( C ) Left panels : growth in EMM without ( red ) or with ( blue ) arginine supplementation for five selected respiratory mutants as indicated ( graphs selected from Fig EV3 ) . Right panels : normalised intracellular - acid concentrations as in ( A ) for same 5 respiratory mutants ( blue ) wild - type control ( grey ) . For each mutant wild type , four independent samples were analysed . * Ornithine ( Orn ) is abbreviated as O . Boxplot features as in ( A ) .

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

(C) Left panels: cell growth in EMM without (red) or with (blue) arginine supplementation for five selected respiratory mutants as indicated (graphs selected from Fig EV3). Right panels: normalised intracellular amino-acid concentrations as in (A) for the same 5 respiratory mutants (blue) and wild-type control cells (grey). For each mutant and the wild type strain, four independent samples were analysed. *Ornithine (Orn) is abbreviated as O. Boxplot features as in (A).


( A ) Normalised - acid concentrations in grown in EMM without ( grey ) or with arginine ( red ) . For each condition , four independent samples were analysed . Concentrations are presented relative to mean value for given acid in EMM medium . * Ornithine ( Orn ) is abbreviated as O . central band indicates median , upper lower limits of box indicate first third quartiles , whiskers show most extreme data , limited to 1 . 5 - time interquartile range .

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

(A) Normalised amino-acid concentrations in cells grown in EMM without (grey) or with arginine (red). For each condition, four independent samples were analysed. Concentrations are presented relative to the mean value for the given amino acid in EMM medium. *Ornithine (Orn) is abbreviated as O. The central band indicates the median, the upper and lower limits of the box indicate first and third quartiles, and the whiskers show the most extreme data, limited to 1.5-time interquartile range.


( C ) Growth curves of strains deleted for car1 , car2 or car3 , a wild - type control . Strains were grown in EMM ( black ) , EMM with antimycin A ( red ) , EMM with arginine ( blue ) or EMM with both arginine antimycin A ( blue ) as indicated .

O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Growth curves of strains deleted for car1, car2 or car3, and a wild-type control. Strains were grown in EMM (black), EMM with antimycin A (red), EMM with arginine (light blue) or EMM with both arginine and antimycin A (dark blue) as indicated.


( D ) Growth curves of strains deleted for car2 ( top ) or car3 ( bottom ) . Strains were grown in EMM ( black ) , EMM with antimycin A ( red ) , EMM with arginine antimycin A ( blue ) , EMM with mix of antimycin A ( bright blue ) , EMM with antimycin A each acid from mix ( grey ) .

O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Growth curves of strains deleted for car2 (top) or car3 (bottom). Strains were grown in EMM (black), EMM with antimycin A (red), EMM with arginine and antimycin A (dark blue), EMM with mix of all amino acids and antimycin A (bright blue), and EMM with antimycin A and each individual amino acid from the mix (grey).


( E ) Growth curves of wild - type control ( WT ) car2 deletion ( Δcar2 ) . Strains were grown in EMM media with antimycin A with either a mix of ( + AA mix ) or different combinations of lysine ( K ) , glutamine ( Q ) , glutamic acid ( E ) , or arginine ( R ) as indicated . Dashed : time point where wild - type saturates in EMM with - acid mix .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Growth curves of wild-type control (WT) and car2 deletion strain (Δcar2). Strains were grown in EMM media with antimycin A and with either a mix of all amino acids (+AA mix) or different combinations of lysine (K), glutamine (Q), glutamic acid (E), or arginine (R) as indicated. Dashed line: time point where wild-type strain saturates in EMM with amino-acid mix.


( A ) Changes in transcript abundance 30 120 min after addition of antimycin A to exponentially growing without ( EMM ) or with arginine ( EMM + R ) . Transcripts annotated as up - or down - regulated Environmental Stress Response ( CESR ) are indicated in red blue , respectively . data is normalised to EMM time point 0 .

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

(A) Changes in transcript abundance 30 and 120 min after addition of antimycin A to exponentially growing cells without (EMM) or with arginine (EMM+R). Transcripts annotated as up- or down-regulated Core Environmental Stress Response (CESR) are indicated in red and blue, respectively. All data is normalised to EMM time point 0.


( B ) Graphs as in ( A ) showing expression profiles of transcripts encoding enzymes for arginine catabolism ( blue ) or arginine biosynthesis ( red ) .

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

(B) Graphs as in (A) showing expression profiles of transcripts encoding enzymes for arginine catabolism (blue) or arginine biosynthesis (red).


( C ) Heat map of expression changes for transcripts repressed over 2 - fold at 30 min in supplemented with arginine ( EMM + R ) relative to without supplementation ( EMM ) . Values for timepoints 0 30 min after antimycin A addition are shown for grown in EMM , EMM with arginine ssp1 deletion mutant grown in EMM . data are normalised to EMM timepoint 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

(C) Heat map of expression changes for transcripts repressed over 2-fold at 30 min in cells supplemented with arginine (EMM +R) relative to cells without supplementation (EMM). Values for timepoints 0 and 30 min after antimycin A addition are shown for cells grown in EMM, EMM with arginine and ssp1 deletion mutant grown in EMM. The data are normalised to EMM timepoint 0.


( D ) Heat map of expression changes of same genes as in ( B ) after one hour of inhibiting TOR kinase with torin1 or caffeine / rapamycin treatment relative to before treatment ; were grown in rich media - YES ( data from ( Rodríguez - López et al . , 2019 ) )

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

(D) Heat map of expression changes of the same genes as in (B) after one hour of inhibiting TOR kinase with torin1 or caffeine/rapamycin treatment relative to cell before treatment; cells were grown in rich media - YES (data from (Rodríguez-López et al., 2019))


A , B Immunoblot analyses of abundance of METTL3 / METTL14 / WTAP heterotrimer in PBMCs ( A ) BMDMs ( B ) upon challenge with VSV - eGFP , poly ( I : C ) , HSV - 1 or HSV60 at indicated time points .

O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B Immunoblot analyses of the protein abundance of the METTL3/METTL14/WTAP heterotrimer in PBMCs (A) and BMDMs (B) upon challenge with VSV-eGFP, poly(I:C), HSV-1 or HSV60 at the indicated time points.


E A549 were stimulated with poly ( I : C ) for 16 hrs then treated with PR - 171 ( 10 μM ) or PS - 341 ( 10 μM ) for additional 4 hrs 8 hrs . lysates were analyzed by immunoblotting . Representative from 3 independent biological experiments are shown ( left ) , relative of WTAP are presented as mean ± SD ( right ) . F A549 were stimulated with poly ( I : C ) for 16 hrs then treated with CQ ( 50 mM ) or Baf A1 ( 0 . 2 mM ) for additional 4 hrs 8 hrs . lysates were analyzed by immunoblotting . LC3B served as a good marker of autophagy process . Representative from 3 independent biological experiments are shown ( left ) , relative of WTAP are presented as mean ± SD ( right ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O

E A549 cells were stimulated with poly (I:C) for 16 hrs and then treated with PR-171 (10 μM) or PS-341 (10 μM) for additional 4 hrs and 8 hrs. The cell lysates were analyzed by immunoblotting. Representative images from 3 independent biological experiments are shown (left), and the relative levels of WTAP are presented as the mean ± SD (right). F A549 cells were stimulated with poly (I:C) for 16 hrs and then treated with CQ (50 mM) or Baf A1 (0.2 mM) for additional 4 hrs and 8 hrs. The cell lysates were analyzed by immunoblotting. LC3B served as a good marker of the autophagy process. Representative images from 3 independent biological experiments are shown (left), and the relative levels of WTAP are presented as the mean ± SD (right).


H Coimmunoprecipitation immunoblot analyses of extracts of 293T transfected with various combinations of plasmids encoding FLAG - tagged WTAP , HA - tagged K48 - linked , K63 - linked or wild - type ubiquitin treated with DMSO or PR171 ( 10 μM ) .

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

H Coimmunoprecipitation and immunoblot analyses of extracts of 293T cells transfected with various combinations of plasmids encoding FLAG-tagged WTAP, and HA-tagged K48-linked, K63-linked or wild-type ubiquitin and treated with DMSO or PR171 (10 μM).


C qRT - PCR analyses to detect mRNA abundances of IFNB1 , RANTES , DDX58 , ISG15 in WT WTAP - / - 293T followed by treatments with intracellular poly ( I : C ) , poly ( dA : dT ) , or VSV - eGFP .

O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C qRT-PCR analyses to detect the mRNA abundances of IFNB1, RANTES, DDX58, and ISG15 in WT and WTAP-/- 293T cells followed by treatments with intracellular poly(I:C), poly(dA:dT), or VSV-eGFP.


I , J Immunoblot analyses to detect expression of ISGs in shCtrl shWTAP A549 that were stimulated with poly ( I : C ) - LMW ( I ) or poly ( dA : dT ) ( J ) at different time points with indicated antibodies .

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

I, J Immunoblot analyses to detect the protein expression of ISGs in shCtrl and shWTAP A549 cells that were stimulated with poly(I:C)-LMW (I) or poly(dA:dT) (J) at different time points with the indicated antibodies.


A , B Immunoblot analyses of total phosphorylated ( p - ) TBK1 IRF3 in A549 transfected with Ctrl or WTAP - specific siRNA , followed by stimulation with poly ( I : C ) ( A ) or poly ( dA : dT ) ( B ) at different time points .

O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B Immunoblot analyses of total and phosphorylated (p-) TBK1 and IRF3 in A549 cells transfected with Ctrl or WTAP-specific siRNA, followed by stimulation with poly (I:C) (A) or poly (dA:dT) (B) at different time points.


E , F Immunoblot analyses of total phosphorylated ( p - ) STAT1 in shCtrl shWTAP A549 , followed by stimulation with IFN - α ( E ) or - β ( F ) at different time points .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O

E, F Immunoblot analyses of total and phosphorylated (p-) STAT1 in shCtrl and shWTAP A549 cells, followed by stimulation with IFN-α (E) or -β (F) at different time points.


A , B Immunoblot analyses to detect expression of critical adaptors in IFN - I Jak - STAT signaling pathway in A549 after transfection with Ctrl or WTAP - specific siRNAs for 48 hrs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O

A, B Immunoblot analyses to detect the protein expression of critical adaptors in the IFN-I and Jak-STAT signaling pathway in A549 cells after transfection with Ctrl or WTAP-specific siRNAs for 48 hrs.


F , G Abundance of IRF3 ( F ) IFNAR1 ( G ) transcripts ( detection site - 1 - 2 ) among mRNA immunoprecipitated with anti - m6A antibody from shCtrl shWTAP A549 . n = 3 independent biological replicates , error bars represent standard deviations .

O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F, G Abundance of IRF3 (F) and IFNAR1 (G) transcripts (detection site-1 and -2) among mRNA immunoprecipitated with anti-m6A antibody from shCtrl and shWTAP A549 cells. n = 3 independent biological replicates, and error bars represent standard deviations.


A Immunoblot analyses to detect IRF3 IFNAR1 expression in A549 stimulated with poly ( I : C ) or poly ( dA : dT ) at indicated time points . B Immunoblot analyses to detect IRF3 IFNAR1 expression in A549 L929 infected with VSV - eGFP at indicated time points .

O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O

A Immunoblot analyses to detect IRF3 and IFNAR1 protein expression in A549 cells stimulated with poly (I:C) or poly (dA:dT) at the indicated time points. B Immunoblot analyses to detect IRF3 and IFNAR1 protein expression in A549 and L929 cells infected with VSV-eGFP at the indicated time points.


H , Immunoblot of IRF3 IFNAR1 in A549 transfected with poly ( I : C ) - LMW or infected with VSV - GFP for 16 hrs then treated with PR - 171 or DMSO for additional 4 8 hrs . Representative from 3 independent biological experiments are shown ( H ) ,

O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H, Immunoblot analysis of IRF3 and IFNAR1 in A549 cells transfected with poly (I:C)-LMW or infected with VSV-GFP for 16 hrs and then treated with PR-171 or DMSO for additional 4 and 8 hrs. Representative images from 3 independent biological experiments are shown (H),


abundance of IRF3 IFNAR1 in A549 transfected with poly ( I : C ) - LMW or infected with VSV - GFP for 16 hrs then treated with PR - 171 or DMSO for additional 4 8 hrs . relative of IFNAR1 ( left ) IRF3 ( right ) are presented as mean ± SD ( I ) .

O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O

protein abundance of IRF3 and IFNAR1 in A549 cells transfected with poly (I:C)-LMW or infected with VSV-GFP for 16 hrs and then treated with PR-171 or DMSO for additional 4 and 8 hrs. the relative levels of IFNAR1 (left) and IRF3 (right) are presented as the mean ± SD (I).


mRNA expression of normalized to 18S - rRNA relative to a pooled organ donor sample . Lines between dots connect samples from same . CP : chronic pancreatitis ; PDAC : pancreatic adenocarcinoma ; significant difference ( P < 0 . 05 ) between normal tissue P = 9 . 39 × 10 − 5 determined by paired two - Student ' s t - , n = 17 .

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

mRNA expression of individual patients normalized to 18S-rRNA and relative to a pooled organ donor sample. Lines between dots connect samples from the same patient. CP: chronic pancreatitis; PDAC: pancreatic ductal adenocarcinoma; significant difference (P < 0.05) between tumor and normal tissue P = 9.39 × 10−5 determined by paired two-tailed Student's t-test, n = 17 patients.


Association of microarray - TBL1 expression survival in 105 with PDAC : High TBL1 expression is significantly associated with shorter post - resection survival . P - value determined by log - rank as described in Materials Methods .

O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

Association of microarray-based TBL1 expression and survival in 105 patients with PDAC: High TBL1 expression is significantly associated with shorter post-resection survival. P-value determined by log-rank test as described in Materials and Methods.


Immunohistochemistrystaining for TBL1 in human paraffin - embedded tissue sections . 1 , 2 : healthy tissue with acini showing no or only very weak . 3 , 4 : PanIN - 1 lesions with occasional cytoplasmicstaining . 5 , 6 : PanIN - 2 lesion with occasional cytoplasmicstaining . 7 - 10 : PanIN - 3 lesion with increased occurrence of cytoplasmaticstaining . 11 , 12 : invasive pancreatic adenocarcinoma with cytoplasmicstaining . Scale bars : 100 μm . are representative of 12 samples .

O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunohistochemistrystaining for TBL1 in human paraffin-embedded tissue sections. 1, 2: healthy tissue with acini and duct showing no or only very weak staining. 3, 4: PanIN-1 lesions with occasional nuclear and cytoplasmicstaining. 5, 6: PanIN-2 lesion with nuclear and occasional cytoplasmicstaining. 7-10: PanIN-3 lesion with nuclear and increased occurrence and intensity of cytoplasmaticstaining. 11, 12: invasive pancreatic ductal adenocarcinoma cells with nuclear and cytoplasmicstaining. Scale bars: 100 μm. Images are representative of 12 patient samples.


Immunohistochemistrystaining for TBL1 in paraffin - embedded tissue sections of wild - type or p48 + / Cre ; Kras + / LSL - G12Dmice . 1 , 2 : healthy tissue with acini , islet of Langerhans , ( zoom - in in panel 2 ) showing occasional weak nuclearstaining . 3 , 4 : PanIN - 1 lesions with nuclearstaining . 5 , 6 : PanIN - 2 lesions with nuclearstaining . 7 , 8 : PanIN - 3 lesion with nuclearstaining . 9 - 12 : invasive pancreatic adenocarcinoma with occasional faint cytoplasmicstaining . Scale bars : 100 μm . are representative of 32 wild - type 29 p48 + / Cre ; Kras + / LSL - G12Dmice .

O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O

Immunohistochemistrystaining for TBL1 in paraffin-embedded tissue sections of wild-type or p48+/Cre; Kras+/LSL-G12Dmice. 1, 2: healthy tissue with acini, islet of Langerhans, and duct (zoom-in in panel 2) showing occasional weak nuclearstaining. 3, 4: PanIN-1 lesions with nuclearstaining. 5, 6: PanIN-2 lesions with nuclearstaining. 7, 8: PanIN-3 lesion with nuclearstaining. 9-12: invasive pancreatic ductal adenocarcinoma cells with nuclear and occasional faint cytoplasmicstaining. Scale bars: 100 μm. Images are representative of 32 wild-type and 29 p48+/Cre; Kras+/LSL-G12Dmice.


Left panel : EdU incorporation in siRNA - transfected Capan - 1 ( 72 h post - transfection ) , n = 6 culture wells per group ; significantly different ( P < 0 . 05 ) from control ( no siRNA ) * , siNC # 1 # , siNC # 2 $ ; P - values : siTBL1 # 1 8 . 24 × 10 − 4 ( * ) , 5 . 43 × 10 − 4 ( # ) , 7 . 93 × 10 − 6 ( $ ) ; siTBL1 # 2 1 . 66 × 10 − 11 ( * ) , 1 . 27 × 10 − 11 ( # ) , 8 . 79 × 10 − 13 ( $ ) , one - way ANOVA with Bonferroni post - . Right panel : EdU incorporation of Panc02 with stable expression of shRNA ; n = 6 culture wells per group ; significantly different ( P < 0 . 05 ) from control ( shNC ) # P = 0 . 0398 ; Welch ' s t - .

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

Left panel: EdU incorporation in siRNA-transfected Capan-1 cells (72 h post-transfection), n = 6 cell culture wells per group; significantly different (P < 0.05) from control (no siRNA) *, siNC #1 #, siNC #2 $; P-values: siTBL1#1 8.24 × 10−4 (*), 5.43 × 10−4 (#), 7.93 × 10−6 ($); siTBL1 #2 1.66 × 10−11 (*), 1.27 × 10−11 (#), 8.79 × 10−13 ($), one-way ANOVA with Bonferroni post-test. Right panel: EdU incorporation of Panc02 cells with stable expression of shRNA; n = 6 cell culture wells per group; significantly different (P < 0.05) from control (shNC) #P = 0.0398; Welch's t-test.


Cellgrowth of siRNA - transfected Capan - 1 or Panc8680 ; n = 3 culture wells per group ; significantly different ( P < 0 . 05 ) from control ( no siRNA ) * , siNC # 1 # , siNC # 2 $ ; P - values : Capan - 1 48 h time - point : 0 . 0408 ( * ) for siTBL1 # 1 / 0 . 0464 for siTBL1 # 2 , Capan - 1 72 h time - point : 8 . 60 × 10 − 5 ( * ) , 1 . 82 × 10 − 3 ( # ) , 3 . 24 × 10 − 5 ( $ ) for siTBL1 # 1 / 2 . 39 × 10 − 10 ( * ) , 3 . 34 × 10 − 9 ( # ) , 1 . 08 × 10 − 10 ( $ ) for siTBL1 # 2 ; Panc8680 72 h time - point : 1 . 55 × 10 − 4 ( * ) , 2 . 73 × 10 − 5 ( $ ) ; two - way ANOVA with Bonferroni post - .

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

Cellgrowth of siRNA-transfected Capan-1 or Panc8680 cells; n = 3 cell culture wells per group; significantly different (P < 0.05) from control (no siRNA) *, siNC #1 #, siNC #2 $; P-values: Capan-1 cells 48 h time-point: 0.0408 (*) for siTBL1 #1 / 0.0464 for siTBL1 #2, Capan-1 cells 72 h time-point: 8.60 × 10−5 (*), 1.82 × 10−3 (#), 3.24 × 10−5 ($) for siTBL1 #1 / 2.39 × 10−10 (*), 3.34 × 10−9 (#), 1.08 × 10−10 ($) for siTBL1 #2; Panc8680 72 h time-point: 1.55 × 10−4 (*), 2.73 × 10−5 ($); two-way ANOVA with Bonferroni post-test.


Uptake of radio - labeled 3H - 2 - deoxyglucose in siRNA - transfected Capan - 1 ( 48 h post - transfection ) ; n = 6 culture wells per group ; statistically different ( P < 0 . 05 ) from control ( siNC # 2 ) $ P = 0 . 03 ; Welch ' s t - .

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

Uptake of radio-labeled 3H-2-deoxyglucose in siRNA-transfected Capan-1 cells (48 h post-transfection); n = 6 cell culture wells per group; statistically different (P < 0.05) from control (siNC #2) $P = 0.03; Welch's t-test.


Extracellular flux measurement of siRNA - transfected Capan - 1 with glycolysis stress ( 48 h post - transfection ) ; 2 - DG : 2 - deoxyglucose ; ECAR : extracellular acidification rate . n = 6 - 8 culture wells per group ; significantly different ( P < 0 . 05 ) from control ( no siRNA ) * , siNC # 1 # , siNC # 2 ) $ . Upper symbols : statistics for siTBL # 1 , P - values : 2 . 18 × 10 − 3 ( * ) time - point 8 , 9 . 83 × 10 − 3 ( * ) time - point 9 , 0 . 0102 ( * ) time - point 10 , 0 . 044 ( * ) time - point 11 ; 2 . 42 × 10 − 6 ( # ) time - point 8 , 1 . 47 × 10 − 4 ( # ) time - point 9 , 3 . 17 × 10 − 4 ( # ) time - point 10 , 0 . 0277 ( # ) time - point 11 ; 9 . 44 × 10 − 7 ( $ ) time - point 8 , 1 . 23 × 10 − 6 ( $ ) time - point 9 , 1 . 33 × 10 − 6 ( $ ) time - point 10 , 7 . 14 × 10 − 5 ( $ ) time - point 11 . Lower symbols : statistics for siTBL # 2 , P - values : 3 . 94 × 10 − 3 ( * ) time - point 5 , 0 . 0143 ( * ) time - point 6 , 0 . 0342 ( * ) time - point 7 , 1 . 45 × 10 − 3 ( * ) time - point 8 , 1 . 27 × 10 − 3 ( * ) time - point 9 , 2 . 38 × 10 − 3 ( * ) time - point 10 ; 0 . 01 ( # ) time - point 5 , 0 . 0362 ( # ) time - point 6 , 1 . 22 × 10 − 6 ( # ) time - point 8 , 1 . 28 × 10 − 5 ( # ) time - point 9 , 5 . 84 × 10 − 5 ( # ) time - point 10 ; 0 . 0118 ( $ ) time - point 5 , 0 . 0208 ( $ ) time - point 6 , 0 . 0291 ( $ ) time - point 7 , 4 . 52 × 10 − 7 ( $ ) time - point 8 , 6 . 04 × 10 − 8 ( $ ) time - point 9 , 1 . 43 × 10 − 7 ( $ ) time - point 10 , 4 . 83 × 10 − 3 ( $ ) time - point 11 . Two - way ANOVA with Bonferroni post - .

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

Extracellular flux measurement of siRNA-transfected Capan-1 cells with glycolysis stress test assay (48 h post-transfection); 2-DG: 2-deoxyglucose; ECAR: extracellular acidification rate. n = 6-8 cell culture wells per group; significantly different (P < 0.05) from control (no siRNA) *, siNC #1 #, siNC #2) $. Upper symbols: statistics for siTBL#1, P-values: 2.18 × 10−3 (*) time-point 8, 9.83 × 10−3 (*) time-point 9, 0.0102 (*) time-point 10, 0.044 (*) time-point 11; 2.42 × 10−6 (#) time-point 8, 1.47 × 10−4 (#) time-point 9, 3.17 × 10−4 (#) time-point 10, 0.0277 (#) time-point 11; 9.44 × 10−7 ($) time-point 8, 1.23 × 10−6 ($) time-point 9, 1.33 × 10−6 ($) time-point 10, 7.14 × 10−5 ($) time-point 11. Lower symbols: statistics for siTBL#2, P-values: 3.94 × 10−3 (*) time-point 5, 0.0143 (*) time-point 6, 0.0342 (*) time-point 7, 1.45 × 10−3 (*) time-point 8, 1.27 × 10−3 (*) time-point 9, 2.38 × 10−3 (*) time-point 10; 0.01 (#) time-point 5, 0.0362 (#) time-point 6, 1.22 × 10−6 (#) time-point 8, 1.28 × 10−5 (#) time-point 9, 5.84 × 10−5 (#) time-point 10; 0.0118 ($) time-point 5, 0.0208 ($) time-point 6, 0.0291 ($) time-point 7, 4.52 × 10−7 ($) time-point 8, 6.04 × 10−8 ($) time-point 9, 1.43 × 10−7 ($) time-point 10, 4.83 × 10−3 ($) time-point 11. Two-way ANOVA with Bonferroni post-test.


Gene expression microarray from Capan - 1 transfected with siRNA . shows genes annotated to KEGG pathway hsa04110 " Cycle " / or gene ontology cluster GO : 0007049 " Cycle " .

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

Gene expression microarray from Capan-1 cells transfected with siRNA. Heatmap shows genes annotated to KEGG pathway hsa04110 "Cell Cycle" and/or gene ontology cluster GO:0007049 "Cell Cycle".


expression of Panc02 with stable shRNA expression prior to implantation into mice .

Protein expression of Panc02 cells with stable shRNA expression prior to implantation into mice.


immunoprecipitation from Capan - 1 with or without siRNA - mediated knockdown of TBL1 . IgG primers for PIK3CA coding region served as a negative control ; histone H3 served as a positive control .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

Chromatin immunoprecipitation from Capan-1 cells with or without siRNA-mediated knockdown of TBL1. IgG and primers for PIK3CA coding region served as a negative control; histone H3 served as a positive control.


Proliferation time course in control TBL1 shRNA - transfected Panc02 overexpressing constitutively active PI3K p100α mutant E545K . were stained with crystal violet , absorbance was measured at 595 nm . n = 9 culture wells per group ; statistically significant at P < 0 . 05 for indicated comparisons : shNC pBabe - puro vs . shNC pBabe - PI3K ( * ) , shTBL1 pBabe - puro vs . sbTBL1 pBabe - PI3K ( # ) , shNC pBabe - puro vs . shTBL1 pBabe - puro ( $ ) , shNC pBabe - PI3K vs . shTBL1 pBabe - PI3K ( † ) ; P - values : day 2 : 2 . 01 × 10 − 3 ( # ) , 4 . 11 × 10 − 9 ( $ ) , 5 . 77 × 10 − 3 ( † ) ; day 3 : 4 . 50 × 10 − 5 ( * ) , 1 . 01 × 10 − 9 ( # ) , 1 . 71 × 10 − 30 ( $ ) , 1 . 22 × 10 − 6 ( † ) ; day 4 : 5 . 61 × 10 − 22 ( * ) , 1 . 10 × 10 − 45 ( # ) , 6 . 65 × 10 − 62 ( $ ) ; two - way ANOVA with Bonferroni post - .

O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Proliferation time course assay in control and TBL1 shRNA-transfected Panc02 cells overexpressing constitutively active PI3K p100α mutant E545K. Cells were stained with crystal violet, and absorbance was measured at 595 nm. n = 9 cell culture wells per group; statistically significant at P < 0.05 for indicated comparisons: shNC pBabe-puro vs. shNC pBabe-PI3K (*), shTBL1 pBabe-puro vs. sbTBL1 pBabe-PI3K (#), shNC pBabe-puro vs. shTBL1 pBabe-puro ($), shNC pBabe-PI3K vs. shTBL1 pBabe-PI3K (†); P-values: day 2: 2.01 × 10−3 (#), 4.11 × 10−9 ($), 5.77 × 10−3 (†); day 3: 4.50 × 10−5 (*), 1.01 × 10−9 (#), 1.71 × 10−30 ($), 1.22 × 10−6 (†); day 4: 5.61 × 10−22 (*), 1.10 × 10−45 (#), 6.65 × 10−62 ($); two-way ANOVA with Bonferroni post-test.


Matrigel invasion in same as in ( D ) . Invading per microscopy field were counted . n = 6 microscopy fields per group ; statistically significant at P < 0 . 05 ; P - values : 6 . 64 × 10 − 4 ( * ) , 1 . 35 × 10 − 7 ( # ) , 6 . 94 × 10 − 5 ( $ ) ; two - way ANOVA with Bonferroni post - .

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

Matrigel invasion assay in same cells as in (D). Invading cells per microscopy field were counted. n = 6 microscopy fields per group; statistically significant at P < 0.05; P-values: 6.64 × 10−4 (*), 1.35 × 10−7 (#), 6.94 × 10−5 ($); two-way ANOVA with Bonferroni post-test.


mRNA expression of PIK3CA in human samples ; significant difference ( P < 0 . 05 ) between normal tissue * P = 2 . 13 × 10 − 4 determined by paired two - Student ' s t - , n = 18 .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

mRNA expression of PIK3CA in human patient samples; significant difference (P < 0.05) between tumor and normal tissue *P = 2.13 × 10−4 determined by paired two-tailed Student's t-test, n = 18 patients.


Correlation of mRNA expression of TBL1 PIK3CA in human tissue ( normalized to 18S rRNA relative to a pooled organ donor sample ) ; n = 17 ; P - value determined by testing for slope of regression ≠ 0 .

O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Correlation of mRNA expression of TBL1 and PIK3CA in human patient tumor tissue (normalized to 18S rRNA and relative to a pooled organ donor sample); n = 17 patients; P-value determined by testing for slope of regression line ≠ 0.


Survival in 105 with PDAC : High PIK3CA expression is significantly associated with shorter post - resection survival . P - value determined by log - rank as described in Materials Methods .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

Survival analysis in 105 patients with PDAC: High PIK3CA expression is significantly associated with shorter post-resection survival. P-value determined by log-rank test as described in Materials and Methods.


A - D Panc02 with stable expression of luciferase were implanted subcutaneously into C57Bl6 / N mice . Six days later , tumors were injected three times per week with 108 ifu of adenovirus encoding shRNA . ( A - C ) : Data plotted as mean ± ; n = 4 in control ( shNC ) group n = 3 in knockdown ( shTBL1 ) group ; significantly different ( P < 0 . 05 ) between control ( shNC ) knockdown ( shTBL1 ) * . ( A ) volume determined by transcutaneous measurement with digital calipers ; P - values : 9 . 21 × 10 − 3 at day 15 , 6 . 02 × 10 − 4 at day 18 , 2 . 49 × 10 − 4 at day 20 ; two - way ANOVA with Bonferroni post - . ( B ) Luminescence of tumors after intraperitoneal injection of firefly d - luciferin ; P = 0 . 0133 at day 20 ; two - way ANOVA with Bonferroni post - . ( C ) mass at necropsy ; P = 0 . 0299 , one - sided Welch ' s t - . ( D ) Luminescence of one representative mouse in each group .

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

A-D Panc02 cells with stable expression of luciferase were implanted subcutaneously into C57Bl6/N mice. Six days later, tumors were injected three times per week with 108 ifu of adenovirus encoding shRNA. (A-C): Data plotted as mean ± SEM; n = 4 animals in control (shNC) group and n = 3 animals in knockdown (shTBL1) group; significantly different (P < 0.05) between control (shNC) and knockdown (shTBL1) *. (A) Tumor volume determined by transcutaneous measurement with digital calipers; P-values: 9.21 × 10−3 at day 15, 6.02 × 10−4 at day 18, 2.49 × 10−4 at day 20; two-way ANOVA with Bonferroni post-test. (B) Luminescence of tumors after intraperitoneal injection of firefly d-luciferin; P = 0.0133 at day 20; two-way ANOVA with Bonferroni post-test. (C) Tumor mass at necropsy; P = 0.0299, one-sided Welch's t-test. (D) Luminescence images of one representative mouse in each group.


E - G Panc02 with stable expression of shRNA were implanted subcutaneously into C57Bl6 / Nmice . Seven days later , mice were treated with 20 mg / kg gemcitabine delivered by intraperitoneal injection ; n = 4 in shNC + GEM group , n = 5 in other groups ; significantly different ( P < 0 . 05 ) between groups : shNC vs . shNC + GEM ( * ) , shNC vs . shTBL1 ( # ) , shNC vs . shTBL1 + GEM ( $ ) , shNC + GEM vs . shTBL1 ( % ) , shNC + GEM vs . shTBL + GEM ( † ) ; shTBL1 vs . shTBL1 + GEM ( ‡ ) ; two - way ANOVA with Bonferroni post - . ( E ) volume determined by transcutaneous measurement with digital calipers . P - values : day 17 : 8 . 48 × 10 − 3 ( # ) , 1 . 07 × 10 − 3 ( $ ) , day 20 : 3 . 07 × 10 − 3 ( * ) , 9 . 37 × 10 − 6 ( # ) , 1 . 56 × 10 − 8 ( $ ) , 0 . 0239 ( † ) . ( F ) volume relative to volume at day 7 post - implantation ; P - values : day 17 : 3 . 94 × 10 − 3 ( $ ) , day 20 : 1 . 09 × 10 − 3 ( * ) , 1 . 39 × 10 − 7 ( $ ) , 0 . 01195 ( % ) , 3 . 12 × 10 − 6 ( ‡ ) . ( G ) under curve for relative volume ; P = 0 . 0195 ( ‡ ) .

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

E-G Panc02 cells with stable expression of shRNA were implanted subcutaneously into C57Bl6/Nmice. Seven days later, mice were treated with 20 mg/kg gemcitabine delivered by intraperitoneal injection; n = 4 animals in shNC+GEM group, n = 5 animals in all other groups; significantly different (P < 0.05) between groups: shNC vs. shNC+GEM (*), shNC vs. shTBL1 (#), shNC vs. shTBL1+GEM ($), shNC+GEM vs. shTBL1 (%), shNC+GEM vs. shTBL+GEM (†); shTBL1 vs. shTBL1+GEM (‡); two-way ANOVA with Bonferroni post-test. (E) Tumor volume determined by transcutaneous measurement with digital calipers. P-values: day 17: 8.48 × 10−3 (#), 1.07 × 10−3 ($), day 20: 3.07 × 10−3 (*), 9.37 × 10−6 (#), 1.56 × 10−8 ($), 0.0239 (†). (F) Tumor volume relative to the volume at day 7 post-implantation; P-values: day 17: 3.94 × 10−3 ($), day 20: 1.09 × 10−3 (*), 1.39 × 10−7 ($), 0.01195 (%), 3.12 × 10−6 (‡). (G) Area under curve for relative tumor volume; P = 0.0195 (‡).


TBL1 - depletion leads to reduction in PI3 kinase p110α cycle - associated proteinsPanc02 with stable expression of shRNA were implanted subcutaneously into C57Bl6 / Nmice . Seven days later , mice were treated with 20 mg / kg gemcitabine delivered by intraperitoneal injection . After 21 days , mice were sacrificed , tumors were removed were extracted immunoblotted .

B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

TBL1-depletion leads to reduction in PI3 kinase p110α and cell cycle-associated proteinsPanc02 cells with stable expression of shRNA were implanted subcutaneously into C57Bl6/Nmice. Seven days later, mice were treated with 20 mg/kg gemcitabine delivered by intraperitoneal injection. After 21 days, mice were sacrificed, tumors were removed and proteins were extracted and immunoblotted.


DU145 were transfected with indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool or siUBTD1single1 or single2 ) for 24 h plated on fibronectin - coated glass coverslips . ( A ) scatter plot shows apparent Young ' s moduli obtained by AFM . Bars represent median values of distributions . immunoblot shows UBTD1 siRNA knock - down efficiency .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O

DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. (A) The scatter plot shows the apparent Young's moduli obtained by AFM analysis. Bars represent the median values of the distributions. The immunoblot shows UBTD1 siRNA knock-down efficiency.


DU145 were transfected with indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool or siUBTD1single1 or single2 ) for 24 h plated on fibronectin - coated glass coverslips . ( B ) Immunoblots ( left ) ( right ) show of GST - ROCK associated GTP - bound RhoA ( RhoA - GTP ) total RhoA in lysates ( RhoA ) in confluent . Immunoblot of UBTD1 shows level of siRNA depletion .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O

DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. (B) Immunoblots (left) and quantification (right) show levels of GST-ROCK associated GTP-bound RhoA (RhoA-GTP) and total RhoA in lysates (RhoA) in confluent cells. Immunoblot of UBTD1 shows the level of siRNA depletion.


DU145 were transfected with indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool or siUBTD1single1 or single2 ) for 24 h plated on fibronectin - coated glass coverslips . ( C ) Representative confocal of focal adhesion ( paxillin ) actin cytoskeleton ( phalloidin ) outlines of focal adhesions using threshold - image . Immunoblot of UBTD1 shows level of siRNA - mediated depletion . ( D ) , number of focal adhesion were quantified ( ImageJ ) using as shown ( C ) .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. (C) Representative confocal images of focal adhesion (paxillin) and actin cytoskeleton (phalloidin) and outlines of focal adhesions using threshold-based image. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. (D) Cell area, number and area of focal adhesion were quantified (ImageJ) using the analysis as shown (C).


DU145 were transfected with indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool or siUBTD1single1 or single2 ) for 24 h plated on fibronectin - coated glass coverslips . Sub - confluent DU145 ( E ) were seeded on two matrices of different stiffnesses ( 4 12 kPa ) . Representative heat map ( left ) ( right ) showing contractile forces generate by plated on 4kPa or 12kPa hydrogel .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. Sub-confluent DU145 (E) cells were seeded on two matrices of different stiffnesses (4 and 12 kPa). Representative heat map (left) and quantification (right) showing contractile forces generate by cells plated on 4kPa or 12kPa hydrogel.


A549 were transfected with indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool or siUBTD1single1 or single2 ) for 24 h plated on fibronectin - coated glass coverslips . Sub - confluent A549 ( F ) were seeded on two matrices of different stiffnesses ( 4 12 kPa ) . Representative heat map ( left ) ( right ) showing contractile forces generate by plated on 4kPa or 12kPa hydrogel .

A549 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. Sub-confluent A549 (F) cells were seeded on two matrices of different stiffnesses (4 and 12 kPa). Representative heat map (left) and quantification (right) showing contractile forces generate by cells plated on 4kPa or 12kPa hydrogel.


DU145 were transfected with indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool or siUBTD1single1 or single2 ) for 24 h plated on fibronectin - coated glass coverslips . ( G ) Confluent DU145 were seeded on 12 kPa matrix . Representative heat map ( left ) ( right ) showing contractile forces generate by plated on 12kPa hydrogel .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. (G) Confluent DU145 cells were seeded on 12 kPa matrix. Representative heat map (left) and quantification (right) showing contractile forces generate by cells plated on 12kPa hydrogel.


DU145 were transfected with indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool or siUBTD1single1 or single2 ) for 24 h plated on fibronectin - coated glass coverslips . ( H ) Representative confocal of YAP UBTD1 localization on two matrices of different stiffness . DU145 were grown on collagen / acrylamide - coated glass coverslips ( soft ) or directly on glass coverslips ( stiff ) . Nuclei were stained with DAPI ( Blue ) on MERGE image .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. (H) Representative confocal images of YAP and UBTD1 localization on two matrices of different stiffness. DU145 cells were grown on collagen/acrylamide-coated glass coverslips (soft) or directly on glass coverslips (stiff). Nuclei were stained with DAPI (Blue) on the MERGE image.


( A ) Immunoblot of DU145 A549 at various densities ranging from 20 % to 100 % confluence showing increased expression of UBTD1 β - catenin with confluency . E - cadherin was used as a positive control . Tubulin was used as a loading control .

O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O

(A) Immunoblot of DU145 and A549 cells at various cell densities ranging from 20% to 100% confluence showing the increased expression of UBTD1 and β-catenin with cell confluency. E-cadherin was used as a positive control. Tubulin was used as a loading control.


, immunofluorescent labelling of UBTD1 E - cadherin ( B ) at - contact in confluent DU145 analyzed by confocal microscopy . Right , representative histogram of a co - localization profile prepared with RGB profiler plugin of ImageJ .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

, immunofluorescent labelling of UBTD1 and E-cadherin (B) at the cell-cell contact in confluent DU145 cells and analyzed by confocal microscopy. Right, representative histogram of a co-localization profile prepared with RGB profiler plugin of ImageJ.


immunofluorescent labelling of UBTD1 β - catenin ( C ) at - contact in confluent DU145 analyzed by confocal microscopy . Right , representative histogram of a co - localization profile prepared with RGB profiler plugin of ImageJ .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

immunofluorescent labelling of UBTD1 and β-catenin (C) at the cell-cell contact in confluent DU145 cells and analyzed by confocal microscopy. Right, representative histogram of a co-localization profile prepared with RGB profiler plugin of ImageJ.


Proximal ligation monitoring ( left ) ( right ) of UBTD1 association with E - cadherin or β - catenin in DU145 transfected with indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool ) . Nuclei were stained with DAPI ( Blue ) on MERGE image .

O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

Proximal ligation assay monitoring (left) and quantification (right) of UBTD1 association with E-cadherin or β-catenin in DU145 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image.


Immunoblots of UBTD1 , β - catenin YAP after subcellular fractionation of confluent DU145 transfected with indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool ) . EGFR , RhoGDI Lamin A / C were used as controls for ( M ) , cytosol ( C ) nucleus ( N ) compartments , respectively .

O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

Immunoblots of UBTD1, β-catenin and YAP after subcellular fractionation of confluent DU145 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool). EGFR, RhoGDI and Lamin A/C were used as controls for the membrane (M), cytosol (C) and nucleus (N) compartments, respectively.


Co - immunoprecipitation between endogenous β - catenin UBTD1 was performed in confluent DU145 . IgG isotype was used as a negative control .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O

Co-immunoprecipitation between endogenous β-catenin and UBTD1 was performed in confluent DU145 cells. IgG isotype was used as a negative control.


( G ) Representative confocal of β - catenin UBTD1 in DU145 under resting ( Ctrl ) , after calcium chelator treatment ( EGTA ) recovery ( Rec ) conditions . Nuclei were stained with DAPI ( Blue ) on MERGE image .

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(G) Representative confocal images of β-catenin and UBTD1 in DU145 under resting (Ctrl), after calcium chelator treatment (EGTA) and recovery (Rec) conditions. Nuclei were stained with DAPI (Blue) on the MERGE image.


( H ) Representative confocal of β - catenin UBTD1 in DU145 after scattering induced by HGF treatment ( 24 h ) . Nuclei were stained with DAPI ( Blue ) on MERGE image .

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(H) Representative confocal images of β-catenin and UBTD1 in DU145 after cell scattering induced by HGF treatment (24 h). Nuclei were stained with DAPI (Blue) on the MERGE image.


Immunoblot of DU145 A549 at various densities ranging from 20 % to 100 % confluence showing increased expression of UBTD1 , p - YAP ( ser127 ) YAP with confluency . Tubulin was used as a loading control .

O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O

Immunoblot of DU145 and A549 cells at various cell densities ranging from 20% to 100% confluence showing the increased expression of UBTD1, p-YAP (ser127) and YAP levels with the cell confluency. Tubulin was used as a loading control.


DU145 were transfected for 48h with indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool ) . ( B ) Immunoblots of p - YAP ( ser127 ) , YAP UBTD1 ( left ) of YAP p - YAP / YAP ( right ) in control or UBTD1 - depleted DU145 at low high density . Immunoblot of UBTD1 shows level of siRNA - mediated depletion . Actin was used as a loading controls . , immunoblots for loading control ( actin ) UBTD1 knock - down efficiency ( UBTD1 ) are duplicated because they belong to same experiment shown in both panels .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

DU145 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). (B) Immunoblots of p-YAP (ser127), YAP and UBTD1 (left) and quantification of YAP and p-YAP/YAP levels (right) in control or UBTD1-depleted DU145 at low and high cell density. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin was used as a loading controls. , immunoblots for the loading control (actin) and UBTD1 knock-down efficiency (UBTD1) are duplicated because they belong to the same experiment shown in both panels.


DU145 were transfected for 48h with indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool ) . ( C ) Immunoblots of Hippo signaling pathway using phosphorylated ( p - ser909LATS p - thr183MST1 / p - thr180MST2 ) total LATS , MST at low high DU145 density . Immunoblot of UBTD1 shows level of siRNA - mediated depletion . Actin Rho - GDI were used as loading controls . , immunoblots for loading control ( actin ) UBTD1 knock - down efficiency ( UBTD1 ) are duplicated because they belong to same experiment shown in both panels .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

DU145 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). (C) Immunoblots of the Hippo signaling pathway using phosphorylated (p-ser909LATS and p-thr183MST1/p-thr180MST2) and total LATS, MST at low and high DU145 cells density. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin and Rho-GDI were used as loading controls. , immunoblots for the loading control (actin) and UBTD1 knock-down efficiency (UBTD1) are duplicated because they belong to the same experiment shown in both panels.


DU145 were transfected for 48h with indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool ) . Immunoblots ( up ) ( down ) of YAP in confluent ( D ) DU145 UBTD1 depleted treated or not with cycloheximide ( CHX ) for 2h . Immunoblot of UBTD1 shows level of siRNA - mediated depletion . Actin was used as a loading control .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

DU145 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). Immunoblots (up) and quantification (down) of YAP levels in confluent (D) DU145 UBTD1 depleted cells treated or not with cycloheximide (CHX) for 2h. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin was used as a loading control.


A549 were transfected for 48h with indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool ) . Immunoblots ( up ) ( down ) of YAP in confluent ( E ) A549 UBTD1 depleted treated or not with cycloheximide ( CHX ) for 2h . Immunoblot of UBTD1 shows level of siRNA - mediated depletion . Actin was used as a loading control .

A549 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). Immunoblots (up) and quantification (down) of YAP levels in confluent (E) A549 UBTD1 depleted cells treated or not with cycloheximide (CHX) for 2h. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin was used as a loading control.


DU145 or A549 were transfected for 48h with indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool ) . Immunoblots ( up ) ( down ) showing YAP in UBTD1 depleted confluent DU145 A549 treated or not with MG132 for 6h . Immunoblot of UBTD1 shows level of siRNA - mediated depletion . Actin was used as a loading control .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

DU145 cells or A549 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). Immunoblots (up) and quantification (down) showing YAP levels in UBTD1 depleted confluent DU145 A549 cells treated or not with MG132 for 6h. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin was used as a loading control.


( A ) Co - immunoprecipitation in DU145 A549 between endogenous YAP UBTD1 . YAP was used as bait . IgG isotype was used as a negative control .

O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O

(A) Co-immunoprecipitation in DU145 and A549 cells between endogenous YAP and UBTD1. YAP was used as bait. The IgG isotype was used as a negative control.


( B ) Co - immunoprecipitation in DU145 A549 between endogenous YAP UBTD1 . UBTD1 was used as bait . IgG isotype was used as a negative control .

(B) Co-immunoprecipitation in DU145 and A549 cells between endogenous YAP and UBTD1. UBTD1 was used as bait. The IgG isotype was used as a negative control.


( C ) Proximal ligation monitoring ( left ) ( right ) of UBTD1 associated with YAP of DU145 A549 transfected with indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool ) . Nuclei were stained with DAPI ( Blue ) on MERGE image .

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(C) Proximal ligation assay monitoring (left) and quantification (right) of UBTD1 associated with YAP of DU145 and A549 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image.


( D ) Co - immunoprecipitation in confluent DU145 between endogenous β - TrCP , Ubch5c UBTD1 . UBTD1 was used as bait . IgG isotype was used as a negative control .

O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O

(D) Co-immunoprecipitation in confluent DU145 cells between endogenous β-TrCP, Ubch5c and UBTD1. UBTD1 was used as bait. IgG isotype was used as a negative control.


( E ) Co - immunoprecipitation in confluent DU145 between endogenous β - TrCP , Ubch5c , YAP β - catenin . DU145 were transfected with indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool or siUBTD1single ) . β - TrCP was used as bait . IgG isotype was used as a negative control . Immunoblot of UBTD1 shows level of siRNA - mediated depletion .

O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O

(E) Co-immunoprecipitation in confluent DU145 cells between endogenous β-TrCP, Ubch5c, YAP and β-catenin. DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool or siUBTD1single). β-TrCP was used as bait. IgG isotype was used as a negative control. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion.


Proximal ligation monitoring ( F ) of β - TrCP associated with Ubch5 , YAP or β - catenin in DU145 transfected with indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool ) . Nuclei were stained with DAPI ( Blue ) on MERGE image .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

Proximal ligation assay monitoring (F) of β-TrCP associated with Ubch5, YAP or β-catenin in DU145 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image.


( G ) of β - TrCP associated with Ubch5 , YAP or β - catenin in DU145 transfected with indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool ) .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O

quantification (G) of β-TrCP associated with Ubch5, YAP or β-catenin in DU145 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool).


( H ) Immunoblots show YAP ubiquitylation in HEK in different experimental conditions . were transfected , as indicated , with expression vectors of histidine - tagged ubiquitin ( His - Ub ) together with control siRNA or UBTD1 siRNA . His - Ub crosslinked forms of YAP were purified ( IP : His ) immunoblot of YAP showed YAP ubiquitylation . immunoblot of YAP ( lower panel ) was performed in parallel to verify amounts of YAP engaged in His - Ub purifications . immunoblot of UBTD1 shows level of siRNA depletion .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O

(H) Immunoblots show YAP ubiquitylation in HEK cells in different experimental conditions. Cells were transfected, as indicated, with expression vectors of histidine-tagged ubiquitin (His-Ub) together with control siRNA or UBTD1 siRNA. His-Ub crosslinked forms of YAP were purified (IP: His) and the immunoblot of YAP showed YAP ubiquitylation. The immunoblot of YAP (lower panel) was performed in parallel to verify the amounts of YAP protein engaged in the His-Ub purifications. The immunoblot of UBTD1 shows the level of siRNA depletion.


DU145 were transfected with indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool ) . ( A ) Representative wide - field immunofluorescence ( left ) ( right ) showing endogenous YAP translocation in DU145 after UBTD1 depletion . Nuclei were stained with DAPI ( Blue ) on MERGE image .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). (A) Representative wide-field immunofluorescence images (left) and quantification (right) showing endogenous YAP nuclear translocation in DU145 cells after UBTD1 depletion. Nuclei were stained with DAPI (Blue) on the MERGE image.


DU145 were transfected with indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool ) . ( B ) of CTGF1 , CYR61 BIRC5 mRNA .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O

DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). (B) Quantification of CTGF1, CYR61 and BIRC5 mRNA levels.


( C ) Immunoblots of YAP in DU145 depleted ( siUBTD1single2 ) or not ( siCTRL ) for UBTD1 transfected or not with GFP - UBTD1 ( + ) . Immunoblot of UBTD1 shows level of siRNA - mediated depletion . Immunoblot of GFP shows level of UBTD1 transfection . CHC was used as a loading control .

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(C) Immunoblots of YAP in DU145 cells depleted (siUBTD1single2) or not (siCTRL) for UBTD1 and transfected or not with GFP-UBTD1 (+). Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Immunoblot of GFP shows the level of UBTD1 transfection. CHC was used as a loading control.


( D ) Representative confocal immunofluorescence ( left ) ( right ) showing endogenous YAP translocation in DU145 depleted ( siUBTD1single2 ) or not ( siCTRL ) for UBTD1 transfected or not with GFP - UBTD1 . Nuclei were stained with DAPI ( Blue ) on MERGE image .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

(D) Representative confocal immunofluorescence images (left) and quantification (right) showing endogenous YAP nuclear translocation in DU145 cells depleted (siUBTD1single2) or not (siCTRL) for UBTD1 and transfected or not with GFP-UBTD1. Nuclei were stained with DAPI (Blue) on the MERGE image.


( E ) Immunoblots of p - YAP YAP in DU145 control ( siCTRL ) or depleted for LATS1 LATS2 ( siLATS1 + siLATS2 ) / or UBTD1 ( siUBTD1pool ) . Immunoblot of p - YAP reflects activity of Hippo pathway . Immunoblot of UBTD1 LATS show siRNA knock - down efficiency . Actin was used as a loading control .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O

(E) Immunoblots of p-YAP and YAP in DU145 control cells (siCTRL) or depleted for LATS1 and LATS2 (siLATS1+siLATS2) and/or UBTD1 (siUBTD1pool). Immunoblot of p-YAP reflects the activity of the Hippo pathway. Immunoblot of UBTD1 and LATS show siRNA knock-down efficiency. Actin was used as a loading control.


Representative confocal immunofluorescence ( F ) showing endogenous YAP translocation in DU145 control ( siCTRL ) or depleted for LATS1 LATS2 ( siLATS1 + siLATS2 ) / or UBTD1 ( siUBTD1pool ) . Nuclei were stained with DAPI ( Blue ) on MERGE image .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

Representative confocal immunofluorescence images (F) showing endogenous YAP nuclear translocation in DU145 control cells (siCTRL) or depleted for LATS1 and LATS2 (siLATS1+siLATS2) and/or UBTD1 (siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image.


( G ) showing endogenous YAP translocation in DU145 control ( siCTRL ) or depleted for LATS1 LATS2 ( siLATS1 + siLATS2 ) / or UBTD1 ( siUBTD1pool ) .

O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O

quantification (G) showing endogenous YAP nuclear translocation in DU145 control cells (siCTRL) or depleted for LATS1 and LATS2 (siLATS1+siLATS2) and/or UBTD1 (siUBTD1pool).


( H ) Immunoblots of YAP in DU145 control ( siCTRL ) or depleted for ROCK1 ( siROCK1 ) or ROCK2 ( siROCK2 ) / or UBTD1 ( siUBTD1pool ) . Immunoblots of UBTD1 , ROCK1 ROCK2 show level of siRNA knock - down efficiency . Actin was used as a loading control .

O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O

(H) Immunoblots of YAP in DU145 control (siCTRL) or depleted for ROCK1 (siROCK1) or ROCK2 (siROCK2) and/or UBTD1 (siUBTD1pool). Immunoblots of UBTD1, ROCK1 and ROCK2 show the level of siRNA knock-down efficiency. Actin was used as a loading control.


Representative confocal immunofluorescence ( I ) showing endogenous YAP localization in DU145 control ( siCTRL ) or depleted for ROCK1 ( siROCK1 ) or ROCK2 ( siROCK2 ) / or UBTD1 ( siUBTD1pool ) . Nuclei were stained with DAPI ( Blue ) on MERGE image .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

Representative confocal immunofluorescence images (I) showing endogenous YAP localization in DU145 control cells (siCTRL) or depleted for ROCK1 (siROCK1) or ROCK2 (siROCK2) and/or UBTD1 (siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image.


( J ) showing endogenous YAP localization in DU145 control ( siCTRL ) or depleted for ROCK1 ( siROCK1 ) or ROCK2 ( siROCK2 ) / or UBTD1 ( siUBTD1pool ) .

O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O

quantification (J) showing endogenous YAP localization in DU145 control cells (siCTRL) or depleted for ROCK1 (siROCK1) or ROCK2 (siROCK2) and/or UBTD1 (siUBTD1pool).


( K ) of CTGF1 , CYR61 , UBTD1 BIRC5 mRNA in DU145 control ( siCTRL ) or depleted for ROCK1 ( siROCK1 ) or ROCK2 ( siROCK2 ) / or UBTD1 ( siUBTD1pool ) .

O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O

(K) Quantification of CTGF1, CYR61, UBTD1 and BIRC5 mRNA in DU145 control cells (siCTRL) or depleted for ROCK1 (siROCK1) or ROCK2 (siROCK2) and/or UBTD1 (siUBTD1pool).


DU145 were transfected with indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool or siUBTD1single1 ; UBTD1 + YAP + TAZ , siUBTD1single1 + siYAPsingle1 + siTAZsingle1 or siUBTD1single1 + siYAPsingle2 + siTAZsingle2 ) . ( A ) of EMT marker genes ( vimentin , slug , snail , fibronectin , α - SMA , E - cadherin ) UBTD1 .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O

DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool or siUBTD1single1; UBTD1 +YAP+TAZ, siUBTD1single1+siYAPsingle1+ siTAZsingle1 or siUBTD1single1+siYAPsingle2+ siTAZsingle2). (A) Quantification of EMT marker genes (vimentin, slug, snail, fibronectin, α-SMA, E-cadherin) and UBTD1.


( B ) Immunoblots of EMT markers ( vimentin , E - cadherin , slug ) . Immunoblot of UBTD1 shows siRNA knock - down efficiency . Hsp90 tubulin were used as loading controls .

O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

(B) Immunoblots of EMT markers (vimentin, E-cadherin, slug). Immunoblot of UBTD1 shows siRNA knock-down efficiency. Hsp90 and tubulin were used as loading controls.


DU145 were transfected with indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool or siUBTD1single1 ; UBTD1 + YAP + TAZ , siUBTD1single1 + siYAPsingle1 + siTAZsingle1 or siUBTD1single1 + siYAPsingle2 + siTAZsingle2 ) . ( C ) of DU145 migration in Transwell chamber inserts .

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DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool or siUBTD1single1; UBTD1 +YAP+TAZ, siUBTD1single1+siYAPsingle1+ siTAZsingle1 or siUBTD1single1+siYAPsingle2+ siTAZsingle2). (C) Quantification of DU145 cell migration in Transwell chamber inserts.


DU145 were transfected with indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool or siUBTD1single1 ; UBTD1 + YAP + TAZ , siUBTD1single1 + siYAPsingle1 + siTAZsingle1 or siUBTD1single1 + siYAPsingle2 + siTAZsingle2 ) . ( D ) of DU145 invasion in Transwell chamber inserts .

DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool or siUBTD1single1; UBTD1 +YAP+TAZ, siUBTD1single1+siYAPsingle1+ siTAZsingle1 or siUBTD1single1+siYAPsingle2+ siTAZsingle2). (D) Quantification of DU145 cell invasion in Transwell chamber inserts.


( E ) Invasive properties of human prostate tumoroids depleted for UBTD1 .

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(E) Invasive properties of human prostate tumoroids depleted for UBTD1.


( F ) of UBTD1 mRNA in prostate ( n = 70 ) adjacent normal tissues ( n = 47 ) .

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(F) Quantification of UBTD1 mRNA levels in prostate tumor (n=70) and adjacent normal tissues (n=47).


( G ) Representative UBTD1 immunohistochemistry on sections of human prostate normal tissues ( n = 8 ) .

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(G) Representative UBTD1 immunohistochemistry staining on sections of human prostate tumor and normal tissues (n=8).


( H ) Representative UBTD1 immunohistochemistry on sections of human lung normal tissues ( n = 6 ) .

(H) Representative UBTD1 immunohistochemistry staining on sections of human lung tumor and normal tissues (n=6).


( I ) of UBTD1 mRNA in grade 3 prostate adenocarcinoma ( 56 ) are correlated with overall survival ( OS ) . OS was calculated from subgroups with mRNA that were less or greater than first quartile value .

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(I) The levels of UBTD1 mRNA in grade 3 prostate adenocarcinoma patients (56 patients) are correlated with overall survival (OS). OS was calculated from patient subgroups with mRNA levels that were less or greater than the first quartile value.


( J ) of UBTD1 mRNA in lung adenocarcinoma ( 522 ) are correlated with OS .

(J) The levels of UBTD1 mRNA in lung adenocarcinoma patients (522 patients) are correlated with OS.


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

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(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.


( A ) Translocation of ectopically expressed mCherry - hGBP1 to GFP + S . flexneri ΔipaH9 . 8 in HeLa hGBP1 - KO was monitored by time - lapse microscopy . time frames of Movie EV1 starting at 55 min post infection ( mpi ) are shown .

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(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 presence or absence of formaldehyde fixed GFP + S . flexneri . time frames of Movie EV2 depict hGBP1F . Merged of hGBP1F S . flexneri are shown for 60 min time points .

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(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 ) were taken at 45 min after addition of 10 µM Alexa - Fluor647 - hGBP1F to formaldehyde fixed GFP + S . flexneri in 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 after 45 min were quantified . Combined data from two independent experiments are shown as mean ± . Significance was determined by one - way ANOVA with Tukey ' s multiple comparison . * * * * , P ≤ 0 . 0001 .

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(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.


( A ) Polymerization of 10 µM hGBP1F hGBP1FR584 - 586A in presence of 2 mM GTP was monitored over time by absorption spectroscopy at 350 nm . Absorbance signals were superimposed with nucleotide composition of same solution analyzed at defined time points , revealing characteristic first phase of slow polymer nucleation second phase of fast polymer growth cooperative hydrolysis .

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(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 of live S . flexneri incubated with no or with 5 µM of either hGBP1F , hGBP1FR584 - 586A , or hGBP1FR48A ( non - polymerizing mutant ) in presence of 2 mM GTP for 4 min . Arrowheads point to unattached hGBP1 polymers , arrows point to hGBP1 polymers attached to , asterisks mark polymeric structures that appear to fuse with surfaces .

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(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 2 mM GTP . Binding of hGBP1F polymers to at 5 min enclosure of with hGBP1F coats after 60 min were quantified . Mean frequencies ± of combined data from at least three independent experiments are shown . Significance was determined by by unpaired t - , two - . * * * , P ≤ 0 . 001 ; * * * * , P ≤ 0 . 0001 .

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(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.


( A ) Representative confocal taken at 60 min after admixture of 2 mM GTP 10 µM Alexa - Fluor647 - or Alexa - Fluor488 - hGBP1F to formaldehyde fixed gram - negative gram - positive 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 ] ) .

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(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 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 of Alexa - Fluor488 - LPS - O55 : B5 supplemented with 2 mM GTP 5 µM of indicated hGBP1 variant . Mean ± of combined data from three independent experiments . Significance was determined by one - way ANOVA with Tukey ' s multiple comparison . . n . s . , not significant ; * * * , P ≤ 0 . 001 ; * * * * , P ≤ 0 . 0001 .

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(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 presence absence of 5 µM LPS - O55 : B5 was monitored by absorption spectroscopy at 350 nm . absorbance signal was superimposed with nucleotide composition of same solution analyzed at defined time points . Maximal hydrolysis rates ( turnover numbers ) were determined for hGBP1F variants in presence absence of 5 µM LPS - O55 : B5 . Graphs show mean turnover numbers ± of combined data from at least three independent experiments . Significance was determined by two - way ANOVA with Tukey ' s multiple comparison . n . s . , not significant ; * * * , P ≤ 0 . 001 ; * * * * , P ≤ 0 . 0001 .

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(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 2 mM GTP . After 60 min hGBP1F - enclosed S . flexneri were quantified . Combined data from three independent experiments are shown as mean ± . Significance was determined by one - way ANOVA with Tukey ' s multiple comparison . * * * * , P ≤ 0 . 0001 .

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(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 of formaldehyde fixed GFP - , RFP - , or dsRed - expressing gram - negative 60 min after addition of 5 µM Alexa - Fluor647 - or Alexa - Fluor488 - hGBP1F 2 mM GTP in presence absence of 100 µM LPS - O55 : B5 . After 60 min hGBP1F - enclosed gram - negative were quantified . Combined data from three independent experiments are shown as mean ± . Significance was determined by two - way ANOVA with Tukey ' s multiple comparison . * * * * , P ≤ 0 . 0001 .

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(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.


( B ) Time - lapse microscopy of fixed GFP + co - isogenic S . flexneri wild type rfaL mutant after adding 10 µM Alexa - Fluor647 - hGBP1F 2 mM GTP . bound by hGBP1F at 5 min hGBP1F - enclosed after 60 min were quantified . Combined data from three independent experiments are shown as mean ± . Significance was determined by unpaired t - , two - . n . s . , not significant ; * * * * , P ≤ 0 . 0001 .

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(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 of live wild type rfaL mutant strains incubated with no or with 5 µM of hGBP1F in presence of 2 mM GTP for 4 min . Arrowheads point to unattached hGBP1 polymers , arrows point to hGBP1 polymers attached to , asterisks mark polymeric structures that appear to fuse with

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(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 of formaldehyde fixed , co - isogenic GFP + wild type mutant S . flexneri strains harboring rfb regions of E . coli serotypes O8 O25 after 60 min of incubation time in presence of 10 µM Alexa - Fluor647 - hGBP1F 2 mM GTP .

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(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.


( A ) Following incubation for 1 . 5 h with buffer in absence or presence of 10 µM hGBP1F 5 mM GTP , live were treated with ( 2 . 5 µg / ml PMB or 5 mg / ml lysozyme ) for 30 min or left untreated . number of viable was subsequently determined by colony forming unit ( CFU ) . Graphs show mean CFUs ± of combined data from at least three independent experiments . Significance was determined by two - way ANOVA with Tukey ' s multiple comparison . n . s . , not significant ; * * * * , P ≤ 0 . 0001 .

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(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 ± of combined data from three independent experiments . Significance was determined by unpaired t - , two - . * * * * , P ≤ 0 . 0001 .

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(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 2 mM GTP . Samples were fixed after 60 min incubation stained with an antibody against inner / lipid A fragment of E . coli LPS ( anti - LPS ) . frequency of anti - LPS stained UPEC was quantified . Graphs show mean ± of combined data from three independent experiments . Significance was determined by two - way ANOVA with Tukey ' s multiple comparison . n . s . , not significant ; * * * * , P ≤ 0 . 0001 .

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(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 hGBP1 - KO HeLa 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 lacking Caspase - 4 antagonist OspC3 as well as hGBP1 antagonist IpaH9 . 8 . were fixed at 4 hpi ( ST ΔsifA ) or at 2 hpi ( Sf ΔospC3ΔipaH9 . 8 ) stained for endogenous hGBP1 . YFP - Caspase - 4C258S associated were quantified mean ± of combined data from three independent experiments are shown . Significance was determined by two - way ANOVA with Tukey ' s multiple comparison . * * * * , P ≤ 0 . 0001 .

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(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.


( B were infected with poly - D - lysine treated GFP + S . flexneri strains at an MOI of 6 . were stained for indicated Z - stacks were recorded using confocal microscopy . Actin tails were classified as tails when ≥ 2 . 5 µm . graphs show mean values ± of combined data from three independent experiments . scale bars are 5 µm . Arrows point to associated with indicated , arrowheads point to lacking indicated , asterisks mark lacking unipolar IcsA localization . ( B ) Unprimed HeLa were infected with wild type or rfaL S . flexneri . Total unipolar localization of IcsA as well as actin tail formation were quantified at 1 hpi . Significance was determined by unpaired two - t - . n . s . , not significant ; * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


were infected with poly - D - lysine treated GFP + S . flexneri strains at an MOI of 6 . were stained for indicated Z - stacks were recorded using confocal microscopy . Actin tails were classified as tails when ≥ 2 . 5 µm . graphs show mean values ± of combined data from three independent experiments . scale bars are 5 µm . Arrows point to associated with indicated , arrowheads point to lacking indicated , asterisks mark lacking unipolar IcsA localization . ( C - mCherry - hGBP1 was expressed in HeLa hGBP1 - KO infected with S . flexneri ΔipaH9 . 8 stained for IcsA , N - WASP Significance was determined by unpaired t - , two - . n . s . , not significant ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ; * * * * , P ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.