Figure 4HSATIII 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 ) .

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Figure 4HSATIII 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).


Figure 5Scatter 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 .

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Figure 5Scatter 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.


Figure 6RT - 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 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 .

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

Figure 6RT-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 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.


Figure 7In 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 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 ) .

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 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

Figure 7In 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 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).


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

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


Figure 3 . 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 . 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 .

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Figure 3.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.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.


Figure 1PCP - 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 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 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 .

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 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 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

Figure 1PCP-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 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 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.


Figure 2Protein 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 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 .

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Figure 2Protein 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 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.


Figure 3A 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 and α6 ( B ) , Rpn3 and Rpn1 ( C ) , PA28β and PA28α ( D ) , and Rpn3 and PA28β ( E ) .

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Figure 3A 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 and α6 (B), Rpn3 and Rpn1 (C), PA28β and PA28α (D), and Rpn3 and PA28β (E).


Figure 4Heat - 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4Heat-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.


Figure 5The 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 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 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 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 ) .

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Figure 5The 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 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 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 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).


Figure 1Dual - 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 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 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 ) .

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 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 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

Figure 1Dual-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 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 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).


Figure 2A ) 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 ) 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 ) .

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 O O O O O O O O O O O O O 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) 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) 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).


Figure 3C ) 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 ) 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 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 .

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 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 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

Figure 3C) 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) 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 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.


Figure 4BAX 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 .

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Figure 4BAX 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.


Figure 5B ) 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 ) 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 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 ) 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 .

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Figure 5B) 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) 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 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) 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.


Figure 6C ) 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 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 . GG - 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 .

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

Figure 6C) 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 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. GG-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.


Figure 1Representative H & E and immunohistochemical stainings of CK5 / 6 and p63 in pulmonary and head - and - neck squamous - cell carcinomas . Scale bar : 500 µm . 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 ) and SQCLC ( n = 178 ) cases from TCGA . Genes exhibiting a mutation rate > 0 . 25 in both cancer types are labelled .

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Figure 1Representative H&E and immunohistochemical stainings of CK5/6 and p63 in pulmonary and head-and-neck squamous-cell carcinomas. Scale bar: 500 µm.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) and SQCLC (n=178) cases from TCGA. Genes exhibiting a mutation rate >0.25 in both cancer types are labelled.


Figure 2Representative histogram showing the SILAC ratio distribution between the Super - SILAC standard and SQCLC ( left ) and HNSCC ( right ) samples . 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 - based protein signal intensities in all 74 tissue samples of the training cohort . 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 .

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

Figure 2Representative histogram showing the SILAC ratio distribution between the Super-SILAC standard and SQCLC (left) and HNSCC (right) samples.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-based protein signal intensities in all 74 tissue samples of the training cohort.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.


Figure 3Volcano 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 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 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 .

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Figure 3Volcano 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 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 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.


Figure 4Boxplot 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 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 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 .

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 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

Figure 4Boxplot 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 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 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.


Figure 2A 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 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 analysis for selected markers in mouse prostate tissue and organoid sections ( scale bar = 50 μm ) . 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 mmE 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 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 mmH 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 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 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 ) .

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

Figure 2A 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 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 analysis for selected markers in mouse prostate tissue and organoid sections (scale bar = 50 μm).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 mmE 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 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 mmH 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 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 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).


Figure 3A 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 images of C57BL / 6J DLP # 1 and # 3 mouse prostate organoid lines during adaptation in the absence of A83 - 01 . Scale bar = 1 mmD 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 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 ) 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 ) .

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

Figure 3A 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 images of C57BL/6J DLP #1 and #3 mouse prostate organoid lines during adaptation in the absence of A83-01. Scale bar = 1 mmD 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 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) 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).


Figure 1 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 .

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Figure 1 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.


Figure 3 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 ) 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 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 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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) 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 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)


Figure 5 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 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 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 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

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

Figure 5 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 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 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 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


Figure 1Immunoprecipitation 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 .

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Figure 1Immunoprecipitation 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.


Figure 2Application 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 ] " .

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Figure 2Application 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]".


Figure 3A ) 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 resultsB ) 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 ] " .

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

Figure 3A) 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 resultsB) 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]".


Figure 4A ) 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 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 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 ) .

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 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 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

Figure 4A) 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 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 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).


Figure 5A ) 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

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

Figure 5A) 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


Figure 6A ) 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 ) 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 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 .

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

Figure 6A) 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) 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 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.


Figure 7A ) 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 the ROS burst measured in 5 week old plants of Col - 0 and two independent inducible MKKK7AA transgenic lines . C ) Effect of 100 nM flg22 treatment on the ROS burst measured in 5 weeks old plants of Col - 0 and two independent inducible MKKK7DD transgenic lines . Graphs represent means with error bars ± SEM ( n = 24 ) . The vertical axis represents the relative increase in ROS production ( photon counts ) after PAMP treatment . At least three biological replicate experiments were done with similar results .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 the ROS burst measured in 5 week old plants of Col-0 and two independent inducible MKKK7AA transgenic lines.C) Effect of 100 nM flg22 treatment on the ROS burst measured in 5 weeks old plants of Col-0 and two independent inducible MKKK7DD transgenic lines. Graphs represent means with error bars ± SEM (n=24). The vertical axis represents the relative increase in ROS production (photon counts) after PAMP treatment. At least three biological replicate experiments were done with similar results.


Figure 1 ( A ) Kaplan - Meier survival curves ( n = 12 - 16 ) . ( B ) Heart gross morphology . ( C ) Body weight ; ( D ) Heart weight and ( E ) Heart - to - body weight ratio ( n = 28 - 40 ) . ( F ) Relative expression levels of cardiac hypertrophy markers ( Nppa and Nppb ) ( n = 5 ) . Bars represent mean ± S . E . M . ( Student ' s t test ; * * p < 0 . 01 , * * * * p < 0 . 0001 ) . ( G ) Enzyme histochemistry double staining for COX and SDH activities . ( n = 4 ) . ( H ) Assessment of cardiac fibrosis by Masson ' s trichrome staining ( n = 4 ) . White scale bars , 100μm ; black scale bars , 1 mm .

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

Figure 1(A) Kaplan-Meier survival curves (n = 12 - 16).(B) Heart gross morphology.(C) Body weight; (D) Heart weight and (E) Heart-to-body weight ratio (n = 28-40).(F) Relative expression levels of cardiac hypertrophy markers (Nppa and Nppb) (n = 5). Bars represent mean ± S.E.M. (Student's t test; **p < 0.01, ****p < 0.0001).(G) Enzyme histochemistry double staining for COX and SDH activities. (n = 4).(H) Assessment of cardiac fibrosis by Masson's trichrome staining (n = 4). White scale bars, 100μm; black scale bars, 1 mm.


Figure 2 ( A ) Western blot analysis and ( B ) Relative quantification of UPRmt markers in heart extracts . HSC70 is used as a loading control ( CTRL ) , ( n = 3 ) . ( C and D ) Relative expression levels normalized to WT of ( C ) established UPRmt markers in 3 - 4 week old mice ( n = 3 - 4 ) . ( C and D ) Relative expression levels normalized to WT of ( D ) mitochondrial chaperones , proteases and TFAM in 3 - 4 week old mice ( n = 3 - 4 ) . ( E ) Western blot analysis of cellular stress markers . HSC70 is used as a loading control ( CTRL ) , ( n = 3 ) ( F ) Relative expression levels of transcription factors involved in UPRmt and ISR ( left ) and Fgf21 ( right ) ( n = 5 ) . Bars represent mean ± S . E . M . ( Student ' s t test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ) .

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

Figure 2(A) Western blot analysis and (B) Relative quantification of UPRmt markers in heart extracts. HSC70 is used as a loading control (CTRL), (n = 3).(C and D) Relative expression levels normalized to WT of (C) established UPRmt markers in 3 - 4 week old mice (n = 3 - 4).(C and D) Relative expression levels normalized to WT of (D) mitochondrial chaperones, proteases and TFAM in 3 - 4 week old mice (n = 3 - 4).(E) Western blot analysis of cellular stress markers. HSC70 is used as a loading control (CTRL), (n = 3)(F) Relative expression levels of transcription factors involved in UPRmt and ISR (left) and Fgf21 (right) (n = 5). Bars represent mean ± S.E.M. (Student's t test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).


Figure 3 ( A ) Western blot analysis of individual OXPHOS subunits in heart extracts . HSC70 and Calnexin are used as loading controls , ( n = 3 ) . Antibodies against individual OXPHOS subunits ( on the right ) were used to detect OXPHOS complexes ( on the left ) . ( B ) BN - PAGE and subsequent Western blot analysis for OXPHOS complexes . Antibodies against individual OXPHOS subunits ( on the right ) were used to detect OXPHOS complexes ( on the left ) . ( C , D ) In gel activity of complexes I ( C ) and IV ( D ) performed after BN - PAGE . ( E ) Oxygen consumption rates in intact heart mitochondria in the presence of pyruvate - glutamate - malate ( C I ) and pyruvate - glutamate - malate + succinate ( C I + C II ) as substrates . State 3 ( substrates + ADP ) ; State 4 ( + oligomycin ) ; Uncoupled ( + FCCP ) . ( n = 3 ) . Bars represent mean ± S . E . M . ( Student ' s t test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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

Figure 3(A) Western blot analysis of individual OXPHOS subunits in heart extracts. HSC70 and Calnexin are used as loading controls, (n = 3). Antibodies against individual OXPHOS subunits (on the right) were used to detect OXPHOS complexes (on the left).(B) BN-PAGE and subsequent Western blot analysis for OXPHOS complexes. Antibodies against individual OXPHOS subunits (on the right) were used to detect OXPHOS complexes (on the left).(C, D) In gel activity of complexes I (C) and IV (D) performed after BN-PAGE.(E) Oxygen consumption rates in intact heart mitochondria in the presence of pyruvate-glutamate-malate (C I) and pyruvate-glutamate-malate + succinate (C I + C II) as substrates. State 3 (substrates+ADP); State 4 (+oligomycin); Uncoupled (+FCCP). (n = 3). Bars represent mean ± S.E.M. (Student's t test, *p < 0.05, **p < 0.01, ***p < 0.001).


Figure 4 ( A ) Representative gel of the In organello translation analysis of heart mitochondria . De novo synthetized proteins are isolated after labeling with 35S - met ( 1 hour Pulse ) or after cold chase ( 3h Chase ) . Positions of individual proteins are indicated on the left . Position of full - length proteins ( Proficient ) and low molecular weight polypeptides ( Abortive ) are indicated on the right . Note that in WT and ClpP KO low molecular weight polypeptides include ATP8 / ND4L proteins . ( B ) Relative overall protein synthesis and turnover rate . ( C ) Western blot analysis of DARS2 levels . HSC70 is used as a loading control ( CTRL ) . ( D ) Relative levels of the individual de novo synthesized OXPHOS subunits in DKO mitochondria normalized to the corresponding Dars2 KO polypeptide level . ( E ) Relative levels of protein synthesis of Proficient and Abortive polypeptides . ( F ) Quantification of the turnover rate of full - length ( Proficient ) de novo synthesized proteins . Bars represent mean ± S . E . M . ( Student ' s t test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ) , ( n = 3 - 4 ) .

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

Figure 4(A) Representative gel of the In organello translation analysis of heart mitochondria. De novo synthetized proteins are isolated after labeling with 35S-met (1 hour Pulse) or after cold chase (3h Chase). Positions of individual proteins are indicated on the left. Position of full-length proteins (Proficient) and low molecular weight polypeptides (Abortive) are indicated on the right. Note that in WT and ClpP KO low molecular weight polypeptides include ATP8/ND4L proteins.(B) Relative overall protein synthesis and turnover rate.(C) Western blot analysis of DARS2 levels. HSC70 is used as a loading control (CTRL).(D) Relative levels of the individual de novo synthesized OXPHOS subunits in DKO mitochondria normalized to the corresponding Dars2 KO polypeptide level.(E) Relative levels of protein synthesis of Proficient and Abortive polypeptides. (F) Quantification of the turnover rate of full-length (Proficient) de novo synthesized proteins. Bars represent mean ± S.E.M. (Student's t test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001), (n=3-4).


Figure 1 ( A - C ) Western blot analysis of THP - 1 cells infected with ( A ) HSV - 1 at MOI 5 for 3 h , 6 h , 12 h , 18 h , and 24 h ( B ) CHIKV at MOI 5 for 3 h , 6 h , 12 h , 18 h , and 24 h ( C ) JEV at MOI 5 for 3 h , 6 h , 12 h , and 24 h . ( I - L ) Control and IRGM knockdown THP - 1 IFN reporter cells were treated with ( I ) Poly I : C ( 1 μg / ml ) for 8 h , 12 h and 24 h or ( J ) Poly dA : dT ( 1 μg / ml ) for 12 h and 24 h or ( K ) IFN - β ( 500 ng / ml ) for 8 h and 12 h or ( L ) cGAMP ( 1 μg / ml ) for 12 h and 24 h and the supernatant was subjected to luciferase reporter assay using QuantiLuc reagent ( M - P ) Control and IRGM knockdown THP - 1 cells were untreated and treated with Poly I : C ( 1 μg / ml ) for 8 h and the total RNA was subjected to qRT PCR with primers of ( M ) MX2 ( N ) ISG15 ( O ) OAS1 and ( P ) IFN - β . ( Q - T ) Control and IRGM knockdown THP - 1 cells were untreated and treated with Poly dA : dT ( 1 μg / ml ) for 8 h and the total RNA was subjected to qRT PCR with primers of ( Q ) MX2 ( R ) ISG15 ( S ) OAS1 and ( T ) IFN - β .

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 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 1(A-C) Western blot analysis of THP-1 cells infected with (A) HSV-1 at MOI 5 for 3 h, 6 h, 12 h, 18 h, and 24 h (B) CHIKV at MOI 5 for 3 h, 6 h, 12 h, 18 h, and 24 h (C) JEV at MOI 5 for 3 h, 6 h, 12 h, and 24 h.(I-L) Control and IRGM knockdown THP-1 IFN reporter cells were treated with (I) Poly I:C (1 μg/ml) for 8 h, 12 h and 24 h or (J) Poly dA:dT (1 μg/ml) for 12 h and 24 h or (K) IFN-β (500 ng/ml) for 8 h and 12 h or (L) cGAMP (1 μg/ml) for 12 h and 24 h and the supernatant was subjected to luciferase reporter assay using QuantiLuc reagent(M-P) Control and IRGM knockdown THP-1 cells were untreated and treated with Poly I:C (1 μg/ml) for 8 h and the total RNA was subjected to qRT PCR with primers of (M) MX2 (N) ISG15 (O) OAS1 and (P) IFN-β. (Q-T) Control and IRGM knockdown THP-1 cells were untreated and treated with Poly dA:dT (1 μg/ml) for 8 h and the total RNA was subjected to qRT PCR with primers of (Q) MX2 (R) ISG15 (S) OAS1 and (T) IFN-β.