( a ) Changes in groups ( carbohydrates , fatty ) of storage metabolites measured in circulating haemolymph with progressing wasting , shown as log2 changes measured by LC - MS calculated per larvae relative to RasV12 ; ctrl at day 6 .

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(a) Changes in groups (carbohydrates, amino acids and fatty acids) of storage metabolites measured in circulating haemolymph with progressing wasting, shown as log2 changes measured by LC-MS and calculated per larvae relative to RasV12; ctrl at day 6.


( b ) Volcano plot showing autophagy - dependent changes to amounts of circulating metabolites at day 6 . X - axis shows log2 of fold change of RasV12 ; atg13 - / - scrib - / - / / atg13 - / - vs . RasV12 ; scrib - / - , y - axis shows - log10 p - value , calculated by t - . Metabolite names are shown for metabolites with log2 ( FC ) > ± 1 / or - log10 ( P ) < 2 . Green points indicate log2 ( FC ) > ± 1 , blue indicate - log10 ( P ) < 2 red indicates for above both thresholds .

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(b) Volcano plot showing autophagy-dependent changes to amounts of circulating metabolites at day 6. X-axis shows the log2 of fold change of RasV12;atg13 -/-scrib-/- // atg13 -/- vs. RasV12; scrib-/- , y-axis shows -log10 p-value, calculated by t-test. Metabolite names are shown for metabolites with log2(FC) >± 1 and/or -log10(P) < 2. Green points indicate log2(FC) >± 1, blue indicate -log10(P) < 2 and red indicates for above both thresholds.


( c ) Autophagy - driven wasting releases metabolites into circulation of 113 reliably detected metabolites , those showing significant differences in any of three comparisons are shown . Color indicates log2 ( fold change ) difference numbers show p - value of comparison . statistical to define significance was FDR - adjusted t - p - value < 0 .

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(c) Autophagy-driven wasting releases metabolites into circulation of 113 reliably detected metabolites, those showing significant differences in any of the three comparisons are shown. Color indicates the log2(fold change) difference and the numbers show the p-value of the comparison. The statistical test to define significance was FDR-adjusted t-test p-value < 0.05


( a ) amount of glycogen in whole larvae measured by biochemical ( n = 4 ) , normalized to RasV12 ; ctrl at day 6 , per number of larvae .

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(a) The amount of glycogen in the whole larvae measured by biochemical assay (n=4), normalized to RasV12; ctrl at day 6, and per number of larvae.


( b - e ) Representative confocal of muscle adipose tissue of larvae carrying RasV12 ; ctrl , RasV12 ; scrib - / - RasV12 ; scrib - / - , atg13 - / - / / atg13 - / - showing glycogen ( ) at day 6 day 8 .

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(b-e) Representative confocal images of muscle and adipose tissue of larvae carrying RasV12; ctrl, RasV12; scrib-/- and RasV12; scrib-/-, atg13-/-//atg13-/- showing glycogen levels (white) at day 6 and day 8.


( g ) Sources of carbon incorporated into biomass were differentiated by changing isotopic carbon content of food source 25 hours before measuring total carbon content isotopic of . A similar , but independent experiment is reported in Holland P . , et al . , 2021 BMC Biol .

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(g) Sources of carbon incorporated into tumor biomass were differentiated by changing the isotopic carbon content of the food source 25 hours before measuring the total carbon content and isotopic ratio of the tumor. A similar, but independent experiment is reported in Holland P., et al., 2021 BMC Biol.


HSATIII lncRNA - dependent localization of m6A - related factors . Control HSATIII knockdown HeLa were exposed to thermal stress ( 42 ° C for 2 h recovery for 1 h at 37 ° C ) stained by HSATIII - FISH immunofluorescence using an anti - WTAP antibody ( B ) or anti - YTHDC1 antibody ( C ) . nuclei were stained with DAPI . Scale bar : 10 µm .

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HSATIII lncRNA-dependent localization of m6A-related factors. Control and HSATIII knockdown HeLa cells were exposed to thermal stress (42°C for 2 h and recovery for 1 h at 37°C) and stained by HSATIII-FISH and immunofluorescence using an anti-WTAP antibody (B) or anti-YTHDC1 antibody (C). The nuclei were stained with DAPI. Scale bar: 10 µm.


YTHDC1 accumulation within nSBs was arrested by METTL3 knockdown . HeLa were stained as described in Figure 1C . Scale bar : 10 µm . Box plot of relative intensities of YTHDC1 to HSATIII within nSBs in nuclei ( n = 30 ) . mean is indicated with X . first third quartiles are ends of box , median is indicated with a vertical in box , minimum maximum except for outliers are ends of whiskers . outliers are indicated with open circles . P - values ( Kruskal - Wallis , followed by Dunn ' s multiple comparison ) are shown above .

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YTHDC1 accumulation within nSBs was arrested by METTL3 knockdown. HeLa cells were stained as described in Figure 1C. Scale bar: 10 µm. Box plot of relative intensities of YTHDC1 to HSATIII within nSBs in individual nuclei (n=30). The mean is indicated with X. The first and third quartiles are the ends of the box, the median is indicated with a vertical line in the box, and the minimum and maximum except for the outliers are the ends of the whiskers. The outliers are indicated with open circles. P-values (Kruskal-Wallis test, followed by Dunn's multiple comparison test) are shown above.


- MS of HSATIII lncRNAs digested by RNase T1 . Base peak chromatogram ( BPC , top panel ) extracted chromatograms ( XIC , lower panels ) for RNase T1 - digested fragments of isolated HSATIII lncRNAs . XICs detecting for negatively - charged ions of AAUGp tetramer without modification ( second panel ) , with one methylation ( third panel ) , two methylations ( bottom panel ) . , mass to charge ( m / z ) charge state are indicated . NL ; normalization level .

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RNA-MS analysis of HSATIII lncRNAs digested by RNase T1. Base peak chromatogram (BPC, top panel) and extracted chromatograms (XIC, lower panels) for the RNase T1-digested fragments of the isolated HSATIII lncRNAs. XICs detecting for the negatively-charged ions of AAUGp tetramer without modification (second panel), with one methylation (third panel), and two methylations (bottom panel) . The sequence, mass to charge ratio (m/z) and charge state are indicated. NL; normalization level.


Changes of nSB localization of m6A - related factors at various time points during after thermal stress exposure . HeLa were exposed to thermal stress ( 42 ° C for 2 h ) cultured at 37 ° C for indicated periods . m6A - related factors were visualized by HSATIII - FISH IF using anti - WTAP antibody ( D ) or anti - YTHDC1 antibody ( F ) . Nuclei were stained with DAPI . Scale bar : 10 µm . ( E , G ) Box plot of relative intensities of WTAP ( D ) YTHDC1 ( F ) to HSATIII within nSB areas in nuclei . nSB areas were defined by binarized of HSATIII - FISH . mean is indicated with X . first third quartiles are ends of box , median is indicated with a vertical in box , minimum maximum except for outliers are ends of whiskers . outliers are indicated with open circles . P - values ( Kruskal - Wallis , followed by Dunn ' s multiple comparison ) are shown above ( n = 30 ) .

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Changes of nSB localization of m6A-related factors at various time points during and after thermal stress exposure. HeLa cells were exposed to thermal stress (42°C for 2 h) and cultured at 37°C for the indicated periods. m6A-related factors were visualized by HSATIII-FISH and IF using anti-WTAP antibody (D) or anti-YTHDC1 antibody (F). Nuclei were stained with DAPI. Scale bar: 10 µm. (E, G) Box plot of relative intensities of WTAP (D) and YTHDC1 (F) to HSATIII within nSB areas in individual nuclei. nSB areas were defined by binarized images of HSATIII-FISH. The mean is indicated with X. The first and third quartiles are the ends of the box, the median is indicated with a vertical line in the box, and the minimum and maximum except for the outliers are the ends of the whiskers. The outliers are indicated with open circles. P-values (Kruskal-Wallis test, followed by Dunn's multiple comparison test) are shown above (n=30).


HSATIII dependent sequestration of nucleoplasmic YTHDC1 ( G ) WTAP ( H ) at four time points during thermal stress recovery . experiments same as A C were performed at indicated timepoints . mean is indicated with X . first third quartiles are ends of box , median is indicated with a vertical in box , minimum maximum except for outliers are ends of whiskers . outliers are indicated with open circles . P - values ( Kruskal - Wallis , followed by Dunn ' s multiple comparison ) are shown above ( n = 40 ) .

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HSATIII dependent sequestration of nucleoplasmic YTHDC1 (G) and WTAP (H) at four time points during thermal stress and recovery. The experiments same as A and C were performed at the indicated timepoints. The mean is indicated with X. The first and third quartiles are the ends of the box, the median is indicated with a vertical line in the box, and the minimum and maximum except for the outliers are the ends of the whiskers. The outliers are indicated with open circles. P-values (Kruskal-Wallis test, followed by Dunn's multiple comparison test) are shown above (n=40).


Scatter plot of LogFC of whole introns ( grey ) HSATIII - target introns ( blue ) upon SRSF9 KD ( X - axis ) HSATIII KD ( Y - axis ) . 51 , 469 548 introns are plotted as whole introns previously - reported HSATIII - target introns , respectively . Pearson ' s correlation ( r ) is indicated above .

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Scatter plot of LogFC of whole introns (grey) and HSATIII-target introns (blue) upon SRSF9 KD (X-axis) and HSATIII KD (Y-axis). 51,469 and 548 introns are plotted as whole introns and previously-reported HSATIII-target introns, respectively. Pearson's correlation (r) is indicated above.


RT - qPCR validation of splicing of nascent RNAs . Splicing efficiency of nascent RNAs was quantified as of spliced form to intron retaining isoform ( IR ) form normalized to control . Control siRNA HSATIII control oligonucleotide are indicated with ( - ) . Data are shown as mean ± SD ( n = 3 ) ; p - values ( Tukey ' s multiple comparison ) are shown above .

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RT-qPCR validation of splicing of nascent RNAs. Splicing efficiency of nascent RNAs was quantified as the ratio of the spliced form to the intron retaining isoform (IR) form and normalized to the control. Control siRNA and HSATIII control oligonucleotide are indicated with (-). Data are shown as the mean ± SD (n=3); p-values (Tukey's multiple comparison test) are shown above.


Read maps represent - seq data of poly ( A ) + RNAs ( upper panel , biological triplicate ) m6A - RIP RNAs ( lower panel , biological duplicate ) in control HSATIII KD . exon numbers of FAM214A gene are indicated below . retaining intron m6A peak are marked by a red arrow , respectively . For entire FAM214A region , see also Figure EV4B .

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Read maps represent RNA-seq data of nuclear poly (A)+ RNAs (upper panel, biological triplicate) and m6A-RIP RNAs (lower panel, biological duplicate) in control and HSATIII KD cells. The exon numbers of the FAM214A gene are indicated below. The retaining intron and the m6A peak are marked by a red line and arrow, respectively. For the entire FAM214A region, see also Figure EV4B.


In vitro pulldown of RNP complexes with various repeat RNAs . In vitro pulldown was performed using biotinylated ( GGAAU ) × 20 other three repeat RNAs incubated in HeLa extract . co - precipitated were detected by western blotting using specific antibodies . ALYREF , a non - specific - binding , was used as a control . of data in A . Relative co - precipitation efficiency of each was normalized by maximum value . Data are shown as mean ± SD ( n = 3 ) .

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In vitro pulldown assay of RNP complexes with various repeat RNAs. In vitro RNA pulldown was performed using biotinylated (GGAAU)×20 and other three repeat RNAs incubated in HeLa nuclear extract. The co-precipitated proteins were detected by western blotting using specific antibodies. ALYREF, a non-specific RNA-binding protein, was used as a control. Quantification of data in A. Relative co-precipitation efficiency of each protein was normalized by the maximum value. Data are shown as the mean ± SD (n=3).


In vitro pulldown with methylated GGAAU repeat . In vitro pulldown was performed using biotinylated GGAAU GGm6Am6AU repeat RNAs as in A . of data in C . Relative co - precipitation efficiency of each was normalized by maximum value . Data are shown as mean ± SD ( n = 3 ) .

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In vitro pulldown assay with methylated GGAAU repeat RNA. In vitro RNA pulldown assay was performed using biotinylated GGAAU and GGm6Am6AU repeat RNAs as in A. Quantification of data in C. Relative co-precipitation efficiency of each protein was normalized by the maximum value. Data are shown as the mean ± SD (n=3).


Serial diluted recombinant S1 of SARS - CoV - 2 , SARS - CoV MERS - CoV were coated on 96 well plates , incubated with recombinant Fc - tagged ACE2 ( ACE2 - Fc ) for binding evaluation .

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Serial diluted recombinant S1 proteins of SARS-CoV-2, SARS-CoV and MERS-CoV were coated on 96 well plates, incubated with the recombinant Fc-tagged ACE2 (ACE2-Fc) for binding evaluation.


Recombinant S1 of SARS - CoV - 2 SARS - CoV were incubated with 293T - ACE2 subjected to FACS evaluation for binding .

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Recombinant S1 proteins of SARS-CoV-2 and SARS-CoV were incubated with 293T-ACE2 cells and subjected to FACS evaluation for binding.


Binding of SARS nAbs to SARS - CoV S1 were tested by ELISA . Recombinant S1 of SARS - CoV were coated on plates , serial diluted nAbs were added for binding to recombinant S1 . B . Binding of SARS nAbs to SARS - CoV - 2 S1 were tested by ELSIA . Recombinant S1 of SARS - CoV - 2 were coated on plates , serial diluted nAbs were added for binding to recombinant S1 .

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Binding of SARS nAbs to SARS-CoV S1 protein were tested by ELISA. Recombinant S1 protein of SARS-CoV were coated on plates, serial diluted nAbs were added for binding to recombinant S1 protein.B. Binding of SARS nAbs to SARS-CoV-2 S1 protein were tested by ELSIA. Recombinant S1 protein of SARS-CoV-2 were coated on plates, serial diluted nAbs were added for binding to recombinant S1 protein.


Neutralization of SARS - CoV nAbs against SARS - CoV - 2 PSV .

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Neutralization of SARS-CoV nAbs against SARS-CoV-2 PSV.


Antibody competition with SARS - CoV RBD binding to ACE2 . Recombinant SARS - CoV RBD was coated on plates , nAbs recombinant ACE2 were then added for RBD binding competition measurements .

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Antibody competition with SARS-CoV RBD binding to ACE2. Recombinant SARS-CoV RBD protein was coated on plates, nAbs and recombinant ACE2 were then added for RBD binding competition measurements.


PCP - MS strategy to identify interacting with specific proteasome subtypes . U937 were cross - linked with formaldehyde lysed , were concentrated ultrafiltrated on a 100 kDa cutoff device . complexes were then separated on a 15 - 40 % glycerol gradient . Each fraction of gradient was analyzed by nano - LC - MS / MS . was performed using mean XIC of three most intense validated peptides for each , after internal standard calibration using a mix of 8 isotopically labeled peptides . PCP was performed as described in Materials Methods section .

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PCP-MS strategy to identify proteins interacting with specific proteasome subtypes. U937 cells were cross-linked with formaldehyde and lysed, and proteins were concentrated and ultrafiltrated on a 100 kDa cutoff device. Protein complexes were then separated on a 15-40% glycerol gradient. Each fraction of the gradient was analyzed by nano-LC-MS/MS. Protein quantification was performed using the mean XIC of the three most intense validated peptides for each protein, after internal standard calibration using a mix of 8 isotopically labeled peptides. The PCP analysis was performed as described in the Materials and Methods section.


PCP of 19S regulatory . abundance profiles of 16 of 19S RP ( Rpt1 - 6 , Rpn1 - 3 , Rpn5 , 7 - 9 , 11 - 13 , gray lanes ) of their median abundance ( black lane ) ( left panel ) . PCP is performed by plotting χ2 values ( representing Euclidian between abundance profile of each reference profile ) of experimental replicate 2 as a function of χ2 values of experimental replicate 1 ( middle left panel ) . median profile of 19S subunits was used as reference profile for calculation of χ2 values . Different zooms of graph are represented ( middle right right panels ) . gray dots represent quantified in fractions of density gradient blue dots represent 19S subunits ( right panel ) .

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PCP analysis of the 19S regulatory complex. Protein abundance profiles of 16 proteins of the 19S RP (Rpt1-6, Rpn1-3, Rpn5, 7-9, 11-13, gray lanes) and of their median abundance (black lane) (left panel). PCP analysis is performed by plotting the χ2 values (representing the Euclidian distance between the abundance profile of each protein and the reference profile) of the experimental replicate 2 as a function of the χ2 values of the experimental replicate 1 (middle left panel). The median profile of the 19S complex subunits was used as the reference profile for the calculation of the χ2 values. Different zooms of the graph are represented (middle right and right panels). Light gray dots represent the proteins quantified in all the fractions of the density gradient and blue dots represent 19S subunits (right panel).


PCP of proteasome 20S . abundance profiles of 17 of 20S CP ( α1 - α7 , β1 - β7 , β1i , β2i , β5i , gray lanes ) of their median abundance ( black lane ) ( left panel ) . PCP is performed by plotting χ2 values of experimental replicate 2 as a function of χ2 values of experimental replicate 1 ( middle left panel ) . median profile of 20S subunits was used as reference profile for calculation of χ2 values . Different zooms of graph are represented ( middle right right panels ) . gray dots represent quantified in fractions of density gradient red dots represent 20S subunits .

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PCP analysis of proteasome 20S complex. Protein abundance profiles of 17 proteins of the 20S CP (α1-α7, β1-β7, β1i, β2i, β5i, gray lanes) and of their median abundance (black lane) (left panel). PCP analysis is performed by plotting the χ2 values of the experimental replicate 2 as a function of the χ2 values of the experimental replicate 1 (middle left panel). The median profile of the 20S complex subunits was used as the reference profile for the calculation of the χ2 values. Different zooms of the graph are represented (middle right and right panels). Light gray dots represent the proteins quantified in all the fractions of the density gradient and red dots represent 20S subunits.


correlation profiling ( PCP ) using median profile of PA28αβ regulator as reference profileProfiles of PA28α , PA28β , β2i ( blue , red green lines , respectively ) .

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Protein correlation profiling (PCP) analysis using the median profile of the PA28αβ regulator as the reference profileProfiles of the PA28α, PA28β, and the β2i proteins (blue, red and green lines, respectively).


Plot of χ2 values of experimental replicate 2 as a function of χ2 values of experimental replicate 1 . A zoom of graph in ( B ) is represented χ2 coordinates for PA28α , PA28β , β2i are highlighted as blue , red , green dots , respectively . gray dots represent χ2 coordinates of quantified in fractions of gradient . median profile of PA28α PA28β subunits was used as reference profile for calculation of χ2 values .

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Plot of the χ2 values of the experimental replicate 2 as a function of the χ2 values of the experimental replicate 1.A zoom of the graph in (B) is represented and χ2 coordinates for PA28α, PA28β, and β2i proteins are highlighted as blue, red, and green dots, respectively. Light gray dots represent the χ2 coordinates of the proteins quantified in all the fractions of the gradient. The median profile of the PA28α and PA28β subunits was used as the reference profile for the calculation of the χ2 values.


A Proteasome complexes were immunopurified from nine formaldehyde - crosslinked human lines analyzed by nano - LC - MS / MS . abundance indexes ( PAIs ) were used to represent abundance of in purified proteasome samples . correlation between two different was quantified using coefficients of determination ( R2 ) .

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A Proteasome complexes were immunopurified from nine formaldehyde-crosslinked human cell lines and analyzed by nano-LC-MS/MS. Protein abundance indexes (PAIs) were used to represent the abundance of proteins in purified proteasome samples. The correlation between two different proteins was quantified using coefficients of determination (R2).


B - E Correlations of abundances of α7 α6 ( B ) , Rpn3 Rpn1 ( C ) , PA28β PA28α ( D ) , Rpn3 PA28β ( E ) .

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

B-E Correlations of abundances of α7 and α6 (B), Rpn3 and Rpn1 (C), PA28β and PA28α (D), and Rpn3 and PA28β (E).


Heat - map representing correlations ( expressed as R2 ) between abundances of 73 known proteasome - interacting ( PIPs ) abundances of 8 reference or complexes , PA28γ , β2i ( representing iP20S ) , PA28αβ , ncP20S ( median of α1 - α7 , β3 , β4 , β6 , β7 profiles ) , 19S ( median of Rpt1 - 6 , Rpn1 - 3 , 5 - 14 profiles ) , PI31 , β5 ( representing sP20S ) , PA200 . For complexes , median PAI of their subunits in each of 24 AP - MS experiments was used : α1 - α7 , β3 , β4 , β6 , β7 subunits for ncP20S , Rpt1 - 6 , Rpn1 - 3 , 5 - 14 for 19S RP , PA28α PA28β subunits for PA28αβ RP . R2 values were hierarchically clustered . Three distinct clusters of composition detailed hereafter could be obtained . Cluster 1 ( from top to bottom ) : Rpt3 , Rpn13 , α2 , Rpn7 , USP14 , hHR23B , α1 , β6 , β3 , α4 , α7 , Rpn6 , Rpn3 , Rpt4 , Rpn10 , Rpn5 , Rpt5 , Rpn1 , Rpn11 , Rpt1 , Rpn9 , Rpn2 , Rpn8 , α3 , α6 , β4 , Rpt6 , PDC6 , Rpn12 , APEH , Ubiquilin - 1 , α5 , β7 , CCT7 , CCT4 , CCT2 , CCT3 , CCT5 , CCT6A , DNAJA1 , HSP90AB1 , HSP90AA1 , PNP , Rpt2 . Cluster 2 ( from top to bottom ) : 14 - 3 - 3ζ / δ , CAND1 , GSR , UBE3C , β2 , ATP5A1 , ATP5B , β1 , PA200 , β5 , FBXO7 , UCHL5 , TXNL1 , ECM29 , PI31 . Cluster 3 ( from top to bottom ) : PA28β , PITH1 , β2i , PA28α , PA28γ , β5i , β1i .

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

Heat-map representing the correlations (expressed as the R2) between the abundances of 73 known proteasome-interacting proteins (PIPs) and the abundances of 8 reference proteins or protein complexes, PA28γ, β2i (representing the iP20S), PA28αβ, ncP20S (median of α1-α7, β3, β4, β6, and β7 profiles), 19S (median of Rpt1-6, Rpn1-3, 5-14 profiles), PI31, β5 (representing the sP20S), and PA200. For protein complexes, the median PAI of their subunits in each of the 24 AP-MS experiments was used: α1-α7, β3, β4, β6, and β7 subunits for the ncP20S, Rpt1-6, Rpn1-3, 5-14 for the 19S RP, and PA28α and PA28β subunits for the PA28αβ RP. The R2 values were hierarchically clustered. Three distinct clusters of composition detailed hereafter could be obtained. Cluster 1 (from top to bottom): Rpt3, Rpn13, α2, Rpn7, USP14, hHR23B, α1, β6, β3, α4, α7, Rpn6, Rpn3, Rpt4, Rpn10, Rpn5, Rpt5, Rpn1, Rpn11, Rpt1, Rpn9, Rpn2, Rpn8, α3, α6, β4, Rpt6, PDC6, Rpn12, APEH, Ubiquilin-1, α5, β7, CCT7, CCT4, CCT2, CCT3, CCT5, CCT6A, DNAJA1, HSP90AB1, HSP90AA1, PNP, Rpt2. Cluster 2 (from top to bottom): 14-3-3ζ/δ, CAND1, GSR, UBE3C, β2, ATP5A1, ATP5B, β1, PA200, β5, FBXO7, UCHL5, TXNL1, ECM29, PI31. Cluster 3 (from top to bottom): PA28β, PITH1, β2i, PA28α, PA28γ, β5i, β1i.


two HEK EBNA lines express only standard proteasome or immunoproteasome subunits . Western blots against immuno - ( β1i , β2i , β5i ) standard ( β1 , β2 , β5 ) catalytic subunits of 20S proteasome . Calnexin is used as a loading control . Black lines delineate boundary between vertically sliced that juxtapose lanes that were non - adjacent in gel . Importantly , bands were assembled from same blot .

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The two HEK EBNA cell lines express only standard proteasome or immunoproteasome subunits. Western blots against the immuno- (β1i, β2i, β5i) and standard (β1, β2, β5) catalytic subunits of the 20S proteasome. Calnexin is used as a loading control. Black lines delineate the boundary between vertically sliced images that juxtapose lanes that were non-adjacent in the gel. Importantly, the bands were assembled from the same blot.


Relative normalized abundance indexes of proteasome regulators in HEK EBNA containing only immunoproteasome compared to HEK EBNA containing only standard proteasome . normalized abundance indexes for each regulator were set to 1 for standard proteasome conditions ( n = 4 ) .

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

Relative normalized abundance indexes of proteasome regulators in HEK EBNA cells containing only immunoproteasome compared to HEK EBNA cells containing only standard proteasome. The normalized abundance indexes for each regulator were set to 1 for standard proteasome conditions (n = 4).


Kinetics of IFN - γ treatment on HeLa . HeLa were stimulated for 0 , 24 , 48 , or 72 h with IFN - γ . Western blots were performed on total lysates with antibodies against β2i , α2 , α5 subunits . IRF - 1 was used to control IFN - γ treatment efficiency , GAPDH was used as a loading control .

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Kinetics of IFN-γ treatment on HeLa cells. HeLa cells were stimulated for 0, 24, 48, or 72 h with IFN-γ. Western blots were performed on total cell lysates with antibodies against the β2i, α2, and α5 subunits. IRF-1 was used to control IFN-γ treatment efficiency, and GAPDH was used as a loading control.


For each time point of IFN - γ treatment , proteasome complexes were purified analyzed by LC - MS / MS . Proteasome complexes dynamics was measured by label - free quantitative proteomics . normalized abundance index of each or obtained at each time point was compared to one obtained at 0 h time point to obtain a regulator relative normalized PAI ( n = 3 ) .

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

For each time point of the IFN-γ treatment, proteasome complexes were purified and analyzed by LC-MS/MS. Proteasome complexes dynamics was measured by label-free quantitative proteomics. The normalized abundance index of each protein or protein complex obtained at each time point was compared to the one obtained at the 0 h time point to obtain a regulator relative normalized PAI (n = 3).


Dual - color SMLM shows co - localization of BAX DRP1 at nanoscale . B ) Magnified SMLM of GFP - BAX DRP1 assemblies co - localizing up to 30 nm during apoptosis . Scale bar 100 nm . C ) Co - localization control of DRP1 immunostaining with same different secondary antibodies ( labelled with AF647 CF680 ) in HeLa . Scale bar 100 nm . D ) of from GFP - BAX to DRP1 structures ( measured at center of each structure ) from dual - color SMLM . Data are quantified from n = 4 independent experiments with a total of 720 BAX structure .

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

Dual-color SMLM shows co-localization of BAX and DRP1 at the nanoscale. B) Magnified SMLM images of GFP-BAX and DRP1 assemblies co-localizing up to 30 nm during apoptosis. Scale bar 100 nm. C) Co-localization control of DRP1 immunostaining with the same primary and different secondary antibodies (labelled with AF647 and CF680) in HeLa cells. Scale bar 100 nm. D) Quantification of the distance from GFP-BAX to DRP1 structures (distance measured at the center of each structure) from dual-color SMLM images. Data are quantified from n=4 independent experiments with a total of 720 BAX structure.


E , F ) Representative confocal microscopy of HeLa transfected with ddFP RA - BAX GB - DRP1 ( E ) in untreated or apoptotic ( STS ) conditions , or with RA - BAX GB - BAX during apoptosis ( F ) . RA - BAX / GB - DRP1 RA - BAX / GB - BAX complexes shown in green , mitochondria labeled with mito - BFP in magenta . Scale bar 10 μm . Right panels are zoomed areas representing merged channels . Scale bar 5 μm .

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

E,F) Representative confocal microscopy images of HeLa cells transfected with ddFP RA-BAX and GB-DRP1 (E) in untreated or apoptotic (STS) conditions, or with RA-BAX and GB-BAX during apoptosis (F). RA-BAX/GB-DRP1 and RA-BAX/GB-BAX complexes shown in green, mitochondria labeled with mito-BFP in magenta. Scale bar 10 μm. Right panels are zoomed areas representing individual and merged channels. Scale bar 5 μm.


G ) BAX / DRP1 interaction compared to BAX / BAX BCL ‑ xL / DRP1 by ddFP . % mito - BFP transfected that show RA / GB signal was quantified in HeLa untreated ( untr . ) 3 h after apoptosis induction ( STS ) . Box plots represent interquartile ( box ) , median ( ) SD ( whiskers ) of n = 3 independent experiments ( with n = 100 each ) . of significance were determined by paired two - Student ´ s t - ( * p < 0 . 05 , * * * p < 0 . 001 ) compared to BAX / DRP1 after apoptotic induction ( STS ) .

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

G) BAX/DRP1 interaction compared to BAX/BAX and BCL‑xL/DRP1 by ddFP. % mito-BFP transfected cells that show RA/GB signal was quantified in HeLa cells untreated (untr.) and 3 h after apoptosis induction (STS). Box plots represent the interquartile (box), median (line) and SD (whiskers) of n=3 independent experiments (with n=100 cells each). Levels of significance were determined by paired two-tailed Student´s t-test (* p < 0.05, *** p < 0.001) compared to BAX/DRP1 after apoptotic induction (STS).


A ) Confocal microscopy time - series of increase in Smac - GFP ( magenta ) in relation to detection of RA - BAX / GB - DRP1 complexes ( green ) during apoptosis induction in MEF DRP1 KO . Scale bar 10 μm . are representative for n = 3 independent experiments .

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

A) Confocal microscopy time-series images of the increase in cytosolic Smac-GFP (magenta) in relation to the detection of RA-BAX/GB-DRP1 complexes (green) during apoptosis induction in MEF DRP1 KO cells. Scale bar 10 μm. Images are representative for n=3 independent experiments.


C ) of BAX / DRP1 interaction ( % with RA - BAX / GB - DRP1 foci normalized to mito - BFP positive , n = 100 per experiment ) during inhibition of effector caspases ( Q - VD - OPh ) , mitochondrial permeability transition pore ( CsA ) versus control ( DMSO ) , with or without apoptosis induction ( + / - STS ) . Box plots represent interquartile ( box ) , median ( ) SD ( whiskers ) . Significance was determined from n = 3 independent experiments ( with n = 100 each ) by paired two - Student ´ s t - ( n . s . indicating p > 0 . 05 ) compared to DMSO control after apoptotic induction ( STS ) .

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

C) Quantification of BAX/DRP1 interaction (% cells with RA-BAX/GB-DRP1 foci normalized to the mito-BFP positive cells, n=100 cells per experiment) during inhibition of effector caspases (Q-VD-OPh), the mitochondrial permeability transition pore (CsA) versus control (DMSO), with or without apoptosis induction (+/- STS). Box plots represent the interquartile (box), median (line) and SD (whiskers). Significance was determined from n=3 independent experiments (with n=100 cells each) by paired two-tailed Student´s t-test (n.s. indicating p > 0.05) compared to DMSO control after apoptotic induction (STS).


C ) of % CC between DRP1 - AF488 BAX - AF633 in solution ( grey ) , in ( violet ) , in ( beige ) in presence of excess unlabeled cBID . Box plots represent interquartile ( outer box ) , mean ( inner box ) , median ( ) range ( whiskers ) . of significance were determined by paired two - Student ´ s t - ( * p < 0 . 05 , * * p < 0 . 01 ) from n = 9 measurements in solution , n = 46 individually measured GUVs in or n = 17 GUVs in presence of cBID .

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

C) Quantification of %CC between DRP1-AF488 and BAX-AF633 in solution (grey), in the membrane (violet), and in the membrane (beige) in presence of excess unlabeled cBID. Box plots represent the interquartile (outer box), mean (inner box), median (line) and range (whiskers). Levels of significance were determined by paired two-tailed Student´s t-test (* p < 0.05, ** p < 0.01) from n=9 measurements in solution, n=46 individually measured GUVs in the membrane or n=17 GUVs in presence of cBID.


E ) Effect of DRP1 on BAX - induced GUV permeabilization . Left : % GUVs permeabilized to Cytochrome c488 ( Cyt c , 12 kDa , beige ) allophycocianin ( APC , 104 kDa , blue ) in absence or presence of cBID , BAX DRP1 combined as indicated . Data are presented as mean ± SD of n = 4 independent experiments . * p < 0 . 05 ( paired two - Student ´ s t - ) . Right : representative confocal microscopy showing GUVs ( grey ) in a solution of Cyt c ( green ) APC ( magenta ) . Scale bar 10 µm .

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

E) Effect of DRP1 on BAX-induced GUV permeabilization. Left: % GUVs permeabilized to Cytochrome c488 (Cyt c, 12 kDa, beige) and allophycocianin (APC, 104 kDa, blue) in the absence or presence of cBID, BAX and DRP1 combined as indicated. Data are presented as mean ± SD of n=4 independent experiments. * p < 0.05 (paired two-tailed Student´s t-test). Right: representative confocal microscopy images showing GUVs (grey) in a solution of Cyt c (green) and APC (magenta). Scale bar 10 µm.


Effect of BAX on DRP1 density DRP1 - induced shape alterations of liposomes measured by flow cytometry . G ) density of DRP1 ( corrected units , cFU , left graph ) DRP1 - induced tethering ( indicated by a shape index > 1 , right graph ) in LUVs in absence or presence of different concentrations of BAX . Data are presented as mean ± SD of n = 3 independent experiments . * p < 0 . 05 ( paired two - Student ´ s t - ) vs . DRP1 without BAX .

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

Effect of BAX on DRP1 membrane density and DRP1-induced shape alterations of liposomes measured by flow cytometry. G) Membrane density of DRP1 (corrected fluorescence units, cFU, left graph) and DRP1-induced membrane tethering (indicated by a shape index > 1, right graph) in LUVs in the absence or presence of different concentrations of BAX. Data are presented as mean ± SD of n=3 independent experiments. * p < 0.05 (paired two-tailed Student´s t-test) vs. DRP1 without BAX.


BAX peptide array to define interaction - site with DRP1 . D ) of relative of DRP1 - AF488 at rim of GUVs for each BAX peptide of n = 3 experiments ( with n = 100 vesicles each ) . Black lines indicate mean value . Interacting BAX peptides are highlighted in beige .

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BAX peptide array to define interaction-site with DRP1. D) Quantification of the relative fluorescence intensity of DRP1-AF488 at the rim of the GUVs for each BAX peptide of n=3 individual experiments (with n=100 vesicles each). Black lines indicate the mean value. Interacting BAX peptides are highlighted in beige.


B ) Representative confocal microscopy of U2OS BAX / BAK DKO of interactions between RA - BAX variants GB - DRP1 , shown as green signal , 3 h after apoptosis induction . Mitochondria labeled with mito - BFP in magenta . Scale bar 10 μm .

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B) Representative confocal microscopy images of U2OS BAX/BAK DKO cells of the interactions between RA-BAX variants and GB-DRP1, shown as green signal, 3 h after apoptosis induction. Mitochondria labeled with mito-BFP in magenta. Scale bar 10 μm.


C - E ) of interaction of DRP1 with variants of BAX on % with detectable RA - BAX / GB - DRP1 signal ( interacting ) normalized to number of mito - BFP positive . Box plots represent interquartile ( box ) , median ( ) SD ( whiskers ) of n = 3 independent experiments ( with n = 100 each ) . of significance were determined by paired two - Student ´ s t - ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ) compared to BAX wild type ( WT ) .

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

C-E) Quantification of the interaction of DRP1 with variants of BAX based on the % cells with detectable RA-BAX/GB-DRP1 fluorescence signal (interacting cells) normalized to the number of mito-BFP positive cells. Box plots represent the interquartile (box), median (line) and SD (whiskers) of n=3 independent experiments (with n=100 cells each). Levels of significance were determined by paired two-tailed Student´s t-test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001) compared to BAX wild type (WT).


G ) Representative confocal microscopy of U2OS BAX / BAK DKO of interactions between RA - BAX GB - DRP1 variants , shown as green signal , 3 h after apoptosis induction . Mitochondria labeled with mito - BFP in magenta . Scale bar 10 μm .

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

G) Representative confocal microscopy images of U2OS BAX/BAK DKO cells of the interactions between RA-BAX and GB-DRP1 variants, shown as green signal, 3 h after apoptosis induction. Mitochondria labeled with mito-BFP in magenta. Scale bar 10 μm.


H ) of interaction of DRP1 variants with BAX on % with detectable RA - BAX / GB - DRP1 signal ( interacting ) normalized to number of mito - BFP positive . Box plots represent interquartile ( box ) , median ( ) SD ( whiskers ) of n = 3 independent experiments ( with n = 100 each ) . Significance was tested using paired two - Student ´ s t - ( n . s . p > 0 . 05 ) compared to DRP1 WT .

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H) Quantification of the interaction of DRP1 variants with BAX based on the % cells with detectable RA-BAX/GB-DRP1 fluorescence signal (interacting cells) normalized to the number of mito-BFP positive cells. Box plots represent the interquartile (box), median (line) and SD (whiskers) of n=3 independent experiments (with n=100 cells each). Significance was tested using paired two-tailed Student´s t-test (n.s. p > 0.05) compared to DRP1 WT.


C ) Forced dimerization of BAX DRP1 in healthy induces their translocation to mitochondria foci formation . Confocal microscopy of U2OS BAX / BAK DKO transfected with FKBP - mCherry - DRP1 ( red ) FRB - EGFP - BAX ( green ) stained with MitoTracker Deep Red FM ( cyan ) were acquired before ( 0 min ) after induction of BAX / DRP1 dimerization ( 5 - 60 min ) . From top to down , rows correspond to signal of BAX DRP1 merge of both together with MitoTracker signal , respectively . Scale bar 20 µm .

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C) Forced dimerization of BAX and DRP1 in healthy cells induces their translocation to mitochondria and foci formation. Confocal microscopy images of U2OS BAX/BAK DKO cells transfected with FKBP-mCherry-DRP1 (red) and FRB-EGFP-BAX (green) and stained with MitoTracker Deep Red FM (cyan) were acquired before (0 min) and after induction of BAX/DRP1 dimerization (5-60 min). From top to down, the individual rows correspond to fluorescence signal of BAX and DRP1 and the merge of both together with the MitoTracker signal, respectively. Scale bar 20 µm.


E ) Induced dimerization of BAX DRP1 causes mitochondrial depolarization . U2OS BAX / BAK DKO were transfected as described in C ) stained with mitochondrial potential - sensitive dye MitoSpy NIR ( cyan ) . Confocal microscopy were acquired before ( 0 min ) after induced dimerization of BAX DRP1 ( 10 min ) . Scale Bar 20 µm . F ) of mitochondrial depolarization normalized to number of transfected before ( Ctrl ) after induced dimerization of BAX DRP1 ( Dim ) . Significance was tested using paired two - Student ´ s t - ( * p < 0 . 05 ) compared to non - induced control .

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E) Induced dimerization of BAX and DRP1 causes mitochondrial depolarization. U2OS BAX/BAK DKO cells were transfected as described in C) and stained with the mitochondrial membrane potential-sensitive dye MitoSpy NIR (cyan). Confocal microscopy images were acquired before (0 min) and after induced dimerization of BAX and DRP1 (10 min). Scale Bar 20 µm. F) Quantification of mitochondrial depolarization normalized to the number of transfected cells before (Ctrl) and after induced dimerization of BAX and DRP1 (Dim). Significance was tested using paired two-tailed Student´s t-test (* p < 0.05) compared to non-induced control. G


G - K ) Induced dimerization of DRP1 with itself ( G ) , BAX with itself ( H ) , BAX with TOM20 ( I ) in U2OS BAX / BAK DKO , or BAX with DRP1 in HCT OctaKO ( J , K ) , transected with FKBP - FRB - mCherry DRP1 ( G , red ) , FKBP - FRB - EGFP - BAX ( H , green ) , TOM20 - mCherry - FKBP ( red ) FRB - EGFP - BAX ( green , I ) , respectively . Mitochondria were stained using MitoTracker Deep Red FM or MitoSpy NIR ( cyan ) as indicated . Scale bar 20 µm ( G - I ) 10 µm ( J , K ) . are representative of n = 3 independent experiments .

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G-K) Induced dimerization of DRP1 with itself (G), BAX with itself (H), BAX with TOM20 (I) in U2OS BAX/BAK DKO cells, or BAX with DRP1 in HCT OctaKO cells (J,K), transected with FKBP- and FRB-mCherry DRP1 (G, red), FKBP- and FRB-EGFP-BAX (H, green), TOM20-mCherry-FKBP (red) and FRB-EGFP-BAX (green, I), respectively. Mitochondria were stained using MitoTracker Deep Red FM or MitoSpy NIR (cyan) as indicated. Scale bar 20 µm (G-I) and 10 µm (J,K ). All images are representative of n=3 independent experiments.


Representative H & E immunohistochemical stainings of CK5 / 6 p63 in pulmonary head - - neck squamous - carcinomas . Scale bar : 500 µm .

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Representative H&E and immunohistochemical stainings of CK5/6 and p63 in pulmonary and head-and-neck squamous-cell carcinomas. Scale bar: 500 µm.


showing somatic mutations in defined pathways HPV status for HNSCC SQCLC cases analysed . ( Blue , no mutation / no HPV detected ; red , mutation / HPV detected ; , no data available )

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Heatmap showing somatic mutations in defined pathways and HPV status for all HNSCC and SQCLC cases analysed. (Blue, no mutation/ no HPV detected; red, mutation/HPV detected; white, no data available)


Comparison of mutation rates in HNSCC ( n = 279 ) SQCLC ( n = 178 ) cases from TCGA . Genes exhibiting a mutation rate > 0 . 25 in both cancer types are labelled .

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Comparison of mutation rates in HNSCC (n=279) and SQCLC (n=178) cases from TCGA. Genes exhibiting a mutation rate >0.25 in both cancer types are labelled.


Representative histogram showing SILAC distribution between Super - SILAC standard SQCLC ( left ) HNSCC ( right ) samples .

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Representative histogram showing the SILAC ratio distribution between the Super-SILAC standard and SQCLC (left) and HNSCC (right) samples.


Boxplot showing numbers of quantified , derived from 44 SQCLC 30 HNSCC tissue samples . central in boxes represents median number of over samples , upper lower borders of boxes correspond to 25 % 75 % quantiles . Whiskers indicate minimum maximum .

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Boxplot showing the numbers of quantified proteins, derived from 44 SQCLC and 30 HNSCC tissue samples. The central line in the boxes represents the median number of proteins over all samples, upper and lower borders of boxes correspond to 25% and 75% quantiles. Whiskers indicate minimum and maximum.


Distribution of MS - signal intensities in 74 tissue samples of training cohort .

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Distribution of MS-based protein signal intensities in all 74 tissue samples of the training cohort.


Principal - component of SQCLC ( n = 44 ; blue ) HNSCC ( n = 30 ; red ) expression . Shown are first two principal components , accounting for 8 . 51 % 8 . 03 % of total variance in data , respectively .

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Principal-component analysis of SQCLC (n=44; blue) and HNSCC (n=30; red) protein expression. Shown are the first two principal components, accounting for 8.51% and 8.03% of the total variance in the data, respectively.


Volcano plot adjusted p values for differential expression to averaged normalised SILAC ratios of two replicates . Red ( higher expression in HNSCC ) blue ( higher expression in SQCLC ) dots indicate significantly regulated ( p < 0 . 05 ) .

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Volcano plot adjusted p values for differential protein expression to averaged normalised SILAC ratios of two replicates. Red (higher expression in HNSCC) and blue (higher expression in SQCLC) dots indicate significantly regulated proteins (p < 0.05).


Two - class comparison of genetic dependencies from a publically available genome - scale CRISPR - Cas9 screen of HNSCC ( 12 ) versus SQCLC ( 10 lines ) identified a subset of differentially expressed that were also differential dependencies in these types . x - axis represents effect of mean difference of dependency scores in HNSCC compared to SQCLC lines . Positive effect indicates a greater mean dependency in HNSCC ; negative effect indicates a greater mean dependency in SQCLC . y - axis represents statistical significance of differential enrichment calculated as - log10 ( p - value ) from a two - sided t - . p - values used for this plot are uncorrected for multiple hypothesis testing . Highlighted in green are genetic dependencies that were also identified as differentially expressed in our study .

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Two-class comparison of genetic dependencies from a publically available genome-scale CRISPR-Cas9 screen of HNSCC (12 cell line) versus SQCLC (10 cell lines) identified a subset of differentially expressed proteins that were also differential dependencies in these tumor types. The x-axis represents the effect size of the mean difference of dependency scores in HNSCC compared to SQCLC cell lines. Positive effect size indicates a greater mean dependency in HNSCC; negative effect size indicates a greater mean dependency in SQCLC. The y-axis represents the statistical significance of differential enrichment calculated as -log10(p-value) from a two-sided t-test. The p-values used for this plot are uncorrected for multiple hypothesis testing. Highlighted in green are genetic dependencies that were also identified as differentially expressed proteins in our study.


Immunohistochemical of expression of HMGCS - 1 ( p = 0 . 0014 ) , FTL ( p = 0 . 0001 ) ( C ) , LGALS7 ( p = 0 . 0001 ) FAM83H ( p = 0 . 0001 ) ( D ) in an independent cohort of 212 SQCLC 343 HNSCC cases . Scale bar indicates 100µm . Shown are mean values standard deviation . Statistical significance was assessed using Wilcoxon - Mann - Whitney .

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Immunohistochemical analysis of the expression of HMGCS-1 (p=0.0014),FTL (p=0.0001) (C),LGALS7 (p=0.0001) and FAM83H (p=0.0001)(D) in an independent cohort of 212 SQCLC and 343 HNSCC cases. Scale bar indicates 100µm. Shown are mean values and standard deviation. Statistical significance was assessed using Wilcoxon-Mann-Whitney test.


Boxplot showing number of quantified derived from 19 SQCLC , 19 HNSCC 51 squamous - lung tumours of unknown origin derived from HNSCC . central in boxes represents median number of over samples , upper lower borders of boxes correspond to 25 % 75 % quantiles . Whiskers indicate minimum maximum .

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Boxplot showing the number of quantified proteins derived from 19 SQCLC, 19 HNSCC and 51 squamous-cell lung tumours of unknown origin derived from HNSCC patients. The central line in the boxes represents the median number of proteins over all samples, upper and lower borders of boxes correspond to 25% and 75% quantiles. Whiskers indicate minimum and maximum.


Representative H & E immunohistochemical stainings of CK5 / 6 p63 in squamous - lung tumours of unknown origin derived from HNSCC . Scale bar : 500 µm .

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Representative H&E and immunohistochemical stainings of CK5/6 and p63 in squamous-cell lung tumours of unknown origin derived from HNSCC patients. Scale bar: 500 µm.


Kaplan - Meier of overall survival ( OS ) in which with lung tumours were grouped according to clinical classification ( left , median survival SQCLC : 55 months ; metHNSCC : 31 months ) or proteomic classification ( right , median survival SQCLC : 55 months ; metHNSCC : 8 months ) of tumour as metHNSCC or SQCLC . p value is from a log - rank . Kaplan - Meier of overall survival ( OS ) in which only that were independent from training cohort were grouped according to clinical classification ( left , median survival SQCLC : 84 months ; metHNSCC : 35 months ) or proteomic classification ( right , median survival SQCLC : 84 months ; metHNSCC : 5 months ) of tumour as metHNSCC or SQCLC . p value is from a log - rank .

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Kaplan-Meier analysis of overall survival (OS) in which all patients with lung tumours were grouped according to the clinical classification (left, median survival SQCLC: 55 months; metHNSCC: 31 months) or proteomic classification (right, median survival SQCLC: 55 months; metHNSCC: 8 months) of the tumour as metHNSCC or SQCLC. The p value is from a log-rank test. Kaplan-Meier analysis of overall survival (OS) in which only the patients that were independent from the training cohort were grouped according to the clinical classification (left, median survival SQCLC: 84 months; metHNSCC: 35 months) or proteomic classification (right, median survival SQCLC: 84 months; metHNSCC: 5 months) of the tumour as metHNSCC or SQCLC. The p value is from a log-rank test.


A cycle by flow cytometry ( EdU vs . TO - PRO - 3 ) in complete medium ( ENRAD ) or in absence of A83 - 01 ( 24 hours ) .

A Cell cycle analysis by flow cytometry (EdU vs. TO-PRO-3) in complete medium (ENRAD) or in the absence of A83-01 (24 hours).


B Detection of secreted by mouse prostate organoids in culture on click - chemistry enrichment mass spectrometry ( n = 4 biological replicates ; data points are shown with crossing representing mean value ) .

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B Detection of proteins secreted by mouse prostate organoids in culture based on click-chemistry enrichment and mass spectrometry analysis (n=4 biological replicates; data points are shown with crossing line representing mean value).


C IF for selected markers in mouse prostate tissue organoid sections ( scale bar = 50 μm ) .

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C IF analysis for selected markers in mouse prostate tissue and organoid sections (scale bar = 50 μm).


D Representative stereoscopic of mouse prostate organoids cultured in absence of A83 - 01 , with or without Follistatin ( Fst , 500 ng / mL , 6 days ) , or with or without Activin A neutralizing antibody MAB3381 ( 1 μg / mL , 6 days ) . Scale bar = 1

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D Representative stereoscopic images of mouse prostate organoids cultured in the absence of A83-01, with or without Follistatin (Fst, 500 ng/mL, 6 days), or with or without the Activin A neutralizing antibody MAB3381 (1 μg/mL, 6 days). Scale bar = 1 mm


E Western blot ( WB ) in mouse prostate organoids for selected canonical non - canonical Tgf - β signalling mediators , cycle inhibitor p21 ( Activin A , 50 ng / mL ; Tgfb1 , 500 ng / mL ; 24 hours ) .

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E Western blot (WB) analysis in mouse prostate organoids for selected canonical and non-canonical Tgf-β signalling mediators, and the cell cycle inhibitor p21 (Activin A, 50 ng/mL; Tgfb1, 500 ng/mL; 24 hours).


G Representative stereoscopic of mouse prostate organoids following treatment with Takinib ( Tak1 inhibitor ; 5 μM , 6 days ) , SB203580 ( p38α / β inhibitor ; 10 μM , 6 days ) or structurally unrelated Ralimetinib ( p38α / β inhibitor ; 1 μM , 7 days ) . Scale bar = 1

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G Representative stereoscopic images of mouse prostate organoids following treatment with Takinib (Tak1 inhibitor; 5 μM, 6 days), SB203580 (p38α/β inhibitor; 10 μM, 6 days) or the structurally unrelated Ralimetinib (p38α/β inhibitor; 1 μM, 7 days). Scale bar = 1 mm


H / fractionation western blot in mouse prostate organoids for selected signalling mediators in presence or absence of SB203580 ( p38α / β inhibitor ; 10 μM , 24 hours ) .

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H Nuclear/Cytoplasmic fractionation and western blot analysis in mouse prostate organoids for selected signalling mediators in the presence or absence of SB203580 (p38α/β inhibitor; 10 μM, 24 hours).


I Gene set enrichment ( GSEA ) plots displaying significant enrichment for inflammatory response Necrosis Factor ( TNF ) signalling in mouse prostate organoids cultured without A83 - 01 ( 24 hours ) versus complete medium ( ENRAD ) .

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I Gene set enrichment analysis (GSEA) plots displaying significant enrichment for inflammatory response and Tumor Necrosis Factor (TNF) signalling in mouse prostate organoids cultured without A83-01 (24 hours) versus complete medium (ENRAD).


J / fractionation western blot in mouse prostate organoids for selected cycle regulators , in presence or absence of SB203580 ( p38α / β inhibitor ; 10 μM , 24 or 48 hours ) .

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J Nuclear/Cytoplasmic fractionation and western blot analysis in mouse prostate organoids for selected cell cycle regulators, in the presence or absence of SB203580 (p38α/β inhibitor; 10 μM, 24 or 48 hours).


A Diagram depicting expansion of mouse prostate organoid cultures in absence of Tgf - β ligand inhibitor A83 - 01 ( n = 3 biological replicates per lobe - specific organoid culture ; arrow = continuous expansion ; dot = passage ; cross = culture loss ) .

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A Diagram depicting the expansion of mouse prostate organoid cultures in the absence of the Tgf-β ligand inhibitor A83-01 (n = 3 biological replicates per lobe-specific organoid culture; arrow = continuous expansion; dot = passage; cross = culture loss).


B Representative stereoscopic of C57BL / 6J DLP # 1 # 3 mouse prostate organoid lines during adaptation in absence of A83 - 01 . Scale bar = 1

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B Representative stereoscopic images of C57BL/6J DLP #1 and #3 mouse prostate organoid lines during adaptation in the absence of A83-01. Scale bar = 1 mm


D Gene set enrichment ( GSEA ) plot displaying significant enrichment for activation of ATR signalling in mouse prostate organoid lines ( C57 # 1 DLP , top ; C57 # 3 DLP , bottom ) adapted to grow in absence of A83 - 01 vs . normal control organoids cultured in complete medium ( ENRAD ) .

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D Gene set enrichment analysis (GSEA) plot displaying significant enrichment for activation of ATR signalling in mouse prostate organoid lines (C57#1 DLP, top; C57#3 DLP, bottom) adapted to grow in the absence of A83-01 vs. normal control organoids cultured in complete medium (ENRAD).


E Western blot in C57 # 1 C57 # 3 DLP mouse prostate organoid lines adapted to grow in absence of A83 - 01 vs . normal control organoids cultured in complete medium ( ENRAD ) . Immunoblots are displayed for Chek1 ( ATR signalling mediator ) , Trp53 p21 .

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E Western blot analysis in C57#1 and C57#3 DLP mouse prostate organoid lines adapted to grow in the absence of A83-01 vs. normal control organoids cultured in complete medium (ENRAD). Immunoblots are displayed for Chek1 (ATR signalling mediator), Trp53 and p21.


F Representative spectral karyotype ( SKY ) of metaphase spreads obtained from C57 # 1 DLP mouse prostate organoids cultured in normal conditions ( ENRAD ) or upon adaptation without A83 - 01 . Widespread genomic instability is observed following adaptation . Scale bars = 5 µm . G - H Representative telomere FISH - ( n = 2 biological replicates , 20 metaphases per condition ) - in C57 # 1 DLP mouse prostate organoids cultured in normal conditions ( ENRAD ) or upon adaptation without A83 - 01 . Widespread telomeric instability is observed following adaptation ( arrows indicate telomere doublets , One - way ANOVA , scale bars = 10 µm ) .

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

F Representative spectral karyotype (SKY) images of metaphase spreads obtained from C57#1 DLP mouse prostate organoids cultured in normal conditions (ENRAD) or upon adaptation without A83-01. Widespread genomic instability is observed following adaptation. Scale bars = 5 µm. G-H Representative telomere FISH images - and quantification (n = 2 biological replicates, 20 metaphases per condition) - in C57#1 DLP mouse prostate organoids cultured in normal conditions (ENRAD) or upon adaptation without A83-01. Widespread telomeric instability is observed following adaptation (arrows indicate telomere doublets, One-way ANOVA, scale bars = 10 µm).


Plasma of 13 biomarkers belonging to clusters 3 , 4 , 6 ( see Table 2 ) in healthy subjects ( n = 16 ) PDAC ( n = 25 ) , * p < 0 . 01 ( Mann - Whitney ) . Data are expressed as a scatter - plot , mean ± . p - values ( for each cluster ) were calculated with Wilcoxon Rank Sum indicate a significant association between each cluster PDAC .

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

Plasma levels of the 13 biomarkers belonging to clusters 3, 4, 6 (see Table 2) in healthy subjects (n=16) and PDAC patients (n=25), *p<0.01 (Mann-Whitney). Data are expressed as a scatter-plot, mean ± SEM. p-values (for each cluster) were calculated with the Wilcoxon Rank Sum Test and indicate a significant association between each cluster and PDAC.


A ) of TIMP1 , MMP7 TSP2 in plasma of KC mice ( n = 7 - 9 ) at 60 , 120 , 180 , 240 330 days of KPC mice ( n = 3 - 8 ) at 30 , 90 150 days of ( mean ± ) . * p < 0 . 05 ( Mann - Whitney ) . exact n p - values are indicated in Appendix Table S3

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

A) Levels of TIMP1, MMP7 and TSP2 in plasma of KC mice (n=7-9) at 60, 120, 180, 240 and 330 days of age and KPC mice (n=3-8) at 30, 90 and 150 days of age (mean ± SEM). *p<0.05 (Mann-Whitney). The exact n and p-values are indicated in Appendix Table S3


B ) of TIMP1 , MMP7 TSP2 in plasma of healthy mice ( n = 15 ) , mice with chronic pancreatitis at 150 days of ( n = 19 ) , KC mice at 330 days of ( control PdxCre n = 3 - 4 ; KC n = 7 - 8 ) , KPC mice at 150 days of ( control PdxCre n = 4 - 7 ; KPC n = 4 ) . exact n is indicated in Appendix Table S3B . Box plots extends from 25th to 75th percentiles , whiskers extends from min to max horizontal lines indicate median . p - values were calculated with 1 - way ANOVA with Tukey " s multiple comparison

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

B) Levels of TIMP1, MMP7 and TSP2 in plasma of healthy mice (n=15), mice with chronic pancreatitis at 150 days of age (n=19), KC mice at 330 days of age (control PdxCre n=3-4; KC n=7-8), KPC mice at 150 days of age (control PdxCre n=4-7; KPC n=4). The exact n is indicated in Appendix Table S3B. Box plots extends from 25th to 75th percentiles, whiskers extends from min to max and horizontal lines indicate median. p-values were calculated with 1-way ANOVA with Tukey"s multiple comparison test


C ) Histological of pancreas from PdxCre KC mice with different grades of PanIN lesions at 330 days of . Anti - TIMP1 , anti - MMP7 , anti - TSP2 anti - CCN2 of a representative KC PanIN lesion ( 200X , scale bars : 100μm )

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

C) Histological analysis of pancreas from PdxCre and KC mice with different grades of PanIN lesions at 330 days of age. Anti-TIMP1, anti-MMP7, anti-TSP2 and anti-CCN2 staining of a representative KC PanIN lesion (200X, scale bars: 100μm)


Responsiveness to gemcitabine ( GEM ) , either alone or combined with Nab - paclitaxel ( NAB - P ) , was evaluated in mice bearing orthotopic HuPa8 in pancreas . Nab - paclitaxel ( 25mg / kg ) gemcitabine ( 150mg / kg ) were given i . v . on days 1 8 of each 21 - day cycle ( for a total of 8 treatments ) . A ) of TIMP1 , MMP7 TSP2 in plasma of mice bearing orthotopic HuPa8 at 30 , 90 150 days after transplantation ( mean ± ; n ≥ 4 for each group ) . * p < 0 . 05 , ( Mann - Whitney ) . exact n p - values are indicated in Table S5A

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

Responsiveness to gemcitabine (GEM), either alone or combined with Nab-paclitaxel (NAB-P), was evaluated in mice bearing orthotopic HuPa8 in the pancreas. Nab-paclitaxel (25mg/kg) and gemcitabine (150mg/kg) were given i.v. on days 1 and 8 of each 21-day cycle (for a total of 8 treatments). A) Levels of TIMP1, MMP7 and TSP2 in plasma of mice bearing orthotopic HuPa8 at 30, 90 and 150 days after tumor transplantation (mean ± SEM; n≥4 for each group). *p<0.05, (Mann-Whitney). The exact n and p-values are indicated in Table S5A


Responsiveness to gemcitabine ( GEM ) , either alone or combined with Nab - paclitaxel ( NAB - P ) , was evaluated in mice bearing orthotopic HuPa8 in pancreas . Nab - paclitaxel ( 25mg / kg ) gemcitabine ( 150mg / kg ) were given i . v . on days 1 8 of each 21 - day cycle ( for a total of 8 treatments ) B ) Magnetic resonance imaging of HuPa8 . Representative are shown , dotted lines indicate masses

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

Responsiveness to gemcitabine (GEM), either alone or combined with Nab-paclitaxel (NAB-P), was evaluated in mice bearing orthotopic HuPa8 in the pancreas. Nab-paclitaxel (25mg/kg) and gemcitabine (150mg/kg) were given i.v. on days 1 and 8 of each 21-day cycle (for a total of 8 treatments) B) Magnetic resonance imaging of HuPa8. Representative images are shown, the white dotted lines indicate tumor masses


. Responsiveness to gemcitabine ( GEM ) , either alone or combined with Nab - paclitaxel ( NAB - P ) , was evaluated in mice bearing orthotopic HuPa8 in pancreas . Nab - paclitaxel ( 25mg / kg ) gemcitabine ( 150mg / kg ) were given i . v . on days 1 8 of each 21 - day cycle ( for a total of 8 treatments ) C ) growth over time measured by MRI ; each black arrow indicates one treatment ( mean ± ; n = 4 for each group ; 2 - way ANOVA with Tukey " s multiple comparison )

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

. Responsiveness to gemcitabine (GEM), either alone or combined with Nab-paclitaxel (NAB-P), was evaluated in mice bearing orthotopic HuPa8 in the pancreas. Nab-paclitaxel (25mg/kg) and gemcitabine (150mg/kg) were given i.v. on days 1 and 8 of each 21-day cycle (for a total of 8 treatments) C) Tumor growth over time measured by MRI; each black arrow indicates one treatment (mean ± SEM; n=4 for each group; 2-way ANOVA with Tukey"s multiple comparison test)


Responsiveness to gemcitabine ( GEM ) , either alone or combined with Nab - paclitaxel ( NAB - P ) , was evaluated in mice bearing orthotopic HuPa8 in pancreas . Nab - paclitaxel ( 25mg / kg ) gemcitabine ( 150mg / kg ) were given i . v . on days 1 8 of each 21 - day cycle ( for a total of 8 treatments ) D ) of 3 selected biomarkers in plasma of HuPa8 bearing mice , prior to ( day 80 ; at randomization ) , during ( day 120 ) after ( day 165 ) treatments ( mean ± ; n ≥ 4 for each group ; 2 - way ANOVA with Tukey " s multiple comparison ) . exact n is indicated in Appendix Table S5B

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

Responsiveness to gemcitabine (GEM), either alone or combined with Nab-paclitaxel (NAB-P), was evaluated in mice bearing orthotopic HuPa8 in the pancreas. Nab-paclitaxel (25mg/kg) and gemcitabine (150mg/kg) were given i.v. on days 1 and 8 of each 21-day cycle (for a total of 8 treatments) D) Levels of the 3 selected biomarkers in plasma of HuPa8 bearing mice, prior to (day 80; at randomization), during (day 120) and after (day 165) the treatments (mean ± SEM; n≥4 for each group; 2-way ANOVA with Tukey"s multiple comparison test). The exact n is indicated in Appendix Table S5B


Immunoprecipitation was performed with GFP - binding immobilized on magnetic beads using extracts of Arabidopsis seedlings , expressing either YFP - MKKK7 or Lti6B . Seedlings were treated with 1 μM flg22 for indicated time . YFP - MKKK7 Lti6B - GFP were detected with an anti - GFP antibody while FLS2 was detected with an FLS - specific antibody . Upper panel shows ( co ) - immunoprecipated , lower panel shows input of . Arrowheads indicate position of of interest .

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

Immunoprecipitation was performed with GFP-binding protein immobilized on magnetic beads using extracts of Arabidopsis seedlings, expressing either YFP-MKKK7 or Lti6B. Seedlings were treated with 1 μM flg22 for the indicated time. YFP-MKKK7 and Lti6B-GFP were detected with an anti-GFP antibody while FLS2 was detected with an FLS-specific antibody. Upper panel shows (co)-immunoprecipated proteins, lower panel shows input levels of protein. Arrowheads indicate the position of proteins of interest.


Application of selected reaction monitoring ( SRM ) mass spectrometry to quantify phosphorylated peptides in extracts treated with 1 μM flg22 . Bars represent mean of endogenous phosphopeptide versus spiked - in synthetic phosphopeptide normalized to t = 0 with error bars ± ( n = 3 . ) . Asterisks indicate significant difference level compared to t = 0 ( student t - , * > 0 . 05 , * * > 0 . 01 * * * > 0 . 001 ) . color of each bar corresponds to different time points ( 0 minutes = blue , 5 minutes = red , 10 minutes = green , 20 minutes = purple , 30 minutes = blue ) . Above each graph is name phosphorylated residue ( in brackets ) is indicated below corresponding phosphopeptide is shown with Serine ( S ) , Threonine ( T ) or Tyrosine ( Y ) phosphorylation site indicated by " [ + 80 ] " .

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

Application of selected reaction monitoring (SRM) mass spectrometry to quantify phosphorylated peptides in cell extracts treated with 1 μM flg22. Bars represent the mean ratio of endogenous phosphopeptide versus spiked-in synthetic phosphopeptide normalized to t = 0 with error bars ± SEM (n=3.). Asterisks indicate significant difference level compared to t = 0 (student t-test, *>0.05, **>0.01 and ***>0.001). The color of each bar corresponds to the different time points (0 minutes = dark blue, 5 minutes = red, 10 minutes = green, 20 minutes = purple, 30 minutes = light blue). Above each graph is the protein name and the phosphorylated residue (in brackets) is indicated and below the corresponding phosphopeptide is shown with the Serine (S), Threonine (T) or Tyrosine (Y) phosphorylation site indicated by "[+80]".


A ) Immunoblot analyses showing MAPK phosphorylation after flg22 induction in Col - 0 in mkkk7 . extracts were made from seedlings treated with 1 μM flg22 samples were taken at t = 0 , 10 30 min post induction . p44 / 42 antibody was used to detect phosphorylated MAPKs . Position of phosphorylated MAPKs is indicated at right . Equal loading of is shown with an α - Actin antibody as a loading control ( bottom panel ) . Three biological replicates were done with identical

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

A) Immunoblot analyses showing MAPK phosphorylation after flg22 induction in Col-0 and in mkkk7. Protein extracts were made from seedlings treated with 1 μM flg22 and samples were taken at t=0, 10 and 30 min post induction. The p44/42 antibody was used to detect phosphorylated MAPKs. Position of the individual phosphorylated MAPKs is indicated at the right. Equal loading of proteins is shown with an α-Actin antibody as a loading control (bottom panel). Three biological replicates were done with identical results


B ) MPK6 phosphorylation is specifically enhanced in mkkk7 . Comparison of phosphopeptide abundances from selected MAP kinases in Col - 0 ( blue ) mkkk7 ( red ) seedlings at t = 0 min t = 10 min after 1 μM flagellin treatment by selected reaction monitoring ( SRM ) mass spectrometry . Phosphopeptide corresponding to MKKK7 are only detectable in Col - 0 seedlings are non - detectable ( ND ) in mkkk7 . Bars represent means of measured peptide areas ( sum of transition areas ) for three biological replicates , with error bars ± ( n = 3 ) . Asterisks indicate significant difference between Col - 0 mkkk7 at time points ( student t - , * > 0 . 05 , * * > 0 . 01 * * * > 0 . 001 ) . ND indicates integration of an without transitions significantly above background . Above each graph name phosphorylated residue ( in brackets ) is indicated as well as corresponding phosphopeptide . Serine ( S ) , Threonine ( T ) or Tyrosine ( Y ) phosphorylation is indicated by " [ + 80 ] " .

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

B) MPK6 phosphorylation is specifically enhanced in mkkk7. Comparison of phosphopeptide abundances from selected MAP kinases in Col-0 (blue) and mkkk7 (red) seedlings at t = 0 min and t = 10 min after 1 μM flagellin treatment by selected reaction monitoring (SRM) mass spectrometry. Phosphopeptide corresponding to MKKK7 are only detectable in Col-0 seedlings and are non-detectable (ND) in mkkk7. Bars represent means of measured peptide areas (sum of all transition areas) for three biological replicates, with error bars ± SEM (n=3). Asterisks indicate significant difference between Col-0 and mkkk7 at individual time points (student t-test, *>0.05, **>0.01 and ***>0.001). ND indicates integration of an area without transitions significantly above background. Above each graph the protein name and the phosphorylated residue (in brackets) is indicated as well as the corresponding phosphopeptide sequence. Serine (S), Threonine (T) or Tyrosine (Y) phosphorylation is indicated by "[+80]".


A ) Transient expression in Arabidopsis mesophyll protoplasts shows enhanced defense gene expression in mkkk7 protoplasts after flg22 treatment . Protoplasts were isolated from 4 week old transfected with pWRKY29 : fLUC ( WRKY29 ) or pFRK1 : fLUC ( FRK1 ) constructs together with 35S : rLUC , as indicated in graph . Protoplasts were treated for 4 hrs with 10 µM flg22 or mock treated as indicated . horizontal axis indicates treatment while vertical axis represents expression relative to mock treated control sample , as fold induction . measurements were normalized to rLUC activity . Bars represent means ± STDEV ( n = 2 ) . Experiment was repeated 6 times with similar results .

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

A) Transient expression analysis in Arabidopsis mesophyll protoplasts shows enhanced defense gene expression in mkkk7 protoplasts after flg22 treatment. Protoplasts were isolated from 4 week old plants and transfected with pWRKY29:fLUC (WRKY29) or pFRK1:fLUC(FRK1) constructs together with 35S:rLUC, as indicated in the graph. Protoplasts were treated for 4 hrs with 10 µM flg22 or mock treated as indicated. The horizontal axis indicates the treatment while the vertical axis represents expression levels relative to the mock treated control sample, as fold induction. All measurements were normalized to the rLUC activity. Bars represent means ± STDEV (n=2). Experiment was repeated 6 times with similar results.


B ) WRKY29 transcripts measured by qRT - PCR in flg22 treated leaf material . Leaf strips of Col - 0 mkkk7 were treated with 1 µM flg22 for t = 0 , 1 , 2 , 4h . WRKY29 transcripts were normalized against Ubiquitin transcript as described before [ 62 ] . Bars represent mean value error bars show SE ( n = 3 ) . ( * p < 0 . 05 , * * p < 0 . 01 , student t - ) .

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

B) WRKY29 transcripts measured by qRT-PCR in flg22 treated leaf material. Leaf strips of Col-0 and mkkk7 were treated with 1 µM flg22 for t=0, 1, 2, and 4h. WRKY29 transcripts were normalized against Ubiquitin transcript as described before [62]. Bars represent mean value and error bars show SE (n=3). (* p<0.05, ** p<0.01, student t-test).


C ) FRK1 transcripts measured by qRT - PCR in flg22 treated leaf material . Leaf strips of Col - 0 mkkk7 were treated with 1 µM flg22 fort = 0 , 1 , 2 , 4h . FRK1 transcripts were normalized against Ubiquitin transcript as described before [ 62 ] . Bars represent mean value error bars show SE ( n = 3 ) . ( * p < 0 . 05 , * * p < 0 . 01 , student t - ) .

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

C) FRK1 transcripts measured by qRT-PCR in flg22 treated leaf material. Leaf strips of Col-0 and mkkk7 were treated with 1 µM flg22 fort=0, 1, 2, and 4h. FRK1 transcripts were normalized against Ubiquitin transcript as described before [62]. Bars represent mean value and error bars show SE (n=3). (* p<0.05, ** p<0.01, student t-test).


A ) Transient co - expression of MKKK7 in Arabidopsis mesophyll protoplasts shows suppression of flg22 - induced WRKY29 gene expression . Protoplasts were transfected with pWRKY29 : fLUC , 35S : rLUC indicated overexpression constructs of MKKK7 ( OE - MKKK7 , OE - MKKK7AA or OE - MKKK7DD ) as indicated on horizontal axis . Protoplasts were treated with 10 μM flg22 or mock treated for 4 hrs . measurements were normalized to rLUC activity expression is relative to mock treated control sample , shown as fold induction on vertical axis . Results shown are means ± STDEV ( n = 2 ) . At least two biological replicates were done with similar

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

A) Transient co-expression of MKKK7 in Arabidopsis mesophyll protoplasts shows suppression of flg22-induced WRKY29 gene expression. Protoplasts were transfected with pWRKY29:fLUC, 35S:rLUC and indicated overexpression constructs of MKKK7 (OE-MKKK7, OE-MKKK7AA or OE-MKKK7DD) as indicated on the horizontal axis. Protoplasts were treated with 10 μM flg22 or mock treated for 4 hrs. All measurements were normalized to the rLUC activity and expression is relative to the mock treated control sample, shown as fold induction on the vertical axis. Results shown are means ± STDEV (n=2). At least two biological replicates were done with similar results


A ) Four - week - old seedlings were dipped into a suspension containing virulent Pst DC3000 72 h later disease symptoms were scored . Data represents mean values ± ( n = 20 ; * * * , p < 0 . 001 ; paired t - ) . Three biological experiments were done showing similar results .

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

A) Four-week-old seedlings were dipped into a suspension containing virulent Pst DC3000 and 72 h later the disease symptoms were scored. Data represents mean values ± SEM (n=20;***, p<0.001; paired t-test). Three biological experiments were done showing similar results.


B ) of growth in Arabidopsis lines Col - 0 , mkkk7 p35S : MKKK7 - GFP in mkkk7 background . Four to five - week - old were pressure - infiltrated with virulent Pst DC3000 at indicated time points 6 samples were harvested re - isolated on selective media . number of colony forming units ( cfu / cm2 ) was determined at t = 0 , 2 3days post inoculation ( dpi ) . Data represents mean values ± ( n = 6 ; * * , p < 0 . 01 ; paired t - ) . Experiments were done at least twice with similar results .

O O O B-EXP_ASSAY O B-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of bacterial growth in Arabidopsis lines Col-0, mkkk7 and p35S:MKKK7-GFP in the mkkk7 background. Four to five-week-old plants were pressure-infiltrated with virulent Pst DC3000 and at indicated time points 6 samples were harvested and bacteria re-isolated on selective media. The number of colony forming units (cfu/cm2) was determined at t=0, 2 and 3days post inoculation (dpi). Data represents mean values ± SEM (n=6; **, p<0.01; paired t-test). Experiments were done at least twice with similar results.


C ) Disease symptom development in Pst - infected lines with estradiol inducible constructs of ind - MKKK7AA L8 , ind - MKKK7AA L10 , ind - MKKK7DD L1 ind - MKKK7DD L3 . Two independent transgenic lines for each construct were grown under short - day conditions disease symptoms were scored 3 dpi . Data represents mean values ± ( n = 20 ; * , p < 0 . 05 ; * * , p < 0 . 01 ; paired t - ) . vertical axis represents disease symptoms . Experiments were done at least twice with similar results .

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

C) Disease symptom development in Pst-infected lines with estradiol inducible constructs of ind-MKKK7AA L8, ind-MKKK7AA L10, ind-MKKK7DD L1 and ind-MKKK7DD L3. Two independent transgenic lines for each construct were grown under short-day conditions and disease symptoms were scored 3 dpi. Data represents mean values ± SEM (n=20; *, p<0.05; **, p<0.01; paired t-test). The vertical axis represents the percentage disease symptoms. Experiments were done at least twice with similar results.


A ) Effect of 100 nM flg22 treatment on ROS burst measured in 5 week old of Col - 0 mkkk7 .

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

A) Effect of 100 nM flg22 treatment on ROS burst measured in 5 week old plants of Col-0 and mkkk7.


B ) Effect of 100 nM flg22 treatment on ROS burst measured in 5 week old of Col - 0 two independent inducible MKKK7AA transgenic lines .

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

B) Effect of 100 nM flg22 treatment on the ROS burst measured in 5 week old plants of Col-0 and two independent inducible MKKK7AA transgenic lines.