Figure 6Mice were pre - treated with pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Plasma was collected 24h post - surgery and ( A ) Alanine - amino transferase ( ALT ) , were measured as described in methods . P - values were calculated with 2 - way ANOVA tests . n = 6 - 7 / group , combined data of 2 independent experiments . Mice were pre - treated with pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Plasma was collected 24h post - surgery and creatinine concentrations , were measured as described in methods . P - values were calculated with 2 - way ANOVA tests . n = 6 - 7 / group , combined data of 2 independent experiments . Mice were pre - treated with pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Plasma was collected 24h post - surgery and IL - 6 levels were measured as described in methods . P - values were calculated with 2 - way ANOVA tests . n = 6 - 7 / group , combined data of 2 independent experiments . Mice were pre - treated with pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Apoptosis in liver paraffin - fixated sections 24h after sepsis , measured with TUNEL staining and depicted as % of PI positive cells / µm² tissue area . P - values were calculated with 2 - way ANOVA tests . n = 6 - 7 / group , combined data of 2 independent experiments . Mice were pre - treated with pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Apoptosis in kidney paraffin - fixated sections 24h after sepsis , measured with TUNEL staining and depicted as % of PI positive cells / µm² tissue area . P - values were calculated with 2 - way ANOVA tests . n = 6 - 7 / group , combined data of 2 independent experiments . Mice were pre - treated with pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Systemic bacterial load ( CFU / ml blood ) 24h post - sepsis in vehicle or pemafibrate treated mice . P - values were calculated with 2 - way Student ' s t - test . Pemafibrate ( 1 mg / kg ) or vehicle ( 0 , 9 % NaCl ) was administered at different timepoints before and after the induction of sepsis and survival was monitored during 9 days , after which no further deaths occurred . Survival curve was analyzed via Log rank testsMice were injected with the PPARα antagonist GW6471 ( 10µg / g ) or vehicle ( DMSO ) 3h pre - and 3h post - CLP . Survival was monitored during 9 days , after which no further deaths occurred . Survival curve was analyzed via a Log rank test . Combined data of 2 independent experiments , n = 16 / group .

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

Figure 6Mice were pre-treated with pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Plasma was collected 24h post-surgery and (A) Alanine-amino transferase (ALT), were measured as described in methods. P-values were calculated with 2-way ANOVA tests. n = 6-7/group, combined data of 2 independent experiments.Mice were pre-treated with pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Plasma was collected 24h post-surgery and creatinine concentrations, were measured as described in methods. P-values were calculated with 2-way ANOVA tests. n = 6-7/group, combined data of 2 independent experiments.Mice were pre-treated with pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Plasma was collected 24h post-surgery and IL-6 levels were measured as described in methods. P-values were calculated with 2-way ANOVA tests. n = 6-7/group, combined data of 2 independent experiments.Mice were pre-treated with pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Apoptosis in liver paraffin-fixated sections 24h after sepsis, measured with TUNEL staining and depicted as % of PI positive cells/µm² tissue area. P-values were calculated with 2-way ANOVA tests. n = 6-7/group, combined data of 2 independent experiments.Mice were pre-treated with pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Apoptosis in kidney paraffin-fixated sections 24h after sepsis, measured with TUNEL staining and depicted as % of PI positive cells/µm² tissue area. P-values were calculated with 2-way ANOVA tests. n = 6-7/group, combined data of 2 independent experiments.Mice were pre-treated with pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Systemic bacterial load (CFU/ml blood) 24h post-sepsis in vehicle or pemafibrate treated mice. P-values were calculated with 2-way Student's t-test.Pemafibrate (1 mg/kg) or vehicle (0,9% NaCl) was administered at different timepoints before and after the induction of sepsis and survival was monitored during 9 days, after which no further deaths occurred. Survival curve was analyzed via Log rank testsMice were injected with the PPARα antagonist GW6471 (10µg/g) or vehicle (DMSO) 3h pre- and 3h post-CLP. Survival was monitored during 9 days, after which no further deaths occurred. Survival curve was analyzed via a Log rank test. Combined data of 2 independent experiments, n = 16/group.


Figure 7Blood samples were collected from healthy volunteers and septic patients ( n = 13 septic patients , = 15 healthy controls ) . Plasma was prepared and ( A ) FFA and ( B ) glycerol concentrations were determined as described in the methods . P - values were calculated with two - way Student t - tests .

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

Figure 7Blood samples were collected from healthy volunteers and septic patients (n= 13 septic patients, = 15 healthy controls). Plasma was prepared and (A) FFA and (B) glycerol concentrations were determined as described in the methods. P-values were calculated with two-way Student t-tests.


Figure 1 ( C ) Confocal images of formaldehyde fixed GFP + S . flexneri following 20 min incubation with 2 mM GTP and 10 µM Alexa - Fluor647 - labeled protein . Bacteria associated with hGBP1 mutants after 20 min were quantified . Mean frequencies ± SEM of combined data from two independent experiments are shown . Significance was determined by one - way ANOVA with Tukey ' s multiple comparison test . * * * , P ≤ 0 . 001 . Scale bars equal 5 µm . Flow diagram depicts effects of K51A , R48A , and H74A hGBP1 mutations on nucleotide binding and hydrolysis .

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

Figure 1(C) Confocal images of formaldehyde fixed GFP+ S. flexneri following 20 min incubation with 2 mM GTP and 10 µM Alexa-Fluor647-labeled protein. Bacteria associated with hGBP1 mutants after 20 min were quantified. Mean frequencies ± SEM of combined data from two independent experiments are shown. Significance was determined by one-way ANOVA with Tukey's multiple comparison test. ***, P ≤ 0.001. Scale bars equal 5 µm. Flow diagram depicts effects of K51A, R48A, and H74A hGBP1 mutations on nucleotide binding and hydrolysis.


Figure 2 ( A ) Translocation of ectopically expressed mCherry - hGBP1 to cytosolic GFP + S . flexneri ΔipaH9 . 8 in HeLa hGBP1 - KO cells was monitored by time - lapse microscopy . Individual time frames of Movie EV1 starting at 55 min post infection ( mpi ) are shown . ( B ) Confocal time - lapse microscopy was used to image 10 µM Alexa - Fluor647 - hGBP1F supplemented with 2 mM GTP in the presence or absence of formaldehyde fixed GFP + S . flexneri . Individual time frames of Movie EV2 depict hGBP1F fluorescence intensity . Merged images of hGBP1F and S . flexneri fluorescence are shown for the 60 min time points . ( C ) Images were taken at 45 min after addition of 10 µM Alexa - Fluor647 - hGBP1F to formaldehyde fixed GFP + S . flexneri in the presence of indicated nucleotides ( GTP , natural substrate ; GppNHp , non - hydrolysable GTP analog ; GTPγS , slowly hydrolysable GTP analog ; GDP · AlFX , GTP transition state analog ) . hGBP1 - associated bacteria after 45 min were quantified . Combined data from two independent experiments are shown as mean ± SEM . Significance was determined by one - way ANOVA with Tukey ' s multiple comparison test . * * * * , 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 O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Translocation of ectopically expressed mCherry-hGBP1 to cytosolic GFP+ S. flexneri ΔipaH9.8 in HeLa hGBP1-KO cells was monitored by time-lapse microscopy. Individual time frames of Movie EV1 starting at 55 min post infection (mpi) are shown.(B) Confocal time-lapse microscopy was used to image 10 µM Alexa-Fluor647-hGBP1F supplemented with 2 mM GTP in the presence or absence of formaldehyde fixed GFP+ S. flexneri. Individual time frames of Movie EV2 depict hGBP1F fluorescence intensity. Merged images of hGBP1F and S. flexneri fluorescence are shown for the 60 min time points.(C) Images were taken at 45 min after addition of 10 µM Alexa-Fluor647-hGBP1F to formaldehyde fixed GFP+ S. flexneri in the presence of indicated nucleotides (GTP, natural substrate; GppNHp, non-hydrolysable GTP analog; GTPγS, slowly hydrolysable GTP analog; GDP·AlFX, GTP transition state analog). hGBP1-associated bacteria after 45 min were quantified. Combined data from two independent experiments are shown as mean ± SEM. Significance was determined by one-way ANOVA with Tukey's multiple comparison test. ****, P ≤ 0.0001.


Figure 3 ( A ) Polymerization of 10 µM hGBP1F and hGBP1FR584 - 586A in the presence of 2 mM GTP was monitored over time by absorption spectroscopy at 350 nm . Absorbance signals were superimposed with nucleotide composition of the same solution analyzed at defined time points , revealing the characteristic first phase of slow polymer nucleation and the second phase of fast polymer growth and cooperative hydrolysis . ( B ) Scanning - electron micrographs of live S . flexneri incubated with no protein or with 5 µM of either hGBP1F , hGBP1FR584 - 586A , or hGBP1FR48A ( non - polymerizing mutant ) in the presence of 2 mM GTP for 4 min . Arrowheads point to unattached hGBP1 polymers , arrows point to hGBP1 polymers attached to bacteria , and asterisks mark polymeric structures that appear to fuse with bacterial surfaces . ( C ) Confocal time - lapse microscopy frames of formaldehyde fixed GFP + S . flexneri following admixture of 10 µM Alexa - Fluor647 - labeled hGBP1F or hGBP1FR584 - 586A and 2 mM GTP . Binding of hGBP1F polymers to bacteria at 5 min and enclosure of bacteria with hGBP1F protein coats after 60 min were quantified . Mean frequencies ± SEM of combined data from at least three independent experiments are shown . Significance was determined by by unpaired t - tests , two - tailed . * * * , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Polymerization of 10 µM hGBP1F and hGBP1FR584-586A in the presence of 2 mM GTP was monitored over time by absorption spectroscopy at 350 nm. Absorbance signals were superimposed with nucleotide composition of the same solution analyzed at defined time points, revealing the characteristic first phase of slow polymer nucleation and the second phase of fast polymer growth and cooperative hydrolysis.(B) Scanning-electron micrographs of live S. flexneri incubated with no protein or with 5 µM of either hGBP1F, hGBP1FR584-586A, or hGBP1FR48A (non-polymerizing mutant) in the presence of 2 mM GTP for 4 min. Arrowheads point to unattached hGBP1 polymers, arrows point to hGBP1 polymers attached to bacteria, and asterisks mark polymeric structures that appear to fuse with bacterial surfaces.(C) Confocal time-lapse microscopy frames of formaldehyde fixed GFP+ S. flexneri following admixture of 10 µM Alexa-Fluor647-labeled hGBP1F or hGBP1FR584-586A and 2 mM GTP. Binding of hGBP1F polymers to bacteria at 5 min and enclosure of bacteria with hGBP1F protein coats after 60 min were quantified. Mean frequencies ± SEM of combined data from at least three independent experiments are shown. Significance was determined by by unpaired t-tests, two-tailed. ***, P ≤ 0.001; ****, P ≤ 0.0001.


Figure 4 ( A ) Representative confocal images taken at 60 min after admixture of 2 mM GTP and 10 µM Alexa - Fluor647 - or Alexa - Fluor488 - hGBP1F to formaldehyde fixed gram - negative and gram - positive bacteria expressing GFP ( S . enterica Typhimurium [ ST ] , L . monocytogenes [ Lm ] ) , or RFP ( S . flexneri [ Sf ] , uropathogenic E . coli [ UPEC ] ) , or dsRed ( L . pneumophila [ Lp ] , S . aureus [ Sa ] ) . ( B ) ( Left panel ) Confocal time - lapse microscopy frames of Alexa - Fluor488 - LPS - O55 : B5 after addition of 5 µM Alexa - Fluor647 - hGBP1F supplemented with 2 mM GTP . ( Right panel ) Confocal images taken of 5 µM Alexa - Fluor488 - LPS - O55 : B5 20 min after addition of 5 µM Alexa - Fluor647 - hGBP1F or - hGBP1FR48A supplemented with 2 mM GTP . Graphs depict average aggregate area of Alexa - Fluor488 - LPS - O55 : B5 supplemented with 2 mM GTP and 5 µM of the indicated hGBP1 variant . Mean area ± SEM of combined data from three independent experiments . Significance was determined by one - way ANOVA with Tukey ' s multiple comparison test . . n . s . , not significant ; * * * , P ≤ 0 . 001 ; * * * * , P ≤ 0 . 0001 . ( C ) Polymerization of 5 µM hGBP1F induced by addition of 2 mM GTP in the presence and absence of 5 µM LPS - O55 : B5 was monitored by absorption spectroscopy at 350 nm . The absorbance signal was superimposed with nucleotide composition of the same solution analyzed at defined time points . Maximal hydrolysis rates ( turnover numbers ) were determined for hGBP1F variants in the presence and absence of 5 µM LPS - O55 : B5 . Graphs show mean turnover numbers ± SEM of combined data from at least three independent experiments . Significance was determined by two - way ANOVA with Tukey ' s multiple comparison test . n . s . , not significant ; * * * , P ≤ 0 . 001 ; * * * * , P ≤ 0 . 0001 . ( D ) Formaldehyde fixed GFP + S . flexneri supplemented with varying LPS - O55 : B5 concentrations were mixed with 5 µM hGBP1F and 2 mM GTP . After 60 min hGBP1F - enclosed S . flexneri were quantified . Combined data from three independent experiments are shown as mean ± SEM . Significance was determined by one - way ANOVA with Tukey ' s multiple comparison test . * * * * , P ≤ 0 . 0001 . ( E ) Confocal images of formaldehyde fixed GFP - , RFP - , or dsRed - expressing gram - negative bacteria 60 min after addition of 5 µM Alexa - Fluor647 - or Alexa - Fluor488 - hGBP1F and 2 mM GTP in the presence and absence of 100 µM LPS - O55 : B5 . After 60 min hGBP1F - enclosed gram - negative bacteria were quantified . Combined data from three independent experiments are shown as mean ± SEM . Significance was determined by two - way ANOVA with Tukey ' s multiple comparison test . * * * * , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 4(A) Representative confocal images taken at 60 min after admixture of 2 mM GTP and 10 µM Alexa-Fluor647- or Alexa-Fluor488-hGBP1F to formaldehyde fixed gram-negative and gram-positive bacteria expressing GFP (S. enterica Typhimurium [ST], L. monocytogenes [Lm]), or RFP (S. flexneri [Sf], uropathogenic E. coli [UPEC]), or dsRed (L. pneumophila [Lp], S. aureus [Sa]).(B) (Left panel) Confocal time-lapse microscopy frames of Alexa-Fluor488-LPS-O55:B5 after addition of 5 µM Alexa-Fluor647-hGBP1F supplemented with 2 mM GTP. (Right panel) Confocal images taken of 5 µM Alexa-Fluor488-LPS-O55:B5 20 min after addition of 5 µM Alexa-Fluor647-hGBP1F or -hGBP1FR48A supplemented with 2 mM GTP. Graphs depict average aggregate area of Alexa-Fluor488-LPS-O55:B5 supplemented with 2 mM GTP and 5 µM of the indicated hGBP1 variant. Mean area ± SEM of combined data from three independent experiments. Significance was determined by one-way ANOVA with Tukey's multiple comparison test.. n.s., not significant; ***, P ≤ 0.001; ****, P ≤ 0.0001.(C) Polymerization of 5 µM hGBP1F induced by addition of 2 mM GTP in the presence and absence of 5 µM LPS-O55:B5 was monitored by absorption spectroscopy at 350 nm. The absorbance signal was superimposed with nucleotide composition of the same solution analyzed at defined time points. Maximal hydrolysis rates (turnover numbers) were determined for hGBP1F variants in the presence and absence of 5 µM LPS-O55:B5. Graphs show mean turnover numbers ± SEM of combined data from at least three independent experiments. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. n.s., not significant; ***, P ≤ 0.001; ****, P ≤ 0.0001.(D) Formaldehyde fixed GFP+ S. flexneri supplemented with varying LPS-O55:B5 concentrations were mixed with 5 µM hGBP1F and 2 mM GTP. After 60 min hGBP1F-enclosed S. flexneri were quantified. Combined data from three independent experiments are shown as mean ± SEM. Significance was determined by one-way ANOVA with Tukey's multiple comparison test. ****, P ≤ 0.0001.(E) Confocal images of formaldehyde fixed GFP-, RFP-, or dsRed-expressing gram-negative bacteria 60 min after addition of 5 µM Alexa-Fluor647- or Alexa-Fluor488-hGBP1F and 2 mM GTP in the presence and absence of 100 µM LPS-O55:B5. After 60 min hGBP1F-enclosed gram-negative bacteria were quantified. Combined data from three independent experiments are shown as mean ± SEM. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. ****, P ≤ 0.0001.


Figure 5 ( B ) Time - lapse microscopy of fixed GFP + co - isogenic S . flexneri wild type and rfaL mutant after adding 10 µM Alexa - Fluor647 - hGBP1F and 2 mM GTP . Bacteria bound by hGBP1F at 5 min and hGBP1F - enclosed bacteria after 60 min were quantified . Combined data from three independent experiments are shown as mean ± SEM . Significance was determined by unpaired t - tests , two - tailed . n . s . , not significant ; * * * * , P ≤ 0 . 0001 . ( C ) Scanning - electron micrographs of live wild type and rfaL mutant strains incubated with no protein or with 5 µM of hGBP1F in the presence of 2 mM GTP for 4 min . Arrowheads point to unattached hGBP1 polymers , arrows point to hGBP1 polymers attached to bacteria , and asterisks mark polymeric structures that appear to fuse with bacterial surfaces ( E ) Confocal images of formaldehyde fixed , co - isogenic GFP + wild type and mutant S . flexneri strains harboring rfb regions of E . coli serotypes O8 and O25 after 60 min of incubation time in the presence of 10 µM Alexa - Fluor647 - hGBP1F and 2 mM GTP .

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

Figure 5(B) Time-lapse microscopy of fixed GFP+ co-isogenic S. flexneri wild type and rfaL mutant after adding 10 µM Alexa-Fluor647-hGBP1F and 2 mM GTP. Bacteria bound by hGBP1F at 5 min and hGBP1F-enclosed bacteria after 60 min were quantified. Combined data from three independent experiments are shown as mean ± SEM. Significance was determined by unpaired t-tests, two-tailed. n.s., not significant; ****, P ≤ 0.0001.(C) Scanning-electron micrographs of live wild type and rfaL mutant strains incubated with no protein or with 5 µM of hGBP1F in the presence of 2 mM GTP for 4 min. Arrowheads point to unattached hGBP1 polymers, arrows point to hGBP1 polymers attached to bacteria, and asterisks mark polymeric structures that appear to fuse with bacterial surfaces(E) Confocal images of formaldehyde fixed, co-isogenic GFP+ wild type and mutant S. flexneri strains harboring rfb regions of E. coli serotypes O8 and O25 after 60 min of incubation time in the presence of 10 µM Alexa-Fluor647-hGBP1F and 2 mM GTP.


Figure 6 ( A ) Following incubation for 1 . 5 h with buffer in the absence or presence of 10 µM hGBP1F and 5 mM GTP , live bacteria were treated with antibiotics ( 2 . 5 µg / ml PMB or 5 mg / ml lysozyme ) for 30 min or left untreated . The number of viable bacteria was subsequently determined by colony forming unit ( CFU ) counts . Graphs show mean CFUs ± SEM of combined data from at least three independent experiments . Significance was determined by two - way ANOVA with Tukey ' s multiple comparison test . n . s . , not significant ; * * * * , P ≤ 0 . 0001 . ( B ) Formaldehyde fixed RFP + UPEC were mixed with 10 µM Alexa - Fluor488 - hGBP1F or - hGBP1FR584 - 586A supplemented with 2 mM GTP . Following 60 min of incubation hGBP1F - enclosed UPEC were quantified . Graphs depict mean ± SEM of combined data from three independent experiments . Significance was determined by unpaired t - test , two - tailed . * * * * , P ≤ 0 . 0001 . ( C ) Formaldehyde fixed RFP + UPEC were mixed with 10 µM Alexa - Fluor488 - hGBP1F or - hGBP1FR584 - 586A and 2 mM GTP . Samples were fixed after 60 min incubation and stained with an antibody against the inner core / lipid A fragment of E . coli LPS ( anti - LPS ) . The frequency of anti - LPS stained UPEC was quantified . Graphs show mean ± SEM of combined data from three independent experiments . Significance was determined by two - way ANOVA with Tukey ' s multiple comparison test . n . s . , not significant ; * * * * , P ≤ 0 . 0001 . ( D ) IFNγ - primed wild type and hGBP1 - KO HeLa cells stably expressing inactive mutant YFP - Caspase - 4C258S were infected with either cytosol - entering S . enterica Typhimurium mutant ΔsifA ( MOI = 25 ) or with S . flexneri ΔospC3ΔipaH9 . 8 ( MOI = 6 ) , a mutant strain lacking Caspase - 4 antagonist OspC3 as well as hGBP1 antagonist IpaH9 . 8 . Cells were fixed at 4 hpi ( ST ΔsifA ) or at 2 hpi ( Sf ΔospC3ΔipaH9 . 8 ) and stained for endogenous hGBP1 . YFP - Caspase - 4C258S associated bacteria were quantified and mean ± SEM of combined data from three independent experiments are shown . Significance was determined by two - way ANOVA with Tukey ' s multiple comparison test . * * * * , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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

Figure 6(A) Following incubation for 1.5 h with buffer in the absence or presence of 10 µM hGBP1F and 5 mM GTP, live bacteria were treated with antibiotics (2.5 µg/ml PMB or 5 mg/ml lysozyme) for 30 min or left untreated. The number of viable bacteria was subsequently determined by colony forming unit (CFU) counts. Graphs show mean CFUs ± SEM of combined data from at least three independent experiments. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. n.s., not significant; ****, P ≤ 0.0001.(B) Formaldehyde fixed RFP+ UPEC were mixed with 10 µM Alexa-Fluor488-hGBP1F or -hGBP1FR584-586A supplemented with 2 mM GTP. Following 60 min of incubation hGBP1F-enclosed UPEC were quantified. Graphs depict mean ± SEM of combined data from three independent experiments. Significance was determined by unpaired t-test, two-tailed. ****, P ≤ 0.0001.(C) Formaldehyde fixed RFP+ UPEC were mixed with 10 µM Alexa-Fluor488-hGBP1F or -hGBP1FR584-586A and 2 mM GTP. Samples were fixed after 60 min incubation and stained with an antibody against the inner core/ lipid A fragment of E. coli LPS (anti-LPS). The frequency of anti-LPS stained UPEC was quantified. Graphs show mean ± SEM of combined data from three independent experiments. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. n.s., not significant; ****, P ≤ 0.0001.(D) IFNγ-primed wild type and hGBP1-KO HeLa cells stably expressing inactive mutant YFP-Caspase-4C258S were infected with either cytosol-entering S. enterica Typhimurium mutant ΔsifA (MOI = 25) or with S. flexneri ΔospC3ΔipaH9.8 (MOI = 6), a mutant strain lacking Caspase-4 antagonist OspC3 as well as hGBP1 antagonist IpaH9.8. Cells were fixed at 4 hpi (ST ΔsifA) or at 2 hpi (Sf ΔospC3ΔipaH9.8) and stained for endogenous hGBP1. YFP-Caspase-4C258S associated bacteria were quantified and mean ± SEM of combined data from three independent experiments are shown. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. ****, P ≤ 0.0001.


Figure 7 ( B Cells were infected with poly - D - lysine treated GFP + S . flexneri strains at an MOI of 6 . Cells were stained for indicated proteins and Z - stacks were recorded using confocal fluorescence microscopy . Actin tails were classified as tails when ≥ 2 . 5 µm . All graphs show mean values ± SEM of combined data from three independent experiments . All scale bars are 5 µm . Arrows point to bacteria associated with indicated proteins , arrowheads point to bacteria lacking indicated proteins , and asterisks mark bacteria lacking unipolar IcsA localization . ( B ) Unprimed HeLa cells were infected with wild type or rfaL S . flexneri . Total and unipolar localization of IcsA as well as the actin tail formation were quantified at 1 hpi . Significance was determined by unpaired two - tailed t - tests . n . s . , not significant ; * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 . Cells were infected with poly - D - lysine treated GFP + S . flexneri strains at an MOI of 6 . Cells were stained for indicated proteins and Z - stacks were recorded using confocal fluorescence microscopy . Actin tails were classified as tails when ≥ 2 . 5 µm . All graphs show mean values ± SEM of combined data from three independent experiments . All scale bars are 5 µm . Arrows point to bacteria associated with indicated proteins , arrowheads point to bacteria lacking indicated proteins , and asterisks mark bacteria lacking unipolar IcsA localization . ( C - mCherry - hGBP1 was expressed in HeLa hGBP1 - KO cells infected with S . flexneri ΔipaH9 . 8 and stained for IcsA , N - WASP Significance was determined by unpaired t - tests , two - tailed . n . s . , not significant ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ; * * * * , P ≤ 0 . 0001 . Cells were infected with poly - D - lysine treated GFP + S . flexneri strains at an MOI of 6 . Cells were stained for indicated proteins and Z - stacks were recorded using confocal fluorescence microscopy . Actin tails were classified as tails when ≥ 2 . 5 µm . All graphs show mean values ± SEM of combined data from three independent experiments . All scale bars are 5 µm . Arrows point to bacteria associated with indicated proteins , arrowheads point to bacteria lacking indicated proteins , and asterisks mark bacteria lacking unipolar IcsA localization . D ) mCherry - hGBP1 was expressed in HeLa hGBP1 - KO cells infected with S . flexneri ΔipaH9 . 8 and stained for Arp2 , and F - actin at 2 . 5 hpi . Significance was determined by unpaired t - tests , two - tailed . n . s . , not significant ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ; * * * * , P ≤ 0 . 0001 . Cells were infected with poly - D - lysine treated GFP + S . flexneri strains at an MOI of 6 . Cells were stained for indicated proteins and Z - stacks were recorded using confocal fluorescence microscopy . Actin tails were classified as tails when ≥ 2 . 5 µm . All graphs show mean values ± SEM of combined data from three independent experiments . All scale bars are 5 µm . Arrows point to bacteria associated with indicated proteins , arrowheads point to bacteria lacking indicated proteins , and asterisks mark bacteria lacking unipolar IcsA localization . ( E ) IFNγ - primed and unprimed wild type and hGBP1 - KO HeLa cells were infected with S . flexneri ΔipaH9 . 8 and subcellular localization of IcsA , N - WASP , Arp2 , and F - actin was assessed and quantified at 2 . 5 hpi . Significance was determined by two - way ANOVA with Tukey ' s multiple comparison 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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

Figure 7(B Cells were infected with poly-D-lysine treated GFP+ S. flexneri strains at an MOI of 6. Cells were stained for indicated proteins and Z-stacks were recorded using confocal fluorescence microscopy. Actin tails were classified as tails when ≥ 2.5 µm. All graphs show mean values ± SEM of combined data from three independent experiments. All scale bars are 5 µm. Arrows point to bacteria associated with indicated proteins, arrowheads point to bacteria lacking indicated proteins, and asterisks mark bacteria lacking unipolar IcsA localization. (B) Unprimed HeLa cells were infected with wild type or rfaL S. flexneri. Total and unipolar localization of IcsA as well as the actin tail formation were quantified at 1 hpi. Significance was determined by unpaired two-tailed t-tests. n.s., not significant; *, P ≤ 0.05; **, P ≤ 0.01.Cells were infected with poly-D-lysine treated GFP+ S. flexneri strains at an MOI of 6. Cells were stained for indicated proteins and Z-stacks were recorded using confocal fluorescence microscopy. Actin tails were classified as tails when ≥ 2.5 µm. All graphs show mean values ± SEM of combined data from three independent experiments. All scale bars are 5 µm. Arrows point to bacteria associated with indicated proteins, arrowheads point to bacteria lacking indicated proteins, and asterisks mark bacteria lacking unipolar IcsA localization. (C- mCherry-hGBP1 was expressed in HeLa hGBP1-KO cells infected with S. flexneri ΔipaH9.8 and stained for IcsA, N-WASP Significance was determined by unpaired t-tests, two-tailed. n.s., not significant; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.Cells were infected with poly-D-lysine treated GFP+ S. flexneri strains at an MOI of 6. Cells were stained for indicated proteins and Z-stacks were recorded using confocal fluorescence microscopy. Actin tails were classified as tails when ≥ 2.5 µm. All graphs show mean values ± SEM of combined data from three independent experiments. All scale bars are 5 µm. Arrows point to bacteria associated with indicated proteins, arrowheads point to bacteria lacking indicated proteins, and asterisks mark bacteria lacking unipolar IcsA localization. D) mCherry-hGBP1 was expressed in HeLa hGBP1-KO cells infected with S. flexneri ΔipaH9.8 and stained for Arp2, and F-actin at 2.5 hpi. Significance was determined by unpaired t-tests, two-tailed. n.s., not significant; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.Cells were infected with poly-D-lysine treated GFP+ S. flexneri strains at an MOI of 6. Cells were stained for indicated proteins and Z-stacks were recorded using confocal fluorescence microscopy. Actin tails were classified as tails when ≥ 2.5 µm. All graphs show mean values ± SEM of combined data from three independent experiments. All scale bars are 5 µm. Arrows point to bacteria associated with indicated proteins, arrowheads point to bacteria lacking indicated proteins, and asterisks mark bacteria lacking unipolar IcsA localization. (E) IFNγ-primed and unprimed wild type and hGBP1-KO HeLa cells were infected with S. flexneri ΔipaH9.8 and subcellular localization of IcsA, N-WASP, Arp2, and F-actin was assessed and quantified at 2.5 hpi. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.


Figure 2A ) IL - 8 production after 6 h of treatment with digitonin and βHBP at indicated concentrations or digitonin and wt S . flexneri lysate in HeLa cells ( Hoechst in blue , IL - 8 in red ) after 6 h of treatment with digitonin and βHBP at indicated concentrations or digitonin and wt S . flexneri lysate in HeLa cell B ) Quantification of IL - 8 awith digitonin and βHBP at indicated concentrations or digitonin and wt S . flexneri lysat C ) TIFA oligomerization at 6 h in TIFA - GFP - expressing HeLa cells treatewith digitonin and βHBP at indicated concentrations or digitonin and wt S . flexneri lysat at 6 h in TIFA - GFP - expressing HeLa cells treate D ) Quantification of TIFA oligomerizatiowith digitonin and βHBP at indicated concentrations or digitonin and wt S . flexneri lysat E ) TIFA oligomerization at 30 min in cells treateF ) Kinetics of TIFA - GFP oligomerization upon treatment with digitonin alone or in combination with βHBP or S . flexneri

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

Figure 2A) IL-8 production after 6 h of treatment with digitonin and βHBP at indicated concentrations or digitonin and wt S. flexneri lysate in HeLa cells (Hoechst in blue, IL-8 in red)after 6 h of treatment with digitonin and βHBP at indicated concentrations or digitonin and wt S. flexneri lysate in HeLa cell B) Quantification of IL-8 awith digitonin and βHBP at indicated concentrations or digitonin and wt S. flexneri lysat C) TIFA oligomerization at 6 h in TIFA-GFP-expressing HeLa cells treatewith digitonin and βHBP at indicated concentrations or digitonin and wt S. flexneri lysat at 6 h in TIFA-GFP-expressing HeLa cells treate D) Quantification of TIFA oligomerizatiowith digitonin and βHBP at indicated concentrations or digitonin and wt S. flexneri lysat E) TIFA oligomerization at 30 min in cells treateF) Kinetics of TIFA-GFP oligomerization upon treatment with digitonin alone or in combination with βHBP or S. flexneri lysate


Figure 3B ) TIFA - GFP oligomerization at 30 min in HeLa cells treated with digitonin ( mock ) or digitonin and a lysate from indicated S . flexneri strains ( pHldA / C means plasmid - encoded HldA and HldC ) with digitonin ( mock ) or digitonin and a lysate from indicated S . flexneri strain ( pHldA / C means plasmid - encoded HldA and HldC ) C ) TIFA - GFP oligomerization at 6 h in cells treatewith digitonin ( mock ) or digitonin and a lysate from indicated S . flexneri strains ( pHldA / C means plasmid - encoded HldA and HldC ) D ) IL - 8 production at 6 h in cells were treateE ) TIFA - GFP oligomerization in cells treated for 30 min with digitonin alone , digitonin and wild - type S . flexneri lysate or digitonin and ADP - L - β - d - heptose at indicated concentrationswith digitonin alone , digitonin and wild - type S . flexneri lysate or digitonin and ADP - L - β - d - heptose at indicated concentrations . F ) IL - 8 production after 6 h in cells

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

Figure 3B) TIFA-GFP oligomerization at 30 min in HeLa cells treated with digitonin (mock) or digitonin and a lysate from indicated S. flexneri strains (pHldA/C means plasmid-encoded HldA and HldC)with digitonin (mock) or digitonin and a lysate from indicated S. flexneri strain (pHldA/C means plasmid-encoded HldA and HldC) C) TIFA-GFP oligomerization at 6 h in cells treatewith digitonin (mock) or digitonin and a lysate from indicated S. flexneri strains (pHldA/C means plasmid-encoded HldA and HldC) D) IL-8 production at 6 h in cells were treateE) TIFA-GFP oligomerization in cells treated for 30 min with digitonin alone, digitonin and wild-type S. flexneri lysate or digitonin and ADP-L-β-d-heptose at indicated concentrationswith digitonin alone, digitonin and wild-type S. flexneri lysate or digitonin and ADP-L-β-d-heptose at indicated concentrations. F) IL-8 production after 6 h in cells treate


Figure 4A ) Representative images of the formation of TIFA oligomers in HeLa cells infected for 30 min with dsRed - expressing wt or indicated mutants of S . flexneri ( MOI 50 ) . pHldA / C means plasmid - encoded HldA and HldC . Fluorescence intensity was adjusted between strains to optimize visualization of bacteria . Scale bar , 20 μmB ) Infectivity of indicated S . flexneri strainsC ) TIFA - GFP oligomerization after 30 min of infection with selected mutants of the LPS biosynthesis pathway ( MOI 10 ) of the LPS biosynthesis pathway ( MOI 10 ) . D ) TIFA - GFP oligomerization after 4 h of infection with the S . flexneri mutantE ) IL - 8 production 4 h post infection ( MOI 5 )

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

Figure 4A) Representative images of the formation of TIFA oligomers in HeLa cells infected for 30 min with dsRed-expressing wt or indicated mutants of S. flexneri (MOI 50). pHldA/C means plasmid-encoded HldA and HldC. Fluorescence intensity was adjusted between strains to optimize visualization of bacteria. Scale bar, 20 μmB) Infectivity of indicated S. flexneri strainsC) TIFA-GFP oligomerization after 30 min of infection with selected mutants of the LPS biosynthesis pathway (MOI 10)of the LPS biosynthesis pathway (MOI 10). D) TIFA-GFP oligomerization after 4 h of infection with the S. flexneri mutantE) IL-8 production 4 h post infection (MOI 5)


Figure 5A ) ADP - heptose - induced TIFA - GFP oligomerization depends on ALPK1 . After siRNA transfection , HeLa cells were transfected with empty pCMV or pCMV - ALPK1 as indicated . They were then infected with wt S . flexneri ( MOI 10 ) or treated with digitonin and ADP - heptose ( 10 - 5 M ) for 30 min . As control , cells were left untreated ( mock ) or treated with digitoninADP - heptose - induced cytokine secretion is ALPK1 - dependent . HeLa cells were transfected with control or ALPK1 - targeting siRNAs and infected with wt S . flexneri ( MOI 2 ) or treated with ADP - heptose ( 10 - 5 M ) . As control , cells were left untreated ( mock ) or treated with digitonin . Cytokines were measured in cell culture supernatants after 8

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 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

Figure 5A) ADP-heptose-induced TIFA-GFP oligomerization depends on ALPK1. After siRNA transfection, HeLa cells were transfected with empty pCMV or pCMV-ALPK1 as indicated. They were then infected with wt S. flexneri (MOI 10) or treated with digitonin and ADP-heptose (10-5 M) for 30 min. As control, cells were left untreated (mock) or treated with digitoninADP-heptose-induced cytokine secretion is ALPK1-dependent. HeLa cells were transfected with control or ALPK1-targeting siRNAs and infected with wt S. flexneri (MOI 2) or treated with ADP-heptose (10-5 M). As control, cells were left untreated (mock) or treated with digitonin. Cytokines were measured in cell culture supernatants after 8 h


Figure 1 ( C ) Decreased levels of free carnitine and modified carnitine species in PKM2 KO cell lines as compared with their parental counterparts ( n = 4 ) . Data information : Data are displayed as means ± SD . Statistical significance was assessed using two - tailed Student ' s t - test , ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 and ∗ ∗ ∗ p < 0 . 001 . n . s means no significance . ( E ) Increased acetyl - CoA in PKM2 KO MCF7 and MDA - MB 231cells as compared with their parental counterparts ( n = 4 ) . ( F ) Decreased total cholesterol in PKM2 KO MCF7 and MDA - MB 231cells as compared with their parental counterparts ( n = 3 ) . ( Data information : Data are displayed as means ± SD . Statistical significance was assessed using two - tailed Student ' s t - test , ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 and ∗ ∗ ∗ p < 0 . 001 . n . s means no significance .

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 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

Figure 1(C) Decreased levels of free carnitine and modified carnitine species in PKM2 KO cell lines as compared with their parental counterparts (n=4). Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001. n.s means no significance.(E) Increased acetyl-CoA in PKM2 KO MCF7 and MDA-MB 231cells as compared with their parental counterparts (n=4). (F) Decreased total cholesterol in PKM2 KO MCF7 and MDA-MB 231cells as compared with their parental counterparts (n=3). ( Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001. n.s means no significance.


Figure 2 ( G ) Western blot analysis of TMEM33 , ABCA1 , SREBP1 , HMGCR , SCAP , FASN , LDLR , FDFT1 , I DI1 , RNF5 , INSIG 1 and 2 , and Calnexin in PKM2 KO MCF7 and MDA - MB - 231 cells , and their respective parental cells . Arrow indicates nuclear SREBP1 . P : precursor , N : nuclear .

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

Figure 2(G) Western blot analysis of TMEM33, ABCA1, SREBP1, HMGCR, SCAP, FASN, LDLR, FDFT1, I DI1, RNF5, INSIG 1 and 2, and Calnexin in PKM2 KO MCF7 and MDA-MB-231 cells, and their respective parental cells. Arrow indicates nuclear SREBP1. P: precursor, N: nuclear.


Figure 3 ( A ) Immunofluorescence of SREBP1 in MCF7 parental and PKM2 KO cells by confocal imaging . Scale bar , 5μm . ( B ) Quantitation of nuclear SREBP1 intensity in A . n > = 25 cells as in ( A ) from three different fields . ( Data information : Data are displayed as means ± SD . Statistical significance was assessed using two - tailed Student ' s t - test , ∗ p < 0 . 05 and ∗ ∗ ∗ p < 0 . 001 . ( G ) Western blot analysis of TMEM33 , RNF5 , SCAP , SREBP1 , HMGCR , LDLR , FDFT1 , IDI1 , and PKM2 in wild type , PKM2 KO , TMEM33 OE ( pool ) , and two TMEM33OE clones ( clones # 1 and # 8 ) MDA - MB - 231 cells . Arrow indicates nuclear SREBP1 . N : nucleus and P : precursor .

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

Figure 3(A) Immunofluorescence of SREBP1 in MCF7 parental and PKM2 KO cells by confocal imaging. Scale bar, 5μm. (B) Quantitation of nuclear SREBP1 intensity in A. n>=25 cells as in (A) from three different fields. ( Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05 and ∗∗∗p < 0.001.(G) Western blot analysis of TMEM33, RNF5, SCAP, SREBP1, HMGCR, LDLR, FDFT1, IDI1, and PKM2 in wild type, PKM2 KO, TMEM33 OE (pool), and two TMEM33OE clones (clones #1 and #8) MDA-MB-231 cells. Arrow indicates nuclear SREBP1. N: nucleus and P: precursor.


Figure 4 ( B ) Western blot analysis of PKM2 , PKM1 , NRF1 , TMEM33 , RNF5 and SCAP levels in MEF ( PKM2fl / fl , Cre - ERT2 ) cells treated with 4 - OHT in a time course experiment . ( C ) Co - immunoprecipitation of endogenous SCAP and RNF5 in Flag - tagged TMEM33 stably expressed MCF7 cells . SEC61β is an ER protein , which is found to interact with TMEM33 . ( H ) RNF5 is involved in TMEM33 mediated SCAP polyubiquitination in 293T cells . 293T cells were stably transfected with shCtrl or shRNF5 constructs , then the indicated plasmids were co - transfected into shCtrl or shRNF5 expressing 293T cells for 18 hours followed by MG - 132 treatment for 8 hours . FLAG M2 beads were used for pull - down .

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

Figure 4(B) Western blot analysis of PKM2, PKM1, NRF1, TMEM33, RNF5 and SCAP levels in MEF (PKM2fl/fl, Cre-ERT2) cells treated with 4-OHT in a time course experiment.(C) Co-immunoprecipitation of endogenous SCAP and RNF5 in Flag-tagged TMEM33 stably expressed MCF7 cells. SEC61β is an ER protein, which is found to interact with TMEM33.(H) RNF5 is involved in TMEM33 mediated SCAP polyubiquitination in 293T cells. 293T cells were stably transfected with shCtrl or shRNF5 constructs, then the indicated plasmids were co-transfected into shCtrl or shRNF5 expressing 293T cells for 18 hours followed by MG-132 treatment for 8 hours. FLAG M2 beads were used for pull-down.


Figure 5 ( D ) qRT - PCR analyses of selected SREBPs downstream genes in shCtrl and shRNF5 PKM2 KO MDA - MB - 231 cells ( n = 3 ) . Data information : Data are displayed as means ± SD . Statistical significance was assessed using two - tailed Student ' s t - test , ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 and ∗ ∗ ∗ p < 0 . 001 . ( G ) Cell proliferation of Ctrl and HA - RNF5 OE MCF7 cells measured by MTT assays ( n = 8 ) . Data information : Data are displayed as means ± SD . Statistical significance was assessed using two - tailed Student ' s t - test , ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 and ∗ ∗ ∗ p < 0 . 001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5(D) qRT-PCR analyses of selected SREBPs downstream genes in shCtrl and shRNF5 PKM2 KO MDA-MB-231 cells (n=3). Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.(G) Cell proliferation of Ctrl and HA-RNF5 OE MCF7 cells measured by MTT assays (n=8). Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.


Figure 6 ( E ) Western blot analyses of TMEM33 and RNF5 in NRF1 overexpressed MCF7 clones . ( F ) Western blot analyses of TMEM33 and RNF5 in shCtrl and shNRF1 PKM2 KO MCF7 cells . ( ( G ) Co - immunoprecipitation of VCP with FLAG - tagged PKM1 / 2 in PKM2 KO 293T cells . FLAG - tagged PKM1 or PKM2 construct was transiently transfected into PKM2 KO or parental 293T cells for 24 hours , followed by pull - down using FLAG - M2 beads . PKM1 , PKM2 and VCP antibodies were used for immunoblotting .

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

Figure 6(E) Western blot analyses of TMEM33 and RNF5 in NRF1 overexpressed MCF7 clones. (F) Western blot analyses of TMEM33 and RNF5 in shCtrl and shNRF1 PKM2 KO MCF7 cells. ( (G) Co-immunoprecipitation of VCP with FLAG-tagged PKM1/2 in PKM2 KO 293T cells. FLAG-tagged PKM1 or PKM2 construct was transiently transfected into PKM2 KO or parental 293T cells for 24 hours, followed by pull-down using FLAG-M2 beads. PKM1, PKM2 and VCP antibodies were used for immunoblotting.


Figure 7 ( E ) Representative H & E and IHC staining images of TMEM33 and SCAP in WT and TMEM33 KO mouse livers . Scale bars , 100µm . ( F , G ) Significant decrease of total plasma cholesterol ( F ) and LDL - C ( G ) levels in TMEM33 - / - and + / - mice relative to WT counterparts ( n > = 5 ) . Data information : Data are displayed as means ± SD . Statistical significance was assessed using two - tailed Student ' s t - test , ∗ p < 0 . 05 and ∗ ∗ ∗ p < 0 . 001 . n . s denotes no significance .

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

Figure 7(E) Representative H&E and IHC staining images of TMEM33 and SCAP in WT and TMEM33 KO mouse livers. Scale bars, 100µm.(F, G) Significant decrease of total plasma cholesterol (F) and LDL-C (G) levels in TMEM33-/- and +/- mice relative to WT counterparts (n>=5). Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05 and ∗∗∗p < 0.001. n.s denotes no significance.


Figure 8 ( B ) Tumor growth curves of PKM2 WT or KO py8119 cell line allografts in systemic PKM2 KO or WT mice ( n = 5 ) . Data information : Data are displayed as means ± SD . Statistical significance was assessed using a two - tailed Student ' s t - test , ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 and ∗ ∗ ∗ p < 0 . 001 . ( E ) The growth curves of py8119 allografts treated with vehicle and TEPP - 46 ( 50mg / kg ) for 17 days in mice ( n = 7 ) . Data information : Data are displayed as means ± SD . Statistical significance was assessed using a two - tailed Student ' s t - test , ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 and ∗ ∗ ∗ p < 0 . 001 .

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

Figure 8(B) Tumor growth curves of PKM2 WT or KO py8119 cell line allografts in systemic PKM2 KO or WT mice (n=5). Data information: Data are displayed as means ± SD. Statistical significance was assessed using a two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.(E) The growth curves of py8119 allografts treated with vehicle and TEPP-46 (50mg/kg) for 17 days in mice (n=7). Data information: Data are displayed as means ± SD. Statistical significance was assessed using a two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.


Figure 1Representative low ( left ) and high ( right ) power images of H & E - stained lung sections from KrasG12D , KrasG12D : Adam17ex / + and KrasG12D : Adam17ex / ex mice at 6 weeks post Ad - Cre inhalation . Scale bars , 3mm ( left ) and 300μm ( right ) . Quantification of lung parenchyma area occupied by tumor lesions ( B ) , and tumor incidence ( C ) , per whole mouse lung in the indicated genotypes ( n = 6 per genotype ) . Representative images of TTF - 1 - stained lung sections from the indicated genotypes . Scale bar , 100μm . Quantification of TTF - 1 - positive cells / high power field ( HPF ) in lungs of the indicated genotypes ( n = 6 per genotype ) .

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

Figure 1Representative low (left) and high (right) power images of H&E-stained lung sections from KrasG12D, KrasG12D:Adam17ex/+ and KrasG12D:Adam17ex/ex mice at 6 weeks post Ad-Cre inhalation. Scale bars, 3mm (left) and 300μm (right). Quantification of lung parenchyma area occupied by tumor lesions (B), and tumor incidence (C), per whole mouse lung in the indicated genotypes (n = 6 per genotype).Representative images of TTF-1-stained lung sections from the indicated genotypes. Scale bar, 100μm. Quantification of TTF-1-positive cells/high power field (HPF) in lungs of the indicated genotypes (n = 6 per genotype).


Figure 2Representative images of PCNA - stained lung sections from KrasWT , KrasG12D and KrasG12D : Adam17ex / ex mice at 6 weeks post Ad - Cre ( for KrasG12D ) or vehicle ( for KrasWT ) inhalation . Scale bar , 100μm . Quantification of PCNA - positive cells / high power field ( HPF ) in the indicated mouse lungs ( n = 6 per genotype ) . qPCR expression analyses of cell cycle regulation genes ( normalized against 18SrRNA ) in lungs from the indicated mice ( n = 6 per genotype ) . * Representative immunoblots of KrasG12D and KrasG12D : Adam17ex / ex lung lysates with the indicated antibodies . Each lane is an individual mouse sample . Semi - quantitative densitometry of Myc protein levels ( relative to Actin ) in lung lysates from ( D ) . n = 6 per genotype . Representative images ( F ) and quantification of CD45 - positive cells / HPF ( G ) in CD45 - stained lung sections from the indicated genotypes at 6 weeks post inhalations . In ( F ) , scale bar , 100μm . In ( G ) , n = 6 per genotype .

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

Figure 2Representative images of PCNA-stained lung sections from KrasWT, KrasG12D and KrasG12D:Adam17ex/ex mice at 6 weeks post Ad-Cre (for KrasG12D) or vehicle (for KrasWT) inhalation. Scale bar, 100μm. Quantification of PCNA-positive cells/high power field (HPF) in the indicated mouse lungs (n = 6 per genotype).qPCR expression analyses of cell cycle regulation genes (normalized against 18SrRNA) in lungs from the indicated mice (n = 6 per genotype). *Representative immunoblots of KrasG12D and KrasG12D:Adam17ex/ex lung lysates with the indicated antibodies. Each lane is an individual mouse sample. Semi-quantitative densitometry of Myc protein levels (relative to Actin) in lung lysates from (D). n = 6 per genotype.Representative images (F) and quantification of CD45-positive cells/HPF (G) in CD45-stained lung sections from the indicated genotypes at 6 weeks post inhalations. In (F), scale bar, 100μm. In (G), n = 6 per genotype.


Figure 3Representative immunofluorescence images of lung sections of KrasG12D mice ( 6 weeks post Ad - Cre inhalation ) co - stained for ADAM17 and markers for club cells ( CC10 ) ( A ) , alveolar type II ( surfactant protein - C , SP - C ) ( B ) and type I ( Podoplanin , Pdpn ) ( C ) cells , total immune cells ( CD45 ) ( D ) and endothelial cells ( CD31 ) ( E ) . DAPI nuclear staining is blue . Scale bars , 100μm . Arrowheads in merged images indicate dual - positive ADAM17 - expressing cells . Quantification of immunofluorescence staining from A - E ( n = 5 mice per stain ) . Representative images of H & E - stained lung sections from KrasG12D recipient mice reconstituted with KrasG12D ( KrasKras ) or KrasG12D : Adam17ex / ex bone marrow ( KrasKras : ex / ex ) . Scale bar , 3mm . Quantification of lung parenchyma area occupied by tumor lesions in the indicated bone marrow chimeras ( n = 6 mice per group ) .

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

Figure 3Representative immunofluorescence images of lung sections of KrasG12D mice (6 weeks post Ad-Cre inhalation) co-stained for ADAM17 and markers for club cells (CC10) (A), alveolar type II (surfactant protein-C, SP-C) (B) and type I (Podoplanin, Pdpn) (C) cells, total immune cells (CD45) (D) and endothelial cells (CD31) (E). DAPI nuclear staining is blue. Scale bars, 100μm. Arrowheads in merged images indicate dual-positive ADAM17-expressing cells. Quantification of immunofluorescence staining from A-E (n = 5 mice per stain).Representative images of H&E-stained lung sections from KrasG12D recipient mice reconstituted with KrasG12D (KrasKras) or KrasG12D:Adam17ex/ex bone marrow (KrasKras:ex/ex). Scale bar, 3mm. Quantification of lung parenchyma area occupied by tumor lesions in the indicated bone marrow chimeras (n = 6 mice per group).


Figure 4Representative images of pADAM17 - stained lung sections from KrasWT and KrasG12D ( 6 weeks post inhalation ) mice . Scale bar , 100μm . Quantification of pADAM17 - positive cells / high power field ( HPF ) in lungs of KrasWT and KrasG12D mice ( n = 6 per genotype ) . Representative immunoblots of individual KrasWT and KrasG12D mouse lung lysates with the indicated antibodies . Semi - quantitative densitometry of pADAM17 in lung lysates ( n = 6 per genotype ) . Representative immunoblots of individual KrasWT and KrasG12D lung lysates with the indicated antibodies . Semi - quantitative densitometry of pp38 and pERK1 / 2 MAPK protein levels in KrasWT and KrasG12D lung lysates ( n = 6 per genotype ) . Representative images of pADAM17 - stained lung sections from KrasG12D mice injected with a single dose of vehicle ( control ) , SB203580 p38 MAPK or U0126 ERK1 / 2 inhibitors . Scale bar , 100μm . Quantification of pADAM17 - positive cells / HPF in lungs of the indicated groups ( n = 5 per group ) . Immunoblots with the indicated antibodies on lysates from A549 and NIH - H23 LAC cell lines treated with SB203580 for 24 hours . MTT viability assays of A549 and NCI - H23 LAC cells treated with SB203580 . ATP proliferation ( K ) assays of A549 and NCI - H23 LAC cells treated with SB203580 . Representative immunoblots of individual KrasG12D and KrasG12D : Adam17ex / ex lung lysates with the indicated antibodies . Representative images of pERK1 / 2 - stained lung sections from the indicated genotypes . Scale bars , 100μm . Quantification of pERK1 / 2 - positive cells / high power field ( HPF ) in lungs of the indicated genotypes ( n = 6 per genotype ) .

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

Figure 4Representative images of pADAM17-stained lung sections from KrasWT and KrasG12D (6 weeks post inhalation) mice. Scale bar, 100μm. Quantification of pADAM17-positive cells/high power field (HPF) in lungs of KrasWT and KrasG12D mice (n = 6 per genotype).Representative immunoblots of individual KrasWT and KrasG12D mouse lung lysates with the indicated antibodies. Semi-quantitative densitometry of pADAM17 in lung lysates (n = 6 per genotype).Representative immunoblots of individual KrasWT and KrasG12D lung lysates with the indicated antibodies. Semi-quantitative densitometry of pp38 and pERK1/2 MAPK protein levels in KrasWT and KrasG12D lung lysates (n = 6 per genotype).Representative images of pADAM17-stained lung sections from KrasG12D mice injected with a single dose of vehicle (control), SB203580 p38 MAPK or U0126 ERK1/2 inhibitors. Scale bar, 100μm.Quantification of pADAM17-positive cells/HPF in lungs of the indicated groups (n = 5 per group).Immunoblots with the indicated antibodies on lysates from A549 and NIH-H23 LAC cell lines treated with SB203580 for 24 hours.MTT viability assays of A549 and NCI-H23 LAC cells treated with SB203580.ATP proliferation (K) assays of A549 and NCI-H23 LAC cells treated with SB203580.Representative immunoblots of individual KrasG12D and KrasG12D:Adam17ex/ex lung lysates with the indicated antibodies.Representative images of pERK1/2-stained lung sections from the indicated genotypes. Scale bars, 100μm. Quantification of pERK1/2-positive cells/high power field (HPF) in lungs of the indicated genotypes (n = 6 per genotype).


Figure 5ELISA of sIL - 6R and TNFα protein levels in serum from KrasG12D and KrasG12D : Adam17ex / ex mice at 6 weeks post Ad - Cre inhalation , along with control KrasWT mice at 6 weeks post vehicle inhalation ( n = 6 per genotype ) . Immunoblots of representative individual KrasWT , KrasG12D and KrasG12D : Adam17ex / ex lung lysates with the indicated antibodies . Representative immunofluorescence staining of KrasG12D mouse lung sections ( n = 5 mice ) for ADAM17 and either ( panels from left to right ) IL - 6R , TGFα , Notch1 , Nrg1 and EGF . Sections were counterstained with DAPI ( blue ) . Scale bars , 100μm . Representative images of pERK1 / 2 - stained lung sections from KrasG12D mice treated with either vehicle ( control , Ctl ) or anti - IL - 6R antibodies 1F7 or 25F10 over 6 weeks post Ad - Cre inhalation . Scale bar , 100μm . Quantification of pERK1 / 2 - positive cells / high power field ( HPF ) in lungs of the indicated groups ( n = 5 per group ) . Representative immunoblots with the indicated antibodies on whole cell lysates from primary immune and epithelial cells isolated from the lungs of mice ( n = 3 ) harbouring the oncogenic KrasG12D allele that were stimulated with either PBS ( - ) or 100ng / ml Hyper - IL - 6 ( + ) . Graphs depict semi - quantitative densitometry of pERK1 / 2 MAPK protein levels in stimulated immune and epithelial cells ( n = 2 experiments ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5ELISA of sIL-6R and TNFα protein levels in serum from KrasG12D and KrasG12D:Adam17ex/ex mice at 6 weeks post Ad-Cre inhalation, along with control KrasWT mice at 6 weeks post vehicle inhalation (n = 6 per genotype).Immunoblots of representative individual KrasWT, KrasG12D and KrasG12D:Adam17ex/ex lung lysates with the indicated antibodies.Representative immunofluorescence staining of KrasG12D mouse lung sections (n = 5 mice) for ADAM17 and either (panels from left to right) IL-6R, TGFα, Notch1, Nrg1 and EGF. Sections were counterstained with DAPI (blue). Scale bars, 100μm.Representative images of pERK1/2-stained lung sections from KrasG12D mice treated with either vehicle (control, Ctl) or anti-IL-6R antibodies 1F7 or 25F10 over 6 weeks post Ad-Cre inhalation. Scale bar, 100μm. Quantification of pERK1/2-positive cells/high power field (HPF) in lungs of the indicated groups (n = 5 per group).Representative immunoblots with the indicated antibodies on whole cell lysates from primary immune and epithelial cells isolated from the lungs of mice (n = 3) harbouring the oncogenic KrasG12D allele that were stimulated with either PBS (-) or 100ng/ml Hyper-IL-6 (+). Graphs depict semi-quantitative densitometry of pERK1/2 MAPK protein levels in stimulated immune and epithelial cells (n = 2 experiments).


Figure 6Representative images of pADAM17 - stained lung sections from normal ( cancer - free ) control and LAC patients . Scale bar , 100μm . Quantification of pADAM17 - positive cells in ( B ) normal ( cancer - free ) control individuals ( n = 8 ) and LAC patients ( n = 18 ) Quantification of pADAM17 - positive cells and pERK1 / 2 MAPK - positive cells in LAC patients either KRAS wild - type ( WT , n = 13 ) or mutant ( MUT , n = 5 ) . Linear regression analyses of pADAM17 with pERK1 / 2 ( D ) and pp38 ( E ) MAPK staining in serial lung sections from LAC KRAS wild - type and mutant patients . Immunoblots of lysates from normal human bronchial epithelial cells ( HBEC ) and LAC cell lines A549 , NIH - H23 and NIH - H2228 with the indicated antibodies . Shown are lysates from 2 separate passages per cell line . Pro , proADAM17 ; Mat , mature ADAM17 . Densitometric semi - quantification of immunoblots showing the relative expression levels of pADAM17 ( relative to Actin ) , pro and mature forms of ADAM17 ( relative to Actin ) , and pERK1 / 2 and pp38 MAPK ( relative to their total counterparts ) protein levels . ELISA of sIL - 6R protein levels in culture supernatants from matching cells in ( F ) cultured over 24 hours . Data are from 4 independent experiments . MTT viability assays of A549 cells transduced with nontargeted control sgRNA ( Ctl ) and ADAM17 sgRNA ( knockout , KO ) . ATP proliferation ( J ) assays of A549 cells transduced with nontargeted control sgRNA ( Ctl ) and ADAM17 sgRNA ( knockout , KO ) . ELISA of sIL - 6R protein levels in culture supernatants from matching A549 cells ( I and J ) cultured over 24 hours . Data are from 3 independent experiments . Tumor volume ( mm3 ) of KO and Ctl A549 xenografts assessed every 3 days starting at one week post injection of 106 cells into NSG mice ( n = 5 per group ) . Weight ( grams ) ( M ) of tumor xenograftsrepresentative images ( N ) of tumor xenograftsELISA of human sIL - 6R protein levels in serum of the indicated mice ( n = 5 per group ) . Levels of sIL - 6R are below detection in serum from KO xenograft mice . ND , not detected . Representative images of pADAM17 and pERK1 / 2 MAPK immunohistochemical staining on KO and Ctl A549 xenograft tumors . Scale bars , 100μm . Immunoblots of lysates from individual A549 KO and Ctl xenograft tumors with the indicated antibodies .

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

Figure 6Representative images of pADAM17-stained lung sections from normal (cancer-free) control and LAC patients. Scale bar, 100μm. Quantification of pADAM17-positive cells in (B) normal (cancer-free) control individuals (n = 8) and LAC patients (n = 18)Quantification of pADAM17-positive cells and pERK1/2 MAPK-positive cells in LAC patients either KRAS wild-type (WT, n = 13) or mutant (MUT, n = 5).Linear regression analyses of pADAM17 with pERK1/2 (D) and pp38 (E) MAPK staining in serial lung sections from LAC KRAS wild-type and mutant patients.Immunoblots of lysates from normal human bronchial epithelial cells (HBEC) and LAC cell lines A549, NIH-H23 and NIH-H2228 with the indicated antibodies. Shown are lysates from 2 separate passages per cell line. Pro, proADAM17; Mat, mature ADAM17. Densitometric semi-quantification of immunoblots showing the relative expression levels of pADAM17 (relative to Actin), pro and mature forms of ADAM17 (relative to Actin), and pERK1/2 and pp38 MAPK (relative to their total counterparts) protein levels.ELISA of sIL-6R protein levels in culture supernatants from matching cells in (F) cultured over 24 hours. Data are from 4 independent experiments.MTT viability assays of A549 cells transduced with nontargeted control sgRNA (Ctl) and ADAM17 sgRNA (knockout, KO).ATP proliferation (J) assays of A549 cells transduced with nontargeted control sgRNA (Ctl) and ADAM17 sgRNA (knockout, KO).ELISA of sIL-6R protein levels in culture supernatants from matching A549 cells (I and J) cultured over 24 hours. Data are from 3 independent experiments.Tumor volume (mm3) of KO and Ctl A549 xenografts assessed every 3 days starting at one week post injection of 106 cells into NSG mice (n = 5 per group).Weight (grams) (M) of tumor xenograftsrepresentative images (N) of tumor xenograftsELISA of human sIL-6R protein levels in serum of the indicated mice (n = 5 per group). Levels of sIL-6R are below detection in serum from KO xenograft mice. ND, not detected.Representative images of pADAM17 and pERK1/2 MAPK immunohistochemical staining on KO and Ctl A549 xenograft tumors. Scale bars, 100μm.Immunoblots of lysates from individual A549 KO and Ctl xenograft tumors with the indicated antibodies.


Figure 7Representative images of H & E - stained lung sections from KrasG12D mice treated with either vehicle ( control ) or the ADAM17 prodomain inhibitor , A17pro ( 1mg / kg , 3 times a week ) , for 6 weeks starting on the day after Cre inhalation . Scale bars , 3mm . Quantification of lung parenchyma area occupied by tumor lesions in the indicated groups ( n = 6 per group ) Representative images of lung sections from the indicated mice from ( A ) stained with antibodies against TTF - 1 ( C ) , PCNA ( E ) and pERK1 / 2 ( G ) . Scale bars , 100μm . Quantification of positive cells / high power field ( HPF ) in the lungs of mice from ( C ) , ( E ) and ( G ) stained for TTF - 1 ( D ) , PCNA ( F ) and pERK1 / 2 ( H ) . n = 6 per group . ELISA of serum sIL - 6R protein levels in the indicated mice ( n = 6 per group ) Tumor volume ( mm3 ) of a KRAS mutant PDX treated with vehicle control ( Ctl ) or A17pro ( 1mg / kg ) every second day . Treatment started when tumors reached a volume of 100 - 150mm3 after injection of 2 × 106 cells in NSG mice . n = 5 per group . Weight ( grams ) of tumor PDXsrepresentative images ( L ) of tumor PDXsELISA of human sIL - 6R protein levels in serum of the indicated mice ( n = 5 per group ) . * Immunoblots with the indicated antibodies of representative lysates from individual PDX tumors treated with vehicle control or A17pro . Representative image of pADAM17 immunohistochemical staining on an untreated KRAS mutant PDX tumor . Scale bar , 100μm .

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

Figure 7Representative images of H&E-stained lung sections from KrasG12D mice treated with either vehicle (control) or the ADAM17 prodomain inhibitor, A17pro (1mg/kg, 3 times a week), for 6 weeks starting on the day after Cre inhalation. Scale bars, 3mm. Quantification of lung parenchyma area occupied by tumor lesions in the indicated groups (n = 6 per group)Representative images of lung sections from the indicated mice from (A) stained with antibodies against TTF-1 (C), PCNA (E) and pERK1/2 (G). Scale bars, 100μm. Quantification of positive cells/high power field (HPF) in the lungs of mice from (C), (E) and (G) stained for TTF-1 (D), PCNA (F) and pERK1/2 (H). n = 6 per group.ELISA of serum sIL-6R protein levels in the indicated mice (n = 6 per group)Tumor volume (mm3) of a KRAS mutant PDX treated with vehicle control (Ctl) or A17pro (1mg/kg) every second day. Treatment started when tumors reached a volume of 100-150mm3 after injection of 2 × 106 cells in NSG mice. n = 5 per group.Weight (grams) of tumor PDXsrepresentative images (L) of tumor PDXsELISA of human sIL-6R protein levels in serum of the indicated mice (n = 5 per group). *Immunoblots with the indicated antibodies of representative lysates from individual PDX tumors treated with vehicle control or A17pro.Representative image of pADAM17 immunohistochemical staining on an untreated KRAS mutant PDX tumor. Scale bar, 100μm.


Figure 1D Representative confocal images of HEK293 cells expressing various GFP - fused proteins ( green ) and supplemented with TopFluor ® TMR - PS ( red ) ; scale bar = 10 µm . Yellow arrows indicate high intensity PS fluorescent regions . E % PM with PS clusters = area of high intensity fluorescent PS clusters over total plasma membrane area from images in panel D . Black bars are control proteins and blue bars are eVP40 proteins . Values are reported as mean ± s . d . ; N > 18 cells per biological replicate , n = 3 for 3 biological replicates ; A one - way ANOVA was performed with multiple comparisons compared to the control GFP % PS clustering ( * * * p = 0 . 0007 , * * p = 0 . 004 ) . Data information : PS : phosphatidylserine ; PM : plasma membrane .

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

Figure 1D Representative confocal images of HEK293 cells expressing various GFP-fused proteins (green) and supplemented with TopFluor® TMR-PS (red); scale bar = 10 µm. Yellow arrows indicate high intensity PS fluorescent regions. E %PM with PS clusters = area of high intensity fluorescent PS clusters over total plasma membrane area from images in panel D. Black bars are control proteins and blue bars are eVP40 proteins. Values are reported as mean ± s.d.; N>18 cells per biological replicate, n=3 for 3 biological replicates; A one-way ANOVA was performed with multiple comparisons compared to the control GFP %PS clustering (***p=0.0007, **p=0.004). Data information: PS: phosphatidylserine; PM: plasma membrane.


Figure 2D , E PS concentration in LUVs enhances the ability of His6 - eVP40 to oligomerize on membranes . D Representative western blot of chemical crosslinking performed on His6 - WT - eVP40 following incubation with LUVs of varying PS content ( detected by Mouse α - His antibody & HRP - Sheep α - Mouse ) . E Oligomerization capacity was determined from the western blot band density ratio of oligomers / ( monomer + dimer ) from chemical crosslinking experiments . A one - way ANOVA was performed with multiple comparisons compared to the control 0 % PS LUVs control ( 30 % PS * p = 0 . 02 ; 60 % PS * p = 0 . 017 ) . n = 3 biological replicates . Values are reported as mean ± s . d . ; Data information : LUVs : large unilamellar vesicles ; PS : phosphatidylserine ; HRP : horseradish peroxidase .

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

Figure 2D, E PS concentration in LUVs enhances the ability of His6-eVP40 to oligomerize on membranes. D Representative western blot of chemical crosslinking performed on His6-WT-eVP40 following incubation with LUVs of varying PS content (detected by Mouse α-His antibody & HRP-Sheep α-Mouse). E Oligomerization capacity was determined from the western blot band density ratio of oligomers/(monomer + dimer) from chemical crosslinking experiments. A one-way ANOVA was performed with multiple comparisons compared to the control 0% PS LUVs control (30% PS *p= 0.02; 60% PS *p=0.017). n=3 biological replicates. Values are reported as mean ± s.d.; Data information: LUVs: large unilamellar vesicles; PS: phosphatidylserine; HRP: horseradish peroxidase.


Figure 3D , E Analysis of PS clustering in HEK293 cells in response to fendiline treatment through N & B analysis . D Left panel : Representative images from time - lapse ( 30 frames ) imaging of HEK293 expressing GFP - LactC2 and treated with fendiline for 48 hours ; scale bar = 5 µm . Middle panel : Brightness and Intensity plots for each representative image . Right panel : Selection map correlating each pixel in the representative image to an oligomerization state ( b value ) ( red : monomer - 5mer , green : 5mer - 10mer , blue : > 10mer ) . E Average % pixels quantification from panel ( d ) = Percentage of GFP - LactC2 with brightness values corresponding to monomer - 5mer ( ~ 1 . - 1 . 5 ) , 5mer - 10mer ( ~ 1 . 5 - 1 . 9 ) and > 10mer ( > 1 . 9 ) over the total pixels within each image . Values are reported as mean ± s . d . ; N ≥ 9 , n = 3 biological replicates ; A two - way ANOVA was performed with Dunnett ' s multiple comparisons comparted to the control DMSO % average pixels ( * * * * p < 0 . 0001 , * * p = 0 . 0043 ) . Data information : GFP - LactC2 : phosphatidylserine sensor ; N & B : Number & Brightness analysis ; PS : phosphatidylserine .

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

Figure 3D, E Analysis of PS clustering in HEK293 cells in response to fendiline treatment through N&B analysis. D Left panel: Representative images from time-lapse (30 frames) imaging of HEK293 expressing GFP-LactC2 and treated with fendiline for 48 hours; scale bar= 5 µm. Middle panel: Brightness and Intensity plots for each representative image. Right panel: Selection map correlating each pixel in the representative image to an oligomerization state (b value) (red: monomer-5mer, green: 5mer-10mer, blue: >10mer). E Average % pixels quantification from panel (d)= Percentage of GFP-LactC2 with brightness values corresponding to monomer-5mer (~1.-1.5), 5mer-10mer (~1.5-1.9) and >10mer (>1.9) over the total pixels within each image. Values are reported as mean ± s.d.; N≥9, n=3 biological replicates; A two-way ANOVA was performed with Dunnett's multiple comparisons comparted to the control DMSO % average pixels (****p<0.0001, **p=0.0043). Data information: GFP-LactC2: phosphatidylserine sensor; N&B: Number & Brightness analysis; PS: phosphatidylserine.


Figure 4Effect of fendiline on EBOV infection . A Representative confocal images of Vero E6 cells infected with EBOV ( Kikwit ) at the indicated MOI and treated with the indicated concentration of fendiline . Cells were pretreated 24 hours prior to infection with the indicated concentration of fendiline . Post infection , cells were treated 1 hour later ( d - 1 / 0 ) , treated every day ( e . d ) , or treated every other day ( e . o . d ) and fixed at either 48 hours , 72 hours or 96 hours post infection . ( green = EBOV ; blue = nuclei ) . White numbering in top right corner indicates % infection . Scale bar = 100 μm . Data information : EBOV : Ebola virus ; MOI : multiplicity of infectionEffect of fendiline on EBOV infection . B - D Quantification of % inhibition of EBOV by fendiline . B 48 hours ( MOI 1 . 0 ) C 72 hours ( MOI 0 . 1 ) D 96 hours ( MOI 0 . 1 ) . Values are reported as mean ± s . d . A one - way ANOVA was performed with multiple comparisons was performed . n = 3 biological replicates . Data information : EBOV : Ebola virus ; MOI : multiplicity of infection ; d . - 1 / 0 : treatment 1 hour after infection ; e . d . : treatment every day ; e . o . d . : treatment every other day .

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 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

Figure 4Effect of fendiline on EBOV infection. A Representative confocal images of Vero E6 cells infected with EBOV (Kikwit) at the indicated MOI and treated with the indicated concentration of fendiline. Cells were pretreated 24 hours prior to infection with the indicated concentration of fendiline. Post infection, cells were treated 1 hour later (d -1/0), treated every day (e.d), or treated every other day (e.o.d) and fixed at either 48 hours, 72 hours or 96 hours post infection. (green=EBOV; blue= nuclei). White numbering in top right corner indicates %infection. Scale bar = 100 μm. Data information: EBOV: Ebola virus; MOI: multiplicity of infectionEffect of fendiline on EBOV infection. B-D Quantification of % inhibition of EBOV by fendiline. B 48 hours (MOI 1.0) C 72 hours (MOI 0.1) D 96 hours (MOI 0.1). Values are reported as mean ± s.d. A one-way ANOVA was performed with multiple comparisons was performed. n=3 biological replicates. Data information: EBOV: Ebola virus; MOI: multiplicity of infection; d. -1/0: treatment 1 hour after infection; e.d.: treatment every day; e.o.d.: treatment every other day.


Figure 5A - C Effect of fendiline on eVP40 and mVP40 PM localization in HEK293 cells after 48 hours of treatment . A Representative confocal images from live cell imaging experiments of HEK293 cells expressing EGFP - WT - eVP40 ( top panel ) and EGFP - WT - mVP40 ( bottom panel ) after 48 hours of fendiline treatment . scale bars = 10 µm . Effect of fendiline on mVP40 ( B ) and eVP40 ( C ) PM localization was quantified by the ratio of EGFP fluorescence intensity at the PM / total EGFP fluorescence intensity ( and normalized to DMSO control ) . N > 15 cell per biological replicate , n = 3 biological replicates . Values are reported as mean ± s . d . A one - way ANOVA with multiple comparisons was performed compared to the DMSO control . Data information : PM : plasma membraneD , E Analysis of eVP40 oligomerization in HEK293 cells in response to 48 hour fendiline treatment using N & B analysis . D Left panel : Representative images from time - lapse ( 30 frames ) of HEK293 expressing EGFP - WT - eVP40 and treated with fendiline for 48 hours . scale bar = 5 µm . Middle panel : Brightness and Intensity plots for each representative image . Right panel : Selection map correlating each pixel in the representative image to an oligomerization state ( b value ) ( red : monomer - hexamer , green : hexamer - 12mer , blue : 12mer - 24mer , pink : > 24mer ) . E Average % pixel quantification from panel ( d ) = % of GFP - WT - eVP40 with brightness values corresponding to monomer - hexamer ( ~ 1 . - 1 . 6 ) , hexamer - 12mer ( ~ 1 . 6 - 2 . 0 ) , 12mer - 24mer ( 2 . 0 - 3 . 2 ) and > 24mer ( > 3 . 2 ) over the total pixels within each image . Values are reported as mean ± s . d . ; N ≥ 9 cell per biological replicate , n = 3 biological replicates ; A two - way ANOVA was performed with Dunnett ' s multiple comparisons compared to the control DMSO % average pixels ( * * p = 0 . 0035 ) . Data information : N & B : Number & Brightness analysis .

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 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

Figure 5A-C Effect of fendiline on eVP40 and mVP40 PM localization in HEK293 cells after 48 hours of treatment. A Representative confocal images from live cell imaging experiments of HEK293 cells expressing EGFP-WT-eVP40 (top panel) and EGFP-WT-mVP40 (bottom panel) after 48 hours of fendiline treatment. scale bars= 10 µm. Effect of fendiline on mVP40 (B) and eVP40 (C) PM localization was quantified by the ratio of EGFP fluorescence intensity at the PM / total EGFP fluorescence intensity (and normalized to DMSO control). N>15 cell per biological replicate, n=3 biological replicates. Values are reported as mean ± s.d. A one-way ANOVA with multiple comparisons was performed compared to the DMSO control. Data information: PM: plasma membraneD, E Analysis of eVP40 oligomerization in HEK293 cells in response to 48 hour fendiline treatment using N&B analysis. D Left panel: Representative images from time-lapse (30 frames) of HEK293 expressing EGFP-WT-eVP40 and treated with fendiline for 48 hours. scale bar = 5 µm. Middle panel: Brightness and Intensity plots for each representative image. Right panel: Selection map correlating each pixel in the representative image to an oligomerization state (b value) (red: monomer-hexamer, green: hexamer-12mer, blue: 12mer-24mer, pink: >24mer). E Average % pixel quantification from panel (d)= % of GFP-WT-eVP40 with brightness values corresponding to monomer-hexamer (~1.-1.6), hexamer-12mer (~1.6-2.0), 12mer-24mer (2.0-3.2) and >24mer (>3.2) over the total pixels within each image. Values are reported as mean ± s.d.; N≥9 cell per biological replicate, n=3 biological replicates; A two-way ANOVA was performed with Dunnett's multiple comparisons compared to the control DMSO % average pixels (**p=0.0035). Data information: N&B: Number & Brightness analysis.


Figure 6A - D Functional budding assays assessed at 24 hours ( A - B ) and 48 hours ( C - D ) post treatment . A Representative western blot of budding assays performed at 24 hours post transfection with vehicle control ( DMSO ) treatment or 500 nM , 1 μM , or 5 μM fendiline treatment . VLP samples ( top panel ) and cell lysate samples ( bottom panel ) collected from HEK293 cells and immunoblotted for eVP40 expression ; GAPDH served as a loading control . eVP40 detected by ( Rabbit α - eVP40 and HRP - Goat α - Rabbit ) ; GAPDH detected by mouse α - GAPDH and HRP - Sheep α - Mouse ) B Quantification of relative budding index at 24 hours post vehicle or fendiline treatment . Relative budding index was determined by the western blot band density of eVP40 in the VLP fraction / ( total eVP40 cell lysate + eVP40 VLP band density ) and was normalized to the DMSO control . Cell lysate eVP40 band density was normalized to GAPDH band density prior to use in budding index quantification . n = 3 biological replicates . Values are reported as mean ± s . d . A one - way ANOVA was performed with multiple comparisons compared to the DMSO control . C Representative western blot of budding assays performed at 48 hours post vehicle control ( DMSO ) treatment or 500 nM , 1 μM , or 5 μM fendiline treatment . VLP samples ( top panel ) and cell lysate samples ( bottom panel ) collected from HEK293 cells and immunoblotted for eVP40 expression ; GAPDH served as a loading control . eVP40 detected by ( Rabbit α - eVP40 and HRP - Goat α - Rabbit ) ; GAPDH detected by ( Mouse α - GAPDH and HRP - Sheep α - Mouse ) D Quantification of relative budding index at 48 hours post fendiline treatment . Relative budding index was determined by the western blot band density of eVP40 in the VLP fraction / ( total eVP40 cell lysate + eVP40 VLP band density ) and was normalized to the DMSO control . Cell lysate eVP40 band density was normalized to GAPDH band density prior to use in budding index quantification . n = 3 biological replicates . Values are reported as mean ± s . d . A one - way ANOVA was performed with multiple comparisons compared to the DMSO control . ( * p = 0 . 0260 ) Data information : VLPs : virus - like particles ; GAPDH : glyceraldehyde 3 - phosphate dehydrogenase ; HRP : horseradish peroxidase .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D Functional budding assays assessed at 24 hours (A-B) and 48 hours (C-D) post treatment. A Representative western blot of budding assays performed at 24 hours post transfection with vehicle control (DMSO) treatment or 500 nM, 1 μM, or 5 μM fendiline treatment. VLP samples (top panel) and cell lysate samples (bottom panel) collected from HEK293 cells and immunoblotted for eVP40 expression; GAPDH served as a loading control. eVP40 detected by (Rabbit α-eVP40 and HRP-Goat α-Rabbit); GAPDH detected by mouse α-GAPDH and HRP-Sheep α-Mouse) B Quantification of relative budding index at 24 hours post vehicle or fendiline treatment. Relative budding index was determined by the western blot band density of eVP40 in the VLP fraction/(total eVP40 cell lysate + eVP40 VLP band density) and was normalized to the DMSO control. Cell lysate eVP40 band density was normalized to GAPDH band density prior to use in budding index quantification. n=3 biological replicates. Values are reported as mean ± s.d. A one-way ANOVA was performed with multiple comparisons compared to the DMSO control. C Representative western blot of budding assays performed at 48 hours post vehicle control (DMSO) treatment or 500 nM, 1 μM, or 5 μM fendiline treatment. VLP samples (top panel) and cell lysate samples (bottom panel) collected from HEK293 cells and immunoblotted for eVP40 expression; GAPDH served as a loading control. eVP40 detected by (Rabbit α-eVP40 and HRP-Goat α-Rabbit); GAPDH detected by (Mouse α-GAPDH and HRP-Sheep α-Mouse) D Quantification of relative budding index at 48 hours post fendiline treatment. Relative budding index was determined by the western blot band density of eVP40 in the VLP fraction/(total eVP40 cell lysate + eVP40 VLP band density) and was normalized to the DMSO control. Cell lysate eVP40 band density was normalized to GAPDH band density prior to use in budding index quantification. n=3 biological replicates. Values are reported as mean ± s.d. A one-way ANOVA was performed with multiple comparisons compared to the DMSO control. (*p=0.0260) Data information: VLPs: virus-like particles; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; HRP: horseradish peroxidase.