tissues. Scale bar, 50 µm. (D Representative images of immunohistochemical staining showing BRCA1 and γH2AX expression levels in MMC-treated and untreated xenograft tumors driven by control or FBL KD HCT116 cells. panel displays quantification of the (D) panel: showing BRCA1 and γH2AX expression levels. Scale bar, 25 µm. Graphs represent the mean ± SD (n = 3 mice in each group). The statistical significance was determined using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.(F, G) Immunofluorescence for TUNEL staining of tumor tissues. Quantification of the percentage of apoptotic cells is shown on the (G) panel. Scale bar, 50 µm. Graphs represent the mean ± SD (n = 3 mice in each group). The statistical significance was determined using one-way ANOVA. *P < 0.05, ***P < 0.001.


Figure 1 ( A ) HEK293 cells were transfected with DAPK ΔCaM or with a control vector ( pcDNA3 - luciferase , LUC ) , together with shRNAs targeting beclin 1 or HcRed , and with GFP - LC3 plasmid . After 72 h , cells were counted and lysates were prepared . ( A ) The percentage of cells with punctate GFP - LC3 fluorescence per total GFP - LC3 - positive cells was quantified . Data presented are the mean ± s . d . from a triplicate of 100 transfected cells . The asterisks denote a significance level of P = 0 . 001 . ( B ) Western blot analysis was performed using the indicated antibodies . ( C ) Representative GFP - LC3 staining of cells transfected with the control shRNA ( HcRed ) together with pcDNA3 ‐ luciferase or DAPKΔCaM . CaM , calmodulin ; DAPK , death ‐ associated protein kinase ; GFP , green fluorescent protein ; HEK , human embryonic kidney ; shRNA , short hairpin RNA .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) HEK293 cells were transfected with DAPK ΔCaM or with a control vector (pcDNA3-luciferase, LUC), together with shRNAs targeting beclin 1 or HcRed, and with GFP-LC3 plasmid. After 72 h, cells were counted and lysates were prepared. (A) The percentage of cells with punctate GFP-LC3 fluorescence per total GFP-LC3-positive cells was quantified. Data presented are the mean±s.d. from a triplicate of 100 transfected cells. The asterisks denote a significance level of P=0.001.(B) Western blot analysis was performed using the indicated antibodies.(C) Representative GFP-LC3 staining of cells transfected with the control shRNA (HcRed) together with pcDNA3‐luciferase or DAPKΔCaM. CaM, calmodulin; DAPK, death‐associated protein kinase; GFP, green fluorescent protein; HEK, human embryonic kidney; shRNA, short hairpin RNA.


Figure 2 ( A ) Flag ‐ tagged DAPK ( 100 ng ) was incubated with GST ( 900 ng ) or GST - beclin ‐ 1 ( 750 ng ) in the presence of Ca2 + , calmodulin and [ γ ‐ 33P ] ATP for 30 min or 60 min . Phosphorylated proteins were visualized by X ‐ ray film exposure , and GST / GST - beclin ‐ 1 levels were visualized by Ponceau S staining . The autophosphorylation of DAPK indicates that its catalytic activity was intact in all samples . ( B ) Flag ‐ tagged DAPK ( 60 ng ) was incubated with Flag ‐ tagged beclin 1 ( 250 ng ) , which was purified from HEK293T cells , and a kinase assay was performed for 60 min . Where indicated ( + LiCl ) , beclin ‐ 1 ‐ bound beads were first washed stringently in 0 . 5 M LiCl and 0 . 5 M KCl . Phosphorylated proteins were visualized by X ‐ ray film exposure , and the levels of beclin 1 were visualized by Western blot analysis using beclin 1 antibodies . DAPK , death ‐ associated protein kinase ; GST , glutathione S ‐ transferase ; HEK , human embryonic kidney .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Flag‐tagged DAPK (100 ng) was incubated with GST (900 ng) or GST-beclin‐1 (750 ng) in the presence of Ca2+, calmodulin and [γ‐33P]ATP for 30 min or 60 min. Phosphorylated proteins were visualized by X‐ray film exposure, and GST/GST-beclin‐1 levels were visualized by Ponceau S staining. The autophosphorylation of DAPK indicates that its catalytic activity was intact in all samples.(B) Flag‐tagged DAPK (60 ng) was incubated with Flag‐tagged beclin 1 (250 ng), which was purified from HEK293T cells, and a kinase assay was performed for 60 min. Where indicated (+LiCl), beclin‐1‐bound beads were first washed stringently in 0.5 M LiCl and 0.5 M KCl. Phosphorylated proteins were visualized by X‐ray film exposure, and the levels of beclin 1 were visualized by Western blot analysis using beclin 1 antibodies. DAPK, death‐associated protein kinase; GST, glutathione S‐transferase; HEK, human embryonic kidney.


Figure 3 ( Aa ) Protein extracts from COS7 cells , HEK293T cells , or no extracts were added to bacterially produced GST or GST - beclin ‐ 1 . The pulled ‐ down proteins , as well as the total cell extracts , were blotted with the indicated antibodies . ( Ab ) Ponceau S staining of GST and GST - beclin ‐ 1 to which the extracts were added . ( B ) HEK293 cells were co ‐ transfected with Flag ‐ tagged beclin 1 or with Flag ‐ tagged beclin 1 lacking the Bcl ‐ 2 ‐ binding domain ( ΔBD ) together with Bcl ‐ XL and HA ‐ tagged DAPK . Beclin 1 was immunoprecipitated using Flag antibodies , and the co ‐ immunoprecipitated proteins , as well as the total cell extracts , were blotted with DAPK , Bcl ‐ XL and beclin 1 antibodies . ( C ) HEK293 cells were transfected with Flag - beclin ‐ 1 or Flag - GFP , and the Flag ‐ tagged proteins were immunoprecipitated using Flag antibodies , and eluted with an excess of Flag peptides . The blot was reacted with DAPK antibodies or with Flag antibodies at different time

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(Aa) Protein extracts from COS7 cells, HEK293T cells, or no extracts were added to bacterially produced GST or GST-beclin‐1. The pulled‐down proteins, as well as the total cell extracts, were blotted with the indicated antibodies. (Ab) Ponceau S staining of GST and GST-beclin‐1 to which the extracts were added.(B) HEK293 cells were co‐transfected with Flag‐tagged beclin 1 or with Flag‐tagged beclin 1 lacking the Bcl‐2‐binding domain (ΔBD) together with Bcl‐XL and HA‐tagged DAPK. Beclin 1 was immunoprecipitated using Flag antibodies, and the co‐immunoprecipitated proteins, as well as the total cell extracts, were blotted with DAPK, Bcl‐XL and beclin 1 antibodies.(C) HEK293 cells were transfected with Flag-beclin‐1 or Flag-GFP, and the Flag‐tagged proteins were immunoprecipitated using Flag antibodies, and eluted with an excess of Flag peptides. The blot was reacted with DAPK antibodies or with Flag antibodies at different time exposures