D - E ' ' Dissipation of the plasma membrane potential upon treatment of wing discs with gramicidin A ( gA ) . ( D ) Quantification of DiBAC4 ( 3 ) signal intensity in mock - treated ( - gA , n = 7 ) and gA - treated ( + gA , n = 5 ) wing discs . ( D ' , D ' ' ) Representative images of DiBAC4 ( 3 ) assay in mock - ( D ' ) and gA - treated ( D ' ' ) wing discs . Next to the maximal projections are shown sum projections of cross - sections along the A / P axis . ( E ) Quantification of PAC - NAE signal intensity in mock - ( n = 7 ) and gA - treated ( n = 9 ) wing discs , shown as fold change from control ( mock - treated ) . ( E ' - E ' ' ) Representative images of PAC - NAE uptake in mock - ( E ' ) and gA - treated ( E ' ' ) wing discs . GA treatment strongly reduces the uptake of PAC - NAE . Error bars indicate ± SD . t - test , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . Scale bars = 50 μm .

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

D-E'' Dissipation of the plasma membrane potential upon treatment of wing discs with gramicidin A (gA). (D) Quantification of DiBAC4(3) signal intensity in mock-treated (- gA, n=7) and gA-treated (+ gA, n=5) wing discs. (D', D'') Representative images of DiBAC4(3) assay in mock- (D') and gA-treated (D'') wing discs. Next to the maximal projections are shown sum projections of cross-sections along the A/P axis. (E) Quantification of PAC-NAE signal intensity in mock- (n=7) and gA-treated (n=9) wing discs, shown as fold change from control (mock-treated). (E'-E'') Representative images of PAC-NAE uptake in mock- (E') and gA-treated (E'') wing discs. GA treatment strongly reduces the uptake of PAC-NAE. Error bars indicate ± SD. t-test, *p≤0.05, ***p≤0.001. Scale bars= 50 μm.


( B ) Example of bead survival distributions for the 1G4 TCR binding NYE 9V . The survival probability at 1 s is shown as a dotted vertical line and is used to calculate the off - rate under force : − ln ( survival at 1 s ) / 1s . ( C ) Off - rates under force obtained from ( B ) are fitted with Bell ' s model ( solid line ) . Data information : The number of independent experiments performed on different days that were combined to produce the estimated off - rates are : 7 ( 1G4 / 9V )

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(B) Example of bead survival distributions for the 1G4 TCR binding NYE 9V. The survival probability at 1 s is shown as a dotted vertical line and is used to calculate the off-rate under force: −ln(survival at 1 s)/1s. (C) Off-rates under force obtained from (B) are fitted with Bell's model (solid line). Data information: The number of independent experiments performed on different days that were combined to produce the estimated off-rates are: 7 (1G4/9V)


( D ) Off - rates under force for additional pMHCs interacting with the 1G4 TCR fitted to Bell ' s model ( solid line ) . ( E ) Correlation of the dissociation constant ( KD ) measured previously by SPR and the extrapolated zero - force flow chamber off - rate ( k0off ) from fitting Bell ' s model for the 1G4 TCR . ( F ) Correlation of the SPR dissociation constant ( KD ) with the fitted force sensitivity parameter ( xβ ) for the 1G4 TCR . Data information : Error bars ( SE ) in E , F , are obtained from the fit . The number of independent experiments performed on different days that were combined to produce the estimated off - rates are : 7 ( 1G4 / 9V ) , 8 ( 1G4 / 6V ) , 8 ( 1G4 / 4E5E8E ) , 10 ( 1G4 / 3Y ) , 6 ( 1G4 / 4D )

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(D) Off-rates under force for additional pMHCs interacting with the 1G4 TCR fitted to Bell's model (solid line). (E) Correlation of the dissociation constant (KD) measured previously by SPR and the extrapolated zero-force flow chamber off-rate (k0off) from fitting Bell's model for the 1G4 TCR. (F) Correlation of the SPR dissociation constant (KD) with the fitted force sensitivity parameter (xβ) for the 1G4 TCR. Data information: Error bars (SE) in E,F, are obtained from the fit. The number of independent experiments performed on different days that were combined to produce the estimated off-rates are: 7 (1G4/9V), 8 (1G4/6V), 8 (1G4/4E5E8E), 10 (1G4/3Y), 6 (1G4/4D)


( G ) Off - rates under force for pMHCs interacting with the A6 TCR fitted to Bell ' s model ( solid line ) . ( H ) Correlation of the dissociation constant ( KD ) measured previously by SPR and the extrapolated zero - force flow chamber off - rate ( k0off ) from fitting Bell ' s model for the A6 TCR . ( I ) Correlation of the SPR dissociation constant ( KD ) with the fitted force sensitivity parameter ( xβ ) for the A6 TCR . Data information : Error bars ( SE ) in H , I are obtained from the fit . The number of independent experiments performed on different days that were combined to produce the estimated off - rates are : 9 ( A6 / Tax WT ) , 9 ( A6 / 7Q ) , 8 ( A6 / 7R ) , 8 ( A6 / 5H ) .

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(G) Off-rates under force for pMHCs interacting with the A6 TCR fitted to Bell's model (solid line). (H) Correlation of the dissociation constant (KD) measured previously by SPR and the extrapolated zero-force flow chamber off-rate (k0off) from fitting Bell's model for the A6 TCR. (I) Correlation of the SPR dissociation constant (KD) with the fitted force sensitivity parameter (xβ) for the A6 TCR. Data information: Error bars (SE) in H,I are obtained from the fit. The number of independent experiments performed on different days that were combined to produce the estimated off-rates are: 9 (A6/Tax WT), 9 (A6/7Q), 8 (A6/7R), 8 (A6/5H).


( A ) The response force ( F ) on the TCR / pMHC interaction when pulled at acting on the spring attached to the C - terminus of the MHC which is being pulled with constant velocity ( ν ) . A second spring is attached to the C - terminus of the TCRβ and fixed in space . Insets : the TCR is shown in grey , the MHC in blue , and the peptide in red . The sites where the springs are attached are marked in orange . The direction of pulling is marked by a dashed line .

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(A) The response force (F) on the TCR/pMHC interaction when pulled at acting on the spring attached to the C-terminus of the MHC which is being pulled with constant velocity (ν). A second spring is attached to the C-terminus of the TCRβ and fixed in space. Insets: the TCR is shown in grey, the MHC in blue, and the peptide in red. The sites where the springs are attached are marked in orange. The direction of pulling is marked by a dashed line.


( B ) The concentration of productively signalling TCRs ( CN ) over the antigen concentration for antigens with the indicated zero - force off - rates ( koff ) . Shown is the effect of forces at 100 pN ( left ) and 10 pN ( right ) . The horizontal dashed line is the threshold value of CN required for activation in the model ( λ ) . The vertical dashed line shows the antigen concentration ( P ) required to elicit activation by the antigen with the largest value of koff .

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(B) The concentration of productively signalling TCRs (CN) over the antigen concentration for antigens with the indicated zero-force off-rates (koff). Shown is the effect of forces at 100 pN (left) and 10 pN (right). The horizontal dashed line is the threshold value of CN required for activation in the model (λ). The vertical dashed line shows the antigen concentration (P) required to elicit activation by the antigen with the largest value of koff.


( C ) The antigen concentration required to elicit activation ( P ) over koff for the indicated molecular forces on the TCR / pMHC interaction . ( D ) Antigen sensitivity quantified as the value of P from ( C ) over the applied force for a higher - affinity antigen with koff = 0 . 5 s - 1 . ( E ) Antigen discrimination quantified by the discrimination power ( α ) over the applied force . Discrimination power is defined as the ratio of the logarithm of the fold - change in P over the fold - change in koff for a higher - affinity ligand ( koff = 0 . 5 s - 1 ) and a lower - affinity ligand ( koff = 101 . 5 s - 1 ) from panel ( C ) .

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(C) The antigen concentration required to elicit activation (P) over koff for the indicated molecular forces on the TCR/pMHC interaction. (D) Antigen sensitivity quantified as the value of P from (C) over the applied force for a higher-affinity antigen with koff = 0.5 s-1. (E) Antigen discrimination quantified by the discrimination power (α) over the applied force. Discrimination power is defined as the ratio of the logarithm of the fold-change in P over the fold-change in koff for a higher-affinity ligand (koff = 0.5 s-1) and a lower-affinity ligand (koff = 101.5 s-1) from panel (C).


( F ) Experimental value of P ( symbols ) over antigen affinity fitted by the kinetic proofreading model with force ( dashed and solid lines ) . The value of P is defined as the concentration of pMHC required for 15 % upregulation of CD69 above baseline and is shown as the mean with SEM from 4 independent experiments . ( G ) The fitted value of the applied force with SEM . ( H ) The fitted value of λ with SEM . Data information : An F - test is used to produce a p - value for the null hypothesis that the applied force or λ is the same across the three conditions . All data fitting ( F - H ) used N = 2 . 46 and kp = 2 . 15 s − 1 ( average of CD58 and ICAM - 1 conditions )

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(F) Experimental value of P (symbols) over antigen affinity fitted by the kinetic proofreading model with force (dashed and solid lines). The value of P is defined as the concentration of pMHC required for 15% upregulation of CD69 above baseline and is shown as the mean with SEM from 4 independent experiments. (G) The fitted value of the applied force with SEM. (H) The fitted value of λ with SEM. Data information: An F-test is used to produce a p-value for the null hypothesis that the applied force or λ is the same across the three conditions. All data fitting (F-H) used N = 2.46 and kp = 2.15 s−1 (average of CD58 and ICAM-1 conditions) taken


B : Silver nitrate staining of proteins pulled - down using the control peptide ( left ) or the FFAT peptide ( right ) , after SDS - PAGE . The two major differential bands are highlighted by

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B: Silver nitrate staining of proteins pulled-down using the control peptide (left) or the FFAT peptide (right), after SDS-PAGE. The two major differential bands are highlighted by arrows


C : Tandem mass spectrometry result table showing the three top scored proteins identified in the FFAT peptide bound fraction . Score : protein score based on the sum of the ion scores of all peptides identified ; Cov . ; percentage of the protein sequence covered by identified peptides ; Pep . : number of unique peptide sequences identified

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C: Tandem mass spectrometry result table showing the three top scored proteins identified in the FFAT peptide bound fraction. Score: protein score based on the sum of the ion scores of all peptides identified; Cov.; percentage of the protein sequence covered by identified peptides; Pep.: number of unique peptide sequences identified


E : Western blot analysis of proteins pulled - down using the control ( Ctrl ) peptide ( left ) or the FFAT ( FFAT ) peptide ( right ) . The Input , FT ( flow - through ) , and Elution fractions correspond to HeLa cell total protein extract , unbound proteins and bound proteins , respectively . Representative illustration of at least two independent experiments

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E: Western blot analysis of proteins pulled-down using the control (Ctrl) peptide (left) or the FFAT (FFAT) peptide (right). The Input, FT (flow-through), and Elution fractions correspond to HeLa cell total protein extract, unbound proteins and bound proteins, respectively. Representative illustration of at least two independent experiments


A : List of the 5 top scored proteins interacting with MOSPD2 and known to contain a FFAT motif ( see Appendix Table S1 for the full list ) . Proteins co - precipitated with GFP - MOSPD2 and GFP - MOSPD2 RD / LD proteins were identified by mass spectrometry ; proteins were filtered for their ability to bind MOSPD2 and not the RD / LD mutant . The sub - cellular localization of the proteins is indicated [ 53 , 65 - 69 ]

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A: List of the 5 top scored proteins interacting with MOSPD2 and known to contain a FFAT motif (see Appendix Table S1 for the full list). Proteins co-precipitated with GFP-MOSPD2 and GFP-MOSPD2 RD/LD proteins were identified by mass spectrometry; proteins were filtered for their ability to bind MOSPD2 and not the RD/LD mutant. The sub-cellular localization of the proteins is indicated [53,65-69]


B - F : Immunoprecipitation ( GFP - Trap ) experiments between GFP - MOSPD2 ( WT and RD / LD mutant ) and Flag - tagged STARD3 ( B ) Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti - Flag ( B , C - E ) anti - GFP ( B - F ) , and anti - actin ( B - F ) antibodies . Immunoprecipitated material was analyzed using anti - Flag ( B , D - F and anti - GFP ( B - F )

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B-F: Immunoprecipitation (GFP-Trap) experiments between GFP-MOSPD2 (WT and RD/LD mutant) and Flag-tagged STARD3 (B) Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti-Flag (B, C-E) anti-GFP (B-F), and anti-actin (B-F) antibodies. Immunoprecipitated material was analyzed using anti-Flag (B, D-F and anti-GFP (B-F) antibodies


B - F : Immunoprecipitation ( GFP - Trap ) experiments between GFP - MOSPD2 ( WT and RD / LD mutant ) and Flag - tagge STARD3NL ( C ) Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti - Flag ( B , C - E anti - GFP ( B - F ) , and anti - actin ( B - F ) antibodies . Immunoprecipitated material was analyzed usin anti - ORP1 ( C ) , and anti - GFP ( B - F )

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B-F: Immunoprecipitation (GFP-Trap) experiments between GFP-MOSPD2 (WT and RD/LD mutant) and Flag-tagge STARD3NL (C) Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti-Flag (B, C-E anti-GFP (B-F), and anti-actin (B-F) antibodies. Immunoprecipitated material was analyzed usin anti-ORP1 (C), and anti-GFP (B-F) antibodies


B - F : Immunoprecipitation ( GFP - Trap ) experiments between GFP - MOSPD2 ( WT and RD / LD mutant ) and Flag - tagge ORP1L ( D Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti - Flag ( B , C - E ) , anti - ORP1 ( D ) ) , anti - GFP ( B - F ) , and anti - actin ( B - F ) antibodies . Immunoprecipitated material was analyzed using anti - Flag ( B , D - F ) , and anti - GFP ( B - F )

B-F: Immunoprecipitation (GFP-Trap) experiments between GFP-MOSPD2 (WT and RD/LD mutant) and Flag-tagge ORP1L (D Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti-Flag (B, C-E), anti-ORP1 (D) ), anti-GFP (B-F), and anti-actin (B-F) antibodies. Immunoprecipitated material was analyzed using anti-Flag (B, D-F ), and anti-GFP (B-F) antibodies


B - F : Immunoprecipitation ( GFP - Trap ) experiments between GFP - MOSPD2 ( WT and RD / LD mutant ) and Flag - tagge STARD11 ( E Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti - Flag ( B , C - E anti - GFP ( B - F ) , and anti - actin ( B - F ) antibodies Immunoprecipitated material was analyzed using anti - Flag ( B , D - F and anti - GFP ( B - F )

B-F: Immunoprecipitation (GFP-Trap) experiments between GFP-MOSPD2 (WT and RD/LD mutant) and Flag-tagge STARD11 (E Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti-Flag (B, C-E anti-GFP (B-F), and anti-actin (B-F) antibodies Immunoprecipitated material was analyzed using anti-Flag (B, D-F and anti-GFP (B-F) antibodies


B - F : Immunoprecipitation ( GFP - Trap ) experiments between GFP - MOSPD2 ( WT and RD / LD mutant ) an HA - tagged PTPIP51 ( F ) ( WT and FFAT - deficient ) . Approximatively 5 µg of total protein extracts were analyzed by Western blot usin anti - HA ( F ) anti - GFP ( B - F ) , and anti - actin ( B - F ) antibodies . Immunoprecipitated material was analyzed using anti - Flag ( B , D - F ) , and anti - GFP ( B - F )

B-F: Immunoprecipitation (GFP-Trap) experiments between GFP-MOSPD2 (WT and RD/LD mutant) an HA-tagged PTPIP51 (F) (WT and FFAT-deficient). Approximatively 5 µg of total protein extracts were analyzed by Western blot usin anti-HA (F) anti-GFP (B-F), and anti-actin (B-F) antibodies. Immunoprecipitated material was analyzed using anti-Flag (B, D-F ), and anti-GFP (B-F) antibodies


G : Immunoprecipitation ( anti - Flag ) experiment between Flag - tagged STARD3NL ( WT and FFAT - deficient ) and endogenous MOSPD2 . Proteins extracts and immunoprecipitated material were analyzed by Western blot using anti - MOSPD2 , anti - STARD3NL and anti - actin antibodies

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G: Immunoprecipitation (anti-Flag) experiment between Flag-tagged STARD3NL (WT and FFAT-deficient) and endogenous MOSPD2. Proteins extracts and immunoprecipitated material were analyzed by Western blot using anti-MOSPD2, anti-STARD3NL and anti-actin antibodies


A : GFP - tagged MOSPD2 ( green ) expressing cells were labelled with an anti - MOSPD2 antibody ( magenta ) . The superposition of the green and magenta signals indicates that the anti - MOSPD2 antibody recognized efficiently MOSPD2 by immunofluorescence Data information : ( A , D - F ) : The subpanels on the right are higher magnification ( 3 . 5x ) images of the area outlined in white . The Overlay panel shows merged green and magenta images . The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co - localize are shown in white . Right : Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay Scale bars : 10

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A: GFP-tagged MOSPD2 (green) expressing cells were labelled with an anti-MOSPD2 antibody (magenta). The superposition of the green and magenta signals indicates that the anti-MOSPD2 antibody recognized efficiently MOSPD2 by immunofluorescence Data information: (A, D-F): The subpanels on the right are higher magnification (3.5x) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Right: Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay Scale bars: 10 µm


B : Western blot analysis of MOSPD2 and VAP proteins in control HeLa cells ( WT ) and in HeLa cells expressing a control shRNA ( shCtrl ) and two individual shRNAs targeting MOSPD2 ( shMOSPD2 - α or shMOSPD2 - β ) . Quantification of MOSPD2 protein level is shown below . Means and error bars ( SD ) are shown . n : three independent experiments

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B: Western blot analysis of MOSPD2 and VAP proteins in control HeLa cells (WT) and in HeLa cells expressing a control shRNA (shCtrl) and two individual shRNAs targeting MOSPD2 (shMOSPD2-α or shMOSPD2-β). Quantification of MOSPD2 protein level is shown below. Means and error bars (SD) are shown. n: three independent experiments


C : Endogenous MOSPD2 ( green ) was labelled in control HeLa cells ( WT ) , in cells expressing a control shRNA ( shCtrl ) , and in cells expressing two individual shRNAs targeting MOSPD2 ( shMOSPD2 - α or shMOSPD2 - β ) . Scale bar : 10 µm

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C: Endogenous MOSPD2 (green) was labelled in control HeLa cells (WT), in cells expressing a control shRNA (shCtrl), and in cells expressing two individual shRNAs targeting MOSPD2 (shMOSPD2-α or shMOSPD2-β). Scale bar: 10 µm


D : Endogenous MOSPD2 ( green ) staining in HeLa cells expressing the ER marker GFP - ER ( magenta ) Data information : ( A , D - F ) : The subpanels on the right are higher magnification ( 3 . 5x ) images of the area outlined in white . The Overlay panel shows merged green and magenta images . The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co - localize are shown in white . Right : Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay Scale bars : 10

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D: Endogenous MOSPD2 (green) staining in HeLa cells expressing the ER marker GFP-ER (magenta) Data information: (A, D-F): The subpanels on the right are higher magnification (3.5x) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Right: Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay Scale bars: 10 µm


E - F : Endogenous MOSPD2 ( green ) staining in HeLa cells expressing Flag - STARD3NL ( E ( anti - Flag ; magenta ) Data information : ( A , D - F ) : The subpanels on the right are higher magnification ( 3 . 5x ) images of the area outlined in white . The Overlay panel shows merged green and magenta images . The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co - localize are shown in white . Right : Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay . Black rectangles indicate the positions of late endosomes ( E ) . Scale bars : 10 µm

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E-F: Endogenous MOSPD2 (green) staining in HeLa cells expressing Flag-STARD3NL (E (anti-Flag; magenta) Data information: (A, D-F): The subpanels on the right are higher magnification (3.5x) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Right: Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay. Black rectangles indicate the positions of late endosomes (E). Scale bars: 10 µm


E - F : Endogenous MOSPD2 ( green ) staining in HeLa cells expressin Flag - STARD3NL ∆ FFAT ( F ) ( anti - Flag ; magenta ) Data information : ( A , D - F ) : The subpanels on the right are higher magnification ( 3 . 5x ) images of the area outlined in white . The Overlay panel shows merged green and magenta images . The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co - localize are shown in white . Right : Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay Scale bars : 10

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E-F: Endogenous MOSPD2 (green) staining in HeLa cells expressin Flag-STARD3NL ∆FFAT (F) (anti-Flag; magenta) Data information: (A, D-F): The subpanels on the right are higher magnification (3.5x) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Right: Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay Scale bars: 10 µm


G : Pearson correlation coefficients between endogenous MOSPD2 and Flag - STARD3NL ( WT or ΔFFAT ) staining are shown . Each dot represents a single cell ( number of cells : MOSPD2 - Flag - STARD3NL : 27 ; MOSPD2 - Flag - STARD3NL ΔFFAT : 26 ; from three independent experiments ) . Means and error bars ( SD ) are shown . Mann - Whitney test ( two - tailed P value ; * * * * : P < 0 , 0001 )

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G: Pearson correlation coefficients between endogenous MOSPD2 and Flag-STARD3NL (WT or ΔFFAT) staining are shown. Each dot represents a single cell (number of cells: MOSPD2-Flag-STARD3NL: 27; MOSPD2-Flag-STARD3NL ΔFFAT: 26; from three independent experiments). Means and error bars (SD) are shown. Mann-Whitney test (two-tailed P value; ****: P< 0,0001)


A : Western blot analysis of MOSPD2 , VAP - A and VAP - B proteins in control HeLa cells ( WT ) and in HeLa cells transfected with a control siRNA ( siCtrl ) , and with siRNA targeting MOSPD2 ( siMOSPD2 ) , VAP - A and VAP - B ( siVAP - A / VAP - B ) , and MOSPD2 , VAP - A and VAP - B ( siMOSPD2 / VAP - A / VAP - B )

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A: Western blot analysis of MOSPD2, VAP-A and VAP-B proteins in control HeLa cells (WT) and in HeLa cells transfected with a control siRNA (siCtrl), and with siRNA targeting MOSPD2 (siMOSPD2), VAP-A and VAP-B (siVAP-A/VAP-B), and MOSPD2, VAP-A and VAP-B (siMOSPD2/VAP-A/VAP-B)


B - F : TEM images of control HeLa cell ( B : HeLa - silenced HeLa cells . An interpretation scheme representing contacts between organelles is shown on the right ; the ER , endosomes and ILV are in dark , light and medium gray , respectively . Mitochondria and Golgi are in pink and light green , respectively . Scale bars : 500 µm

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B-F: TEM images of control HeLa cell (B: HeLa -silenced HeLa cells. An interpretation scheme representing contacts between organelles is shown on the right; the ER, endosomes and ILV are in dark, light and medium gray, respectively. Mitochondria and Golgi are in pink and light green, respectively. Scale bars: 500 µm


B - F : TEM images of control HeLa cell C : HeLa / siCtrl - silenced HeLa cells . An interpretation scheme representing contacts between organelles is shown on the right ; the ER , endosomes and ILV are in dark , light and medium gray , respectively . Mitochondria and Golgi are in pink and light green , respectively . Scale bars : 500 µm

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F: TEM images of control HeLa cell C: HeLa/siCtrl -silenced HeLa cells. An interpretation scheme representing contacts between organelles is shown on the right; the ER, endosomes and ILV are in dark, light and medium gray, respectively. Mitochondria and Golgi are in pink and light green, respectively. Scale bars: 500 µm


B - F : TEM images o MOSPD2 ( D : siMOSPD2 - silenced HeLa cells . An interpretation scheme representing contacts between organelles is shown on the right ; the ER , endosomes and ILV are in dark , light and medium gray , respectively . Mitochondria and Golgi are in pink and light green , respectively . Scale bars : 500 µm

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F: TEM images o MOSPD2 (D: siMOSPD2 -silenced HeLa cells. An interpretation scheme representing contacts between organelles is shown on the right; the ER, endosomes and ILV are in dark, light and medium gray, respectively. Mitochondria and Golgi are in pink and light green, respectively. Scale bars: 500 µm


B - F : TEM images o VAP - A and VAP - B ( E : siVAP - A / VAP - B - silenced HeLa cells . An interpretation scheme representing contacts between organelles is shown on the right ; the ER , endosomes and ILV are in dark , light and medium gray , respectively . Mitochondria and Golgi are in pink and light green , respectively . Scale bars : 500 µm

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F: TEM images o VAP-A and VAP-B (E: siVAP-A/VAP-B -silenced HeLa cells. An interpretation scheme representing contacts between organelles is shown on the right; the ER, endosomes and ILV are in dark, light and medium gray, respectively. Mitochondria and Golgi are in pink and light green, respectively. Scale bars: 500 µm


B - F : TEM images o MOSPD2 , VAP - A and VAP - B ( F : siVAP - A / VAP - B / MOSPD2 ) - silenced HeLa cells . An interpretation scheme representing contacts between organelles is shown on the right ; the ER , endosomes and ILV are in dark , light and medium gray , respectively . Mitochondria and Golgi are in pink and light green , respectively . Scale bars : 500 µm

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F: TEM images o MOSPD2, VAP-A and VAP-B (F: siVAP-A/VAP-B/MOSPD2) -silenced HeLa cells. An interpretation scheme representing contacts between organelles is shown on the right; the ER, endosomes and ILV are in dark, light and medium gray, respectively. Mitochondria and Golgi are in pink and light green, respectively. Scale bars: 500 µm


G - I : Quantification by stereology of ER - mitochondria ( G contacts . The percentages of mitochondria perimeter in contact with the ER ( G are shown as means and error bars ( SEM ) . ( G ) 200 ( HeLa ) , 208 ( siCtrl ) , 199 ( siMOSPD2 ) , 202 ( siVAP - A / VAP - B ) , 199 ( siMOSPD2 / VAP - A / VAP - B ) mitochondria from 8 , 10 , 8 , 12 and 12 cells , respectively , were

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

G-I: Quantification by stereology of ER-mitochondria (G contacts. The percentages of mitochondria perimeter in contact with the ER (G are shown as means and error bars (SEM). (G) 200 (HeLa), 208 (siCtrl), 199 (siMOSPD2), 202 (siVAP-A/VAP-B), 199 (siMOSPD2/VAP-A/VAP-B) mitochondria from 8, 10, 8, 12 and 12 cells, respectively, were analyzed


G - I : Quantification by stereology o ER - endosome ( H contacts The percentages o ) , endosome perimeter in contact with the ER ( H are shown as means and error bars ( SEM ) ( H , I ) 202 ( HeLa ) , 189 ( siCtrl ) , 148 ( siMOSPD2 ) , 141 ( siVAP - A / VAP - B ) , 221 ( siMOSPD2 / VAP - A / VAP - B ) endosomes from 9 , 19 , 10 , 14 and 12 cells , respectively , were analyzed . Kruskal - Wallis with Dunn " s multiple comparison test ( * : P - values < 0 . 05 ; * * * : 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G-I: Quantification by stereology o ER-endosome (H contacts The percentages o ), endosome perimeter in contact with the ER (H are shown as means and error bars (SEM) (H, I) 202 (HeLa), 189 (siCtrl), 148 (siMOSPD2), 141 (siVAP-A/VAP-B), 221 (siMOSPD2/VAP-A/VAP-B) endosomes from 9, 19, 10, 14 and 12 cells, respectively, were analyzed. Kruskal-Wallis with Dunn"s multiple comparison test (*: P-values <0.05; ***: P< 0,001; ****: P <0.0001)


G - I : Quantification by stereology o endosome - endosome ( I ) contacts . The percentages o endosome perimeter in contact with an endosome ( I ) are shown as means and error bars ( SEM ) ( H , I ) 202 ( HeLa ) , 189 ( siCtrl ) , 148 ( siMOSPD2 ) , 141 ( siVAP - A / VAP - B ) , 221 ( siMOSPD2 / VAP - A / VAP - B ) endosomes from 9 , 19 , 10 , 14 and 12 cells , respectively , were analyzed . Kruskal - Wallis with Dunn " s multiple comparison test ( * : P - values < 0 . 05 ; * * * : 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G-I: Quantification by stereology o endosome-endosome (I) contacts. The percentages o endosome perimeter in contact with an endosome (I) are shown as means and error bars (SEM) (H, I) 202 (HeLa), 189 (siCtrl), 148 (siMOSPD2), 141 (siVAP-A/VAP-B), 221 (siMOSPD2/VAP-A/VAP-B) endosomes from 9, 19, 10, 14 and 12 cells, respectively, were analyzed. Kruskal-Wallis with Dunn"s multiple comparison test (*: P-values <0.05; ***: P< 0,001; ****: P <0.0001)


Negative stain TEM image of B . cereus NVH 0075 - 95 endospore , showing spore body ( SB ) , exosporium ( E ) , and endospore appendages ( Ena ) , which emerge from the endospore individually or as fiber bundles . At the distal end , Enas terminate in a single or multiple thin ruffles ( R ) .

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Negative stain TEM image of B. cereus NVH 0075-95 endospore, showing spore body (SB), exosporium (E), and endospore appendages (Ena), which emerge from the endospore individually or as fiber bundles. At the distal end, Enas terminate in a single or multiple thin ruffles (R).


cryoEM images ( left ) and negative stain 2D class averages of single S - Ena ( E ) and L - Ena ( F ) fibers . S - Ena show ~ 100 Å diameter and a helical or staggered appearance with a ~ 37 Å rise , whereas L - Ena have a ~ 80 Å diameter and appear as stacked disks of ~ 40 Å height and ~ 60 Å translation along the fiber . L - and S - Ena show , respectively , a single or multiple ruffle ( s ) ( black arrow , labeled ' R ' ) at the distal end of fiber .

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cryoEM images (left) and negative stain 2D class averages of single S-Ena (E) and L-Ena (F) fibers. S-Ena show ~100 Å diameter and a helical or staggered appearance with a ~37 Å rise, whereas L-Ena have a ~80 Å diameter and appear as stacked disks of ~40 Å height and ~60 Å translation along the fiber. L- and S-Ena show, respectively, a single or multiple ruffle(s) (black arrow, labeled 'R') at the distal end of fiber.


SDS - PAGE of recEna1B , treated with β - mercaptoethanol or TEV protease ( to remove N - terminal 6xHis tag ) as indicated . Bands with apparent MW of ~ 13 and ~ 15 kDa correspond recEna1B monomer with ( Ena1B6His ) and without ( Ena1BC ) the 6xHis tag and TEV recognition site , respectively . In the uncleaved sample , a band running at ~ 30 kDa ( labeled * ) corresponds to a non - physiological disulfide bound S - Ena1B dimer . Lower intensity in the cleaved recEna1B results from a loss of monomers to SDS - resistant high molecular weight complexes stuck in the stacking gel .

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

SDS-PAGE of recEna1B, treated with β-mercaptoethanol or TEV protease (to remove N-terminal 6xHis tag) as indicated. Bands with apparent MW of ~13 and ~15 kDa correspond recEna1B monomer with (Ena1B6His) and without (Ena1BC) the 6xHis tag and TEV recognition site, respectively. In the uncleaved sample, a band running at ~ 30 kDa (labeled *) corresponds to a non-physiological disulfide bound S-Ena1B dimer. Lower intensity in the cleaved recEna1B results from a loss of monomers to SDS-resistant high molecular weight complexes stuck in the stacking gel.


Negative stain TEM images of rec1Ena1B oligomers formed after refolding , but prior to TEV removal of the N - terminal 6xHis tag . Close - up view that shows recEna1B oligomers form open crescents similar in dimensions and shape to single helical turns or arcs found in the S - Ena fiber ( model - right ) . Steric hindrance by the 6xHis is thought to arrest recEna1B polymerization into single helical arcs .

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Negative stain TEM images of rec1Ena1B oligomers formed after refolding, but prior to TEV removal of the N-terminal 6xHis tag. Close-up view that shows recEna1B oligomers form open crescents similar in dimensions and shape to single helical turns or arcs found in the S-Ena fiber (model - right). Steric hindrance by the 6xHis is thought to arrest recEna1B polymerization into single helical arcs.


Transcription level of ena1A ( x ) , ena1B ( ▲ ) , ena1C ( ○ ) and dedA ( ♦ ) relative to rpoB determined by RT - qPCR during 16 hours of growth of B . cereus strain NVH 0075 - 95 . The dotted line represents the bacterial growth measured by increase in OD600 . Of note , the transcription of ena1C was surprisingly higher than ena1A and ena1B , the major components of the isolated S - Ena ( Fig EV2 ) .

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Transcription level of ena1A (x), ena1B (▲), ena1C (○) and dedA (♦) relative to rpoB determined by RT-qPCR during 16 hours of growth of B. cereus strain NVH 0075-95. The dotted line represents the bacterial growth measured by increase in OD600. Of note, the transcription of ena1C was surprisingly higher than ena1A and ena1B, the major components of the isolated S-Ena (Fig EV2).


Representative negative stain images of endospores of NVH 0075 - 95 mutants lacking ena1A , ena1B , or ena1C , as well as endospores of strains complemented with the respective ena subunit expressed from plasmid ( i . e . pA , pAB and pC ) . The ena1B mutant was complemented with a plasmid carrying both ena1A and 1B ( pAB ) due to repeated failure to transform with a plasmid holding ena1B only . Inset are 2D class averages of Enas observed on the respective mutants . Knockout of ena1A , ena1B , or ena1C results in the loss of S - Ena , a phenotype that is restored by plasmid - based complementation .

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Representative negative stain images of endospores of NVH 0075-95 mutants lacking ena1A, ena1B, or ena1C, as well as endospores of strains complemented with the respective ena subunit expressed from plasmid (i.e. pA, pAB and pC). The ena1B mutant was complemented with a plasmid carrying both ena1A and 1B (pAB) due to repeated failure to transform with a plasmid holding ena1B only. Inset are 2D class averages of Enas observed on the respective mutants. Knockout of ena1A, ena1B, or ena1C results in the loss of S-Ena, a phenotype that is restored by plasmid-based complementation.


Number ( top ) and length ( bottom ) of Enas found on WT , mutant ( ∆ ena1A , ∆ ena1B , or ∆ ena1C ) and plasmid complemented ( ∆ ena1A : pena1A , ∆ ena1B : pena1AB , or ∆ ena1C : pena1C ) NVH 0075 - 95 endospores . Statistics : pair - wise Mann - Whitney U tests against WT ( n : ≥ 18 spores ; n : ≥ 50 Enas ; ns : not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 . - - - : mean ± s . d . )

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Number (top) and length (bottom) of Enas found on WT, mutant (∆ena1A, ∆ena1B, or ∆ena1C) and plasmid complemented (∆ena1A:pena1A, ∆ena1B:pena1AB, or ∆ena1C:pena1C) NVH 0075-95 endospores. Statistics: pair-wise Mann-Whitney U tests against WT (n: ≥18 spores; n: ≥50 Enas; ns: not significant, * p<0.05, ** p<0.01, *** p<0.001 and **** p<0.0001. ---: mean ± s.d.)


A Scatterplot representing differences in ssGSEA immune signatures in TERThigh versus TERTlow tumours ( n = 4 , 632 tumours in each group ) across TCGA . - log10 ( padj ) for enrichment of ssGSEA immune signatures in TERThigh versus TERTlow tumours is shown on the y - axis . Signatures more highly represented in TERThigh tumours are shown on the right , while those in TERTlow tumours are shown on the left .

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A Scatterplot representing differences in ssGSEA immune signatures in TERThigh versus TERTlow tumours (n = 4,632 tumours in each group) across TCGA. -log10(padj) for enrichment of ssGSEA immune signatures in TERThigh versus TERTlow tumours is shown on the y-axis. Signatures more highly represented in TERThigh tumours are shown on the right, while those in TERTlow tumours are shown on the left.


D - F RT - qPCR analysis of interferon - related genes ( D ) , western blot analysis of TERT , TBK1 , pTBK1 , IRF3 , pIRF3 , and GAPDH levels ( E ) , and ELISA of IFNβ and CXCL10 in culture supernatant ( F ) in U2OS and HeLa cells ectopically expressing TERT - WT or TERT - K626A .

D-F RT-qPCR analysis of interferon-related genes (D), western blot analysis of TERT, TBK1, pTBK1, IRF3, pIRF3, and GAPDH levels (E), and ELISA of IFNβ and CXCL10 in culture supernatant (F) in U2OS and HeLa cells ectopically expressing TERT-WT or TERT-K626A.


D RT - qPCR analysis of TA - ERVs in U2OS and HeLa cells ectopically expressing TERT - WT or TERT - K626A . E RT - qPCR analysis of TA - ERVs in HeLa and HCT116 cells with TERT knockdown .

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D RT-qPCR analysis of TA-ERVs in U2OS and HeLa cells ectopically expressing TERT-WT or TERT-K626A. E RT-qPCR analysis of TA-ERVs in HeLa and HCT116 cells with TERT knockdown. F


F - H RT - qPCR analysis of interferon - related genes and TA - ERVs ( F ) , western blot analysis of TERT , TBK1 , pTBK1 , IRF3 , pIRF3 , and GAPDH levels ( G ) , and ELISA of IFNβ and CXCL10 in culture supernatant ( H ) in U2OS cells ectopically expressing TERT - WT or TERT - K626A using Dox - inducible system . GFP was used as negative control . Relative levels of pTBK1 and pIRF3 were quantified with ImageJ software and normalized to GAPDH , as indicated at the bottom of the blot ( G ) .

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F-H RT-qPCR analysis of interferon-related genes and TA-ERVs (F), western blot analysis of TERT, TBK1, pTBK1, IRF3, pIRF3, and GAPDH levels (G), and ELISA of IFNβ and CXCL10 in culture supernatant (H) in U2OS cells ectopically expressing TERT-WT or TERT-K626A using Dox-inducible system. GFP was used as negative control. Relative levels of pTBK1 and pIRF3 were quantified with ImageJ software and normalized to GAPDH, as indicated at the bottom of the blot (G).


D MeDIP - qPCR analysis of TA - ERVs sites in HeLa cells ectopically expressing TERT - WT or TERT - K626A . Primer sets recognizing exon 8 of the human ZC3H13 gene and a gene desert on chromosome 12 were used for positive and negative controls , respectively .

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D MeDIP-qPCR analysis of TA-ERVs sites in HeLa cells ectopically expressing TERT-WT or TERT-K626A. Primer sets recognizing exon 8 of the human ZC3H13 gene and a gene desert on chromosome 12 were used for positive and negative controls, respectively.


E ChIP - qPCR for TERT and Sp1 targets on TA - ERVs in U2OS cells with TERT ectopic expression and in HeLa cells . GAPDH promoter was used as a negative control , and VEGF promoter was used as a positive control .

O O O O O O O O O O O O O O O O O O O O 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 ChIP-qPCR for TERT and Sp1 targets on TA-ERVs in U2OS cells with TERT ectopic expression and in HeLa cells. GAPDH promoter was used as a negative control, and VEGF promoter was used as a positive control.


A Expression of murine ERVs in blood of WT and G1 Tert - / - mice . n = 5 mice per group . B Expression of MMERGLN _ LTR and MMERGLN - int in livers of WT and G1 Tert - / - mice treated with ENU or saline ( control ) . n = 3 mice per group .

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A Expression of murine ERVs in blood of WT and G1 Tert-/- mice. n = 5 mice per group. B Expression of MMERGLN_LTR and MMERGLN-int in livers of WT and G1 Tert-/- mice treated with ENU or saline (control). n = 3 mice per group. C


D Heat map showing log2 fold change differences of interferon - related genes expression between ENU treatment and control ( saline ) in WT and G1 Tert - / - mice by RNA - seq . n = 3 mice per group . E Heat map showing log2 fold change differences of cytokine / chemokine between ENU treatment and control ( saline ) in WT and G1 Tert - / - mice by cytokine / chemokine array . n = 5 mice per group .

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D Heat map showing log2 fold change differences of interferon-related genes expression between ENU treatment and control (saline) in WT and G1 Tert-/- mice by RNA-seq. n = 3 mice per group. E Heat map showing log2 fold change differences of cytokine/chemokine between ENU treatment and control (saline) in WT and G1 Tert-/- mice by cytokine/chemokine array. n = 5 mice per group. F


B TCGA fragments per kilobase million ( FPKM ) values of TERT , CXCL10 , ISG15 , and OASL in tumours grouped in TERThigh ( n = 500 ) and TERTlow groups ( n = 500 ) . Data represent mean ± SD . P - values were analysed using unpaired two - tailed Student ' s t - tests .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 TCGA fragments per kilobase million (FPKM) values of TERT, CXCL10, ISG15, and OASL in tumours grouped in TERThigh (n = 500) and TERTlow groups (n = 500). Data represent mean ± SD. P-values were analysed using unpaired two-tailed Student's t-tests.


C TCGA FPKM values of interferon - related genes in tumours grouped in TERThigh ( n = 500 ) and TERTlow ( n = 500 ) groups . Heat map showing mean FPKM values of each gene .

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C TCGA FPKM values of interferon-related genes in tumours grouped in TERThigh (n = 500) and TERTlow (n = 500) groups. Heat map showing mean FPKM values of each gene.


Expression of TERT ( D ) in TERThigh and TERTlow cancer types ( 6 types in each ) . ACC ( adrenocortical carcinoma ) , n = 79 . KICH ( kidney chromophobe ) , n = 66 . KIRP ( kidney renal papillary cell carcinoma ) , n = 291 . LGG ( brain lower grade glioma ) , n = 532 . PRAD , n = 502 . THCA , n = 513 . BLCA ( bladder urothelial carcinoma ) , n = 414 . CESC ( cervical squamous cell carcinoma and endocervical adenocarcinoma ) , n = 306 . DLBC ( lymphoid neoplasm diffuse large B - cell lymphoma ) , n = 48 . LUSC ( lung squamous cell carcinoma ) , n = 502 . OV ( ovarian serous cystadenocarcinoma ) , n = 430 . All boxplots include the median line , box indicates the interquartile range ( IQR ) and whiskers denote the 1 . 5 × IQR . P - values were analysed using two - tailed Mann - Whitney U test .

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Expression of TERT (D) in TERThigh and TERTlow cancer types (6 types in each). ACC (adrenocortical carcinoma), n = 79. KICH (kidney chromophobe), n = 66. KIRP (kidney renal papillary cell carcinoma), n = 291. LGG (brain lower grade glioma), n = 532. PRAD, n = 502. THCA, n = 513. BLCA (bladder urothelial carcinoma), n = 414. CESC (cervical squamous cell carcinoma and endocervical adenocarcinoma), n = 306. DLBC (lymphoid neoplasm diffuse large B-cell lymphoma), n = 48. LUSC (lung squamous cell carcinoma), n = 502. OV (ovarian serous cystadenocarcinoma), n = 430. All boxplots include the median line, box indicates the interquartile range (IQR) and whiskers denote the 1.5 × IQR. P-values were analysed using two-tailed Mann-Whitney U test.


Expression of DDX58 , IFIH1 , CXCL10 , IFI44 , ISG15 , and OASL ( E ) in TERThigh and TERTlow cancer types ( 6 types in each ) . ACC ( adrenocortical carcinoma ) , n = 79 . KICH ( kidney chromophobe ) , n = 66 . KIRP ( kidney renal papillary cell carcinoma ) , n = 291 . LGG ( brain lower grade glioma ) , n = 532 . PRAD , n = 502 . THCA , n = 513 . BLCA ( bladder urothelial carcinoma ) , n = 414 . CESC ( cervical squamous cell carcinoma and endocervical adenocarcinoma ) , n = 306 . DLBC ( lymphoid neoplasm diffuse large B - cell lymphoma ) , n = 48 . LUSC ( lung squamous cell carcinoma ) , n = 502 . OV ( ovarian serous cystadenocarcinoma ) , n = 430 . All boxplots include the median line , box indicates the interquartile range ( IQR ) and whiskers denote the 1 . 5 × IQR . P - values were analysed using two - tailed Mann - Whitney U test .

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Expression of DDX58, IFIH1, CXCL10, IFI44, ISG15, and OASL (E) in TERThigh and TERTlow cancer types (6 types in each). ACC (adrenocortical carcinoma), n = 79. KICH (kidney chromophobe), n = 66. KIRP (kidney renal papillary cell carcinoma), n = 291. LGG (brain lower grade glioma), n = 532. PRAD, n = 502. THCA, n = 513. BLCA (bladder urothelial carcinoma), n = 414. CESC (cervical squamous cell carcinoma and endocervical adenocarcinoma), n = 306. DLBC (lymphoid neoplasm diffuse large B-cell lymphoma), n = 48. LUSC (lung squamous cell carcinoma), n = 502. OV (ovarian serous cystadenocarcinoma), n = 430. All boxplots include the median line, box indicates the interquartile range (IQR) and whiskers denote the 1.5 × IQR. P-values were analysed using two-tailed Mann-Whitney U test.


G Representative sections of immunohistochemical staining of CD4 and FOXP3 in TERT - ERVshigh and TERT - ERVslow colon tumours . Red arrowheads indicate FOXP3 + cells . Scale bar = 100 μm .

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G Representative sections of immunohistochemical staining of CD4 and FOXP3 in TERT-ERVshigh and TERT-ERVslow colon tumours. Red arrowheads indicate FOXP3+ cells. Scale bar = 100 μm.


( e ) Gene Set Enrichment Analysis ( GSEA ) of the genes differentially expressed between YAP / TAZ - deficient ( siY / T ) and control siRNA ( siCtrl ) transfected HLE cells showed an enrichment for genes involved in the regulation of lipid peroxidation .

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(e) Gene Set Enrichment Analysis (GSEA) of the genes differentially expressed between YAP/TAZ-deficient (siY/T) and control siRNA (siCtrl) transfected HLE cells showed an enrichment for genes involved in the regulation of lipid peroxidation.


( g ) Basal lipid peroxidation levels increased with the loss of function of YAP / TAZ . HLE - shLuc and HLE - shY / T cells were stained with C11 - BODIPY 581 / 591 . Reduced - Bodipy was measured by flow cytometry using a 488 nm laser , and oxidized - Bodipy was measured with a 561 nm laser . A significant shift of oxidized - Bodipy occurred upon depletion of YAP / TAZ . Results represent three independent experiments .

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(g) Basal lipid peroxidation levels increased with the loss of function of YAP/TAZ. HLE-shLuc and HLE-shY/T cells were stained with C11-BODIPY 581/591. Reduced-Bodipy was measured by flow cytometry using a 488 nm laser, and oxidized-Bodipy was measured with a 561 nm laser. A significant shift of oxidized-Bodipy occurred upon depletion of YAP/TAZ. Results represent three independent experiments.


( b ) Quantitative RT - PCR analysis confirmed the dependency of SLC7A11 gene expression on YAP / TAZ . HLE cells were transfected with control siRNA ( siCtrl ) or siRNA against YAP / TAZ ( siY / T ) and cultured with DMSO or 6μM Sorafenib for 18 hours . RNA was extracted and analyzed by quantitative RT - PCR . Data are shown as mean ± standard deviation ( SD ) . Statistical significance was calculated using one - way ANOVA . Results represent three independent experiments .

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(b) Quantitative RT-PCR analysis confirmed the dependency of SLC7A11 gene expression on YAP/TAZ. HLE cells were transfected with control siRNA (siCtrl) or siRNA against YAP/TAZ (siY/T) and cultured with DMSO or 6μM Sorafenib for 18 hours. RNA was extracted and analyzed by quantitative RT-PCR. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one-way ANOVA. Results represent three independent experiments.


( f ) Binding of YAP and TAZ to a DNA fragment containing the TEAD binding motif in the SLC7A11 promoter . ChIP was performed on HLE cell lysate with antibodies against YAP and TAZ and rabbit IgG as control . DNA fragments were amplified using the primers specific for TEAD binding motif in the SLC7A11 promoter region shown in ( e ) . The non - coding region NC10 served as negative control , and the bona fide TEAD target gene CYR61 served as positive control . Data are shown as mean ± standard deviation ( SD ) . Statistical significance was calculated using one - way ANOVA . Results represent three independent experiments .

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(f) Binding of YAP and TAZ to a DNA fragment containing the TEAD binding motif in the SLC7A11 promoter. ChIP was performed on HLE cell lysate with antibodies against YAP and TAZ and rabbit IgG as control. DNA fragments were amplified using the primers specific for TEAD binding motif in the SLC7A11 promoter region shown in (e). The non-coding region NC10 served as negative control, and the bona fide TEAD target gene CYR61 served as positive control. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one-way ANOVA. Results represent three independent experiments.


( i ) Representative images of immuno - histochemical staining of SLC7A11 and YAP proteins in HCC samples from patients . Scale bars , 50μm . ( j ) Quantification of the immunohistochemical stainings shown in ( i ) revealed a positive correlation of YAP and SLC7A11 expression ( N = 10 ) . Statistical significance was calculated using Pearson correlation analysis .

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(i) Representative images of immuno-histochemical staining of SLC7A11 and YAP proteins in HCC samples from patients. Scale bars, 50μm. (j) Quantification of the immunohistochemical stainings shown in (i) revealed a positive correlation of YAP and SLC7A11 expression (N=10). Statistical significance was calculated using Pearson correlation analysis.


( a ) SLC7A11 protein levels decreased upon siRNA - mediated depletion of ATF4 but not of NRF2 either with or without Sorafenib treatment , and Sorafenib promoted the expression of ATF4 . HLE cells were transfected with siCtrl , siATF4 or siNRF2 and treated with or without 6μM Sorafenib for 18 hours . The expression of SLC7A11 , ATF4 and NRF2 was determined by immunoblotting . GAPDH served as loading control . Results represent three independent experiments .

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(a) SLC7A11 protein levels decreased upon siRNA-mediated depletion of ATF4 but not of NRF2 either with or without Sorafenib treatment, and Sorafenib promoted the expression of ATF4. HLE cells were transfected with siCtrl, siATF4 or siNRF2 and treated with or without 6μM Sorafenib for 18 hours. The expression of SLC7A11, ATF4 and NRF2 was determined by immunoblotting. GAPDH served as loading control. Results represent three independent experiments.


( f ) Colony formation assay demonstrating that the ferroptosis inhibitor Ferrostatin - 1 ( Fer ) reversed Sorafenib - induced cell death in ATF4 - deficient HCC cells . HLE cells transfected with siCtrl or siATF4 were treated with Sorafenib ( 8μM ) or DMSO plus either DMSO or Ferrostatin - 1 ( Fer ; 5μM ) for 2 weeks . Results represent three independent experiments .

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(f) Colony formation assay demonstrating that the ferroptosis inhibitor Ferrostatin-1 (Fer) reversed Sorafenib-induced cell death in ATF4-deficient HCC cells. HLE cells transfected with siCtrl or siATF4 were treated with Sorafenib (8μM) or DMSO plus either DMSO or Ferrostatin-1 (Fer; 5μM) for 2 weeks. Results represent three independent experiments.


( g ) The forced expression of SLC7A11 rescued cell death induced by ATF4 ablation . HLE cells were transfected with a construct coding for SLC7A11 ( SLC7A11 OE ) or empty vector ( EV ) and with siCtrl or siATF4 every other day . Cells were then treated with either DMSO or 6μM Sorafenib for 2 weeks . Colony formation was visualized by crystal violet staining . Results represent three independent experiments .

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(g) The forced expression of SLC7A11 rescued cell death induced by ATF4 ablation. HLE cells were transfected with a construct coding for SLC7A11 (SLC7A11 OE) or empty vector (EV) and with siCtrl or siATF4 every other day. Cells were then treated with either DMSO or 6μM Sorafenib for 2 weeks. Colony formation was visualized by crystal violet staining. Results represent three independent experiments.


( h ) Immunohistochemical staining of ATF4 in HCC and adjacent non - neoplastic areas from patients . Tumor tissues showed a higher expression of ATF4 . Scale bar , 100μm . ( i ) Quantification of ATF4 - positive cells in tumor and non - tumor samples showed that HCC tumors present higher ATF4 levels . Data are shown as mean ± standard deviation ( SD ) . Statistical significance was calculated using unpaired t - test . ( j ) Representative images of immuno - histochemical staining of ATF4 and SLC7A11 proteins in HCC samples from patients . Scale bars , 50μm .

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(h) Immunohistochemical staining of ATF4 in HCC and adjacent non-neoplastic areas from patients. Tumor tissues showed a higher expression of ATF4. Scale bar, 100μm. (i) Quantification of ATF4-positive cells in tumor and non-tumor samples showed that HCC tumors present higher ATF4 levels. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using unpaired t-test. (j) Representative images of immuno-histochemical staining of ATF4 and SLC7A11 proteins in HCC samples from patients. Scale bars, 50μm.


( a ) YAP / TAZ deficiency repressed Sorafenib - induced expression of ATF4 , whereas ATF4 depletion had no effect on YAP / TAZ protein levels . HLE cells were transfected with siCtrl or siATF4 or siY / T and treated with 6μM Sorafenib or not for 18 hours . ATF4 and YAP / TAZ protein levels were analyzed by immunoblotting . GAPDH served as loading control . Results represent three independent experiments .

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(a) YAP/TAZ deficiency repressed Sorafenib-induced expression of ATF4, whereas ATF4 depletion had no effect on YAP/TAZ protein levels. HLE cells were transfected with siCtrl or siATF4 or siY/T and treated with 6μM Sorafenib or not for 18 hours. ATF4 and YAP/TAZ protein levels were analyzed by immunoblotting. GAPDH served as loading control. Results represent three independent experiments.


( d ) Cellular fractionation revealed that high cell density , and with it , lower YAP / TAZ activity reduced the levels of nuclear ATF4 . HLE cells were seeded at different cell numbers , and cell lysates of high cell density cells ( HD ) and low cell density ( LD ) were fractionated into cytoplasmic and nuclear fractions and analyzed for ATF4 and YAP / TAZ by immunoblotting . GAPDH served as loading control for total lysate and cytoplasmic proteins , Lamin A / C served as loading control for nuclear proteins . Results represent three independent experiments .

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(d) Cellular fractionation revealed that high cell density, and with it, lower YAP/TAZ activity reduced the levels of nuclear ATF4. HLE cells were seeded at different cell numbers, and cell lysates of high cell density cells (HD) and low cell density (LD) were fractionated into cytoplasmic and nuclear fractions and analyzed for ATF4 and YAP/TAZ by immunoblotting. GAPDH served as loading control for total lysate and cytoplasmic proteins, Lamin A/C served as loading control for nuclear proteins. Results represent three independent experiments.


( h ) Representative images of immuno - histochemical staining of ATF4 and YAP proteins in HCC samples from patients . Scale bars , 50μm . ( i ) Quantification of the immunohistochemical staining in ( j ) showed a positive correlation of ATF4 and YAP expression ( N = 18 ) . Statistical significance was calculated using Pearson correlation analysis .

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(h) Representative images of immuno-histochemical staining of ATF4 and YAP proteins in HCC samples from patients. Scale bars, 50μm. (i) Quantification of the immunohistochemical staining in (j) showed a positive correlation of ATF4 and YAP expression (N=18). Statistical significance was calculated using Pearson correlation analysis.


( b ) Quantitative RT - PCR analysis verified that depletion of YAP / TAZ or ATF4 declined the expression levels of SLC7A11 and CHAC1 . HLE cells were transfected with siCtrl , siY / T or siATF4 and cultured with 6μM Sorafenib for 18 hours . Quantitative RT - PCR was conducted to determine SLC7A11 and CHAC1 mRNA levels . Data are shown as mean ± standard deviation ( SD ) . Statistical significance was calculated using Two - way ANOVA . Results represent 3 independent experiments .

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(b) Quantitative RT-PCR analysis verified that depletion of YAP/TAZ or ATF4 declined the expression levels of SLC7A11 and CHAC1. HLE cells were transfected with siCtrl, siY/T or siATF4 and cultured with 6μM Sorafenib for 18 hours. Quantitative RT-PCR was conducted to determine SLC7A11 and CHAC1 mRNA levels. Data are shown as mean ± standard deviation (SD).Statistical significance was calculated using Two-way ANOVA. Results represent 3 independent experiments.


( e ) Binding of YAP and TAZ to DNA fragments containing the AARE binding motif in the SLC7A11 promoter . ChIP was performed on HLE cell lysate with antibodies against YAP and TAZ and rabbit IgG as control . DNA fragments were amplified using the primers specific for AARE binding motif in the SLC7A11 promoter region . The non - coding region NC10 served as negative control , and the bona fide TEAD target genes CYR61 and ANKRD1 served as positive controls . Data are shown as mean ± standard deviation ( SD ) . Statistical significance was calculated using one - way ANOVA . Results represent three independent experiments .

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(e) Binding of YAP and TAZ to DNA fragments containing the AARE binding motif in the SLC7A11 promoter. ChIP was performed on HLE cell lysate with antibodies against YAP and TAZ and rabbit IgG as control. DNA fragments were amplified using the primers specific for AARE binding motif in the SLC7A11 promoter region. The non-coding region NC10 served as negative control, and the bona fide TEAD target genes CYR61 and ANKRD1 served as positive controls. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one-way ANOVA. Results represent three independent experiments.


( g ) YAP / TAZ bind to DNA fragments containing the AARE binding motif within the SLC7A11 promoter via ATF4 . HLE cells were cultured with 6μM Sorafenib for 18 hours before harvest . In a 1st round ChIP ATF4 was immunoprecipitated with antibody against ATF4 , rabbit IgG was used as control . DNA - protein immunocomplexes were eluted and in a 2nd round ChIP antibody against YAP / TAZ was used to precipitate DNA fragments which were then amplified and analyzed by quantitative PCR for the AARE motif in the SLC7A11 promoter . NC10 served as negative PCR control . Data are shown as mean ± standard deviation ( SD ) . Statistical significance was calculated using one - way ANOVA . Results represent three independent experiments .

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(g) YAP/TAZ bind to DNA fragments containing the AARE binding motif within the SLC7A11 promoter via ATF4. HLE cells were cultured with 6μM Sorafenib for 18 hours before harvest. In a 1st round ChIP ATF4 was immunoprecipitated with antibody against ATF4, rabbit IgG was used as control. DNA-protein immunocomplexes were eluted and in a 2nd round ChIP antibody against YAP/TAZ was used to precipitate DNA fragments which were then amplified and analyzed by quantitative PCR for the AARE motif in the SLC7A11 promoter. NC10 served as negative PCR control. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one-way ANOVA. Results represent three independent experiments.


Combination of YAP / TAZ deficiency and Sorafenib treatment suppressed tumor growth in a HCC xenograft model . SNU398 - shLuc or SNU398 - shYAP / TAZ ( shY / T ) cells were transplanted into the flanks of immunodeficient NSG mice . Once the tumors were palpable , mice were treated with 20mg / kg Sorafenib or vehicle control , and tumor sizes were measured twice a week ( b ) . Tumor weights were also recorded after sacrifice of the mice Data are shown as mean ± standard deviation ( SD ) . Statistical significance was calculated using two - way ANOVA analysis . Mouse numbers of shLuc + Vehicle , shY / T + Vechicle and shY / T + Srf were 4 per cohort , mouse numbers of shLuc + Srf were 3 per cohort .

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Combination of YAP/TAZ deficiency and Sorafenib treatment suppressed tumor growth in a HCC xenograft model. SNU398-shLuc or SNU398-shYAP/TAZ (shY/T) cells were transplanted into the flanks of immunodeficient NSG mice. Once the tumors were palpable, mice were treated with 20mg/kg Sorafenib or vehicle control, and tumor sizes were measured twice a week (b). Tumor weights were also recorded after sacrifice of the mice Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using two-way ANOVA analysis. Mouse numbers of shLuc + Vehicle, shY/T + Vechicle and shY/T + Srf were 4 per cohort, mouse numbers of shLuc + Srf were 3 per cohort.


( e ) Pharmacological inhibition of glutathione synthesis and function sensitizes HCC tumors to Sorafenib therapy . SNU398 cells were transplanted into the flanks of immunodeficient NSG mice . Once the tumors were palpable , mice were treated with vehicle control or with 20mg / kg Sorafenib alone , or with 20mg / kg Sorafenib and 20mM BSO in the drinking water or 120mg / kg SSA per os . Tumor sizes were measured twice a week . Data are shown as mean ± standard deviation ( SD ) . Statistical significance was calculated using two - way ANOVA analysis . Mouse numbers were 8 for the experimental cohorts Vehicle , Srf , and Srf + SSA and 7 for Srf + BSO .

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(e) Pharmacological inhibition of glutathione synthesis and function sensitizes HCC tumors to Sorafenib therapy. SNU398 cells were transplanted into the flanks of immunodeficient NSG mice. Once the tumors were palpable, mice were treated with vehicle control or with 20mg/kg Sorafenib alone, or with 20mg/kg Sorafenib and 20mM BSO in the drinking water or 120mg/kg SSA per os. Tumor sizes were measured twice a week. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using two-way ANOVA analysis. Mouse numbers were 8 for the experimental cohorts Vehicle, Srf, and Srf + SSA and 7 for Srf + BSO.


( g ) Quantification of 4 - HNE - positive cells of the immunohistochemical stainings described in ( f ) . Data are shown as mean ± standard deviation ( SD ) . Statistical significance was calculated using one - way ANOVA . Each data point represents the average of 3 technical replicates of one histological section , the numbers of tumor samples are given in ( e ) .

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(g) Quantification of 4-HNE-positive cells of the immunohistochemical stainings described in (f). Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one-way ANOVA. Each data point represents the average of 3 technical replicates of one histological section, the numbers of tumor samples are given in (e).


Left : Immunofluorescence images of cGAS in the nucleus ( DAPI ) and cytosol ( phalloidin ) in BMDMos cultured at low / high density Scale bar : 50 μm . Right : Corresponding quantification of the nuclear cGAS from 6 different fields with n > 50 cells .

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Left: Immunofluorescence images of cGAS in the nucleus (DAPI) and cytosol (phalloidin) in BMDMos cultured at low/high density Scale bar: 50 μm. Right: Corresponding quantification of the nuclear cGAS from 6 different fields with n > 50 cells.


Left : Immunofluorescence images of cGAS in the nucleus ( DAPI ) and cytosol ( phalloidin ) in BMDMos cultured with / without serum Scale bar : 50 μm . Right : Corresponding quantification of the nuclear cGAS from 6 different fields with n > 50 cells .

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Left: Immunofluorescence images of cGAS in the nucleus (DAPI) and cytosol (phalloidin) in BMDMos cultured with/without serum Scale bar: 50 μm. Right: Corresponding quantification of the nuclear cGAS from 6 different fields with n > 50 cells.


Immunofluorescence images of cGAS in the nucleus ( DAPI ) and cytosol ( phalloidin ) in BMDMos cultured with / without Aphidicolin ( Aphi ) ( C ) . Scale bar : 50 μm . Right : Corresponding quantification of the nuclear cGAS from 6 different fields with n > 50 cells .

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Immunofluorescence images of cGAS in the nucleus (DAPI) and cytosol (phalloidin) in BMDMos cultured with/without Aphidicolin (Aphi) (C). Scale bar: 50 μm. Right: Corresponding quantification of the nuclear cGAS from 6 different fields with n > 50 cells.


Immunoblot estimation of cGAS in nuclear / cytosolic subcellular fraction of BMDMos cultured under indicated conditions . Lamin B and α - Tubulin are nuclear and cytosolic markers respectively .

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Immunoblot estimation of cGAS in nuclear/cytosolic subcellular fraction of BMDMos cultured under indicated conditions. Lamin B and α-Tubulin are nuclear and cytosolic markers respectively.


Flow cytometric analysis of cell cycle of BMDMos


Micronuclei ( indicated by arrow head ) in GFP - NLS - or GFP - hcGAS expressing HEK293 cells before ( 0 h ) or 24 h after γ - irradiation ( IR ; 10 Gy ) . Scale bar : 10 μm ( A ) .

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Micronuclei (indicated by arrow head) in GFP-NLS- or GFP-hcGAS expressing HEK293 cells before (0 h) or 24 h after γ-irradiation (IR; 10 Gy). Scale bar: 10 μm (A).


Micronuclei ( indicated by arrow head ) in GFP - NLS - or GFP - hcGAS expressing HEK293 cells before ( 0 h ) or 24 h after γ - irradiation ( IR ; 10 Gy ) . The average MNs / cell . Graphs show Mean ± SEM ( n = 3 independent experiments ) representing six different microscopic fields with over 200 cells .

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Micronuclei (indicated by arrow head) in GFP-NLS- or GFP-hcGAS expressing HEK293 cells before (0 h) or 24 h after γ-irradiation (IR; 10 Gy). The average MNs/cell. Graphs show Mean ± SEM (n=3 independent experiments) representing six different microscopic fields with over 200 cells.


IFNB1 response in HEK293 cells stimulated with transfected plasmid DNA . Mean ± SEM of n = 3 independent experiments .

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IFNB1 response in HEK293 cells stimulated with transfected plasmid DNA. Mean ± SEM of n=3 independent experiments.


Micronuclei ( indicated by arrow head ) and cGAS staining in WT and cGAS - / - BMDMos exposed to γ - irradiation ( 10 Gy ) . Scale bar : 10 μm .

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Micronuclei (indicated by arrow head) and cGAS staining in WT and cGAS-/- BMDMos exposed to γ-irradiation (10 Gy). Scale bar: 10 μm.


Average MNs / cell in BMDMos . MN graphs show Mean ± SEM ( n = 3 independent experiments ) representing eight different microscopic fields with over 200 cells .

Average MNs/cell in BMDMos. MN graphs show Mean ± SEM (n=3 independent experiments) representing eight different microscopic fields with over 200 cells.


Cell death in WT and cGAS - / - BMDMos that were first synchronized at G2 / M , then γ - irradiated ( 10 Gy ) followed by release and analysis at indicated time points . Mean ± SD , x biological triplicates ( n = 3 ) per treatment group are shown .

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Cell death in WT and cGAS-/- BMDMos that were first synchronized at G2/M, then γ-irradiated (10 Gy) followed by release and analysis at indicated time points. Mean ±SD, x biological triplicates (n=3) per treatment group are shown.


BMDMos from cGAS - / - mice exhibit enhanced DNA repair efficiency than those from WT and Sting - / - mice . ( A ) Representative comet tails of WT , cGAS - / - and Sting - / - BMDMos exposed to γ - irradiation ( IR : 10 Gy ) on ice then incubated at 37 ̊ C for indicated duration . ( B ) Corresponding quantification of the comet tail moments from 20 different fields with n > 200 comets of 3 independent experiments .

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BMDMos from cGAS-/- mice exhibit enhanced DNA repair efficiency than those from WT and Sting-/- mice. (A) Representative comet tails of WT, cGAS-/- and Sting-/-BMDMos exposed to γ-irradiation (IR: 10 Gy) on ice then incubated at 37 ̊C for indicated duration. (B) Corresponding quantification of the comet tail moments from 20 different fields with n > 200 comets of 3 independent experiments.


cGAS promotes micronuclei generation in BMDMos independently of STING . Confocal microscopic visualization of micronuclei ( indicated by arrow head ) and cGAS staining in WT , cGAS - / - and Sting - / - BMDMos exposed to γ - irradiation ( 10Gy ) . Scale bar : 10 μm .

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cGAS promotes micronuclei generation in BMDMos independently of STING. Confocal microscopic visualization of micronuclei (indicated by arrow head) and cGAS staining in WT, cGAS-/- and Sting-/- BMDMos exposed to γ-irradiation (10Gy). Scale bar: 10 μm.


cGAS promotes micronuclei generation in BMDMos independently of STING . Average MNs / cell in corresponding representative images . Bar graphs show mean values from eight different microscopic fields with over 200 cells .

cGAS promotes micronuclei generation in BMDMos independently of STING. Average MNs/cell in corresponding representative images. Bar graphs show mean values from eight different microscopic fields with over 200 cells.


γ - irradiation - induced cell death in WT , cGAS - / -

γ-irradiation-induced cell death in WT, cGAS-/- BMDMos


γ - irradiation - induced cell death in Sting - / - and cGAS - / - Sting - / - BMDMos ( F ) .

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γ-irradiation-induced cell death in Sting-/- and cGAS-/- Sting-/- BMDMos (F).


Kinetics of in vivo depletion of indicated bone marrow cells in WT mice ( n = 3 ) after γ - irradiation ( 9 Gy ) .

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Kinetics of in vivo depletion of indicated bone marrow cells in WT mice (n=3) after γ-irradiation (9 Gy).


Bone marrow cells in WT ( n = 3 ) and cGAS - / - ( n = 3 ) mice 10 hours post γ - irradiation .

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Bone marrow cells in WT (n=3) and cGAS-/- (n=3) mice 10 hours post γ-irradiation.


Bone marrow cells in Sting - / - ( n = 3 ) and cGAS - / - Sting - / - ( n = 3 ) mice 10 hours post γ - irradiation .

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Bone marrow cells in Sting-/- (n=3) and cGAS-/- Sting-/- (n=3) mice 10 hours post γ-irradiation.


Obtained results showing enhanced HR efficiency upon knockdown of endogenous cGAS in U2OS cells . Immunoblot inserts in depict knockdown efficiency of cGAS and Histone H1 . 2 .

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Obtained results showing enhanced HR efficiency upon knockdown of endogenous cGAS in U2OS cells. Immunoblot inserts in depict knockdown efficiency of cGAS and Histone H1.2.


Results showing the effect of hcGAS , hcGAS △ cGAMP , hcGAS △ DNA or hcGAS △ Oligo on HR in HEK293 cells . Corresponding immunoblot inserts depict cGAS expression .

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Results showing the effect of hcGAS, hcGAS△cGAMP, hcGAS△DNA or hcGAS△Oligo on HR in HEK293 cells. Corresponding immunoblot inserts depict cGAS expression.


Results showing the effect of hcGAS , hcGAS △ cGAMP , hcGAS △ DNA or hcGAS △ Oligo on NHEJ in HEK293 cells . Corresponding immunoblot inserts depict cGAS expression .

Results showing the effect of hcGAS, hcGAS△cGAMP, hcGAS△DNA or hcGAS△Oligo on NHEJ in HEK293 cells. Corresponding immunoblot inserts depict cGAS expression.


cGAS is not recruited to DSB sites : Confocal microscopic images of GFP - NLS - or GFP - hcGAS - expressing U2OS - DSB reporter cells incubated ( or not ) with Shield - 1 and 4 - OHT to induce the expression and translocation of mCherry - LacI - FokI ( red ) to specific DSB sites . Scale bar : 10μm . The arrowheads indicate DSB sites .

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cGAS is not recruited to DSB sites: Confocal microscopic images of GFP-NLS- or GFP-hcGAS-expressing U2OS-DSB reporter cells incubated (or not) with Shield-1 and 4-OHT to induce the expression and translocation of mCherry-LacI-FokI (red) to specific DSB sites. Scale bar: 10μm. The arrowheads indicate DSB sites.


cGAS does not colocalize with γ - H2AX at DSB sites : GFP - NLS - or GFP - hcGAS - expressing HEK293 cells exposed ( or not ) to γ - irradiation ( IR : 10 Gy ) then stained for γ - H2AX . Scale bar : 10 μm .

cGAS does not colocalize with γ-H2AX at DSB sites: GFP-NLS- or GFP-hcGAS- expressing HEK293 cells exposed (or not) to γ-irradiation (IR: 10 Gy) then stained for γ-H2AX. Scale bar: 10 μm.


Nuclear cGAS is mainly chromatin - bound and remains unaltered upon γ - irradiation . Cytosolic ( cyto ) and nuclear fractions of γ - irradiated ( 10 Gy , 30 min ) BMDMos analyzed for cGAS and indicated molecules .

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Nuclear cGAS is mainly chromatin-bound and remains unaltered upon γ-irradiation. Cytosolic (cyto) and nuclear fractions of γ-irradiated (10 Gy, 30 min) BMDMos analyzed for cGAS and indicated molecules.


Nuclear cGAS is mainly chromatin - bound and remains unaltered upon γ - irradiation . Cytosolic , soluble nuclear and chromatin fractions from BMDMos were immunoblotted for cGAS and indicated proteins .

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Nuclear cGAS is mainly chromatin-bound and remains unaltered upon γ-irradiation. Cytosolic, soluble nuclear and chromatin fractions from BMDMos were immunoblotted for cGAS and indicated proteins.


cGAS co - isolates with DNA repair proteins because of bound chromatin bridges . Nuclease digestion abrogates the co - isolation of cGAS and DNA repair proteins : Lysates of control ( - IR ) and γ - irradiated ( + IR , 10 Gy , 30 min ) GFP - hcGAS expressing HEK293 cells were treated ( or not ) with Benzonase before cGAS immunoprecipitation and analysis for indicated proteins . Agarose gel analysis of DNA in corresponding cell lysates in ( E ) .

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cGAS co-isolates with DNA repair proteins because of bound chromatin bridges. Nuclease digestion abrogates the co-isolation of cGAS and DNA repair proteins: Lysates of control (-IR) and γ-irradiated (+IR, 10 Gy, 30 min) GFP-hcGAS expressing HEK293 cells were treated (or not) with Benzonase before cGAS immunoprecipitation and analysis for indicated proteins. Agarose gel analysis of DNA in corresponding cell lysates in (E).


cGAS co - isolates with DNA repair proteins because of bound chromatin bridges . Co - isolation of cGAS and DNA repair proteins depends on its binding to DNA : cGAS pulldowns along with lysate inputs of control and γ - irradiated HEK293 cells expressing GFP - hcGAS or GFP - hcGASΔDNA probed for indicated proteins .

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cGAS co-isolates with DNA repair proteins because of bound chromatin bridges. Co-isolation of cGAS and DNA repair proteins depends on its binding to DNA: cGAS pulldowns along with lysate inputs of control and γ-irradiated HEK293 cells expressing GFP-hcGAS or GFP-hcGASΔDNA probed for indicated proteins.


Confocal images of γ - irradiated GFP - NLS - , GFP - hcGAS - expressing HEK293 cells stained for RAD51 ( red ) with or without γ - irradiation . Scale bar : 10 μm .

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Confocal images of γ-irradiated GFP-NLS-, GFP-hcGAS-expressing HEK293 cells stained for RAD51 (red) with or without γ-irradiation. Scale bar: 10 μm.


Pre - incubation of dsDNA with mcGAS - cat prevents D - loop formation by human RAD51 , but does not affect the RAD1 activity once RAD51 filaments are bound to dsDNA . The percentage of D - loop formed in each reaction ( left ) was graphed as the average of triplicates ± SD .

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Pre-incubation of dsDNA with mcGAS-cat prevents D-loop formation by human RAD51, but does not affect the RAD1 activity once RAD51 filaments are bound to dsDNA. The percentage of D-loop formed in each reaction (left) was graphed as the average of triplicates ± SD.


Pre - incubation of template dsDNA with hcGAScat blocks subsequent D - loop formation . The percentage of D - loop formation ( below ) was graphed as the average of triplicates ± SD .

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Pre-incubation of template dsDNA with hcGAScat blocks subsequent D-loop formation. The percentage of D-loop formation (below) was graphed as the average of triplicates±SD.


Negative - stain electron micrographs of cGAS - dsDNA complexes following incubation of dsDNA with indicated cGAS variants . Scale bar : 100 nm .

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Negative-stain electron micrographs of cGAS-dsDNA complexes following incubation of dsDNA with indicated cGAS variants. Scale bar: 100 nm.


Effect of indicated hcGAS variants on D - loop formation when pre - incubated with dsDNA . Percentage of D - loop formed in each reaction ( left ) graphed as the average of triplicates ± SD .

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Effect of indicated hcGAS variants on D-loop formation when pre-incubated with dsDNA. Percentage of D-loop formed in each reaction (left) graphed as the average of triplicates± SD.


DR - GFP assay showing that hcGASΔDNA - Y215E is impaired in HR inhibition .

DR-GFP assay showing that hcGASΔDNA-Y215E is impaired in HR inhibition.


hcGASΔDNA - Y215E but not hcGASΔcGAMP has a decreased affinity to dsDNA24 .

hcGASΔDNA-Y215E but not hcGASΔcGAMP has a decreased affinity to dsDNA24.


hcGASΔDNA - Y215E and hcGASΔcGAMP are defective in synthase activity .

hcGASΔDNA-Y215E and hcGASΔcGAMP are defective in synthase activity.


Negative - stain electron micrographs showing that hcGAScat - ΔDNA - Y215E is defective in inducing cGAS - dsDNA complexes . Scale bar : 100 nm .

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Negative-stain electron micrographs showing that hcGAScat-ΔDNA-Y215E is defective in inducing cGAS-dsDNA complexes. Scale bar: 100 nm.


Effect of indicated hcGAS variants on D - loop formation .

Effect of indicated hcGAS variants on D-loop formation.


1D 1H - NMR spectra of a metabolite mix and the individual metabolites contained therein . Identification of single compound peaks from 1D 1H - NMR spectra of a metabolite mix . Compound detection is exemplified by showing sections of the 1D 1H - NMR spectra of ATP , IMP , proline ( PRO ) , malate ( MAL ) , shikimate ( SKM ) and methionine ( MET ) .

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1D 1H-NMR spectra of a metabolite mix and the individual metabolites contained therein. Identification of single compound peaks from 1D 1H-NMR spectra of a metabolite mix. Compound detection is exemplified by showing sections of the 1D 1H-NMR spectra of ATP, IMP, proline (PRO), malate (MAL), shikimate (SKM) and methionine (MET).


Relative activity of NADP + - dependent glucose - 6 - phosphate dehydrogenase in the presence of potential regulators , error bars represent the s . e . m . . All regulators were tested at two concentrations in four distinct replicates ( n = 4 , ATP : 5mM , 18 mM ; GTP : 2 mM , 10 mM ; IMP and AMP : 1 mM , 5 mM ) , the asterisk denotes significant inhibition ( 18 mM ATP : p - value = 0 . 029 , 10 mM GTP : p - value = 0 . 025 , one - tailed t - test ) .

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Relative activity of NADP+-dependent glucose-6-phosphate dehydrogenase in the presence of potential regulators, error bars represent the s.e.m.. All regulators were tested at two concentrations in four distinct replicates (n=4, ATP: 5mM, 18 mM; GTP: 2 mM, 10 mM; IMP and AMP: 1 mM, 5 mM), the asterisk denotes significant inhibition (18 mM ATP: p-value = 0.029, 10 mM GTP: p-value = 0.025, one-tailed t-test).


Relative activity of phosphate acetyltransferase in the presence of phenylpyruvate and tryptophan , error bars represent the s . e . m . . All regulators were tested at three concentrations in three distinct replicates ( n = 3 , phenylpyruvate : 0 . 1 , 1 , 5 mM , L - tryptophan : 0 . 1 , 1 , 4 . 4 mM ) .

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Relative activity of phosphate acetyltransferase in the presence of phenylpyruvate and tryptophan, error bars represent the s.e.m.. All regulators were tested at three concentrations in three distinct replicates (n=3, phenylpyruvate: 0.1, 1, 5 mM, L-tryptophan: 0.1, 1, 4.4 mM).


Relative activity of fructose - bisphosphate aldolase class II in the presence of 5 mM of potential regulators , except hypoxanthine , which was tested at 3 . 22 mM . All regulators were tested in three distinct replicates ( n = 3 ) , error bars represent the s . e . m . . The asterisk denotes significant activation ( 5 mM 3PG : p - value = 0 . 025 , 5 mM ATP : p - value = 0 . 024 , 5 mM PEP : p - value = 0 . 046 , one - tailed t - test ) .

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Relative activity of fructose-bisphosphate aldolase class II in the presence of 5 mM of potential regulators, except hypoxanthine, which was tested at 3.22 mM. All regulators were tested in three distinct replicates (n=3), error bars represent the s.e.m.. The asterisk denotes significant activation (5 mM 3PG: p-value = 0.025, 5 mM ATP: p-value = 0.024, 5 mM PEP: p-value = 0.046, one-tailed t-test).


( A ) HeLa cells were infected with S . Typhimurium ( Salmonella ) for 1 h or transfected with Effectene - coated latex beads for 3 h and then subjected to immunocytochemistry for LC3 and transferrin receptor ( TfR ) . Bar , 10 µm .

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(A) HeLa cells were infected with S. Typhimurium (Salmonella) for 1 h or transfected with Effectene-coated latex beads for 3 h and then subjected to immunocytochemistry for LC3 and transferrin receptor (TfR). Bar, 10 µm.


( B ) HeLa cells were infected with Salmonella for 1 h or transfected with Effectene - coated latex beads for 3 h and then subjected to immunocytochemistry for LC3 and galectin3 . Bar , 5 µm .

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(B) HeLa cells were infected with Salmonella for 1 h or transfected with Effectene-coated latex beads for 3 h and then subjected to immunocytochemistry for LC3 and galectin3. Bar, 5 µm.