Figure 3SDS - 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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Figure 3SDS-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.


Figure 5Transcription 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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Figure 5Transcription 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).


Figure 6Representative 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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Figure 6Representative 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.)


Figure 1A 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 .

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Figure 1A 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.


Figure 2D 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 . FF - 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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Figure 2D 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. FF-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).


Figure 3D 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 .

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Figure 3D 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.


Figure 4A 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 . CD 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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Figure 4A 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. CD 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


Figure 5B 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 . 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 . 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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Figure 5B 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.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.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.


Figure 1 ( 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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Figure 1(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.


Figure 2 ( 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 . ( 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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Figure 2(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.(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.


Figure 3 ( 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 . ( 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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Figure 3(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.(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.


Figure 4 ( 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 . ( 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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Figure 4(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.(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.


Figure 5 ( 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 . ( 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 .

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

Figure 5(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.(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.


Figure 6Combination 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 . ( 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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Figure 6Combination 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.(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).


Figure 1Left : 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 . 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 . 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 . 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 BMDMosFlow cytometric analysis of cell cycle of BMDMosFlow cytometric analysis of cell cycle of

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Figure 1Left: 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.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.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.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 BMDMosFlow cytometric analysis of cell cycle of BMDMosFlow cytometric analysis of cell cycle of BMDMos


Figure 2Micronuclei ( 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 . 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 . 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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Figure 2Micronuclei (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.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.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.


Figure 3BMDMos 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 . 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 - / - BMDMosγ - irradiation - induced cell death in Sting - / - and cGAS - / - Sting - / - BMDMos ( F ) .

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Figure 3BMDMos 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.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-/- BMDMosγ-irradiation-induced cell death in Sting-/- and cGAS-/- Sting-/- BMDMos (F).


Figure 4Kinetics 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 . Bone marrow cells in Sting - / - ( n = 3 ) and cGAS - / - Sting - / - ( n = 3 ) mice 10 hours post γ - irradiation .

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Figure 4Kinetics 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.Bone marrow cells in Sting-/- (n=3) and cGAS-/- Sting-/- (n=3) mice 10 hours post γ-irradiation.


Figure 5Obtained 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 . 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 . Results showing the effect of hcGAS , hcGAS △ cGAMP , hcGAS △ DNA or hcGAS △ Oligo on NHEJ in HEK293 cells . Corresponding immunoblot inserts depict cGAS expression .

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Figure 5Obtained 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.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.Results showing the effect of hcGAS, hcGAS△cGAMP, hcGAS△DNA or hcGAS△Oligo on NHEJ in HEK293 cells. Corresponding immunoblot inserts depict cGAS expression.


Figure 6cGAS 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 . 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 . 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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Figure 6cGAS 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.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.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.


Figure 7Confocal 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 . 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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Figure 7Confocal 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.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.


Figure 8Negative - 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 . 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 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 . Effect of indicated hcGAS variants on D - loop formation .

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Figure 8Negative-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.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 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.Effect of indicated hcGAS variants on D-loop formation.


Figure 11D 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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Figure 11D 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).


Figure 5Relative 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 ) . 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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Figure 5Relative 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).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).


Figure 1 . ( 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 . ( C ) HeLa cells were transfected with Effectene - coated latex beads for 3 h and subjected to immunocytochemistry for LC3 and Ub ( top ) or LC3 and p62 ( bottom ) . Bar , 5 µm . The percentages of LC3 - or p62 - positive beads per Ub - positive ( Ub + ) or Ub - negative ( Ub − ) beads were enumerated . Statistical analysis was performed by Student ' s unpaired t test . * , P < 0 . 01 . ( D and E ) HeLa cells stably expressing GFP - LC3 were transfected with Effectene - coated latex beads for 3 h . Bead - autophagosomes were fractionated as described in Materials and methods . The bead - autophagosome fraction was observed by confocal microscopy ( D ; bar , 10 µm ) or lysed with RIPA buffer and subjected to Western blot analysis using the indicated antibodies ( E ) . In a control sample , scraped cells were mixed with Effectene - coated beads and immediately homogenized . ( F ) The bead - autophagosome fraction was lysed and subjected to immunoprecipitation with an anti - Ub IgG antibody ( FK2 ) or control IgG . Co - immunoprecipitated molecules were examined by Western blotting using the indicated antibodies . ( G and H ) NIH3T3 cells stably expressing mStrawberry ( mStr ) - Gal3 and GFP - LC3 , mStr - Gal3 , and GFP - p62 , or mStr - Gal3 and GFP - Ub were transfected with Effectene - coated latex beads for 30 min and then washed . Live cells were observed at 1 - min intervals by fluorescence microscopy . Bar , 3 µm . The time after galectin3 localization was measured for at least 30 cases for each combination ( H ) . Statistical analysis was performed by Student ' s unpaired t test . * , P < 0 . 05 ; NS , not significant .

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Figure 1.(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.(C) HeLa cells were transfected with Effectene-coated latex beads for 3 h and subjected to immunocytochemistry for LC3 and Ub (top) or LC3 and p62 (bottom). Bar, 5 µm. The percentages of LC3- or p62-positive beads per Ub-positive (Ub+) or Ub-negative (Ub−) beads were enumerated. Statistical analysis was performed by Student's unpaired t test. *, P < 0.01.(D and E) HeLa cells stably expressing GFP-LC3 were transfected with Effectene-coated latex beads for 3 h. Bead-autophagosomes were fractionated as described in Materials and methods. The bead-autophagosome fraction was observed by confocal microscopy (D; bar, 10 µm) or lysed with RIPA buffer and subjected to Western blot analysis using the indicated antibodies (E). In a control sample, scraped cells were mixed with Effectene-coated beads and immediately homogenized.(F) The bead-autophagosome fraction was lysed and subjected to immunoprecipitation with an anti-Ub IgG antibody (FK2) or control IgG. Co-immunoprecipitated molecules were examined by Western blotting using the indicated antibodies.(G and H) NIH3T3 cells stably expressing mStrawberry (mStr)-Gal3 and GFP-LC3, mStr-Gal3, and GFP-p62, or mStr-Gal3 and GFP-Ub were transfected with Effectene-coated latex beads for 30 min and then washed. Live cells were observed at 1-min intervals by fluorescence microscopy. Bar, 3 µm. The time after galectin3 localization was measured for at least 30 cases for each combination (H). Statistical analysis was performed by Student's unpaired t test. *, P < 0.05; NS, not significant.


Figure 2 . ( A and B ) NIH3T3 cells stably expressing mStr - Ub and GFP - tagged LC3 , Atg5 , WIPI - 1 , Atg14L1 , or ULK1 were transfected with Effectene - coated latex beads for 30 min . Then , live cells were observed at 1 - min intervals by fluorescence microscopy . Bar , 3 µm . The time after Ub localization was measured for at least 30 cases for each combination . Each value represents the mean ± SD . Statistical analysis was performed by Student ' s unpaired t test . * , P < 0 . 05 ; NS , not significant . ( C ) NIH3T3 cells stably expressing GFP - tagged Ub , LC3 , Atg5 , WIPI - 1 , Atg14L1 , Atg9L1 , or ULK1 were transfected with Effectene - coated beads for 3 h in the presence or absence ( mock ) of 30 µM UBEI - 41 ( a ubiquitin E1 - specific inhibitor ) and subjected to immunocytochemistry for galectin3 . The percentages of GFP - positive per galectin3 - positive beads were enumerated . At least 30 beads were counted ( n = 3 ) . The values are the mean ± SD . Statistical analysis was performed by Student ' s unpaired t test . * , P < 0 . 05 . ( D and E ) Parent NIH3T3 cells , Atg4B mutant overexpressing NIH3T3 cells stably expressing GFP - tagged LC3 , Atg5 , WIPI - 1 , Atg14L1 , Atg9L1 , or ULK1 were transfected with Effectene - coated latex beads for 3 h and subjected to immunocytochemistry for galectin3 . The percentages of Atg - positive per galectin3 - positive beads were enumerated . At least 30 beads were counted ( n = 3 ) . The values are the mean ± SD . ( E ) wild - type MEFs , and Atg5 - KO MEFs stably expressing GFP - tagged LC3 , Atg5 , WIPI - 1 , Atg14L1 , Atg9L1 , or ULK1 were transfected with Effectene - coated latex beads for 3 h and subjected to immunocytochemistry for galectin3 . The percentages of Atg - positive per galectin3 - positive beads were enumerated . At least 30 beads were counted ( n = 3 ) . The values are the mean ± SD .

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Figure 2.(A and B) NIH3T3 cells stably expressing mStr-Ub and GFP-tagged LC3, Atg5, WIPI-1, Atg14L1, or ULK1 were transfected with Effectene-coated latex beads for 30 min. Then, live cells were observed at 1-min intervals by fluorescence microscopy. Bar, 3 µm. The time after Ub localization was measured for at least 30 cases for each combination. Each value represents the mean ± SD. Statistical analysis was performed by Student's unpaired t test. *, P < 0.05; NS, not significant.(C) NIH3T3 cells stably expressing GFP-tagged Ub, LC3, Atg5, WIPI-1, Atg14L1, Atg9L1, or ULK1 were transfected with Effectene-coated beads for 3 h in the presence or absence (mock) of 30 µM UBEI-41 (a ubiquitin E1-specific inhibitor) and subjected to immunocytochemistry for galectin3. The percentages of GFP-positive per galectin3-positive beads were enumerated. At least 30 beads were counted (n = 3). The values are the mean ± SD. Statistical analysis was performed by Student's unpaired t test. *, P < 0.05.(D and E) Parent NIH3T3 cells, Atg4B mutant overexpressing NIH3T3 cells stably expressing GFP-tagged LC3, Atg5, WIPI-1, Atg14L1, Atg9L1, or ULK1 were transfected with Effectene-coated latex beads for 3 h and subjected to immunocytochemistry for galectin3. The percentages of Atg-positive per galectin3-positive beads were enumerated. At least 30 beads were counted (n = 3). The values are the mean ± SD.(E) wild-type MEFs, and Atg5-KO MEFs stably expressing GFP-tagged LC3, Atg5, WIPI-1, Atg14L1, Atg9L1, or ULK1 were transfected with Effectene-coated latex beads for 3 h and subjected to immunocytochemistry for galectin3. The percentages of Atg-positive per galectin3-positive beads were enumerated. At least 30 beads were counted (n = 3). The values are the mean ± SD.


Figure 3 . ( A ) HeLa cells were lysed with 1 % Triton X - 100 lysis buffer and subjected to immunoprecipitation with FK2 - or control IgG - immobilized beads . The coimmunoprecipitated molecules were examined by Western blotting using the indicated antibodies . ( C ) Purified recombinant GST or GST - Ub - immobilized glutathione Sepharose beads were incubated with lysates from HEK293T cells transiently expressing FLAG - tagged Atg16L1 constructs for 1 h at 25 ° C with gentle agitation . The beads were washed three times with ice - cold PBS and the bound complexes were eluted with 50 mM reduced glutathione and then subjected to Western blotting for FLAG . ( D ) Purified GST or GST - Ub - immobilized glutathione Sepharose beads were incubated with lysates from bacteria expressing trigger factor ( TF ) or TF - FLAG - WD β - propellers for 1 h at 25 ° C with gentle agitation . Washing and elution were performed as described in C . The eluted samples were subjected to SDS - PAGE and Western blotting for FLAG .

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Figure 3.(A) HeLa cells were lysed with 1% Triton X-100 lysis buffer and subjected to immunoprecipitation with FK2- or control IgG-immobilized beads. The coimmunoprecipitated molecules were examined by Western blotting using the indicated antibodies.(C) Purified recombinant GST or GST-Ub-immobilized glutathione Sepharose beads were incubated with lysates from HEK293T cells transiently expressing FLAG-tagged Atg16L1 constructs for 1 h at 25°C with gentle agitation. The beads were washed three times with ice-cold PBS and the bound complexes were eluted with 50 mM reduced glutathione and then subjected to Western blotting for FLAG.(D) Purified GST or GST-Ub-immobilized glutathione Sepharose beads were incubated with lysates from bacteria expressing trigger factor (TF) or TF-FLAG-WD β-propellers for 1 h at 25°C with gentle agitation. Washing and elution were performed as described in C. The eluted samples were subjected to SDS-PAGE and Western blotting for FLAG.


Figure 4 . ( A ) Yeast two - hybrid interactions of the ULK1 complexes with other human Atg proteins . ULK1 , ULK2 , Atg13 , FIP200 , and Atg101 - AD fusions ( rows 2 - 6 ) or control AD constructs ( row 1 , Empty ) were coexpressed with control DBD constructs ( column 1 , Empty ) or Atg protein - DBD fusions ( columns 2 - 35 ) and tested for positive yeast two - hybrid interactions ( top ) or cotransformation ( control , bottom ) . White lines divide images derived from the same plate , and red lines divide images from different plates . Atg16L1 positively interacted with FIP200 in this assay . ( B ) FIP200 binds to exogenously expressed Atg16L1 . Myc - Atg16L1 coprecipitations with an empty vector control ( lane 1 ) or One - STrEP - FLAG ( OSF ) - FIP200 ( lane 2 ) . ( C ) FIP200 binds endogenous Atg16L1 . Atg16L1 coprecipitations with an empty vector control ( lane 1 ) or OSF - FIP200 ( lane 2 ) . ( D ) Wild - type or FIP200 - KO MEFs stably expressing the empty vector , full - length Atg16L1 , or ΔWD mutant were incubated in growth medium ( − ) or Earle ' s balanced salt solution ( EBSS ) ( + ) for 1 h and examined by Western blotting using the indicated antibodies . We previously reported that stable expression of an exogenous Atg16L1 construct can be used to replace the endogenous Atg16L1 protein with an exogenous Atg16L1 protein ( Fujita et al . , 2009 ) because free Atg16L1 molecules not complexed with the Atg12 - Atg5 conjugate are preferentially degraded by the ubiquitin - proteasome system . In control cells ( Empty ) , two Atg16L1 splicing variants , the α - and β - forms , were detected . In full - length β - form - replaced cells ( Full ) , the α - form was not detected , whereas in ΔWD - replaced cells ( ΔWD ) both the α - and β - forms were minimally detected . Thus , we successfully obtained cells that express only endogenous levels of the full - length or ΔWD mutant . ( E - L ) Atg16L1 - replaced wild - type or FIP200 - KO MEFs were infected with Salmonella ( MOI = 10 ) for 1 h and subjected to immunocytochemistry for Atg16L1 and Ub ( E and F ) or LC3 and Ub ( I and J ) . The percentages of Atg16L1 - positive ( G and H ) or LC3 - positive per Ub - positive bacteria were enumerated ( K and L ) . At least 50 Salmonella were counted . The average ± SD is shown for three independent experiments . Statistical analysis was performed by Student ' s t test . * , P < 0 . 05 ; NS , not significant . Bar , 5 µm .

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

Figure 4.(A) Yeast two-hybrid interactions of the ULK1 complexes with other human Atg proteins. ULK1, ULK2, Atg13, FIP200, and Atg101-AD fusions (rows 2-6) or control AD constructs (row 1, Empty) were coexpressed with control DBD constructs (column 1, Empty) or Atg protein-DBD fusions (columns 2-35) and tested for positive yeast two-hybrid interactions (top) or cotransformation (control, bottom). White lines divide images derived from the same plate, and red lines divide images from different plates. Atg16L1 positively interacted with FIP200 in this assay.(B) FIP200 binds to exogenously expressed Atg16L1. Myc-Atg16L1 coprecipitations with an empty vector control (lane 1) or One-STrEP-FLAG (OSF)-FIP200 (lane 2).(C) FIP200 binds endogenous Atg16L1. Atg16L1 coprecipitations with an empty vector control (lane 1) or OSF-FIP200 (lane 2).(D) Wild-type or FIP200-KO MEFs stably expressing the empty vector, full-length Atg16L1, or ΔWD mutant were incubated in growth medium (−) or Earle's balanced salt solution (EBSS) (+) for 1 h and examined by Western blotting using the indicated antibodies. We previously reported that stable expression of an exogenous Atg16L1 construct can be used to replace the endogenous Atg16L1 protein with an exogenous Atg16L1 protein (Fujita et al., 2009) because free Atg16L1 molecules not complexed with the Atg12-Atg5 conjugate are preferentially degraded by the ubiquitin-proteasome system. In control cells (Empty), two Atg16L1 splicing variants, the α- and β-forms, were detected. In full-length β-form-replaced cells (Full), the α-form was not detected, whereas in ΔWD-replaced cells (ΔWD) both the α- and β-forms were minimally detected. Thus, we successfully obtained cells that express only endogenous levels of the full-length or ΔWD mutant.(E-L) Atg16L1-replaced wild-type or FIP200-KO MEFs were infected with Salmonella (MOI = 10) for 1 h and subjected to immunocytochemistry for Atg16L1 and Ub (E and F) or LC3 and Ub (I and J). The percentages of Atg16L1-positive (G and H) or LC3-positive per Ub-positive bacteria were enumerated (K and L). At least 50 Salmonella were counted. The average ± SD is shown for three independent experiments. Statistical analysis was performed by Student's t test. *, P < 0.05; NS, not significant. Bar, 5 µm.


Figure 5 . ( A ) Mapping of the Atg16L1 - binding site on FIP200 . Yeast two - hybrid mapping experiments showing that Atg16L1 binds to both the N - terminal and C - terminal coiled - coil regions of FIP200 . ( B ) Mapping of the FIP200 - binding site within Atg16L1 . Yeast two - hybrid mapping experiments showing that the 1 - 840 fragment of FIP200 binds to the C - terminal 247 - 607 region , while the 1276 - 1591 fragment of FIP200 binds to the N - terminal 1 - 264 region of Atg16L1 . ( C and D ) Additional yeast two - hybrid mapping experiments showing that the binding site of the FIP200 1276 - 1591 fragment maps to residues 239 - 246 of Atg16L1 . White lines divide images derived from the same plate , and red lines divide images from different plates . ( E ) The N - terminal 1 - 249 fragment of Atg16L1 is sufficient to interact with FIP200 . Full - length ( lanes 1 and 2 ) or the N - terminal 1 - 249 fragments ( lanes 3 and 4 ) of Myc - Atg16L1 coprecipitations with empty vector controls ( lanes 1 and 3 ) or OSF - FIP200 1276 - 1591 ( lanes 2 and 4 ) . ( F ) Co - precipitation experiments confirming that OSF - FIP200 1276 - 1591 coprecipitates with wild - type ( WT ) Myc - Atg16L1 ( 1 - 249 ) fragments , but not with analogous fragments containing a mutant FIP200 - binding site ( Myc - Atg16L1 ΔWD 239 - 242A ) .

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

Figure 5.(A) Mapping of the Atg16L1-binding site on FIP200. Yeast two-hybrid mapping experiments showing that Atg16L1 binds to both the N-terminal and C-terminal coiled-coil regions of FIP200.(B) Mapping of the FIP200-binding site within Atg16L1. Yeast two-hybrid mapping experiments showing that the 1-840 fragment of FIP200 binds to the C-terminal 247-607 region, while the 1276-1591 fragment of FIP200 binds to the N-terminal 1-264 region of Atg16L1.(C and D) Additional yeast two-hybrid mapping experiments showing that the binding site of the FIP200 1276-1591 fragment maps to residues 239-246 of Atg16L1. White lines divide images derived from the same plate, and red lines divide images from different plates.(E) The N-terminal 1-249 fragment of Atg16L1 is sufficient to interact with FIP200. Full-length (lanes 1 and 2) or the N-terminal 1-249 fragments (lanes 3 and 4) of Myc-Atg16L1 coprecipitations with empty vector controls (lanes 1 and 3) or OSF-FIP200 1276-1591 (lanes 2 and 4).(F) Co-precipitation experiments confirming that OSF-FIP200 1276-1591 coprecipitates with wild-type (WT) Myc-Atg16L1 (1-249) fragments, but not with analogous fragments containing a mutant FIP200-binding site (Myc-Atg16L1 ΔWD 239-242A).


Figure 6 . ( B - E ) Atg16L1 - Δ / Δ MEFs stably expressing the indicated constructs were infected with Salmonella ( MOI = 10 ) for 1 h and then analyzed by immunocytochemistry for LC3 ( B ) or Atg16L1 ( D ) . Bar , 5 µm . The percentage of LC3 - or Atg16L1 - positive Salmonella per Ub - positive Salmonella was enumerated by fluorescence microscopy ( C and E ) . At least 50 bacteria were counted . The average ± SD is shown for three independent experiments . ( F ) Atg16L1 - Δ / Δ MEFs stably expressing the indicated constructs were cultured in growth medium ( Fed ) or EBSS ( Starved ) for 1 h and then subjected to immunocytochemistry using an anti - LC3 antibody . The number of LC3 puncta in each cell was counted for more than 50 cells . The average ± SD is shown for three independent experiments .

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

Figure 6.(B-E) Atg16L1-Δ/Δ MEFs stably expressing the indicated constructs were infected with Salmonella (MOI = 10) for 1 h and then analyzed by immunocytochemistry for LC3 (B) or Atg16L1 (D). Bar, 5 µm. The percentage of LC3- or Atg16L1-positive Salmonella per Ub-positive Salmonella was enumerated by fluorescence microscopy (C and E). At least 50 bacteria were counted. The average ± SD is shown for three independent experiments.(F) Atg16L1-Δ/Δ MEFs stably expressing the indicated constructs were cultured in growth medium (Fed) or EBSS (Starved) for 1 h and then subjected to immunocytochemistry using an anti-LC3 antibody. The number of LC3 puncta in each cell was counted for more than 50 cells. The average ± SD is shown for three independent experiments.


Figure 2 ( A ) ELISA titration curves ( OD at 450nm ) of Fab3 ( blue ; left panel ) and Fab71 ( blue ; right panel ) against mouse recPrP23 ‑ 231 compared to their best affinity - matured versions , Fab83 and Fab101 Curves were fitted by non - linear least - squares regression analysis for statistical analysis : log ( agonist ) vs . response . ( B - E ) FT - peptide competition ELISA to map the epitopes of the indicated Fabs . Peptides that strongly inhibit the binding of the Fabs to recPrP23 ‑ 231 are indicated by their residue numbers in the PrP sequence and reflect the respective binding epitopes . Positive control : recPrP23 ‑ 231 ; negative controls : Neu , BSA , and the secondary antibody ( 2 ° Ab ) .

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

Figure 2 (A) ELISA titration curves (OD at 450nm) of Fab3 (blue; left panel) and Fab71 (blue; right panel) against mouse recPrP23‑231 compared to their best affinity-matured versions, Fab83 and Fab101 Curves were fitted by non-linear least-squares regression analysis for statistical analysis: log(agonist) vs. response.  (B-E) FT-peptide competition ELISA to map the epitopes of the indicated Fabs. Peptides that strongly inhibit the binding of the Fabs to recPrP23‑231 are indicated by their residue numbers in the PrP sequence and reflect the respective binding epitopes. Positive control: recPrP23‑231; negative controls: Neu, BSA, and the secondary antibody (2° Ab).


Figure 3 ( A ) Fluorescence micrographs of tga20 slices chronically exposed to prions and cultured in the presence of either Fab3 ( CC123 ‑ 50 ) , Fab71 ( OR51 ‑ 91 ) or Fab1 ( GD ) for 45 days . Scale bar : 500 μm . ( B ) Same as ( A ) , but in the presence of the affinity - maturated antibodies . ( C ) NeuN immunofluorescence coverage of prion - infected slices cultured in the presence of the Fabs . Antibodies targeting the CC123 ‑ 50 and the OR51 ‑ 91 afforded protection against neurodegeneration . NBH : non - infectious brain homogenate . Each dot represents a cerebellar slice for all treatment groups . ( D ) Western blot analysis of COCS lysates . Prion - infected slices treated with Fab100 , but not with Fab83 and Fab25 showed reduced levels of PrPSc . n = 3 biological replicates .

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

Figure 3 (A) Fluorescence micrographs of tga20 slices chronically exposed to prions and cultured in the presence of either Fab3 (CC123‑50), Fab71 (OR51‑91) or Fab1 (GD) for 45 days. Scale bar: 500 μm. (B) Same as (A), but in the presence of the affinity-maturated antibodies. (C) NeuN immunofluorescence coverage of prion-infected slices cultured in the presence of the Fabs. Antibodies targeting the CC123‑50 and the OR51‑91 afforded protection against neurodegeneration. NBH: non-infectious brain homogenate. Each dot represents a cerebellar slice for all treatment groups.  (D) Western blot analysis of COCS lysates. Prion-infected slices treated with Fab100, but not with Fab83 and Fab25 showed reduced levels of PrPSc. n = 3 biological replicates.


Figure 4 ( C ) Left : Heat map showing the binding specificities ( Z - scores ) of selected Fab71 human analogous antibodies to human recPrP23 ‑ 230 compared to the negative controls ( BSA and Neu ) . Right : Heat map representing the reactivity ( - logEC50 ) obtained from dose - dependent ELISA binding curves of the analogous antibodies to human recPrP23 ‑ 230 . Red : low reactivity ; blue : high reactivity .

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

Figure 4 (C) Left: Heat map showing the binding specificities (Z-scores) of selected Fab71 human analogous antibodies to human recPrP23‑230 compared to the negative controls (BSA and Neu). Right: Heat map representing the reactivity (-logEC50) obtained from dose-dependent ELISA binding curves of the analogous antibodies to human recPrP23‑230. Red: low reactivity; blue: high reactivity.


Figure 5 ( C ) Boxplot with half - violin plot displaying the distribution of autoantibody reactivity in 48 , 718 plasma samples . Twenty - seven samples over the reactivity threshold of - log ( EC50 ) ≥ 2 and fitting error < 20 % were considered as hits in the primary screen . Black dots correspond to single samples . The boxplot divides the data set into three quartiles . The box extends from the first to the third quartile values of the data set , with a line at the median . Whiskers show the range of the data ( minimum and maximum ) . ( E ) Heat map representation for the validation of the hits from the primary screen ( 21 out of 27 hits ) . Confirmed hits displayed selective reactivity against human recPrP23 ‑ 230 . Controls : Patients # 11 - 13 , ApoE4 and BSA . Red : high reactivity ; blue : low reactivity . ( F ) Heat maps representing autoantibody clonality of hits analyzed by ELISA . Autoantibodies were mostly constituted by lambda light chains ( λ ) , while the kappa light chains ( κ ) showed prevalence in the total IgG fraction . ( G ) Determination of antibody reactivity ( - log ( EC50 ) ) for three individuals at two different time points . Individuals were selected by displaying strong anti - PrP IgG reactivity in the first test ( Time 1 ) . Reactivity was maintained for even more than one year ( Time 2 ) . ( H ) Purified IgGs from patients with strong anti - PrP reactivity were tested against PrP variants and BSA ( negative control ) in dose - dependent ELISAs . Patient # 5 and # 6 showed binding mostly to the human recPrP121 ‑ 230 . Patient # 1 displayed a polyreactive pattern with binding to the human recPrP121 ‑ 230 and mouse recPrP23 - 110 .

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

Figure 5 (C) Boxplot with half-violin plot displaying the distribution of autoantibody reactivity in 48,718 plasma samples. Twenty-seven samples over the reactivity threshold of -log(EC50) ≥ 2 and fitting error < 20% were considered as hits in the primary screen. Black dots correspond to single samples. The boxplot divides the data set into three quartiles. The box extends from the first to the third quartile values of the data set, with a line at the median. Whiskers show the range of the data (minimum and maximum).  (E) Heat map representation for the validation of the hits from the primary screen (21 out of 27 hits). Confirmed hits displayed selective reactivity against human recPrP23‑230. Controls: Patients #11-13, ApoE4 and BSA. Red: high reactivity; blue: low reactivity.  (F) Heat maps representing autoantibody clonality of hits analyzed by ELISA. Autoantibodies were mostly constituted by lambda light chains (λ), while the kappa light chains (κ) showed prevalence in the total IgG fraction.  (G) Determination of antibody reactivity (-log(EC50)) for three individuals at two different time points. Individuals were selected by displaying strong anti-PrP IgG reactivity in the first test (Time 1). Reactivity was maintained for even more than one year (Time 2).  (H) Purified IgGs from patients with strong anti-PrP reactivity were tested against PrP variants and BSA (negative control) in dose-dependent ELISAs. Patient #5 and #6 showed binding mostly to the human recPrP121‑230. Patient #1 displayed a polyreactive pattern with binding to the human recPrP121‑230 and mouse recPrP23-110.


Figure 1 ( B , Performance in the multi - trail inverted screen test is impaired in ( B ) SUR2 - STOP1149 ( WT n = 9 [ 3 male ; 6 female ] ; STOP n = 9 [ 3 male ; 6 female ] ; p = 0 . 011 according to Mann Whitney U test ) Data information : Bars show mean ( ± SEM ) with individual biological replicates shown as circles / squares . * denotes p value < α = 0 . 05 for all panels . C ) Performance in the multi - trail inverted screen test is impaired in ( C ) SUR2 - STOP475 mice ( WT n = 9 [ 4 male ; 5 female ] ; STOP n = 15 [ 5 male ; 10 female ] ; p = 2 . 9x10 - 4 according to Mann Whitney U test ) . Data information : Bars show mean ( ± SEM ) with individual biological replicates shown as circles / squares . * denotes p value < α = 0 . 05 for all panels . ( D ) Treadmill exercise tolerance is also reduced in SUR2 - STOP475 mice ( WT n = 4 [ 2 male ; 2 female ] ; STOP n = 8 [ 4 male ; 4 female ] ; p = 0 . 008 according to Mann Whitney U test ) . Treadmill workload tolerated was also reduced ( WT 18 . 1 ± 2 . 2 vs STOP475 9 . 2 ± 0 . 9 J ; p = 0 . 008 from Man Whitney U test ) , mean mouse body weight was WT 22 . 1 ± 0 . 9 vs STOP475 21 . 9 ± 0 . 6 g . Data information : Bars show mean ( ± SEM ) with individual biological replicates shown as circles / squares . * denotes p value < α = 0 . 05 for all panels .

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

Figure 1(B, Performance in the multi-trail inverted screen test is impaired in (B) SUR2-STOP1149 (WT n = 9 [3 male; 6 female]; STOP n = 9 [3 male; 6 female]; p = 0.011 according to Mann Whitney U test) Data information: Bars show mean (± SEM) with individual biological replicates shown as circles/squares. * denotes p value < α = 0.05 for all panels.C) Performance in the multi-trail inverted screen test is impaired in (C) SUR2-STOP475 mice (WT n = 9 [4 male; 5 female]; STOP n = 15 [5 male; 10 female]; p = 2.9x10-4 according to Mann Whitney U test). Data information: Bars show mean (± SEM) with individual biological replicates shown as circles/squares. * denotes p value < α = 0.05 for all panels.(D)Treadmill exercise tolerance is also reduced in SUR2-STOP475 mice (WT n = 4 [2 male; 2 female]; STOP n = 8 [4 male; 4 female]; p = 0.008 according to Mann Whitney U test). Treadmill workload tolerated was also reduced (WT 18.1 ± 2.2 vs STOP475 9.2 ± 0.9 J; p = 0.008 from Man Whitney U test), mean mouse body weight was WT 22.1 ± 0.9 vs STOP475 21.9 ± 0.6 g. Data information: Bars show mean (± SEM) with individual biological replicates shown as circles/squares. * denotes p value < α = 0.05 for all panels.