Figure 2A . LITATS1 expression in different breast cells as measured by RT ‒ qPCR . Results from epithelial - like and mesenchymal - like cells are labeled in blue and green , respectively . Representative results from two independent experiments are shown . C . Quantification of LITATS1 expression levels by in situ hybridization in lung adenocarcinoma tissue microarrays . Representative images ( bar = 100 μm ) and zoomed images ( bar = 20 μm ) of in situ hybridization results in lung adenocarcinoma and matched adjacent normal tissues . The comparison of the LITATS1 staining index between the paired tissues is shown in the right panel . Tissue pairs with higher LITATS1 expression in the normal tissue ( normal ) than in the lung adenocarcinoma tissue ( tumor ) are highlighted in red , whereas tissue pairs with lower LITATS1 expression in the normal tissue than in the tumor tissue are highlighted in green . D . Kaplan ‒ Meier survival curves of relapse - free survival in 175 breast cancer patients stratified by LITATS1 expression . LITATS1 expression was measured by in situ hybridization in breast cancer tissue microarrays . Kaplan ‒ Meier survival curves of overall survival ( E ) in breast cancer patients stratified by LITATS1 expression . The data were generated via Kaplan ‒ Meier PlotterKaplan ‒ Meier survival curves of distant metastasis - free survival ( F ) and relapse - free survival ( G ) in breast cancer patients and overall survival ( H ) in non - small cell lung cancer patients stratified by LITATS1 expression . The data were generated via Kaplan ‒ Meier

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 O O O O O O O O O O O O O O O O O O O O O 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 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 2A. LITATS1 expression in different breast cells as measured by RT‒qPCR. Results from epithelial-like and mesenchymal-like cells are labeled in blue and green, respectively. Representative results from two independent experiments are shown.C. Quantification of LITATS1 expression levels by in situ hybridization in lung adenocarcinoma tissue microarrays. Representative images (bar=100 μm) and zoomed images (bar=20 μm) of in situ hybridization results in lung adenocarcinoma and matched adjacent normal tissues. The comparison of the LITATS1 staining index between the paired tissues is shown in the right panel. Tissue pairs with higher LITATS1 expression in the normal tissue (normal) than in the lung adenocarcinoma tissue (tumor) are highlighted in red, whereas tissue pairs with lower LITATS1 expression in the normal tissue than in the tumor tissue are highlighted in green.D. Kaplan‒Meier survival curves of relapse-free survival in 175 breast cancer patients stratified by LITATS1 expression. LITATS1 expression was measured by in situ hybridization in breast cancer tissue microarrays.Kaplan‒Meier survival curves of overall survival (E) in breast cancer patients stratified by LITATS1 expression. The data were generated via Kaplan‒Meier PlotterKaplan‒Meier survival curves of distant metastasis-free survival (F) and relapse-free survival (G) in breast cancer patients and overall survival (H) in non-small cell lung cancer patients stratified by LITATS1 expression. The data were generated via Kaplan‒Meier Plotter


Figure 3Effect of LITATS1 on TGF - β - induced EMT marker expression in MCF10A - M2 upon CRISPRa - mediated LITATS1 overexpression ( A ) GAPDH loading control . Representative results from a minimum of three independent experiments are shown . Effect of LITATS1 on TGF - β - induced EMT marker expression in MCF10A - M2 upon shRNA - mediated knockdown ( B ) . α / β - Tubulin , loading control . The results of LITATS1 overexpression Representative results from a minimum of three independent experiments are shown . D , E . An IncuCyte chemotactic migration assay was performed to evaluate the effect of LITATS1 ectopic expression ( D ) or knockdown ( E ) on TGF - β - induced MDA - MB - 231 cell migration . The results of LITATS1 overexpression and E . Representative results from two independent experiments are shown . F , G . In vivo zebrafish extravasation experiments with MDA - MB - 231 cells upon ectopic LITATS1 expression ( F ) or LITATS1 knockdown ( G ) . Representative zoomed images of the tail fin area are shown in the left panels . Extravasated breast cancer cell clusters are indicated with yellow arrows . Analysis of the extravasated cell cluster numbers in the indicated groups is shown in the right panel . Whole zebrafish image , bar = 309 . 4 μm ; zoomed image , bar = 154 . 7 μm . Representative results from two independent experiments are shown .

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 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 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

Figure 3Effect of LITATS1 on TGF-β-induced EMT marker expression in MCF10A-M2 upon CRISPRa-mediated LITATS1 overexpression (A) GAPDH loading control. Representative results from a minimum of three independent experiments are shown.Effect of LITATS1 on TGF-β-induced EMT marker expression in MCF10A-M2 upon shRNA-mediated knockdown (B). α/β-Tubulin, loading control. The results of LITATS1 overexpression Representative results from a minimum of three independent experiments are shown.D, E. An IncuCyte chemotactic migration assay was performed to evaluate the effect of LITATS1 ectopic expression (D) or knockdown (E) on TGF-β-induced MDA-MB-231 cell migration. The results of LITATS1 overexpression and E. Representative results from two independent experiments are shown.F, G. In vivo zebrafish extravasation experiments with MDA-MB-231 cells upon ectopic LITATS1 expression (F) or LITATS1 knockdown (G). Representative zoomed images of the tail fin area are shown in the left panels. Extravasated breast cancer cell clusters are indicated with yellow arrows. Analysis of the extravasated cell cluster numbers in the indicated groups is shown in the right panel. Whole zebrafish image, bar=309.4 μm; zoomed image, bar=154.7 μm. Representative results from two independent experiments are shown.


Figure 4C . Expression of TGF - β target genes ( as measured by RT ‒ qPCR ) in MDA - MB - 231 cells without ( Co . sh ) or with ( sh # 1 and sh # 2 ) LITATS1 depletion . Cells were serum starved for 16 h and treated with or without TGF - β for 4 h . Representative results from a minimum of three independent experiments are shown . D . Effect of LITATS1 knockdown on TGF - β - induced SMAD2 phosphorylation in MDA - MB - 231 cells . Cells were serum starved for 16 h and stimulated with TGF - β for the indicated durations . The p - SMAD2 and total SMAD2 ( t - SMAD2 ) levels were analyzed by western blotting . GAPDH , loading control . Representative results from a minimum of three independent experiments are shown . E . Effect of LITATS1 knockdown on E - cadherin , N - cadherin and SNAIL expression in A549 cells . Cells were stimulated with vehicle control ( - ) , SB431542 ( SB ; 10 μM ) or TGF - β ( Τβ ) for 24 h , and protein expression was analyzed by western blotting . α / β - Tubulin , loading control . Representative results from a minimum of three independent experiments are shown . FH . In vivo zebrafish extravasation experiments with MDA - MB - 231 cells upon LITATS1 knockdown and blockage of TGF - β signaling . Representative zoomed images of the tail fin area are shown in the left panels . Extravasated breast cancer cell clusters are indicated with yellow arrows . Analysis of the extravasated cell cluster numbers in the indicated groups is shown in the right panel . Whole zebrafish image , bar = 618 . 8 μm ; zoomed image , scale bar = 154 . 7 μm . Representative results from two independent experiments are shown .

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 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 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

Figure 4C. Expression of TGF-β target genes (as measured by RT‒qPCR) in MDA-MB-231 cells without (Co.sh) or with (sh#1 and sh#2) LITATS1 depletion. Cells were serum starved for 16 h and treated with or without TGF-β for 4 h. Representative results from a minimum of three independent experiments are shown.D. Effect of LITATS1 knockdown on TGF-β-induced SMAD2 phosphorylation in MDA-MB-231 cells. Cells were serum starved for 16 h and stimulated with TGF-β for the indicated durations. The p-SMAD2 and total SMAD2 (t-SMAD2) levels were analyzed by western blotting. GAPDH, loading control. Representative results from a minimum of three independent experiments are shown. E. Effect of LITATS1 knockdown on E-cadherin, N-cadherin and SNAIL expression in A549 cells. Cells were stimulated with vehicle control (-), SB431542 (SB; 10 μM) or TGF-β (Τβ) for 24 h, and protein expression was analyzed by western blotting. α/β-Tubulin, loading control. Representative results from a minimum of three independent experiments are shown. FH. In vivo zebrafish extravasation experiments with MDA-MB-231 cells upon LITATS1 knockdown and blockage of TGF-β signaling. Representative zoomed images of the tail fin area are shown in the left panels. Extravasated breast cancer cell clusters are indicated with yellow arrows. Analysis of the extravasated cell cluster numbers in the indicated groups is shown in the right panel. Whole zebrafish image, bar=618.8 μm; zoomed image, scale bar=154.7 μm. Representative results from two independent experiments are shown.


Figure 5A , B . Effect of ectopic LITATS1 expression ( A ) or LITATS knockdown ( B ) on TβRI expression in MDA - MB - 231 cells . Right panel : quantification of relative TβRI protein levels . Vinculin or GAPDH , loading control . Representative blots from a minimum of three independent experiments are shown . C , D . Analysis of TβRI protein stability ( as measured by western blotting ) in MDA - MB - 231 cells with ectopic LITATS1 expression ( C ) or LITATS knockdown ( D ) . Cells were treated with CHX ( 50 µg / mL ) for the indicated durations . Quantification of the relative TβRI protein level is shown in the lower panel . GAPDH , loading control . Representative blots from a minimum of three independent experiments are shown . E . TβRI expression in MDA - MB - 231 cells with ectopic LITATS1 expression in the absence or presence of lysosome or proteasome inhibitors . Cells were incubated with vehicle control DMSO ( - ) , the lysosome inhibitor BafA1 ( 20 nM ) or HCQ ( 20 μM ) , or the proteasome inhibitor MG132 ( 5 μM ) for 8 h . Vinculin , loading control . Representative results from a minimum of three independent experiments are shown . F . Effect of LITATS1 on TβRI ubiquitination . HEK293T cells were transfected with ectopic expression constructs for HA - Ubiquitin ( HA - Ub ) , caTβRI - FLAG and / or LITATS1 . TβRI polyubiquitination was analyzed by western blotting . Representative blots from a minimum of three independent experiments are shown .

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 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 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

Figure 5A, B. Effect of ectopic LITATS1 expression (A) or LITATS knockdown (B) on TβRI expression in MDA-MB-231 cells. Right panel: quantification of relative TβRI protein levels. Vinculin or GAPDH, loading control. Representative blots from a minimum of three independent experiments are shown.C, D. Analysis of TβRI protein stability (as measured by western blotting) in MDA-MB-231 cells with ectopic LITATS1 expression (C) or LITATS knockdown (D). Cells were treated with CHX (50 µg/mL) for the indicated durations. Quantification of the relative TβRI protein level is shown in the lower panel. GAPDH, loading control. Representative blots from a minimum of three independent experiments are shown.E. TβRI expression in MDA-MB-231 cells with ectopic LITATS1 expression in the absence or presence of lysosome or proteasome inhibitors. Cells were incubated with vehicle control DMSO (-), the lysosome inhibitor BafA1 (20 nM) or HCQ (20 μM), or the proteasome inhibitor MG132 (5 μM) for 8 h. Vinculin, loading control. Representative results from a minimum of three independent experiments are shown. F. Effect of LITATS1 on TβRI ubiquitination. HEK293T cells were transfected with ectopic expression constructs for HA-Ubiquitin (HA-Ub), caTβRI-FLAG and/or LITATS1. TβRI polyubiquitination was analyzed by western blotting. Representative blots from a minimum of three independent experiments are shown. D


Figure 6B . Interactions between LITATS1 and TβRI or SMURF2 in MDA - MB - 231 cells were detected by the CARPID approach . Cells with stable expression of TurboID - dCasRx were transduced without ( Co . ) or with ( # 1 and # 2 ) LITATS1 targeting gRNAs . Cells were stimulated with or without TGF - β ( 2 . 5 ng / mL ) for 2 h and were then stimulated with biotin ( 500 μM ) for 30 min . Western blotting was performed to detect SMURF2 and TβRI expression in whole - cell lysates ( Input ) and immunoprecipitates ( IP ) . GAPDH and HA - dCasRx expression levels were measured for equal loading of Input samples and as the negative control or positive control , respectively , for proximity biotinylation in immunoprecipitate ( IP ) samples . Representative results from a minimum of three independent experiments are shown . C . Interactions between LITATS1 and TβRI ( left ) or SMURF2 ( right ) were analyzed by RIP . MDA - MB - 231 cells were stimulated with or without TGF - β ( 5 ng / mL ) for 4 h before RIP . RT ‒ qPCR was performed to detect LITATS1 expression in immunoprecipitates from MDA - MB - 231 cells . IgG was included as the control for immunoprecipitation . Representative results from two independent experiments are shown . D . Interactions between LITATS1 and caTβRI or SMURF2 were analyzed by RNA pull - down . Biotinylated 25x poly ( A ) , antisense LITATS1 ( LITATS1 - AS ) or LITATS1 was incubated with lysates from HEK293T cells transfected with the caTβRI - HA or MYC - SMURF2 expression construct . Western blot analysis was performed to detect HA or MYC expression in whole - cell lysates ( Input ) and immunoprecipitates ( IP ) . Representative blots from a minimum of three independent experiments are shown . E . In vitro RNA pull - down assays were performed to evaluate the interactions between LITATS1 and SMURF1 / 2 . In vitro - transcribed Antisense LITATS1 ( LITATS1 - AS ) or LITATS1 ( LITATS1 - S ) was incubated with recombinant FLAG - tagged SMURF1 or SMURF2 protein . Western blotting analysis was performed to evaluate FLAG expression in input and IP samples . The amounts of RNA probes used for RNA pull - down were evaluated by agarose gel electrophoresis . Representative results from a minimum of three independent experiments are shown . G . Effect of LITATS1 overexpression on SMURF2 - mediated TβRI polyubiquitination . HEK293T cells were transfected with expression constructs for HA - Ubiquitin ( HA - Ub ) and caTβRI - FLAG and ectopic expression constructs for SMURF2 and / or LITATS1 . Polyubiquitination of TβRI was evaluated by western blotting . Representative blots from a minimum of three independent experiments are shown . H . Effect of SMURF2 knockdown on LITATS1 - mediated TβRI polyubiquitination . MDA - MB - 231 cells with stable HA - Ub expression were transduced with expression constructs for LITATS1 and / or two different SMURF2 shRNAs , as indicated . Polyubiquitination of TβRI was evaluated by western blotting . Representative blots from a minimum of three independent experiments are shown .

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 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 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 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 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 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

Figure 6B. Interactions between LITATS1 and TβRI or SMURF2 in MDA-MB-231 cells were detected by the CARPID approach. Cells with stable expression of TurboID-dCasRx were transduced without (Co.) or with (#1 and #2) LITATS1 targeting gRNAs. Cells were stimulated with or without TGF-β (2.5 ng/mL) for 2 h and were then stimulated with biotin (500 μM) for 30 min. Western blotting was performed to detect SMURF2 and TβRI expression in whole-cell lysates (Input) and immunoprecipitates (IP). GAPDH and HA-dCasRx expression levels were measured for equal loading of Input samples and as the negative control or positive control, respectively, for proximity biotinylation in immunoprecipitate (IP) samples. Representative results from a minimum of three independent experiments are shown.C. Interactions between LITATS1 and TβRI (left) or SMURF2 (right) were analyzed by RIP. MDA-MB-231 cells were stimulated with or without TGF-β (5 ng/mL) for 4 h before RIP. RT‒qPCR was performed to detect LITATS1 expression in immunoprecipitates from MDA-MB-231 cells. IgG was included as the control for immunoprecipitation. Representative results from two independent experiments are shown.D. Interactions between LITATS1 and caTβRI or SMURF2 were analyzed by RNA pull-down. Biotinylated 25x poly(A), antisense LITATS1 (LITATS1-AS) or LITATS1 was incubated with lysates from HEK293T cells transfected with the caTβRI-HA or MYC-SMURF2 expression construct. Western blot analysis was performed to detect HA or MYC expression in whole-cell lysates (Input) and immunoprecipitates (IP). Representative blots from a minimum of three independent experiments are shown.E. In vitro RNA pull-down assays were performed to evaluate the interactions between LITATS1 and SMURF1/2. In vitro-transcribed Antisense LITATS1 (LITATS1-AS) or LITATS1 (LITATS1-S) was incubated with recombinant FLAG-tagged SMURF1 or SMURF2 protein. Western blotting analysis was performed to evaluate FLAG expression in input and IP samples. The amounts of RNA probes used for RNA pull-down were evaluated by agarose gel electrophoresis. Representative results from a minimum of three independent experiments are shown.G. Effect of LITATS1 overexpression on SMURF2-mediated TβRI polyubiquitination. HEK293T cells were transfected with expression constructs for HA-Ubiquitin (HA-Ub) and caTβRI-FLAG and ectopic expression constructs for SMURF2 and/or LITATS1. Polyubiquitination of TβRI was evaluated by western blotting. Representative blots from a minimum of three independent experiments are shown.H. Effect of SMURF2 knockdown on LITATS1-mediated TβRI polyubiquitination. MDA-MB-231 cells with stable HA-Ub expression were transduced with expression constructs for LITATS1 and/or two different SMURF2 shRNAs, as indicated. Polyubiquitination of TβRI was evaluated by western blotting. Representative blots from a minimum of three independent experiments are shown.


Figure 7A . An in vitro RNA pull - down assay was performed to evaluate the interaction between LITATS1 truncation mutants and SMURF2 . Recombinant FLAG - SMURF2 protein was incubated with antisense LITATS1 ( LITATS1 - AS ) , LITATS1 ( LITATS1 - S ) , or LITATS1 truncation mutants ( T1 - T4 ) . Western blot analysis was performed to evaluate FLAG expression in immunoprecipitates ( IP ) . The amounts of RNA probes used for RNA pull - down were evaluated by agarose gel electrophoresis . Representative results from a minimum of three independent experiments are shown . E . SMURF2 expression and localization ( as measured by immunofluorescence ) upon LITATS1 depletion in A549 cells . DAPI staining was performed to visualize nuclei . Scale bar = 23 . 2 μm . Representative results from two independent experiments are shown . F . Effect of LITATS1 knockdown on SMURF2 localization in A549 cells . After subcellular protein fractionation , western blotting was performed to detect SMURF2 expression in whole - cell lysates ( Total ) and the cytoplasmic ( Cyto ) and nuclear ( Nuc ) fractions . The levels of the cytoplasmic marker GAPDH and the nuclear marker Lamin A / C are included to demonstrate subcellular protein fractionation . Representative results from two independent experiments are shown .

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

Figure 7A. An in vitro RNA pull-down assay was performed to evaluate the interaction between LITATS1 truncation mutants and SMURF2. Recombinant FLAG-SMURF2 protein was incubated with antisense LITATS1 (LITATS1-AS), LITATS1 (LITATS1-S), or LITATS1 truncation mutants (T1-T4). Western blot analysis was performed to evaluate FLAG expression in immunoprecipitates (IP). The amounts of RNA probes used for RNA pull-down were evaluated by agarose gel electrophoresis. Representative results from a minimum of three independent experiments are shown.E. SMURF2 expression and localization (as measured by immunofluorescence) upon LITATS1 depletion in A549 cells. DAPI staining was performed to visualize nuclei. Scale bar=23.2 μm. Representative results from two independent experiments are shown.F. Effect of LITATS1 knockdown on SMURF2 localization in A549 cells. After subcellular protein fractionation, western blotting was performed to detect SMURF2 expression in whole-cell lysates (Total) and the cytoplasmic (Cyto) and nuclear (Nuc) fractions. The levels of the cytoplasmic marker GAPDH and the nuclear marker Lamin A/C are included to demonstrate subcellular protein fractionation. Representative results from two independent experiments are shown.


Figure 1 ( A ) Confocal live cell imaging of HeLa cells overexpressing GFP - Bax ( green ) and mitochondria marked with TMRE 100 nM ( magenta ) show cytosolic GFP - Bax distribution and healthy mitochondria . Lower panels are zoomed images corresponding to the white rectangle in the upper panel and represent the individual and merged channels . Time shows the minutes after apoptosis induction with Staurosporine ( STS ) 1 µM . Scale bars , 5µm ( overview ) and 2µm ( zooms ) ( B ) HeLa cells shown in A at longer times after STS treatment . Images show sequential GFP - Bax translocation ( green ) , which correlates with mitochondria depolarization ( TMRE , magenta ) . Scale bars , 5 µm ( C ) HeLa cells overexpressing GFP - Bax mutant ( 1 - 2 / L - 6 ) ( green ) and mitochondria marked with TMRE 100 nM ( magenta ) . Images show constitutive GFP - Bax localization to healthy mitochondria without apoptosis induction . Right panels are zoomed images corresponding to the white rectangle in the left panel and represent the individual and overlaid channels . Scale bars , 5µm ( overview ) and 2 µm ( zooms ) ( D ) Standard deviation of the fluorescence intensity of GFP - Bax blue ) versus the corrected total cell fluorescence ( CTCF ) of TMRE ( purple ) in individual cells ( N = 4 ) . CTCF = Integrated Density - ( Area of selected cell X Mean fluorescence of 3 background readings ) . Time 0 corresponds to the normalized time when both events cross in each cell . Black line represents the average of the individual cells .

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Figure 1(A) Confocal live cell imaging of HeLa cells overexpressing GFP-Bax (green) and mitochondria marked with TMRE 100 nM (magenta) show cytosolic GFP-Bax distribution and healthy mitochondria. Lower panels are zoomed images corresponding to the white rectangle in the upper panel and represent the individual and merged channels. Time shows the minutes after apoptosis induction with Staurosporine (STS) 1 µM. Scale bars, 5µm (overview) and 2µm (zooms)(B) HeLa cells shown in A at longer times after STS treatment. Images show sequential GFP-Bax translocation (green), which correlates with mitochondria depolarization (TMRE, magenta). Scale bars, 5 µm(C) HeLa cells overexpressing GFP-Bax mutant (1-2/L-6) (green) and mitochondria marked with TMRE 100 nM (magenta). Images show constitutive GFP-Bax localization to healthy mitochondria without apoptosis induction. Right panels are zoomed images corresponding to the white rectangle in the left panel and represent the individual and overlaid channels. Scale bars, 5µm (overview) and 2 µm (zooms)(D) Standard deviation of the fluorescence intensity of GFP-Bax blue) versus the corrected total cell fluorescence (CTCF) of TMRE (purple) in individual cells (N=4). CTCF= Integrated Density - (Area of selected cell X Mean fluorescence of 3 background readings). Time 0 corresponds to the normalized time when both events cross in each cell. Black line represents the average of the individual cells.


Figure 2 ( A ) Overview of a reconstructed superresolution image of GFP - Bax overexpressing HeLa cells stained with AF647 - anti - GFP - nanobodies . Image was acquired on fixed cells 3 h after apoptosis induction with STS . Dotted line shows the cell shape ( see Fig EV3 ) . Scale bar , 5 µm . ( B ) Magnified reconstructed superresolution image corresponding to the white rectangle in A showing the shapes of GFP - Bax WT structures ( white arrows ) . Scale bar , 500 nm . ( C ) Gallery of typical GFP - Bax WT structures during apoptosis found in all the analyzed cells . Scale bars , 100 nm . ( D ) Overview ( left ) and 3 enlarged insets ( right ) of HeLa cells overexpressing GFP - Bax 1 - 2 / L - 6 stained with AF647 - anti - GFP - nanobodies . Figure shows reconstructed superresolution images acquired without apoptosis induction . Scale bars , 5 µm ( overview ) and 500 nm ( insets ) .

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Figure 2(A) Overview of a reconstructed superresolution image of GFP-Bax overexpressing HeLa cells stained with AF647-anti-GFP-nanobodies. Image was acquired on fixed cells 3 h after apoptosis induction with STS. Dotted line shows the cell shape (see Fig EV3). Scale bar, 5 µm.(B) Magnified reconstructed superresolution image corresponding to the white rectangle in A showing the shapes of GFP-Bax WT structures (white arrows). Scale bar, 500 nm.(C) Gallery of typical GFP-Bax WT structures during apoptosis found in all the analyzed cells. Scale bars, 100 nm.(D) Overview (left) and 3 enlarged insets (right) of HeLa cells overexpressing GFP-Bax 1-2/L-6 stained with AF647-anti-GFP-nanobodies. Figure shows reconstructed superresolution images acquired without apoptosis induction. Scale bars, 5 µm (overview) and 500 nm (insets).


Figure 3 ( A ) Quantification of the structures found on Bax wild type ( purple ) and on Bax 1 - 2 / L - 6 ( grey ) overexpressing cells . Data show the total number of structures in all the measurements . n ( Bax wild type ) = 13 , n ( Bax 1 - 2 / L - 6 ) = 11 . ( B ) Cumulative distribution plots of GFP - Bax wild type ( purple ) and GFP - Bax 1 - 2 / L - 6 ( grey ) showing differences in amplitude and frequency . Each plot represents a single cell . Thick lines represent average cumulative distributions for all the single cells transfected with GFP - Bax WT or mutant GFP - Bax 1 - 2 / L - 6 .

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Figure 3(A) Quantification of the structures found on Bax wild type (purple) and on Bax 1-2/L-6 (grey) overexpressing cells. Data show the total number of structures in all the measurements. n (Bax wild type)= 13, n (Bax 1-2/L-6)=11.(B) Cumulative distribution plots of GFP-Bax wild type (purple) and GFP-Bax 1-2/L-6 (grey) showing differences in amplitude and frequency. Each plot represents a single cell. Thick lines represent average cumulative distributions for all the single cells transfected with GFP-Bax WT or mutant GFP-Bax 1-2/L-6.


Figure 4 ( A ) Overview of a reconstructed superresolution image of GFP - Bax overexpressing HCT116Bax / Bak - / - cells stained with AF647 - anti - GFP - nanobodies Image was acquired on fixed cells 7 h after apoptosis induction with STS . Dotted line shows the cell shape ( see Fig EV3 ) . Scale bar , 5 µm . ( B ) Gallery of typical GFP - Bax WT non - random structures during apoptosis on HCT116Bax / Bak - / - cells . Scale bars , 100 nm . ( C ) Comparison of confocal ( left ) and STED ( right ) images of GFP - Bax overexpressing HeLa cells 2h after STS treatment . STED reveals a notable increase in resolution . Scale bars , 500 nm . ( D ) Representative GFP - Bax non - random structures found with STED ( lines , arcs , rings , double lines ) . Scale bars , 100 nm .

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Figure 4(A) Overview of a reconstructed superresolution image of GFP-Bax overexpressing HCT116Bax/Bak -/- cells stained with AF647-anti-GFP-nanobodies Image was acquired on fixed cells 7 h after apoptosis induction with STS. Dotted line shows the cell shape (see Fig EV3). Scale bar, 5 µm.(B) Gallery of typical GFP-Bax WT non-random structures during apoptosis on HCT116Bax/Bak -/- cells. Scale bars, 100 nm.(C) Comparison of confocal (left) and STED (right) images of GFP-Bax overexpressing HeLa cells 2h after STS treatment. STED reveals a notable increase in resolution. Scale bars, 500 nm.(D) Representative GFP-Bax non-random structures found with STED (lines, arcs, rings, double lines). Scale bars, 100 nm.


Figure 5 ( A ) Representative TEM pictures of HeLa wild - type cells . Bottom panels show healthy mitochondria . Scale bars , 500nm ( overview ) and 200 nm ( zooms ) . ( B ) Representative TEM pictures of apoptotic HeLa wild - type cells 4h after STS treatment ( 1µM ) . Black arrowheads point to defects on the mitochondrial outer membrane . Scale bars , 500nm ( overview ) and 200 nm ( zooms ) ( C ) Representative TEM pictures of apoptotic HeLaGFP - Bax transfected cells 4h after STS treatment ( 1µM ) . Black arrowheads point to defects on the mitochondrial outer membrane . Scale bars , 500nm ( overview ) and 200 nm ( zooms ) .

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Figure 5(A) Representative TEM pictures of HeLa wild-type cells. Bottom panels show healthy mitochondria. Scale bars, 500nm (overview) and 200 nm (zooms).(B) Representative TEM pictures of apoptotic HeLa wild-type cells 4h after STS treatment (1µM). Black arrowheads point to defects on the mitochondrial outer membrane. Scale bars, 500nm (overview) and 200 nm (zooms)(C) Representative TEM pictures of apoptotic HeLaGFP-Bax transfected cells 4h after STS treatment (1µM). Black arrowheads point to defects on the mitochondrial outer membrane. Scale bars, 500nm (overview) and 200 nm (zooms).


Figure 6 ( A ) Left panel shows the individual channels and overlaid images of confocal live cell imaging of HeLa cells overexpressing GFP - Bax ( green ) and mito - dsRed ( magenta ) at 0 and 85 minutes after apoptosis induction with STS 1µM . Right panel shows zoomed overlaid images of HeLa cells following GFP - Bax translocation ( green ) and mitochondrial fragmentation ( magenta ) during apoptosis induction with STS 1µM . Scale bars , 10 µm ( left ) and 2 µm ( right ) ( B ) Reconstructed dual - color superresolution image of GFP - Bax overexpressing HeLa cells . GFP - Bax was imaged by immunosstaining with AF647 - anti - GFP - nanobodies and mitochondria were immunostained with CoxIV ( primary ) and cf680 ( secondary ) antibodies . Image was acquired 3h after STS treatment . Left panel shows the cell overview ( scale bar , 5 µm ) , right panel up shows the overview inset ( scale bar , 500nm ) and right panel down shows a non - random linear GFP - Bax assembly at mitochondria ( scale bar , 100nm ) . ( C ) Reconstructed dual - color superresolution image of HeLa cells co - overexpressing mMaple - mito and GFP - Bax and stained with AF647 - anti - GFP - nanobodies . Image was acquired 3h after STS treatment . Lower panel shows enlarged insets corresponding to the white rectangles in the upper panel . Scale bars , 1 µm ( overview ) and 100 nm ( insets )

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Figure 6(A) Left panel shows the individual channels and overlaid images of confocal live cell imaging of HeLa cells overexpressing GFP-Bax (green) and mito-dsRed (magenta) at 0 and 85 minutes after apoptosis induction with STS 1µM. Right panel shows zoomed overlaid images of HeLa cells following GFP-Bax translocation (green) and mitochondrial fragmentation (magenta) during apoptosis induction with STS 1µM. Scale bars, 10 µm (left) and 2 µm (right)(B) Reconstructed dual-color superresolution image of GFP-Bax overexpressing HeLa cells. GFP-Bax was imaged by immunosstaining with AF647-anti-GFP-nanobodies and mitochondria were immunostained with CoxIV (primary) and cf680 (secondary) antibodies. Image was acquired 3h after STS treatment. Left panel shows the cell overview (scale bar, 5 µm), right panel up shows the overview inset (scale bar, 500nm) and right panel down shows a non-random linear GFP-Bax assembly at mitochondria (scale bar, 100nm).(C) Reconstructed dual-color superresolution image of HeLa cells co-overexpressing mMaple-mito and GFP-Bax and stained with AF647-anti-GFP-nanobodies. Image was acquired 3h after STS treatment. Lower panel shows enlarged insets corresponding to the white rectangles in the upper panel. Scale bars, 1 µm (overview) and 100 nm (insets)


Figure 7 ( A ) AFM image of a control SLB prepared from LUVs with a mitochondrial - like lipid composition . The bilayer looks flat , without aggregates or defects . Scale bar , 1 µm . ( B ) AFM image of a SLB prepared from LUVs with a mitochondrial - like lipid composition pre - incubated with heat - activated Bax . The green arrows indicate the presence of membrane pores , which are heterogeneous in size and shape . The edges of the pores present protrusions corresponding to Bax clusters . Scale bar , 1 µm . ( C ) 3D AFM topography of a Bax arc ( left ) and ring ( right ) . Both images reveal a circular dark hole that spans the lipid membrane ( green ) . Bax molecules around the pore rim ( magenta and white ) protrude 3 , 97 ± 1 , 02 nm above the membrane plane , as confirmed by the height profiles shown below each image ( corresponding to the white line in the 2D image insets ) . The topography of the arc structure reveals a pore only partially surrounded by Bax molecules , while lipids alone form the rest of the pore rim . Images are shown in a 42 ° tilted representation . ( D ) Quantitative analysis of the distinct structures found for activated Bax on SLBs . Data show the total number of structures in all the measurements .

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 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

Figure 7(A) AFM image of a control SLB prepared from LUVs with a mitochondrial-like lipid composition. The bilayer looks flat, without aggregates or defects. Scale bar, 1 µm.(B) AFM image of a SLB prepared from LUVs with a mitochondrial-like lipid composition pre-incubated with heat-activated Bax. The green arrows indicate the presence of membrane pores, which are heterogeneous in size and shape. The edges of the pores present protrusions corresponding to Bax clusters. Scale bar, 1 µm.(C) 3D AFM topography of a Bax arc (left) and ring (right). Both images reveal a circular dark hole that spans the lipid membrane (green). Bax molecules around the pore rim (magenta and white) protrude 3,97 ± 1,02 nm above the membrane plane, as confirmed by the height profiles shown below each image (corresponding to the white line in the 2D image insets). The topography of the arc structure reveals a pore only partially surrounded by Bax molecules, while lipids alone form the rest of the pore rim. Images are shown in a 42° tilted representation.(D) Quantitative analysis of the distinct structures found for activated Bax on SLBs. Data show the total number of structures in all the measurements.


Figure 1F Clonogenic growth assay of indicated HPAF - II cells left untreated or in the presence of 150nM LGK974 for 10 days ; representative of three independent replicates . G Immunoblot of cytoplasmic βcatenin expression from lysates of HPAF - II cells from indicated genotype , treated with vehicle or 100nM LGK974 for 48h ; representative of three biological replicates .

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Figure 1F Clonogenic growth assay of indicated HPAF-II cells left untreated or in the presence of 150nM LGK974 for 10 days; representative of three independent replicates.G Immunoblot of cytoplasmic βcatenin expression from lysates of HPAF-II cells from indicated genotype, treated with vehicle or 100nM LGK974 for 48h; representative of three biological replicates.


Figure 3A Immunoblot of CCNL1 and FBXW7 expression in HPAF - II wild - type and FBXW7 - / - cells , representative blot of 3 independent replicates . B Immunoblot of CCNL1 expression following a cycloheximide chase in HPAF - II wild - type and FBXW7 - / - cells , representative blot of 3 independent replicates . C Quantification of cycloheximide chase in B , mean ± SEM of three independent replicates . D Immunoblot of CCNL1 expression in HPAF - II wild - type and dN - Cul1 expressing cells , representative blot of 3 independent replicates . G Immunoblot of lysates following cycloheximide treatment of HEK293T cells expressing wild - type or degron - mutated CCNL1 , representative blot of 3 independent replicates . H Quantification of cycloheximide chase in G , mean ± SEM of three independent replicates .

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Figure 3A Immunoblot of CCNL1 and FBXW7 expression in HPAF-II wild-type and FBXW7-/- cells, representative blot of 3 independent replicates.B Immunoblot of CCNL1 expression following a cycloheximide chase in HPAF-II wild-type and FBXW7-/- cells , representative blot of 3 independent replicates. C Quantification of cycloheximide chase in B, mean ± SEM of three independent replicates.D Immunoblot of CCNL1 expression in HPAF-II wild-type and dN-Cul1 expressing cells, representative blot of 3 independent replicates.G Immunoblot of lysates following cycloheximide treatment of HEK293T cells expressing wild-type or degron-mutated CCNL1, representative blot of 3 independent replicates. H Quantification of cycloheximide chase in G, mean ± SEM of three independent replicates.


Figure 4A Immunoblot of immunoprecipitation of FLAG - FBXW7 overexpressed in HEK293T cells , detecting endogenous CCNL1 . Representative of three independent replicates . B Immunoblot of immunoprecipitation of FLAG - CCNL1 overexpressed in HEK293T cells , detecting endogenous FBXW7 . Representative of three independent replicates . C Immunoblot of cellular ubiquitination assay demonstrating the requirement of the 325 - TPALS - 329 degron for ubiquitination of CCNL1 . Representative of three independent replicates . D Immunoblot of cellular ubiquitination assay demonstrating the requirement of FBXW7 for ubiquitination of CCNL1 . Representative of three independent replicates . E Immunoblot of cellular ubiquitination assay showing re - expression of FBXW7 in knockout cells leads to increased CCNL1 ubiquitination . Representative of three independent replicates .

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Figure 4A Immunoblot of immunoprecipitation of FLAG-FBXW7 overexpressed in HEK293T cells, detecting endogenous CCNL1. Representative of three independent replicates.B Immunoblot of immunoprecipitation of FLAG-CCNL1 overexpressed in HEK293T cells, detecting endogenous FBXW7. Representative of three independent replicates.C Immunoblot of cellular ubiquitination assay demonstrating the requirement of the 325-TPALS-329 degron for ubiquitination of CCNL1. Representative of three independent replicates.D Immunoblot of cellular ubiquitination assay demonstrating the requirement of FBXW7 for ubiquitination of CCNL1. Representative of three independent replicates.E Immunoblot of cellular ubiquitination assay showing re-expression of FBXW7 in knockout cells leads to increased CCNL1 ubiquitination. Representative of three independent replicates.


Figure 5B Immunoblot of HPAF - II wild - type and FBXW7 - / - released from overnight nocodazole treatment at indicated time points , D Representative images of GFP - tubulin labeled wild - type , FBXW7 - / - , and CCNL1OE HPAF - II cells monitoring cellular progression through cytokinesis following arrest in prometaphase using monastrol ( 150μM for 18 hours ) . E Quantification of cytokinetic timing , n = 20 , 25 , 26 ( wild - type , FBXW7 - / , CCNL1OE ) pooled from three independent experiments F Immunoblot of wild - type , FBXW7 - / - and CCNL1OE HPAF - II cell lines demonstrating varying levels of CCNL1 .

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Figure 5B Immunoblot of HPAF-II wild-type and FBXW7-/- released from overnight nocodazole treatment at indicated time points,D Representative images of GFP-tubulin labeled wild-type, FBXW7-/-, and CCNL1OE HPAF-II cells monitoring cellular progression through cytokinesis following arrest in prometaphase using monastrol (150μM for 18 hours). E Quantification of cytokinetic timing, n=20, 25, 26 (wild-type, FBXW7-/, CCNL1OE ) pooled from three independent experiments F Immunoblot of wild-type, FBXW7-/- and CCNL1OE HPAF-II cell lines demonstrating varying levels of CCNL1.


Figure 6A Clonogenic growth assays for HPAF - II wild - type and FBXW7 - / - polyclonal , and FBXW7 - / - clones in presence of various OTS964 doses for 14 days . Representative images of 4 independent replicates , quantified by crystal violet absorbance at A595 and B Clonogenic growth assays for HPAF - II wild - type and CCNL1OE polyclonal , and CCNL1OE clones in presence of various doses of OTS964 for 14 days . Representative images of 4 independent replicates , quantified by crystal violet absorbance at A595C Immunoblot for the indicated proteins from lysates extracted from the individual FBXW7 - / - and CCNL1OE clones .

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Figure 6A Clonogenic growth assays for HPAF-II wild-type and FBXW7-/- polyclonal, and FBXW7-/- clones in presence of various OTS964 doses for 14 days. Representative images of 4 independent replicates, quantified by crystal violet absorbance at A595 and B Clonogenic growth assays for HPAF-II wild-type and CCNL1OE polyclonal, and CCNL1OE clones in presence of various doses of OTS964 for 14 days. Representative images of 4 independent replicates, quantified by crystal violet absorbance at A595C Immunoblot for the indicated proteins from lysates extracted from the individual FBXW7-/- and CCNL1OE clones.


Figure 7B Immunoblot of lysates from cervical cell lines demonstrating C33A cells ( FBXW7R465H ) express high levels of CCNL1 , representative of three independent replicates . C Clonogenic growth assay of C33A , Caski , and SiHa cells in presence of various doses of OTS964 for 14 days . Representative images of 3 independent replicates , quantified by crystal violet absorbance at

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Figure 7B Immunoblot of lysates from cervical cell lines demonstrating C33A cells (FBXW7R465H) express high levels of CCNL1, representative of three independent replicates.C Clonogenic growth assay of C33A, Caski, and SiHa cells in presence of various doses of OTS964 for 14 days. Representative images of 3 independent replicates, quantified by crystal violet absorbance at A595


Figure 1B ) Bar chart indicating protein quantification , protein localization ( according to UniProt ) in the secretome of primary neurons , comparing the previous SPECS ( blue ) to the new hiSPECS method using DDA ( light green ) or DIA ( dark green ) . Proteins were counted if quantified in at least 9 of the 11 biological replicates of hiSPECS or 4 of 5 biological replicates The category glycoprotein * includes UniProt annotations and proteins annotated as glycoproteins Proteins can have multiple UniProt annotations , e . g . for APP membrane , TM , cytoplasm and nucleus , because distinct proteolytic fragments are found in different organelles , so that some proteins in categories cytoplasm and nucleus may overlap with the categories secreted and TM + GPI . Data information : TM : single - pass transmembrane protein ; GPI : Glycosylphosphatidylinositol - anchored membrane protein .

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Figure 1B) Bar chart indicating protein quantification, protein localization (according to UniProt) in the secretome of primary neurons, comparing the previous SPECS (blue) to the new hiSPECS method using DDA (light green) or DIA (dark green). Proteins were counted if quantified in at least 9 of the 11 biological replicates of hiSPECS or 4 of 5 biological replicates The category glycoprotein* includes UniProt annotations and proteins annotated as glycoproteins Proteins can have multiple UniProt annotations, e.g. for APP membrane, TM, cytoplasm and nucleus, because distinct proteolytic fragments are found in different organelles, so that some proteins in categories cytoplasm and nucleus may overlap with the categories secreted and TM+GPI. Data information: TM: single-pass transmembrane protein; GPI: Glycosylphosphatidylinositol-anchored membrane protein.


Figure 2B ) Number of peptides ( left ) and protein groups ( right ) quantified in the secretome of the investigated brain cell types with the hiSPECS DIA method . Proteins quantified in at least 5 out of 6 biological replicates in at least one cell type are considered . In total 995 protein groups were detected .

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Figure 2B) Number of peptides (left) and protein groups (right) quantified in the secretome of the investigated brain cell types with the hiSPECS DIA method. Proteins quantified in at least 5 out of 6 biological replicates in at least one cell type are considered. In total 995 protein groups were detected.


Figure 4B , C ) Comparison of the hiSPECS secretome resource and lysate The % enrichment is indicated normalized to the average of the most abundant cell type . B ) TREM2 and CD200 are jointly enriched in both secretome and lysate in microglia or neurons , respectively . In C ) two examples , of proteins specifically enriched in the secretome , but not in the lysate are shown . ADIPOQ is specifically secreted from oligodendrocytes , but reveals highest expression in astrocytes . CACHD1 is specifically secreted from neurons , but high protein levels can be found in astrocytes , neurons and oligodendrocytes . The black line indicates the mean .

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Figure 4B,C) Comparison of the hiSPECS secretome resource and lysate The % enrichment is indicated normalized to the average of the most abundant cell type. B) TREM2 and CD200 are jointly enriched in both secretome and lysate in microglia or neurons, respectively. In C) two examples, of proteins specifically enriched in the secretome, but not in the lysate are shown. ADIPOQ is specifically secreted from oligodendrocytes, but reveals highest expression in astrocytes. CACHD1 is specifically secreted from neurons, but high protein levels can be found in astrocytes, neurons and oligodendrocytes. The black line indicates the mean.


Figure 5B ) Volcano plot showing changes in protein levels in the secretome of primary neurons upon BACE1 inhibitor C3 treatment using the hiSPECS DIA method . The negative log10 p - values ( two sample t - test ) of all proteins are plotted against their log2 fold changes ( C3 vs control ) ( N = 11 ) . The grey hyperbolic curves depict a permutation based false discovery rate estimation ( p = 0 . 05 ; s0 = 0 . 1 ) . Significantly regulated proteins ( p < 0 . 05 ) are indicated with a dark blue dot and known BACE1 substrates are indicated with blue letters . The two newly validated BACE1 substrates CD200 and ADAM22 are indicated in red . C ) Independent validation of the novel BACE1 substrate candidates CD200 and ADAM22 by Western blotting in supernatants and lysates of primary neurons incubated with or without the BACE1 ( B1 ) inhibitor C3 for 48h . Full - length ( fl ) ADAM22 ( mainly mature ADAM22 , lower band ) and CD200 levels in the neuronal lysate and were mildly increased upon BACE1 inhibition , as expected due to reduced cleavage by BACE1 . Calnexin served as a loading control . The soluble ectodomain of ADAM22 ( sADAM22 ) was strongly reduced in the conditioned medium upon BACE1 inhibition . Ectodomain levels of the known BACE1 substrate SEZ6 ( sSEZ6 ) were strongly reduced upon BACE1 inhibition and served as positive control . Arrows indicate the mature ( m ) and immature ( im ) ( before prodomain cleavage ) form of ADAM22 . D ) Quantification of the Western blots in ( C ) ( N = 6 ) . Signals were normalized to calnexin levels and quantified relative to the control ( ctr ) condition . Statistical testing was performed with N = 6 biological replicates , using the one sample t - test with the significance criteria of p < 0 . 05 . According to this criterion , ADAM22 and CD200 were significantly increased in total lysates upon C3 treatment ( flADAM22 : * p - value 0 . 0251 , flCD200 : * * p - value 0 . 011 ) . Soluble ADAM22 was significantly reduced in the supernatant upon C3 treatment ( * * * * p - value < 0 . 0001 ) . The black central horizontal line indicates the mean and error bars , mean ± SD . E ) The reduction of the soluble ectodomain of CD200 ( sCD200 ) was detected by ELISA ( one sample t - test , * p - value 0 . 0116 ) , because the available antibodies were not sensitive enough for Western blots of the conditioned medium ( N = 4 ) . The black central horizontal line indicates the mean and error bars , mean ± SD .

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 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 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 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

Figure 5B) Volcano plot showing changes in protein levels in the secretome of primary neurons upon BACE1 inhibitor C3 treatment using the hiSPECS DIA method. The negative log10 p-values (two sample t-test) of all proteins are plotted against their log2 fold changes (C3 vs control) (N=11). The grey hyperbolic curves depict a permutation based false discovery rate estimation (p = 0.05; s0 = 0.1). Significantly regulated proteins (p<0.05) are indicated with a dark blue dot and known BACE1 substrates are indicated with blue letters. The two newly validated BACE1 substrates CD200 and ADAM22 are indicated in red.C) Independent validation of the novel BACE1 substrate candidates CD200 and ADAM22 by Western blotting in supernatants and lysates of primary neurons incubated with or without the BACE1 (B1) inhibitor C3 for 48h. Full-length (fl) ADAM22 (mainly mature ADAM22, lower band) and CD200 levels in the neuronal lysate and were mildly increased upon BACE1 inhibition, as expected due to reduced cleavage by BACE1. Calnexin served as a loading control. The soluble ectodomain of ADAM22 (sADAM22) was strongly reduced in the conditioned medium upon BACE1 inhibition. Ectodomain levels of the known BACE1 substrate SEZ6 (sSEZ6) were strongly reduced upon BACE1 inhibition and served as positive control. Arrows indicate the mature (m) and immature (im) (before prodomain cleavage) form of ADAM22.D) Quantification of the Western blots in (C) (N=6). Signals were normalized to calnexin levels and quantified relative to the control (ctr) condition. Statistical testing was performed with N=6 biological replicates, using the one sample t-test with the significance criteria of p < 0.05. According to this criterion, ADAM22 and CD200 were significantly increased in total lysates upon C3 treatment (flADAM22: *p-value 0.0251, flCD200: **p-value 0.011). Soluble ADAM22 was significantly reduced in the supernatant upon C3 treatment (****p-value <0.0001). The black central horizontal line indicates the mean and error bars, mean ± SD.E) The reduction of the soluble ectodomain of CD200 (sCD200) was detected by ELISA (one sample t-test, *p-value 0.0116), because the available antibodies were not sensitive enough for Western blots of the conditioned medium (N=4). The black central horizontal line indicates the mean and error bars, mean ± SD.


Figure 6A ) Protein dynamic range plot of the log10 transformed LFQ intensities of the murine CSF proteins quantified in at least 3 of 4 biological replicates measured with DIA . The proteins are split into quartiles according to their intensities , with the 1st quartile representing the 25 % most abundant proteins . The percentage of proteins annotated in UniProt with the following subcellular locations / keywords are visualized for : membrane , secreted , cytoplasm , and glycoprotein . Examples of cell type specifically secreted proteins are indicated with circles colored according to the UniProt annotation ( purple : secreted , blue : membrane ) . B ) Protein dynamic range plot of the log10 transformed LFQ intensities specifically of glycoproteins in the murine CSF quantified in at least 3 of 4 biological replicates measured with DIA ( N = 4 ) . The proteins are split into quartiles according to their intensities . The percentage of proteins identified in the secretome of astrocytes , microglia , neurons or oligodendrocytes in at least 5 of 6 biological replicates is illustrated below . Selected proteins specifically secreted from one cell type are indicated with the color code of the corresponding cell type .

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

Figure 6A) Protein dynamic range plot of the log10 transformed LFQ intensities of the murine CSF proteins quantified in at least 3 of 4 biological replicates measured with DIA. The proteins are split into quartiles according to their intensities, with the 1st quartile representing the 25% most abundant proteins. The percentage of proteins annotated in UniProt with the following subcellular locations/keywords are visualized for: membrane, secreted, cytoplasm, and glycoprotein. Examples of cell type specifically secreted proteins are indicated with circles colored according to the UniProt annotation (purple: secreted, blue: membrane). B) Protein dynamic range plot of the log10 transformed LFQ intensities specifically of glycoproteins in the murine CSF quantified in at least 3 of 4 biological replicates measured with DIA (N=4). The proteins are split into quartiles according to their intensities. The percentage of proteins identified in the secretome of astrocytes, microglia, neurons or oligodendrocytes in at least 5 of 6 biological replicates is illustrated below. Selected proteins specifically secreted from one cell type are indicated with the color code of the corresponding cell type.


Figure 7A ) hiSPECS DDA and DIA analysis of brain slice cultures in the presence of 25 % serum . The bar chart comparing the hiSPECS DDA and DIA method indicates the protein number and their localization according to UniProt identified in the secretome of brain slices . Proteins quantified in at least 5 of 6 biological replicates are considered ( N = 6 ) . B ) Protein dynamic range plot of the log10 LFQ intensities of secretome proteins of brain slices in descending order . According to their intensity , proteins are grouped into quartiles and the percentage of proteins with the following UniProt keywords is visualized : membrane , secreted , cytoplasm . The percentage of proteins identified in the secretome of astrocytes , microglia , neurons or oligodendrocytes in at least 5 of 6 biological replicates is illustrated . Selected proteins specifically secreted from one cell type are indicated with the color code of the corresponding cell type . The circle of APOE indicates it as a soluble secreted protein . D ) Volcano plot showing changes in protein levels in the secretome of primary cultured brain slices upon 6 h LPS treatment in a 24 h collection window using the hiSPECS DIA method . The negative log10 p - values ( two sample t - test ) of all proteins are plotted against their log2 fold changes ( LPS vs control ) ( N = 6 ) . The grey hyperbolic curves depict a permutation based false discovery rate estimation ( p = 0 . 05 ; s0 = 0 . 1 ) . Significantly regulated proteins ( p < 0 . 05 ) are indicated with a dark blue dot . Proteins highlighted in red indicate proteins known to increase upon LPS treatment , whereas proteins labeled in blue are not upregulated in this

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 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 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) hiSPECS DDA and DIA analysis of brain slice cultures in the presence of 25% serum. The bar chart comparing the hiSPECS DDA and DIA method indicates the protein number and their localization according to UniProt identified in the secretome of brain slices. Proteins quantified in at least 5 of 6 biological replicates are considered (N=6).B) Protein dynamic range plot of the log10 LFQ intensities of secretome proteins of brain slices in descending order. According to their intensity, proteins are grouped into quartiles and the percentage of proteins with the following UniProt keywords is visualized: membrane, secreted, cytoplasm. The percentage of proteins identified in the secretome of astrocytes, microglia, neurons or oligodendrocytes in at least 5 of 6 biological replicates is illustrated. Selected proteins specifically secreted from one cell type are indicated with the color code of the corresponding cell type. The circle of APOE indicates it as a soluble secreted protein.D) Volcano plot showing changes in protein levels in the secretome of primary cultured brain slices upon 6 h LPS treatment in a 24 h collection window using the hiSPECS DIA method. The negative log10 p-values (two sample t-test) of all proteins are plotted against their log2 fold changes (LPS vs control) (N=6). The grey hyperbolic curves depict a permutation based false discovery rate estimation (p = 0.05; s0 = 0.1). Significantly regulated proteins (p<0.05) are indicated with a dark blue dot. Proteins highlighted in red indicate proteins known to increase upon LPS treatment, whereas proteins labeled in blue are not upregulated in this study


Figure 2 ( A ) RT - qPCR of early passage RMS tumoroid models shows positivity for at least one gene used in standard - of - care pathology analysis ( DES , MYOG , or MYOD1 ) . Conventional RMS cell lines ( RD and RH30 ) were used as positive controls , while two Synovial Sarcoma ( SS000DAZ and SS077 ) tumoroid models were used as negative controls . Gene expression was normalized to the expression of a house - keeping gene and human reference RNA ( HREF ) via the ΔΔCq method . Each tumoroid line was measured once with four technical replicates with the error bars representing the standard deviation of said technical replicates . ( C ) Morphological ( via H & E ) and immunohistochemical ( IHC ) comparison of RMS tumors and derived RMS tumoroid models shows retained marker protein ( Desmin , Myogenin and MYOD1 ) expression and cellular morphology . Scale bars equal 200 µm ( RMS012 , RMS102 ) or 100 µm ( RMS000HQC ) .

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 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

Figure 2(A) RT-qPCR of early passage RMS tumoroid models shows positivity for at least one gene used in standard-of-care pathology analysis (DES, MYOG, or MYOD1). Conventional RMS cell lines (RD and RH30) were used as positive controls, while two Synovial Sarcoma (SS000DAZ and SS077) tumoroid models were used as negative controls. Gene expression was normalized to the expression of a house-keeping gene and human reference RNA (HREF) via the ΔΔCq method. Each tumoroid line was measured once with four technical replicates with the error bars representing the standard deviation of said technical replicates.(C) Morphological (via H&E) and immunohistochemical (IHC) comparison of RMS tumors and derived RMS tumoroid models shows retained marker protein (Desmin, Myogenin and MYOD1) expression and cellular morphology. Scale bars equal 200 µm (RMS012, RMS102) or 100 µm (RMS000HQC).


Figure 3 ( A ) Copy number frequency plots of RMS tumors ( upper row ) and derived RMS tumoroid models ( lower row ) divided by fusion - status ( columns ) . Chromosomes are annotated on the x - axis from left to right while the y - axis shows the percentage of samples in this group carrying a gain ( red ) or loss ( blue ) in this genomic region . ( B ) Contribution of somatic mutational signatures per tumor and tumoroid model . SBS = single base substitution , TMZ = temozolomide , T = tumor , O = tumoroid . ( C ) Table depicting pathogenic single - nucleotide variants ( SNVs ) in RMS tumors ( T ) and tumoroid models ( O ) . Circle color indicates SNV type while circle size indicates variant allele fraction ( VAF ) . Vertical dotted lines separate samples derived from individual patients . Highlighted are genes previously reported for this RMS subtype . ( D ) Correlogram of bulk mRNA sequencing expression profiles of pediatric kidney tumors ( controls ) as well as RMS tumoroid models and RMS tumors . CCRCC = Clear Cell Renal Cell Carcinoma , CMN = Congenital Mesoblastic Nephroma , WT = Wilms Tumor , Cor = correlation .

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 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 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

Figure 3(A) Copy number frequency plots of RMS tumors (upper row) and derived RMS tumoroid models (lower row) divided by fusion-status (columns). Chromosomes are annotated on the x-axis from left to right while the y-axis shows the percentage of samples in this group carrying a gain (red) or loss (blue) in this genomic region.(B) Contribution of somatic mutational signatures per tumor and tumoroid model. SBS = single base substitution, TMZ = temozolomide, T = tumor, O = tumoroid.(C) Table depicting pathogenic single-nucleotide variants (SNVs) in RMS tumors (T) and tumoroid models (O). Circle color indicates SNV type while circle size indicates variant allele fraction (VAF). Vertical dotted lines separate samples derived from individual patients. Highlighted are genes previously reported for this RMS subtype.(D) Correlogram of bulk mRNA sequencing expression profiles of pediatric kidney tumors (controls) as well as RMS tumoroid models and RMS tumors. CCRCC = Clear Cell Renal Cell Carcinoma, CMN = Congenital Mesoblastic Nephroma, WT = Wilms Tumor, Cor = correlation.


Figure 5 ( A ) Clustered heatmap of viability measurements per RMS tumoroid model ( x axis ) and drug ( y axis ) , showing the Area Under the Curve ( AUC ) after treatment of the cells for 120 h with a dose - range of 0 . 1 nM to 10 µM . Low AUC ( red ) indicates high drug efficacy while high AUC ( blue ) indicates low drug efficacy . Annotated clusters of ( 1 ) MEK / ERK and γ - secretase inhibitors showing specific efficacy in RMS tumoroid models without ( RMS007 , RMS012 , RMS444 ) or low ( RMS000HQC ) fusion transcript expression , and ( 2 ) drugs that show broad efficacy across RMS tumoroid models .

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

Figure 5(A) Clustered heatmap of viability measurements per RMS tumoroid model (x axis) and drug (y axis), showing the Area Under the Curve (AUC) after treatment of the cells for 120 h with a dose-range of 0.1 nM to 10 µM. Low AUC (red) indicates high drug efficacy while high AUC (blue) indicates low drug efficacy. Annotated clusters of (1) MEK/ERK and γ-secretase inhibitors showing specific efficacy in RMS tumoroid models without (RMS007, RMS012, RMS444) or low (RMS000HQC) fusion transcript expression, and (2) drugs that show broad efficacy across RMS tumoroid models.


Figure 6 ( B ) Western Blot analysis of TP53 wildtype ( WT ) and knockout ( KO ) RMS tumoroid line RMS012 . Histone 3 ( H3 ) served as loading control . ( C ) Dose - response curve of TP53 WT and KO cells treated with the Chk - 1 inhibitor prexasertib . Thin lines with numbers indicate individual biological replicates ( n = 3 ) while thick lines indicate fitted lines over all replicates . The statistical significance of the differences in fitted IC50 values between WT and KO were obtained using a two - sided t - test ( p = 0 . 008 ) .

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

Figure 6(B) Western Blot analysis of TP53 wildtype (WT) and knockout (KO) RMS tumoroid line RMS012. Histone 3 (H3) served as loading control.(C) Dose-response curve of TP53 WT and KO cells treated with the Chk-1 inhibitor prexasertib. Thin lines with numbers indicate individual biological replicates (n = 3) while thick lines indicate fitted lines over all replicates. The statistical significance of the differences in fitted IC50 values between WT and KO were obtained using a two-sided t-test (p = 0.008).


figf1a ) FBXL2 binds p85α and p85β . HEK293T cells were transfected with either an empty vector ( EV ) or the indicated FLAG - tagged F - box proteins ( FBPs ) . 24 h post - transfection , cells were treated with MG132 for 3 h before collection for immunoprecipitation ( IP ) and immunoblotting as indicated . WCL , whole - cell lysate . ( b ) FBXL2 does not bind p110α and p110β . HEK293T and NIH3T3 cells were transfected with either an empty vector ( EV ) or the indicated FLAG - tagged proteins . The experiment was performed as described in a . ( c ) The CaaX motif of FBXL2 is required to bind p85α and p85β . HEK293T cells were transfected with either an empty vector ( EV ) or the indicated FLAG - tagged F - box proteins . The experiment was performed as described in a .

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 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

figf1a) FBXL2 binds p85α and p85β. HEK293T cells were transfected with either an empty vector (EV) or the indicated FLAG-tagged F-box proteins (FBPs). 24 h post-transfection, cells were treated with MG132 for 3 h before collection for immunoprecipitation (IP) and immunoblotting as indicated. WCL, whole-cell lysate.(b) FBXL2 does not bind p110α and p110β. HEK293T and NIH3T3 cells were transfected with either an empty vector (EV) or the indicated FLAG-tagged proteins. The experiment was performed as described in a.(c) The CaaX motif of FBXL2 is required to bind p85α and p85β. HEK293T cells were transfected with either an empty vector (EV) or the indicated FLAG-tagged F-box proteins. The experiment was performed as described in a.


figf2a ) HEK293T cells were transfected with an empty vector ( EV ) or the indicated FLAG - tagged constructs . 24 h after transfection , cells were treated with either MG132 or solvent for 3 h before collection for immunoblotting as indicated . ( b ) HeLa cells stably expressing FLAG - HA - tagged FBXL2 were transfected with either siRNAs targeting FBXL2 ( four different siRNAs ) or a non - silencing siRNA ( NS ) . Cells were lysed , and proteins were immunoblotted as indicated . NT , non - transfected . ( c ) During a 72 - h serum starvation , NHFs were transfected with either an siRNA targeting FBXL2 ( no . 1 ) or a non - silencing siRNA ( NS ) . Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting . The graph shows FBXL2 mRNA levels in the different samples analysed using real - time PCR in triplicate measurements . The values represent the ratios between FBXL2 and GAPDH mRNAs . SR , serum re - addition . ( d ) HEK293T cells were transfected with HA - tagged p85β , FLAG - tagged FBXL2 , FLAG - tagged FBXL2 ( ΔF - box ) or an empty vector ( EV ) as indicated . After immunopurification with anti - FLAG resin , in vitro ubiquitylation of p85β was performed in the presence of E1 , E2s and ubiquitin ( Ub ) . Where indicated , an excess of methylated ubiquitin ( MeUb ) was also added . Samples were analysed by immunoblotting with the indicated antibodies . The ladder of bands with a relative molecular mass of > 85 , 000 ( lane 3 ) corresponds to ubiquitylated p85β . Immunoblots of whole - cell lysates ( WCL ) are shown at the bottom . Uncropped images of blots are shown in Supplementary Fig .

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 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 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

figf2a) HEK293T cells were transfected with an empty vector (EV) or the indicated FLAG-tagged constructs. 24 h after transfection, cells were treated with either MG132 or solvent for 3 h before collection for immunoblotting as indicated.(b) HeLa cells stably expressing FLAG-HA-tagged FBXL2 were transfected with either siRNAs targeting FBXL2 (four different siRNAs) or a non-silencing siRNA (NS). Cells were lysed, and proteins were immunoblotted as indicated. NT, non-transfected.(c) During a 72-h serum starvation, NHFs were transfected with either an siRNA targeting FBXL2 (no. 1) or a non-silencing siRNA (NS). Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting. The graph shows FBXL2 mRNA levels in the different samples analysed using real-time PCR in triplicate measurements. The values represent the ratios between FBXL2 and GAPDH mRNAs. SR, serum re-addition.(d) HEK293T cells were transfected with HA-tagged p85β, FLAG-tagged FBXL2, FLAG-tagged FBXL2(ΔF-box) or an empty vector (EV) as indicated. After immunopurification with anti-FLAG resin, in vitro ubiquitylation of p85β was performed in the presence of E1, E2s and ubiquitin (Ub). Where indicated, an excess of methylated ubiquitin (MeUb) was also added. Samples were analysed by immunoblotting with the indicated antibodies. The ladder of bands with a relative molecular mass of >85,000 (lane 3) corresponds to ubiquitylated p85β. Immunoblots of whole-cell lysates (WCL) are shown at the bottom. Uncropped images of blots are shown in Supplementary Fig. S8


figf3 ( b ) HEK293T cells were transfected with GFP - tagged FBXL2 and the indicated FLAG - tagged p85β mutants . Whole - cell lysates ( WCL ) were immunoprecipitated ( IP ) with anti - FLAG resin , and immunocomplexes were probed with antibodies against the indicated proteins . ( c ) p85β ( ΔSH2C ) is more stable than wild - type p85β . RPE1 - hTERT cells were infected with either a retrovirus expressing wild - type p85β or p85β ( ΔSH2C ) . Cells were incubated with cycloheximide ( CHX ) for the indicated times , collected and analysed by immunoblotting as indicated . In the graph , the amount of p85β ( wild - type or mutant ) is represented relative to the amount at time 0 . ( ( d ) p85β ( QR / AA ) is more stable than wild - type p85β . HEK293T cells were infected with either a retrovirus expressing wild - type p85β or p85β ( QR / AA ) . Cells were incubated with cycloheximide ( CHX ) for the indicated times , collected and analysed by immunoblotting as indicated . In the graph , the amount of p85β ( wild - type or mutant ) is represented relative to the amount at time 0 . ( e ) p85β ( Y655A ) is less stable than wild - type p85β . The experiment was performed as described in c except that p85β ( Y655A ) was used . In the graph , the amount of p85β ( wild - type or mutant ) is represented relative to the amount at time 0 . Uncropped images of blots are shown in Supplementary Fig . S8 .

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 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 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

figf3(b) HEK293T cells were transfected with GFP-tagged FBXL2 and the indicated FLAG-tagged p85β mutants. Whole-cell lysates (WCL) were immunoprecipitated (IP) with anti-FLAG resin, and immunocomplexes were probed with antibodies against the indicated proteins.(c) p85β(ΔSH2C) is more stable than wild-type p85β. RPE1-hTERT cells were infected with either a retrovirus expressing wild-type p85β or p85β (ΔSH2C). Cells were incubated with cycloheximide (CHX) for the indicated times, collected and analysed by immunoblotting as indicated. In the graph, the amount of p85β (wild-type or mutant) is represented relative to the amount at time 0. ((d) p85β (QR/AA) is more stable than wild-type p85β. HEK293T cells were infected with either a retrovirus expressing wild-type p85β or p85β (QR/AA). Cells were incubated with cycloheximide (CHX) for the indicated times, collected and analysed by immunoblotting as indicated. In the graph, the amount of p85β (wild-type or mutant) is represented relative to the amount at time 0.(e) p85β (Y655A) is less stable than wild-type p85β. The experiment was performed as described in c except that p85β(Y655A) was used. In the graph, the amount of p85β (wild-type or mutant) is represented relative to the amount at time 0. Uncropped images of blots are shown in Supplementary Fig. S8.


figf4 ( a ) Lysates from cells expressing the indicated FLAG - tagged F - box proteins ( FBPs ) were used in binding reactions with beads coupled to either a peptide or a phospho - peptide ( sequences shown on top of the panel ) . Beads were washed with lysis buffer , and bound proteins were eluted and subjected to SDS - PAGE and immunoblotting . ( ( b ) Denatured extracts from HEK293T cells stably expressing either FLAG - tagged wild - type p85β or FLAG - tagged p85β ( Y655A ) were immunoprecipitated ( IP ) with either an anti - FLAG resin or a 4G10 platinum resin and immunoblotted ( IB ) as indicated . Immunoblots of whole - cell lysates ( WCL ) are also shown in the last two lanes . ( c ) Denatured extracts from HEK293T cells expressing either FLAG - tagged wild - type p85β or the indicated FLAG - tagged p85β mutants were immunoprecipitated ( IP ) with an anti - FLAG resin and immunoblotted as indicated . Uncropped images of blots are shown in Supplementary Fig . S8 .

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 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

figf4(a) Lysates from cells expressing the indicated FLAG-tagged F-box proteins (FBPs) were used in binding reactions with beads coupled to either a peptide or a phospho-peptide (sequences shown on top of the panel). Beads were washed with lysis buffer, and bound proteins were eluted and subjected to SDS-PAGE and immunoblotting. ((b) Denatured extracts from HEK293T cells stably expressing either FLAG-tagged wild-type p85β or FLAG-tagged p85β(Y655A) were immunoprecipitated (IP) with either an anti-FLAG resin or a 4G10 platinum resin and immunoblotted (IB) as indicated. Immunoblots of whole-cell lysates (WCL) are also shown in the last two lanes.(c) Denatured extracts from HEK293T cells expressing either FLAG-tagged wild-type p85β or the indicated FLAG-tagged p85β mutants were immunoprecipitated (IP) with an anti-FLAG resin and immunoblotted as indicated. Uncropped images of blots are shown in Supplementary Fig. S8.


figf5 ( a ) The CaaX motif of FBXL2 is not required to bind PTPL1 . HEK293T cells were transfected with either FLAG - tagged wild - type FBXL2 or FLAG - tagged FBXL2 ( CaaX / SaaX ) . 24 h post - transfection , cells were collected and whole - cell lysates ( WCL ) were immunoprecipitated ( IP ) and immunoblotted as indicated . ( c ) PTPL1 stimulates the binding of FBXL2 to wild - type p85β , but not p85β ( Y655A ) . HEK293T cells were transfected with GFP - tagged FBXL2 and either FLAG - tagged p85β or FLAG - tagged p85β ( Y655A ) . Where indicated , HA - tagged PTPL1 or HA - tagged PTPL1 ( C / S ) were also transfected . The experiment was performed as described in a . ( d ) PTPL1 silencing inhibits the FBXL2 - p85β interaction . HeLa cells stably expressing FLAG - tagged FBXL2 were transfected with either an siRNA targeting PTPL1 or a non - silencing siRNA ( NS ) . Forty - eight hours post - transfection , cells were collected and whole - cell lysates ( WCL ) were immunoprecipitated ( IP ) and immunoblotted as indicated . ( e ) During a 72 - h serum starvation , NHFs were transfected twice with either siRNAs targeting FBXL2 or PTPL1 , or a non - silencing siRNA ( NS ) . Cells were subsequently re - stimulated with media containing serum and collected at the indicated time points for immunoblotting . SR , serum re - addition . ( f ) HEK293T cells were transfected twice with either siRNAs targeting FBXL2 or PTPL1 , or a non - silencing siRNA ( NS ) . Cells were transfected with p85β ( Y655A ) and 16 h after cells were incubated with cycloheximide ( CHX ) for the indicated times , collected and analysed by immunoblotting as indicated . g ) During a 48 - h serum starvation , U2OS cells stably transfected with a doxycycline - inducible p85β construct were transfected twice with either an siRNA targeting PTPL1 or a non - silencing siRNA ( NS ) . During the last 16 h before collection , p85β expression was stimulated with doxycycline . Cells were subsequently re - stimulated with media containing serum and collected 30 min later . Denatured cell lysates were immunoprecipitated with an anti - FLAG resin and immunoblotted as indicated . The asterisk indicates a non - specific band . Uncropped images of blots are shown in Supplementary Fig . S8 .

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 O O 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 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 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

figf5(a) The CaaX motif of FBXL2 is not required to bind PTPL1. HEK293T cells were transfected with either FLAG-tagged wild-type FBXL2 or FLAG-tagged FBXL2(CaaX/SaaX). 24 h post-transfection, cells were collected and whole-cell lysates (WCL) were immunoprecipitated (IP) and immunoblotted as indicated.(c) PTPL1 stimulates the binding of FBXL2 to wild-type p85β, but not p85β(Y655A). HEK293T cells were transfected with GFP-tagged FBXL2 and either FLAG-tagged p85β or FLAG-tagged p85β(Y655A). Where indicated, HA-tagged PTPL1 or HA-tagged PTPL1(C/S) were also transfected. The experiment was performed as described in a.(d) PTPL1 silencing inhibits the FBXL2-p85β interaction. HeLa cells stably expressing FLAG-tagged FBXL2 were transfected with either an siRNA targeting PTPL1 or a non-silencing siRNA (NS). Forty-eight hours post-transfection, cells were collected and whole-cell lysates (WCL) were immunoprecipitated (IP) and immunoblotted as indicated.(e) During a 72-h serum starvation, NHFs were transfected twice with either siRNAs targeting FBXL2 or PTPL1, or a non-silencing siRNA (NS). Cells were subsequently re-stimulated with media containing serum and collected at the indicated time points for immunoblotting. SR, serum re-addition.(f) HEK293T cells were transfected twice with either siRNAs targeting FBXL2 or PTPL1, or a non-silencing siRNA (NS). Cells were transfected with p85β(Y655A) and 16 h after cells were incubated with cycloheximide (CHX) for the indicated times, collected and analysed by immunoblotting as indicated.g) During a 48-h serum starvation, U2OS cells stably transfected with a doxycycline-inducible p85β construct were transfected twice with either an siRNA targeting PTPL1 or a non-silencing siRNA (NS). During the last 16 h before collection, p85β expression was stimulated with doxycycline. Cells were subsequently re-stimulated with media containing serum and collected 30 min later. Denatured cell lysates were immunoprecipitated with an anti-FLAG resin and immunoblotted as indicated. The asterisk indicates a non-specific band. Uncropped images of blots are shown in Supplementary Fig. S8.


figf6a ) During a 72 - h serum starvation , NHFs were transfected with either siRNAs targeting FBXL2 , p85β , both FBXL2 and p85β , or a non - silencing siRNA ( NS ) . Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting . The graph at the right shows FBXL2 mRNA levels in the different samples analysed using real - time PCR in triplicate measurements . The values represent the ratios between FBXL2 and GAPDH mRNAs . SR , serum re - addition . ( b ) Cells were treated as in a , except that 3 h after serum re - addition , cells were lysed , immunoprecipitated ( IP ) with an anti - IRS1 antibody and immunoblotted as indicated . Uncropped images of blots are shown in Supplementary Fig . S8 .

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 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

figf6a) During a 72-h serum starvation, NHFs were transfected with either siRNAs targeting FBXL2, p85β, both FBXL2 and p85β, or a non-silencing siRNA (NS). Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting. The graph at the right shows FBXL2 mRNA levels in the different samples analysed using real-time PCR in triplicate measurements. The values represent the ratios between FBXL2 and GAPDH mRNAs. SR, serum re-addition.(b) Cells were treated as in a, except that 3 h after serum re-addition, cells were lysed, immunoprecipitated (IP) with an anti-IRS1 antibody and immunoblotted as indicated. Uncropped images of blots are shown in Supplementary Fig. S8.


figf7 ( a ) During a 72 - h serum starvation , NHFs were transfected with either siRNAs targeting FBXL2 ( no . 1 or 2 ) or a non - silencing siRNA ( NS ) . Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting . SR , serum re - addition . ( b ) During a 72 - h serum starvation , NHFs were transfected with either siRNAs targeting FBXL2 , both FBXL2 and p85β , or a non - silencing siRNA ( NS ) . Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting . SR , serum re - addition . ( c ) U2OS cells stably transfected with a doxycycline - inducible p85β construct were serum starved for 48 h . During the last 16 h before collection , p85β expression was stimulated with doxycycline . Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting . SR , serum re - addition . ( d ) Cells constitutively expressing GFP - LC3 were transfected with HA - tagged p85β , fixed and incubated with an anti - HA p85β antibody ( red ) . Arrows in the top panels point to the enhanced autophagic response ( LC3 - II vesicles ) in cells expressing high levels of p85β , as shown in the bottom panels . Uncropped images of blots are shown in Supplementary Fig . S8 . Scale bars , 10 μm .

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 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 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

figf7(a) During a 72-h serum starvation, NHFs were transfected with either siRNAs targeting FBXL2 (no. 1 or 2) or a non-silencing siRNA (NS). Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting. SR, serum re-addition.(b) During a 72-h serum starvation, NHFs were transfected with either siRNAs targeting FBXL2, both FBXL2 and p85β, or a non-silencing siRNA (NS). Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting. SR, serum re-addition.(c) U2OS cells stably transfected with a doxycycline-inducible p85β construct were serum starved for 48 h. During the last 16 h before collection, p85β expression was stimulated with doxycycline. Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting. SR, serum re-addition.(d) Cells constitutively expressing GFP-LC3 were transfected with HA-tagged p85β, fixed and incubated with an anti-HA p85β antibody (red). Arrows in the top panels point to the enhanced autophagic response (LC3-II vesicles) in cells expressing high levels of p85β, as shown in the bottom panels. Uncropped images of blots are shown in Supplementary Fig. S8. Scale bars, 10 μm.