( G ) Viability of six PTEN - null VS five PTEN - WT TNBC cell lines - not carrying other known mutations in PIK3CA , PIK3CB or PIK3R1 genes - treated with PI3Kβi ( AZD8186 90 nM ) , EGFRi ( gefitinib 3 μM ) alone or in combination for 6 days . Mean of 3 independent experiments ± SD . Statistical significance of two - tailed unpaired student t - test in PTEN - null PI3Kbi versus PI3Kbi + EGFRi * * P = 0 . 0059 , PTEN - null EGFRi versus PI3Kbi + EGFRi * * P = 0 . 0047 , PTEN - null PI3Kbi + EGFRi versus PTEN - WT PI3Kbi + EGFRi * P = 0 . 0459 , PTEN - WT PI3Kbi versus PI3Kbi + EGFRi * P = 0 . 0108 , PTEN - WT EGFRi versus PI3Kbi + EGFRi n . s . P = 0 . 1926 . PTEN - null cell lines used in the experiments were : MDA - MB - 468 , HCC70 , HCC1937 , HCC38 , HCC1395 and BT - 549 ; PTEN - WT cell lines were : MDA - MB - 157 , MDA - MB - 231 , HCC1187 , HCC1428 and HCC1806 .

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(G) Viability of six PTEN-null VS five PTEN-WT TNBC cell lines - not carrying other known mutations in PIK3CA, PIK3CB or PIK3R1 genes - treated with PI3Kβi (AZD8186 90 nM), EGFRi (gefitinib 3 μM) alone or in combination for 6 days. Mean of 3 independent experiments ± SD. Statistical significance of two-tailed unpaired student t-test in PTEN-null PI3Kbi versus PI3Kbi+EGFRi **P=0.0059, PTEN-null EGFRi versus PI3Kbi+EGFRi **P=0.0047, PTEN-null PI3Kbi+EGFRi versus PTEN-WT PI3Kbi+EGFRi *P=0.0459, PTEN-WT PI3Kbi versus PI3Kbi+EGFRi *P=0.0108, PTEN-WT EGFRi versus PI3Kbi+EGFRi n.s. P=0.1926. PTEN-null cell lines used in the experiments were: MDA-MB-468, HCC70, HCC1937, HCC38, HCC1395 and BT-549; PTEN-WT cell lines were: MDA-MB-157, MDA-MB-231, HCC1187, HCC1428 and HCC1806.


( H ) Synergy score for combinations of serial dilutions of PI3Kβi ( AZD8186 ) plus EGFRi ( gefitinib ) tested on six PTEN - null and five PTEN - WT TNBC cell lines for 6 days in three independent experiments . The score was obtained analysing the viability data through the software Chalice Analyser . Mean of the synergy scores ± SD . Statistical significance of Mann Whitney two - tailed test * P = 0 . 0303 .

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(H) Synergy score for combinations of serial dilutions of PI3Kβi (AZD8186) plus EGFRi (gefitinib) tested on six PTEN-null and five PTEN-WT TNBC cell lines for 6 days in three independent experiments. The score was obtained analysing the viability data through the software Chalice Analyser. Mean of the synergy scores ± SD. Statistical significance of Mann Whitney two-tailed test *P=0.0303.


( I ) Patient samples from METABRIC dataset classified as TNBCs were assigned to the groups " PTEN - low " when falling in the lower quartile for PTEN expression , " PTEN - mut " when harbouring a non - synonymous mutation on PTEN gene , " PTEN - WT " in all other cases . Comparison of the expression of EGFR between the PTEN low or mut and the PTEN - WT groups . Data presented in a box and whisker plot with the central band indicating the median , the upper and lower extremes of the box or hinge being the third and first quartiles respectively and the whiskers extending to the most extreme data values Mean ± SD . P value calculated by unpaired t - test .

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(I) Patient samples from METABRIC dataset classified as TNBCs were assigned to the groups "PTEN-low" when falling in the lower quartile for PTEN expression, "PTEN-mut" when harbouring a non-synonymous mutation on PTEN gene, "PTEN-WT" in all other cases. Comparison of the expression of EGFR between the PTEN low or mut and the PTEN-WT groups. Data presented in a box and whisker plot with the central band indicating the median, the upper and lower extremes of the box or hinge being the third and first quartiles respectively and the whiskers extending to the most extreme data values Mean ± SD. P value calculated by unpaired t-test.


( A ) Tumor volume of MDA - MB - 468 mammary fat - pad xenografts treated with vehicle , AZD8186 ( 50 mg / kg , og twice / day ) , erlotinib ( 50 mg / kg IP once / day ) alone or in combination ( 5 - 6 mice per group , mean ± SEM ) . Statistical significance of two - way ANOVA statistical test * * P = 0 . 0028 and * * * P < 0 . 0001 . ( B ) Tumor volume of HCC - 70 mammary fat - pad xenografts treated with vehicle , AZD8186 ( 150 mg / kg , og once / day ) , erlotinib ( 50 mg / kg IP once / day ) alone or in combination ( 6 - 7 mice per group , mean ± SEM ) . Growth curves were compared using two - way ANOVA statistical test . Statistical significance of two - way ANOVA statistical test * * * P = 0 . 0006 and * * * * P < 0 . 0001 .

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(A) Tumor volume of MDA-MB-468 mammary fat-pad xenografts treated with vehicle, AZD8186 (50 mg/kg, og twice/day), erlotinib (50 mg/kg IP once/day) alone or in combination (5-6 mice per group, mean±SEM). Statistical significance of two-way ANOVA statistical test **P=0.0028 and ***P<0.0001. (B) Tumor volume of HCC-70 mammary fat-pad xenografts treated with vehicle, AZD8186 (150 mg/kg, og once/day), erlotinib (50 mg/kg IP once/day) alone or in combination (6-7 mice per group, mean±SEM). Growth curves were compared using two-way ANOVA statistical test. Statistical significance of two-way ANOVA statistical test ***P=0.0006 and ****P<0.0001.


( C ) Waterfall representation of changes in the volume of individual HCC - 70 tumors during the treatment . Statistical comparison between different treatment groups by Mann - Whitney test * * P = 0 . 0023 and * * * P = 0 . 0006 .

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(C) Waterfall representation of changes in the volume of individual HCC-70 tumors during the treatment. Statistical comparison between different treatment groups by Mann-Whitney test **P=0.0023 and ***P=0.0006.


( D ) Change in the body weight of mice harbouring HCC70 xenograft tumors ( 6 - 7 mice per group , mean ± SEM ) during 21 days of treatment .

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(D) Change in the body weight of mice harbouring HCC70 xenograft tumors (6-7 mice per group, mean±SEM) during 21 days of treatment.


( E ) A cell line derived from a mammary tumor spontaneously developed in a Wap - cre : Ptenfl / fl : Tp53fl / fl mouse was cloned and injected in the mammary fat pad of syngeneic C57BL6 / J recipient mice . Tumours grew in 12 out of 35 transplanted mice and only these tumours were selected for the treatments described in the figure . When tumours reached an average volume of 100mm3 , they were treated with vehicle or a combination of AZD8186 ( 150mg / kg , og once / day ) and erlotinib ( 50mg / kg IP once / day ) . Tumours were then measured during the treatment ( 6 mice per group , mean ± SEM ) . Statistical significance of two - way ANOVA statistical test * * * * P < 0 . 0001 .

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(E) A cell line derived from a mammary tumor spontaneously developed in a Wap-cre:Ptenfl/fl:Tp53fl/fl mouse was cloned and injected in the mammary fat pad of syngeneic C57BL6/J recipient mice. Tumours grew in 12 out of 35 transplanted mice and only these tumours were selected for the treatments described in the figure. When tumours reached an average volume of 100mm3, they were treated with vehicle or a combination of AZD8186 (150mg/kg, og once/day) and erlotinib (50mg/kg IP once/day). Tumours were then measured during the treatment (6 mice per group, mean±SEM). Statistical significance of two-way ANOVA statistical test ****P<0.0001.


( A ) MDA - MB - 468 cells were treated for 24h with vehicle , PI3Kβi ( AZD8186 250 nM ) , EGFRi ( gefitinib 3 μM ) or cetuximab ( 100 μg / ml ) , alone or in combination . The cell lysates were probed with the indicated antibodies .

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(A) MDA-MB-468 cells were treated for 24h with vehicle, PI3Kβi (AZD8186 250 nM), EGFRi (gefitinib 3 μM) or cetuximab (100 μg/ml), alone or in combination. The cell lysates were probed with the indicated antibodies.


( B ) MDA - MB - 468 cells were treated for 24h with vehicle , AKTi ( MK2206 450 nM ) , gefitinib ( 3 μM ) or cetuximab ( 100 μg / ml ) , alone or in the indicated combinations . The cell lysates were probed with the indicated antibodies .

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(B) MDA-MB-468 cells were treated for 24h with vehicle, AKTi (MK2206 450 nM), gefitinib (3 μM) or cetuximab (100 μg/ml), alone or in the indicated combinations. The cell lysates were probed with the indicated antibodies.


( C , D ) HCC70 ( C ) and ZR - 75 - 1 ( D ) parental cells or PI3Kβi - Res ( derivative cells with acquired resistance to AZD8186 ) , or AKTi - Res ( acquired resistance models to MK2206 ) were treated with vehicle , PI3Kβi ( AZD8186 250 nM ) or AKTi ( MK2206 1 μM ) for 24 hours . The whole cell lysates were then probed with the indicated antibodies . Spliced images of parental and resistant paired samples were taken from the same original blot and blots showing P - S6 and S6 tot in ( D ) have been cut and reassembled for figure purposes .

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(C, D) HCC70 (C) and ZR-75-1 (D) parental cells or PI3Kβi-Res (derivative cells with acquired resistance to AZD8186), or AKTi-Res (acquired resistance models to MK2206) were treated with vehicle, PI3Kβi (AZD8186 250 nM) or AKTi (MK2206 1 μM) for 24 hours. The whole cell lysates were then probed with the indicated antibodies. Spliced images of parental and resistant paired samples were taken from the same original blot and blots showing P-S6 and S6 tot in (D) have been cut and reassembled for figure purposes.


( E ) HCC70 parental , PI3Kβi - Res or AKTi - Res cells were treated with vehicle , AKTi ( MK2206 250 nM ) or gefitinib ( 3 μM ) , alone or in the indicated combinations . The cell lysates were probed with the indicated antibodies .

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(E) HCC70 parental, PI3Kβi-Res or AKTi-Res cells were treated with vehicle, AKTi (MK2206 250 nM) or gefitinib (3 μM), alone or in the indicated combinations. The cell lysates were probed with the indicated antibodies.


( F ) HCC70 MK res ( acquired resistance models to MK2206 ) were treated with serial dilutions of gefitinib , alone or in combination with MK2206 ( 810 nM ) , as indicated , and viability measured after 4 days of treatment . Average ± SD of triplicates and representative of two independent experiments .

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(F) HCC70 MK res (acquired resistance models to MK2206) were treated with serial dilutions of gefitinib, alone or in combination with MK2206 (810 nM), as indicated, and viability measured after 4 days of treatment. Average ± SD of triplicates and representative of two independent experiments.


( G ) HCC70 AZD res ( acquired resistance models to AZD8186 ) were treated with serial dilutions of gefitinib , alone or in combination with AZD8186 ( 270 nM ) , as indicated , and viability measured after 4 days . Average ± SD of triplicates and representative of two independent experiments .

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(G) HCC70 AZD res (acquired resistance models to AZD8186) were treated with serial dilutions of gefitinib, alone or in combination with AZD8186 (270 nM), as indicated, and viability measured after 4 days. Average ± SD of triplicates and representative of two independent experiments.


( H , I ) p110β co - immunoprecipitates with EGFR in MDA - MB - 468 ( H ) and in HCC70 ( I ) . Cells were pre - treated with different inhibitors , including vehicle , pan - PI3Ki GDC0941 ( 1 μM for MDA - MB - 468 or 0 . 5 μM for HCC70 ) , EGFRi ( gefitinib 3 μM ) , PI3Kβi ( AZD8186 250 nM ) or a combination of EGFRi and PI3Kβi . Cell lysates were incubated with IgG control or anti - EGFR antibody and the immuno - complexes or the total lysates were immune - blotted with the indicated antibodies .

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(H,I) p110β co-immunoprecipitates with EGFR in MDA-MB-468 (H) and in HCC70 (I). Cells were pre-treated with different inhibitors, including vehicle, pan-PI3Ki GDC0941 (1 μM for MDA-MB-468 or 0.5 μM for HCC70), EGFRi (gefitinib 3 μM), PI3Kβi (AZD8186 250 nM) or a combination of EGFRi and PI3Kβi. Cell lysates were incubated with IgG control or anti-EGFR antibody and the immuno-complexes or the total lysates were immune-blotted with the indicated antibodies.


( J ) EGF - induced increase in phospho - AKT is dependent on p110β kinase activity . MDA - MB - 468 were starved in 0 % FBS and pre - treated with vehicle or PI3Kβi ( AZD8186 250 nM ) for 1h . Cell lysates were probed with the indicated antibodies . Phospho - AKT and pan - AKT bands were quantified by the use of ImageLite software : the ratio of phospho - AKT to pan - AKT normalised to the control ( left hand lane ) is shown .

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(J) EGF-induced increase in phospho-AKT is dependent on p110β kinase activity. MDA-MB-468 were starved in 0% FBS and pre-treated with vehicle or PI3Kβi (AZD8186 250 nM) for 1h. Cell lysates were probed with the indicated antibodies. Phospho-AKT and pan-AKT bands were quantified by the use of ImageLite software: the ratio of phospho-AKT to pan-AKT normalised to the control (left hand lane) is shown.


( A Results of CRISPR - Cas9 screening in combination with AZD8186 100nM ( A ) The dot - plots show for each gene knocked - out by sgRNAs the fold change ( log2 ) between treated conditions ( AZD8186 and vehicle in the fluorescence signal ( anti - phosphoS6 immunofluorescence ) versus the p - value of the difference calculated by two sided t - test . The plots represent means of biological triplicates . Genes for which it was calculated a p < 0 . 0001 and Log2 ( fold change ) > 0 . 25 are reported and highlighted in green ; genes having a 0 . 0001 0 . 25 are reported and shown in red . Gefitinib combined with AZD8186 represents the positive control of the experiment ( A ) . < 0 . 05 >

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(A Results of CRISPR-Cas9 screening in combination with AZD8186 100nM (A) The dot-plots show for each gene knocked-out by sgRNAs the fold change (log2) between treated conditions (AZD8186 and vehicle in the fluorescence signal (anti-phosphoS6 immunofluorescence) versus the p-value of the difference calculated by two sided t-test. The plots represent means of biological triplicates. Genes for which it was calculated a p<0.0001 and Log2 (fold change)>0.25 are reported and highlighted in green; genes having a 0.0001 0.25 are reported and shown in red. Gefitinib combined with AZD8186 represents the positive control of the experiment (A). <0.05>


B ) Results of CRISPR - Cas9 screening in combination with GDC0941 400nM ( B ) . The dot - plots show for each gene knocked - out by sgRNAs the fold change ( log2 ) between treated conditions GDC0941 , and vehicle in the fluorescence signal ( anti - phosphoS6 immunofluorescence ) versus the p - value of the difference calculated by two sided t - test . The plots represent means of biological triplicates . Genes for which it was calculated a p < 0 . 0001 and Log2 ( fold change ) > 0 . 25 are reported and highlighted in green ; genes having a 0 . 0001 0 . 25 are reported and shown in red . < 0 . 05 >

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B) Results of CRISPR-Cas9 screening in combination with GDC0941 400nM (B). The dot-plots show for each gene knocked-out by sgRNAs the fold change (log2) between treated conditions GDC0941, and vehicle in the fluorescence signal (anti-phosphoS6 immunofluorescence) versus the p-value of the difference calculated by two sided t-test. The plots represent means of biological triplicates. Genes for which it was calculated a p<0.0001 and Log2 (fold change)>0.25 are reported and highlighted in green; genes having a 0.0001 0.25 are reported and shown in red. <0.05>


( C ) Box & Whisker plot showing the fold change in fluorescence signal between vehicle and GDC0941 - treated conditions for MDA - MB - 468 cells transduced with non - target Control , GNB2 or GNG5 sgRNAs ( N = 2 or 3 ) . Data presented in a box and whisker plot with the central band indicating the median , the upper and lower extremes of the box or hinge being the third and first quartiles respectively and the whiskers extending to the most extreme data values within 1 . 5 times the inter - quartile range . Statistical significance of unpaired t - test * * * * P < 0 . 0001 .

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(C) Box & Whisker plot showing the fold change in fluorescence signal between vehicle and GDC0941-treated conditions for MDA-MB-468 cells transduced with non-target Control, GNB2 or GNG5 sgRNAs (N=2 or 3). Data presented in a box and whisker plot with the central band indicating the median, the upper and lower extremes of the box or hinge being the third and first quartiles respectively and the whiskers extending to the most extreme data values within 1.5 times the inter-quartile range. Statistical significance of unpaired t-test ****P<0.0001.


( D ) Biochemical analysis of GNB2 KO cells . Cell lysates of MDA - MB - 468 parental cells and three MDA - MB - 468 GNB2 KO clones were probed with the indicated antibodies . Quantification of the bands was performed by ImageLite software .

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(D) Biochemical analysis of GNB2 KO cells. Cell lysates of MDA-MB-468 parental cells and three MDA-MB-468 GNB2 KO clones were probed with the indicated antibodies. Quantification of the bands was performed by ImageLite software.


( A ) MDA - MB - 468 parental cells and three MDA - MB - 468 GNB2 KO clones were treated with vehicle , GDC0941 1 μM , gefitinib 3 μM or lapatinib 1 μM for 24 hours . The cell lysates were probed with the indicated antibodies .

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(A) MDA-MB-468 parental cells and three MDA-MB-468 GNB2 KO clones were treated with vehicle, GDC0941 1 μM, gefitinib 3 μM or lapatinib 1 μM for 24 hours. The cell lysates were probed with the indicated antibodies.


( B - D ) Viability assays of MDA - MB - 468 parental cells and three MDA - MB - 468 GNB2 KO clones treated with serial dilutions of the indicated drugs for six days . Mean ± SD of triplicates and representative of two or three independent experiments .

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(B-D) Viability assays of MDA-MB-468 parental cells and three MDA-MB-468 GNB2 KO clones treated with serial dilutions of the indicated drugs for six days. Mean ± SD of triplicates and representative of two or three independent experiments.


( E ) Patient samples from METABRIC dataset classified as TNBCs ( N = 299 ) were assigned to the groups " PTEN - low " when falling in the lower quartile for PTEN expression , " PTEN - mut " when harbouring a non - synonymous mutation on PTEN gene , " PTEN - WT " in all other cases . Comparison of the expression of GNB2 between the PTEN low or mut and the PTEN - WT groups . Box & Whisker plot ( Median / IQR / 1 . 5 * IQR Whiskers ) . P value calculated by unpaired t - test .

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(E) Patient samples from METABRIC dataset classified as TNBCs (N=299) were assigned to the groups "PTEN-low" when falling in the lower quartile for PTEN expression, "PTEN-mut" when harbouring a non-synonymous mutation on PTEN gene, "PTEN-WT" in all other cases. Comparison of the expression of GNB2 between the PTEN low or mut and the PTEN-WT groups. Box & Whisker plot (Median/IQR/1.5*IQR Whiskers). P value calculated by unpaired t-test.


( A ) Schematic of the results from the drug screening with compounds targeting GPCR signaling . MDA - MB - 468 cells were treated with the compounds of the GPCR - targeted library at three different concentrations ( 0 . 1 , 1 or 10 μM ) in combination with vehicle , GDC0941 450 nM or lapatinib 1 μM . The pS6 signal was measured by IF after 24 hours of treatment and normalized to DAPI . Z scores of the normalized fluorescence values for each drug measured in the presence of vehicle , GDC0941 or lapatinib are reported on the y axis or on the left or right x axis of the dot plot , respectively . Readings acquired following treatment with the three different concentrations of the GPCR - targeted drugs are reported in 3 different colours . Dots corresponding to vorapaxar treatments at the three different concentrations are highlighted in the plot . The values reported are mean of a biological triplicate of the experiment .

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(A) Schematic of the results from the drug screening with compounds targeting GPCR signaling. MDA-MB-468 cells were treated with the compounds of the GPCR-targeted library at three different concentrations (0.1, 1 or 10 μM) in combination with vehicle, GDC0941 450 nM or lapatinib 1 μM. The pS6 signal was measured by IF after 24 hours of treatment and normalized to DAPI. Z scores of the normalized fluorescence values for each drug measured in the presence of vehicle, GDC0941 or lapatinib are reported on the y axis or on the left or right x axis of the dot plot, respectively. Readings acquired following treatment with the three different concentrations of the GPCR-targeted drugs are reported in 3 different colours. Dots corresponding to vorapaxar treatments at the three different concentrations are highlighted in the plot. The values reported are mean of a biological triplicate of the experiment.


( B ) MDA - MB - 468 were treated for 24 h with vehicle , vorapaxar ( 10 μM ) , GDC0941 ( 1 μM ) or lapatinib ( 1 μM ) alone or in the indicated combinations . The cell lysates were probed with the indicated antibodies .

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(B) MDA-MB-468 were treated for 24 h with vehicle, vorapaxar (10 μM), GDC0941 (1 μM) or lapatinib (1 μM) alone or in the indicated combinations. The cell lysates were probed with the indicated antibodies.


( C ) MDA - MB - 468 parental cells or GNB2 KO clones were starved and treated with scramble or PAR1 activating peptide for 5 minutes , alone or in combination with vorapaxar . The cell lysates were probed with the indicated antibodies . Phospho - AKT , pan - AKT , phospho - ERK1 / 2 and pan - ERK1 / 2 bands were quantified by the use of ImageLite software : the ratio of phospho - AKT to pan - AKT and phospho - ERK1 / 2 to pan - ERK1 / 2 normalised to the control peptide - treated conditions for WT and GNB2 KO cells ( first and forth lanes , respectively ) is shown .

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(C) MDA-MB-468 parental cells or GNB2 KO clones were starved and treated with scramble or PAR1 activating peptide for 5 minutes, alone or in combination with vorapaxar. The cell lysates were probed with the indicated antibodies. Phospho-AKT, pan-AKT, phospho-ERK1/2 and pan-ERK1/2 bands were quantified by the use of ImageLite software: the ratio of phospho-AKT to pan-AKT and phospho-ERK1/2 to pan-ERK1/2 normalised to the control peptide-treated conditions for WT and GNB2 KO cells (first and forth lanes, respectively) is shown.


( D ) p - S6 / loading control signal from western blots experiments in which a panel of six TNBC PTEN - null cell lines and HCC70 cells that acquired resistance to AZD8186 or MK2206 were treated with vehicle , vorapaxar ( 10 μM ) , GDC0941 ( 1 μM ) or lapatinib ( 1 μM ) alone or in the indicated combinations . The values were normalised to vehicle treatment for all cell lines . Mean of 2 independent experiments ± SD . P values calculated by two - tailed paired student t - test * P = 0 . 0235 and * * P = 0 . 002 . Cell lines used in the experiments were : MDA - MB - 468 , HCC70 , HCC1937 , HCC38 , HCC1395 , BT - 549 , HCC70 AZD8186 - resistant and HCC70 MK2206 - resistant .

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(D) p-S6/loading control signal from western blots experiments in which a panel of six TNBC PTEN-null cell lines and HCC70 cells that acquired resistance to AZD8186 or MK2206 were treated with vehicle, vorapaxar (10 μM), GDC0941 (1 μM) or lapatinib (1 μM) alone or in the indicated combinations. The values were normalised to vehicle treatment for all cell lines. Mean of 2 independent experiments ± SD. P values calculated by two-tailed paired student t-test *P=0.0235 and **P=0.002. Cell lines used in the experiments were: MDA-MB-468, HCC70, HCC1937, HCC38, HCC1395, BT-549, HCC70 AZD8186-resistant and HCC70 MK2206-resistant.


( E ) Long - term proliferation assay of MDA - MB - 468 cells treated with vorapaxar ( 5 or 2 . 5 μM ) , GDC0941 ( 1 μM ) or lapatinib ( 1 μM ) , alone or in the indicated combinations . Cells were treated for two weeks and stained by crystal violet . One representative experiment of three is shown .

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(E) Long-term proliferation assay of MDA-MB-468 cells treated with vorapaxar (5 or 2.5 μM), GDC0941 (1 μM) or lapatinib (1 μM), alone or in the indicated combinations. Cells were treated for two weeks and stained by crystal violet. One representative experiment of three is shown.


b , Carboxy - terminal V5immunoblot of full - length ( 68 kDa ) and cleaved ( 36 kDa ) ATG16L1β . Silver stain ( bottom gels ) depicts total protein . Data represent 3 independent experiments . IB , immunoblot .

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b, Carboxy-terminal V5immunoblot of full-length (68 kDa) and cleaved (36 kDa) ATG16L1β. Silver stain (bottom gels) depicts total protein. Data represent 3 independent experiments. IB, immunoblot.


c , TNF - α - mediated processing of humanATG16L1 in monocyte - derived macrophages . Scatter plot of immunoblot densitometry depicts a ratio of cleaved : full - length ATG16L1 ( Extended Data Fig . 2 ) . n = 7 donors .

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c, TNF-α-mediated processing of humanATG16L1 in monocyte-derived macrophages. Scatter plot of immunoblot densitometry depicts a ratio of cleaved:full-length ATG16L1 (Extended Data Fig. 2). n = 7 donors.


d , TNF - α - mediated processing of murineATG16L1 in macrophages . Immunoblot represents 5 independent experiments . Scatterplot represents data pooled from 5 independent experiments . WT , wild type .

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d, TNF-α-mediated processing of murineATG16L1 in macrophages. Immunoblot represents 5 independent experiments. Scatterplot represents data pooled from 5 independent experiments. WT, wild type.


a , HeLa cells transfected with control siRNA ( siCTL ) or siCaspase 8 were stimulated with 2 . 0 μM staurosporine or 20 ng ml − 1 TNF + 10 μg ml − 1 CHX .

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a, HeLa cells transfected with control siRNA (siCTL) or siCaspase 8 were stimulated with 2.0 μM staurosporine or 20 ng ml−1 TNF + 10 μg ml−1 CHX.


b , HeLa cells transfected with control siRNA or siCaspase 9 were stimulated as in a .

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b, HeLa cells transfected with control siRNA or siCaspase 9 were stimulated as in a.


c , HeLa cells transfected with control , caspase - 3 - or caspase - 7 - specific siRNAs were stimulated with 20 ng ml − 1 TNF + 10 & amp ; amp ; mgr ; g ml − 1 CHX .

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c, HeLa cells transfected with control, caspase-3- or caspase-7-specific siRNAs were stimulated with 20 ng ml−1 TNF + 10 &amp;mgr;g ml−1 CHX.


d , Wild - type or caspase - 3 - knockout macrophages were stimulated with 2 . 0 µM staurosporine or 20 ng ml − 1 TNF + 10 µg ml − 1 CHX . Data in a - c represent 2 independent experiments ; data in d represent 4 independent experiments .

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d, Wild-type or caspase-3-knockout macrophages were stimulated with 2.0 µM staurosporine or 20 ng ml−1 TNF + 10 µg ml−1 CHX. Data in a-c represent 2 independent experiments; data in d represent 4 independent experiments.


a , Intracellular staining for LC3 and ATG16L1 following glucose starvation in the presence of bafilomycin A1 . White mask in LC3 channel illustrates punctate LC3 staining , representing LC3 - II . Histogram depicts mean fluorescence intensity of 10 , 000 cells per sample . b , Quantification of mean ATG16L1 fluorescence intensity in a , n = 4 mice . Glu . , glucose .

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a, Intracellular staining for LC3 and ATG16L1 following glucose starvation in the presence of bafilomycin A1. White mask in LC3 channel illustrates punctate LC3 staining, representing LC3-II. Histogram depicts mean fluorescence intensity of 10,000 cells per sample. b, Quantification of mean ATG16L1 fluorescence intensity in a, n = 4 mice. Glu., glucose.


c , Immunoblot of phosphorylated ( p - ) AMPK & amp ; amp ; agr ; ( Thr 172 ) and lipidated LC3 ( LC3 - II ) .

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c, Immunoblot of phosphorylated (p-) AMPK&amp;agr; (Thr 172) and lipidated LC3 (LC3-II).


d , Quantification of mean LC3 - II area in murine macrophages , n = 4 mice .

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d, Quantification of mean LC3-II area in murine macrophages, n = 4 mice.


e , Caspase 3 activity in murine macrophages following glucose starvation , n = 3 mice . RLU , relative light units .

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e, Caspase 3 activity in murine macrophages following glucose starvation, n = 3 mice. RLU, relative light units.


f , Quantification of mean LC3 - II area in wild - type ( Casp3 + / + ) or Casp3 - knockout ( Casp3 − / − ) macrophages , n = 3 mice . Data in a - f represent 2 independent experiments .

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f, Quantification of mean LC3-II area in wild-type (Casp3+/+) or Casp3-knockout (Casp3−/−) macrophages, n = 3 mice. Data in a-f represent 2 independent experiments.


a , Quantification of ATG16L1 mean fluorescence intensity by image - based flow cytometry of 10 , 000 cells , n = 4 ( wild - type , WT ) , 5 ( T300A ) . Data represent 3 independent experiments . b , Quantification of caspase 3 activity upon glucose starvation of 2 × 104 cells , n = 5 . Data represent 2 independent experiments . c , Quantification of mean LC3 - II area of samples in a . Data represent 3 independent experiments .

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a, Quantification of ATG16L1 mean fluorescence intensity by image-based flow cytometry of 10,000 cells, n = 4 (wild-type, WT), 5 (T300A). Data represent 3 independent experiments. b, Quantification of caspase 3 activity upon glucose starvation of 2 × 104 cells, n = 5. Data represent 2 independent experiments. c, Quantification of mean LC3-II area of samples in a. Data represent 3 independent experiments.


d , Visualization of early autophagosomes by transmission electron micrographs ( TEM ) . Red arrows , early autophagosomes , characterized by vesicular structures with a double - membrane . Blue arrows , multivesicular lysosomal structures , identified by degraded intravesicular cargo . Micrographs representative of 10 - 12 images per donor , n = 4 ( WT ) , n = 3 ( T300A ) . A total of 29 ( WT ) and 26 ( T300A ) images containing autophagosomes was analysed . Histogram quantifies autophagic vesicles as a ratio of autophagosome : cytosolic area .

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d, Visualization of early autophagosomes by transmission electron micrographs (TEM). Red arrows, early autophagosomes, characterized by vesicular structures with a double-membrane. Blue arrows, multivesicular lysosomal structures, identified by degraded intravesicular cargo. Micrographs representative of 10-12 images per donor, n = 4 (WT), n = 3 (T300A). A total of 29 (WT) and 26 (T300A) images containing autophagosomes was analysed. Histogram quantifies autophagic vesicles as a ratio of autophagosome:cytosolic area.


a - c , Y . enterocolitica colony forming units ( c . f . u . ) in human and murine macrophages infected for 6 h ( 20 m . o . i . ) . Data in a are pooled from 4 independent cohorts of 3 or 4 donors per genotype , total n = 16 ( non - risk , WT ) , 15 ( T300A , risk ) . Data in b represent 3 independent experiments , n = 4 mice . Data in c represent 3 independent experiments , n = 6 .

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a-c, Y. enterocolitica colony forming units (c.f.u.) in human and murine macrophages infected for 6 h (20 m.o.i.). Data in a are pooled from 4 independent cohorts of 3 or 4 donors per genotype, total n = 16 (non-risk, WT), 15 (T300A, risk). Data in b represent 3 independent experiments, n = 4 mice. Data in c represent 3 independent experiments, n = 6.


d , TNF - α , IL - 1β and IL - 6 levels in culture media of WT and T316A macrophages infected with 20 m . o . i . Y . enterocolitica . Data represent 2 independent experiments , n = 4 mice .

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d, TNF-α, IL-1β and IL-6 levels in culture media of WT and T316A macrophages infected with 20 m.o.i. Y. enterocolitica. Data represent 2 independent experiments, n = 4 mice.


e , Ileum and colon histology ( haematoxylin and eosin staining ) of WT mice . Images are representative of 3 ( PBS ) and 8 ( Y . enterocolitica ) treated male mice .

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e, Ileum and colon histology (haematoxylin and eosin staining) of WT mice. Images are representative of 3 (PBS) and 8 (Y. enterocolitica) treated male mice.


f , Measurement of cytokine mRNA transcripts in mesenteric lymph nodes . Data are pooled from 2 independent experiments of 3 mice ( PBS ) and 7 mice ( Y . enterocolitica ) per experiment . Transcripts are normalized to Gapdh ( 2 − Dgr ; Ct ) .

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f, Measurement of cytokine mRNA transcripts in mesenteric lymph nodes. Data are pooled from 2 independent experiments of 3 mice (PBS) and 7 mice (Y. enterocolitica) per experiment. Transcripts are normalized to Gapdh (2−Dgr;Ct).


g , Y . enterocolitica c . f . u . in mesenteric lymph nodes of WT and T316A knock - in mice 48 h following oral gavage . Data represent 3 independent experiments , n = 9 male mice .

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g, Y. enterocolitica c.f.u. in mesenteric lymph nodes of WT and T316A knock-in mice 48 h following oral gavage. Data represent 3 independent experiments, n = 9 male mice.


D . Immunoblot analysis of caspase - 1 ( Casp - 1 ) , caspase - 11 ( Casp - 11 ) , gasdermin D ( GSDMD ) and GAPDH ( loading control ) in WT and mutant BMDMs left untreated or assessed 20 h after infection with M . catarrhalis ( Ne11 , MOI 100 ) . Data information : Data are from one three independent experiments (

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D. Immunoblot analysis of caspase-1 (Casp-1), caspase-11 (Casp-11), gasdermin D (GSDMD) and GAPDH (loading control) in WT and mutant BMDMs left untreated or assessed 20 h after infection with M. catarrhalis (Ne11, MOI 100). Data information: Data are from one three independent experiments (D


E . Release of IL - 1β , IL - 18 , TNF and LDH from BMDMs after treatment as in D . Data information : Each symbol represents an independent biological replicate ( E ) . * * * * P < 0 . 0001 ( one - way ANOVA with Dunnett ' s multiple - comparisons test ( E ) . Data are from three independent experiments E ; mean and s . e . m . in

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E. Release of IL-1β, IL-18, TNF and LDH from BMDMs after treatment as in D. Data information: Each symbol represents an independent biological replicate (E). **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test (E). Data are from three independent experiments E; mean and s.e.m. in E


G . IncuCyte live - imaging analysis of WT and Casp11 - / - BMDM viability after infection with M . catarrhalis as in D , or transfection with LPS . Data information : Data are pooled from two ( G ) independent experiments mean and s . e . m . in G ) .

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G. IncuCyte live-imaging analysis of WT and Casp11-/- BMDM viability after infection with M. catarrhalis as in D, or transfection with LPS. Data information: Data are pooled from two (G) independent experiments mean and s.e.m. in G).


A . Immunoblot analysis of caspase - 1 ( Casp - 1 ) , caspase - 11 ( Casp - 11 ) and gasdermin D ( GSDMD ) in WT , Nlrp3 - / - , Casp11 - / - and Aim2 - / - BMDMs left untreated ( Med . ) or 5 h after transfection with 5 µg of LOS from M . catarrhalis ( O35E ) or 5 µg of LPS from E . coli . Data information : Data are from three independent experiments ( A ,

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A. Immunoblot analysis of caspase-1 (Casp-1), caspase-11 (Casp-11) and gasdermin D (GSDMD) in WT, Nlrp3-/-, Casp11-/- and Aim2-/- BMDMs left untreated (Med.) or 5 h after transfection with 5 µg of LOS from M. catarrhalis (O35E) or 5 µg of LPS from E. coli. Data information: Data are from three independent experiments (A,


B . Release of IL - 1β , IL - 18 , TNF and LDH from BMDMs after treatment as in A . Data information : Each symbol represents an independent biological replicate ( B , NS , no statistical significance ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ( one - way ANOVA with Dunnett ' s multiple - comparisons test ( B , Data are pooled from three independent experiments ( B , mean and s . e . m . in B ,

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B. Release of IL-1β, IL-18, TNF and LDH from BMDMs after treatment as in A. Data information: Each symbol represents an independent biological replicate (B, NS, no statistical significance; ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test (B, Data are pooled from three independent experiments (B, mean and s.e.m. in B,


F . Transcript abundance for genes encoding GTPases in WT and Ifnar1 - / - BMDMs infected with M . catarrhalis as in C . Transcript abundance is provided in counts per million ( CPM ) . Data information : Data are pooled from three independent

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F. Transcript abundance for genes encoding GTPases in WT and Ifnar1-/- BMDMs infected with M. catarrhalis as in C. Transcript abundance is provided in counts per million (CPM). Data information: Data are pooled from three independent experiments


G . qRT - PCR analysis of genes encoding the family of guanylate binding proteins ( GBP1 - 11 ) in WT and Ifnar1 - / - BMDMs left untreated ( Med . ) or following treated as in C . Data information : Each symbol represents an independent biological replicate G ) . NS , no statistical significance ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ( one - way ANOVA with Dunnett ' s multiple - comparisons test G ) ) . Data are from one experiment representative of four ( G ) independent experiments ( mean and s . e . m . in G ) .

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G. qRT-PCR analysis of genes encoding the family of guanylate binding proteins (GBP1-11) in WT and Ifnar1-/- BMDMs left untreated (Med.) or following treated as in C. Data information: Each symbol represents an independent biological replicate G). NS, no statistical significance; ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test G)). Data are from one experiment representative of four (G) independent experiments (mean and s.e.m. in G).


A . Confocal microscopy analysis of intracellular M . catarrhalis ( green ) and GBP1 , GBP2 , GBP3 , GBP5 or GBP7 ( red ) in WT BMDMs left untreated ( Med . ) or assessed 12 h after infection with M . catarrhalis ( Ne11 , MOI 20 ) . B . Quantitation of GBP1 - , GBP2 - , GBP3 - , GBP5 - and GBP7 - postive M . catarrhalis in WT BMDMs as treated in A . Data information : Arrowheads indicate bacteria colocalised with GBPs . To quantify the proportion of GBP - positive bacteria in a field of view , we used the single red channel ( anti - GBP staining ) and compared this to the green channel ( anti - M . catarrhalis staining ) . The number of GBP - positive M . catarrhalis was divided by the total number of M . catarrhalis counted to obtain the proportion of GBP - positive bacteria ( B ) . A total of 100 BMDMs were analysed to quantify the proportion of GBP - positive bacteria per cell using confocal microscopy ( B ) Data are from three independent experiments ( A , or are pooled from two ( B ) independent experiments Scale bars , 5 µm ( A ,

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

A. Confocal microscopy analysis of intracellular M. catarrhalis (green) and GBP1, GBP2, GBP3, GBP5 or GBP7 (red) in WT BMDMs left untreated (Med.) or assessed 12 h after infection with M. catarrhalis (Ne11, MOI 20). B. Quantitation of GBP1-, GBP2-, GBP3-, GBP5- and GBP7-postive M. catarrhalis in WT BMDMs as treated in A. Data information: Arrowheads indicate bacteria colocalised with GBPs. To quantify the proportion of GBP-positive bacteria in a field of view, we used the single red channel (anti-GBP staining) and compared this to the green channel (anti-M. catarrhalis staining). The number of GBP-positive M. catarrhalis was divided by the total number of M. catarrhalis counted to obtain the proportion of GBP-positive bacteria (B). A total of 100 BMDMs were analysed to quantify the proportion of GBP-positive bacteria per cell using confocal microscopy (B) Data are from three independent experiments (A, or are pooled from two (B) independent experiments Scale bars, 5 µm (A,


F . Recovery of M . catarrhalis ( as colony - forming units ( CFU ) from primary WT , Gbpchr3 - KO and Ifnar1 - / - lung fibroblasts 4 , 8 and 12 h after infection with M . catarrhalis ( Ne11 , MOI 100 ) . Data information : Each symbol represents an independent biological replicate F ) . NS , no statistical significance ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ( one - way ANOVA with Dunnett ' s multiple - comparisons test Data are from three independent experiments F ; mean and s . e . m .

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F. Recovery of M. catarrhalis (as colony-forming units (CFU) from primary WT, Gbpchr3-KO and Ifnar1-/- lung fibroblasts 4, 8 and 12 h after infection with M. catarrhalis (Ne11, MOI 100). Data information: Each symbol represents an independent biological replicate F). NS, no statistical significance; * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test Data are from three independent experiments F; mean and s.e.m.


G . Quantitation of intracellular bacteria by Transmission electron microscopy ( TEM ) in WT , Gbpchr3 - KO and Ifnar1 - / - BMDMs infected with M . catarrhalis ( Ne11 , MOI 50 ) for 12 h . Each symbol represents an independent experiment ( E and F ) . Data information : A total of 100 BMDMs were analysed to quantify the intracellular bacterial burden using electron microscopy ( G ) . Data are from one experiment representative of two ( G ) independent experiments mean and s . e . m .

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G. Quantitation of intracellular bacteria by Transmission electron microscopy (TEM) in WT, Gbpchr3-KO and Ifnar1-/- BMDMs infected with M. catarrhalis (Ne11, MOI 50) for 12 h. Each symbol represents an independent experiment (E and F). Data information: A total of 100 BMDMs were analysed to quantify the intracellular bacterial burden using electron microscopy (G). Data are from one experiment representative of two (G) independent experiments mean and s.e.m.


A . Immunoblot analysis of caspase - 1 ( Casp - 1 ) , caspase - 11 ( Casp - 11 ) and gasdermin D ( GSDMD ) in WT , Gbp1 - / - , Gbp2 - / - , Gbp3 - / - , Gbp5 - / - , Gbp7 - / - , Gbp4 / 8 / 9 - / - , Gbp11 - / - and Casp11 - / - BMDMs left untreated ( Med . ) or assessed 10 h after infection with M . catarrhalis ( Ne11 , MOI 100 ) , or 5 h after transfection with 5 µg LPS from E . coli .

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A. Immunoblot analysis of caspase-1 (Casp-1), caspase-11 (Casp-11) and gasdermin D (GSDMD) in WT, Gbp1-/-, Gbp2-/-, Gbp3-/-, Gbp5-/-, Gbp7-/-, Gbp4/8/9-/-, Gbp11-/- and Casp11-/- BMDMs left untreated (Med.) or assessed 10 h after infection with M. catarrhalis (Ne11, MOI 100), or 5 h after transfection with 5 µg LPS from E. coli.


B . Release of IL - 1β , IL - 18 , LDH , TNF , IL - 6 and KC from BMDMs after treatment as in A . Data information : Each symbol represents an independent biological replicate ( B ) . NS , no statistical significance ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ( one - way ANOVA with Dunnett ' s multiple - comparisons test ( B ) ) . Data are are pooled from three independent experiments ( B ; mean and s . e . m . in B ) .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Release of IL-1β, IL-18, LDH, TNF, IL-6 and KC from BMDMs after treatment as in A. Data information: Each symbol represents an independent biological replicate (B). NS, no statistical significance; * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test (B)). Data are are pooled from three independent experiments (B; mean and s.e.m. in B).


B . Quantification of OD600 of M . catarrhalis in BHI media over 6 h in the presence of solvent control ( Sol . Ctrl . ) , 80 µg / mL recombinant GBP2 or 50 µg / mL kanamycin . Data information : * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ( one - way ANOVA with Dunnett ' s multiple - comparisons test ( B Data are from one experiment representative of two ( A ) or three ( B , Each independent experiment consists of three ( B , technical replicates .

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B. Quantification of OD600 of M. catarrhalis in BHI media over 6 h in the presence of solvent control (Sol. Ctrl.), 80 µg/mL recombinant GBP2 or 50 µg/mL kanamycin. Data information: ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test (B Data are from one experiment representative of two (A) or three (B, Each independent experiment consists of three (B, technical replicates.


C . Quantification of ATP from M . catarrhalis in BHI media over 6 h in the presence of Sol . Ctrl . , 30 µg / mL recombinant GBP2 or 50 µg / mL kanamycin . Data information : Data are pooled from two ( C ) independent experiments Each independent experiment consists of two ( C ) technical replicates .

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C. Quantification of ATP from M. catarrhalis in BHI media over 6 h in the presence of Sol. Ctrl., 30 µg/mL recombinant GBP2 or 50 µg/mL kanamycin. Data information: Data are pooled from two (C) independent experiments Each independent experiment consists of two (C) technical replicates.


D . Viability of M . catarrhalis ( as a percentage of CFU in relation to Sol . Ctrl . ) assessed 6 h after incubation with recombinant GBP2 at 3 , 30 or 300 µg / mL . Data information : * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ( one - way ANOVA with Dunnett ' s multiple - comparisons test D ) Data are from one experiment representative of three D , independent experiments Each independent experiment consists of three D , technical replicates .

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D. Viability of M. catarrhalis (as a percentage of CFU in relation to Sol. Ctrl.) assessed 6 h after incubation with recombinant GBP2 at 3, 30 or 300 µg/mL. Data information: ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test D) Data are from one experiment representative of three D, independent experiments Each independent experiment consists of three D, technical replicates.


A , B . Bacterial burden and concentration of IL - 18 in the spleen of WT mice ( n = 10 ) and Casp11 - / - mice ( n = 10 ) after M . catarrhalis infection . C , D . Bacterial burden and concentration of IL - 18 in the spleen of WT mice ( n = 10 ) and Nlrp3 - / - mice ( n = 11 ) after M . catarrhalis infection . E , F . Bacterial burden and concentration of IL - 18 in the spleen of WT mice ( n = 10 ) and Gbp1 - / - mice ( n = 9 ) after M . catarrhalis infection . G , H . Bacterial burden and concentration of IL - 18 in the spleen of WT mice ( n = 10 ) and Gbp2 - / - mice ( n = 8 ) after M . catarrhalis infection . I , J . Bacterial burden and concentration of IL - 18 in the spleen of WT mice ( n = 10 ) and Gbp3 - / - mice ( n = 8 ) after M . catarrhalis infection . K , L . Bacterial burden and concentration of IL - 18 in the spleen of WT mice ( n = 5 ) and Gbp5 - / - mice ( n = 8 ) after M . catarrhalis infection . Data information : A mix of male and female mice 6 - 8 weeks old were used in each experiment and were injected i . p . with 2 × 107 CFU of M . catarrhalis and analysed after 6 h . Each symbol represents an individual mouse ( A - L ) . Each panel represents data from a single experiment ( mean and s . e . m . in A - L ) . Each experiment was performed at least two times . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( two - tailed t - test ( A - L ) ) .

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

A, B. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=10) and Casp11-/- mice (n=10) after M. catarrhalis infection. C, D. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=10) and Nlrp3-/- mice (n=11) after M. catarrhalis infection. E, F. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=10) and Gbp1-/- mice (n=9) after M. catarrhalis infection. G, H. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=10) and Gbp2-/- mice (n=8) after M. catarrhalis infection. I, J. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=10) and Gbp3-/- mice (n=8) after M. catarrhalis infection. K, L. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=5) and Gbp5-/- mice (n=8) after M. catarrhalis infection. Data information: A mix of male and female mice 6-8 weeks old were used in each experiment and were injected i.p. with 2×107 CFU of M. catarrhalis and analysed after 6 h. Each symbol represents an individual mouse (A-L). Each panel represents data from a single experiment (mean and s.e.m. in A-L). Each experiment was performed at least two times. * P<0.05; ** P<0.01; *** P<0.001 (two-tailed t-test (A-L)).


C Frequency of C179 + or V5 - tag + 293T cells 24h after transfection of AAV - vector plasmids as measured by flow cytometry . Symbols represent single experiments and bars the mean ± SE ( n = 3 ) .

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C Frequency of C179+ or V5-tag+ 293T cells 24h after transfection of AAV-vector plasmids as measured by flow cytometry. Symbols represent single experiments and bars the mean ± SE (n = 3).


D Immunoblot of 293T cells 72 h after transduction with AAV - vectors at a MOI of 106 . Antigen expression was detected with an anti - V5 - tag antibody . Equal loading was controlled with a GAPDH antibody ( n = 3 ) .

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D Immunoblot of 293T cells 72 h after transduction with AAV-vectors at a MOI of 106. Antigen expression was detected with an anti-V5-tag antibody. Equal loading was controlled with a GAPDH antibody (n = 3).


Total IgG ELISA titers expressed as area under the curve ( AUC ) against homologous Cal / 7 / 9 virus of pre - immune and pre - challenge sera of individual animals of the indicated vaccine groups ( AAV - HA , - cHA , - GFP , WIV n = 18 , AAV - NP n = 11 ) . Background reactivity of pooled AAV - GFP pre - challenge serum is shown as dashed line . ELISAs were performed in technical duplicates .

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Total IgG ELISA titers expressed as area under the curve (AUC) against homologous Cal/7/9 virus of pre-immune and pre-challenge sera of individual animals of the indicated vaccine groups (AAV-HA, -cHA, -GFP, WIV n = 18, AAV-NP n = 11). Background reactivity of pooled AAV-GFP pre-challenge serum is shown as dashed line. ELISAs were performed in technical duplicates.


IgA ELISA titers in post - challenge lung homogenates against Cal / 7 / 9 of individual mice of the indicated vaccine groups ( AAV - HA , - cHA , - GFP , - NP , WIV n = 11 ) . of the Cal / 7 / 9 ( blue symbols ) and PR8 low dose ( red symbols ) challenge groups . ELISAs were performed in technical duplicates .

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IgA ELISA titers in post-challenge lung homogenates against Cal/7/9 of individual mice of the indicated vaccine groups (AAV-HA, -cHA, -GFP, -NP, WIV n = 11). of the Cal/7/9 (blue symbols) and PR8 low dose (red symbols) challenge groups. ELISAs were performed in technical duplicates.


A Immunoblot analysis of purified H1N1 viruses A / California / 7 / 2009 ( Cal / 7 / 9 ) , A / Brevig Mission / 1 / 1918 ( BM / 1 / 1918 ) , A / Puerto Rico / 8 / 1934 ( PR8 ) or A / Brisbane / 59 / 2007 ( Bris / 59 / 7 ) separated under reducing and denaturing conditions . Uncleaved HA0 and cleavage products HA1 and HA2 were detected with pooled pre - challenge sera ( AAV - HA , - cHA , - GFP , WIV n = 18 , AAV - NP n = 11 ) as indicated ( n = 3 ) .

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A Immunoblot analysis of purified H1N1 viruses A/California/7/2009 (Cal/7/9), A/Brevig Mission/1/1918 (BM/1/1918), A/Puerto Rico/8/1934 (PR8) or A/Brisbane/59/2007 (Bris/59/7) separated under reducing and denaturing conditions. Uncleaved HA0 and cleavage products HA1 and HA2 were detected with pooled pre-challenge sera (AAV-HA, -cHA, -GFP , WIV n = 18, AAV-NP n = 11) as indicated (n = 3).


A Immunoblot analysis of purified H1N1 viruses A / California / 7 / 2009 ( Cal / 7 / 9 ) , A / Brevig Mission / 1 / 1918 ( BM / 1 / 1918 ) , A / Puerto Rico / 8 / 1934 ( PR8 ) or A / Brisbane / 59 / 2007 ( Bris / 59 / 7 ) separated under reducing and denaturing conditions . Uncleaved HA0 and cleavage products HA1 and HA2 were detected with pooled pre - challenge sera ( AAV - HA , - cHA , -

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

A Immunoblot analysis of purified H1N1 viruses A/California/7/2009 (Cal/7/9), A/Brevig Mission/1/1918 (BM/1/1918), A/Puerto Rico/8/1934 (PR8) or A/Brisbane/59/2007 (Bris/59/7) separated under reducing and denaturing conditions. Uncleaved HA0 and cleavage products HA1 and HA2 were detected with pooled pre-challenge sera (AAV-HA, -cHA, -GFP


C to F Binding of C179 ( C ) , or antibodies present in AAV - HA ( D ) , AAV - cHA ( E ) or WIV ( F ) pooled pre - challenge sera ( n = 18 mice per group ) to Cal / 7 / 9 or PR8 virus after incubation of the virions at pH = 7 . 2 , 5 . 8 , 5 . 4 , 5 . 0 , 4 . 4 or 4 . 4 + DTT to induce conformational changes in HA or remove the HA1 subdomain . Mean ± SD ( n = 3 , in technical triplicates ) . Statistical significance between pH = 7 . 2 and other conditions was determined using Kruskal - Wallis test with Dunn ' s multiple comparison testing ( * p < 0 . 05 , * * p < 0 . 01 ) .

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C to F Binding of C179 (C), or antibodies present in AAV-HA (D), AAV-cHA (E) or WIV (F) pooled pre-challenge sera (n = 18 mice per group) to Cal/7/9 or PR8 virus after incubation of the virions at pH = 7.2, 5.8, 5.4, 5.0, 4.4 or 4.4+DTT to induce conformational changes in HA or remove the HA1 subdomain. Mean ± SD (n = 3, in technical triplicates). Statistical significance between pH = 7.2 and other conditions was determined using Kruskal-Wallis test with Dunn's multiple comparison testing (*p<0.05, **p<0.01).


A and B Influenza virus specific activation of the murine FcγRI , FcγRII , FcγRIII and FcγRIV by antibodies in pooled pre - challenge sera ( AAV - HA , - cHA , - GFP , WIV n = 18 , AAV - NP n = 11 mice per group ) . MDCKII cells were infected with Cal / 7 / 9 ( A ) or PR8 ( B ) before mouse serum and FcγR - expressing reporter cells were added . Influenza specific Fc - FcγR interaction induces the production of IL - 2 in the FcγR - expressing reporter cell , which was measured by anti - IL2 ELISA ( n = 3 , technical duplicates ) . Mean ± SE .

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A and B Influenza virus specific activation of the murine FcγRI, FcγRII, FcγRIII and FcγRIV by antibodies in pooled pre-challenge sera (AAV-HA, -cHA, -GFP, WIV n = 18, AAV-NP n = 11 mice per group). MDCKII cells were infected with Cal/7/9 (A) or PR8 (B) before mouse serum and FcγR-expressing reporter cells were added. Influenza specific Fc-FcγR interaction induces the production of IL-2 in the FcγR-expressing reporter cell, which was measured by anti-IL2 ELISA (n = 3, technical duplicates). Mean ± SE.


C HA - specific activation of FcγR by AAV - HA , AAV - cHA or WIV pooled pre - challenge sera ( n = 18 mice per group ) . MDCKII cells were transfected with wildtype HA ( pAAV - HA ) or a stalk - only HA ( pAAV - mHL1 ) before mouse serum and FcγR - I - expressing reporter cells were added ( n = 2 , technical duplicates ) . Mean ± SE .

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

C HA-specific activation of FcγR by AAV-HA, AAV-cHA or WIV pooled pre-challenge sera (n = 18 mice per group). MDCKII cells were transfected with wildtype HA (pAAV-HA) or a stalk-only HA (pAAV-mHL1) before mouse serum and FcγR-I-expressing reporter cells were added (n = 2, technical duplicates). Mean ± SE.


A to F Mice were vaccinated three times with AAV - vectors or two times with WIV and then challenged with a lethal dose of homologous influenza virus ( Cal / 7 / 9 ) ( A , B ; n = 5 ) or two different lethal doses of the heterologous PR8 strain ( C , D ; n = 6 and E , F ; n = 7 ) . Survival was monitored over a 14 - day period and is depicted as Kaplan - Meier plot ( A , C , E ) . Weight loss was determined during the 14 - day period , which is shown in relation to the weight at day 0 ( B , D , F ) . Mean ± SE . Statistical significance between negative control group ( AAV - GFP ) or WIV group ( E ) and each vaccine group was determined using Log - Rank ( Mantel - Cox ) test ( * p < 0 . 05 , * * p < 0 . 01 ) .

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A to F Mice were vaccinated three times with AAV-vectors or two times with WIV and then challenged with a lethal dose of homologous influenza virus (Cal/7/9) (A, B; n = 5) or two different lethal doses of the heterologous PR8 strain (C, D; n = 6 and E, F; n = 7). Survival was monitored over a 14-day period and is depicted as Kaplan-Meier plot (A, C, E). Weight loss was determined during the 14-day period, which is shown in relation to the weight at day 0 (B, D, F). Mean ± SE. Statistical significance between negative control group (AAV-GFP) or WIV group (E) and each vaccine group was determined using Log-Rank (Mantel-Cox) test (*p<0.05, **p<0.01).


B Cal / 7 / 9 - specific serum antibody titers in individual sera at indicated time points . Black lines : individual animals , red lines : mean titer . Statistical significance between pre - immune and immune sera was determined using Friedman test with Dunn ' s multiple comparison testing ( * p < 0 . 05 , * * p < 0 . 01 ) . The lower table indicates Cal / 7 / 9 - specific MN50 and HAI mean titers . Assays were performed in technical duplicates .

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B Cal/7/9-specific serum antibody titers in individual sera at indicated time points. Black lines: individual animals, red lines: mean titer. Statistical significance between pre-immune and immune sera was determined using Friedman test with Dunn's multiple comparison testing (*p<0.05, **p<0.01). The lower table indicates Cal/7/9-specific MN50 and HAI mean titers. Assays were performed in technical duplicates.


C to E Clinical score ( C ) , body temperature ( D ) , and relative weight ( E ) during challenge period . Statistical significance between pre - immune and immune sera was determined using Friedman test with Dunn ' s multiple comparison testing ( AAV - HA # # p < 0 . 01 ; AAV - cHA § § p < 0 . 01 ; AAV - GFP $ p < 0 . 05 ; $ $ p < 0 . 01 ; QIV * p < 0 . 05 ; ) . Mean ± SD

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C to E Clinical score (C), body temperature (D), and relative weight (E) during challenge period. Statistical significance between pre-immune and immune sera was determined using Friedman test with Dunn's multiple comparison testing (AAV-HA ##p<0.01; AAV-cHA §§p<0.01; AAV-GFP $p<0.05; $$p<0.01; QIV *p<0.05;). Mean ± SD


C to E Clinical score ( C ) , body temperature ( D ) , and relative weight ( E ) during challenge period . Statistical significance between pre - immune and immune sera was determined using Friedman test with Dunn ' s multiple comparison testing ( AAV - HA # # p < 0 . 01 ; AAV - cHA § § p < 0 . 01 ; AAV - GFP $ p < 0 . 05 ; $ $ p < 0 . 01 ; QIV * p < 0 . 05 ; ) . Mean ±

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F Virus titers in nasal turbinates ( NT ) , trachea or lung homogenates . Statistical significance between AAV - GFP and vaccine groups was determined using Kruskal - Wallis test with Dunn ' s multiple comparison testing ( * p < 0 . 05 ) . Lines indicate the GMT . Assays were performed in technical duplicates .

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F Virus titers in nasal turbinates (NT), trachea or lung homogenates. Statistical significance between AAV-GFP and vaccine groups was determined using Kruskal-Wallis test with Dunn's multiple comparison testing (*p<0.05). Lines indicate the GMT. Assays were performed in technical duplicates.


G and H IHC staining of submucosal glands ( SMG ) ( G ) or bronchi ( H ) with an anti - H1N1 polyclonal antibody ( bar : 100 µm ; red : influenza antigen ( neufuchsin ) ; blue : nuclei ( haematoxylin ) ) .

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G and H IHC staining of submucosal glands (SMG) (G) or bronchi (H) with an anti-H1N1 polyclonal antibody (bar: 100 µm; red: influenza antigen (neufuchsin); blue: nuclei (haematoxylin)).


I and J H & E stained histological sections of bronchi ( I ) and lung parenchyma with alveolar epithelial cells ( AEC , J ) . Arrowheads : areas of suppurative bronchitis / bronchiolitis ; arrows : interstitial pneumonia , necrosis of AEC ( bar : ( I ) 100 µm ; ( J ) 50 µm ) .

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I and J H&E stained histological sections of bronchi (I) and lung parenchyma with alveolar epithelial cells (AEC, J). Arrowheads: areas of suppurative bronchitis/bronchiolitis; arrows: interstitial pneumonia, necrosis of AEC (bar: (I) 100 µm; (J) 50 µm).


( B ) The concentration - effect relationships of PARP1 inhibition by thioparib , talazoparib , and olaparib , as assayed by histone - based ELISA . Data are obtained from three biological replicates and depicted as mean ± SEM . ( C ) Concentration - dependent increase in PARP1 - DNA binding by three PARP inhibitors using DSB FA assays . FA was measured 60 min after adding NAD + , and the ΔFA values represent the changes in PARP1 - DNA binding . Data from four biological replicates are presented as mean ± SEM . (

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(B) The concentration-effect relationships of PARP1 inhibition by thioparib, talazoparib, and olaparib, as assayed by histone-based ELISA. Data are obtained from three biological replicates and depicted as mean ± SEM. (C) Concentration-dependent increase in PARP1-DNA binding by three PARP inhibitors using DSB FA assays. FA was measured 60 min after adding NAD+, and the ΔFA values represent the changes in PARP1-DNA binding. Data from four biological replicates are presented as mean ± SEM. (


( F ) and ( G ) Effects of thioparib on BRCA1 - deficient MDA - MB - 436 ( F ) and BRCA2 - deficient Capan - 1 ( G ) xenografts . Mice bearing MDA - MB - 436 tumors were dosed orally with 10 mg / kg thioparib , 100 mg / kg olaparib , or vehicle ( Veh ) once daily for 3 weeks ; and mice bearing Capan - 1 tumors were dosed orally with 5 or 25 mg / kg thioparib , 0 . 3 mg / kg talazoparib , or vehicle ( Veh ) once daily for 3 weeks ( n = 6 ) . Data are shown as mean ± SEM . Statistical analysis was performed by two - way ANOVA . * * * P < 0 . 0001 . Representative images of xenograft tumors are shown , and mice tails represent complete regression of tumors .

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(F) and (G) Effects of thioparib on BRCA1-deficient MDA-MB-436 (F) and BRCA2-deficient Capan-1 (G) xenografts. Mice bearing MDA-MB-436 tumors were dosed orally with 10 mg/kg thioparib, 100 mg/kg olaparib, or vehicle (Veh) once daily for 3 weeks; and mice bearing Capan-1 tumors were dosed orally with 5 or 25 mg/kg thioparib, 0.3 mg/kg talazoparib, or vehicle (Veh) once daily for 3 weeks (n = 6). Data are shown as mean ± SEM. Statistical analysis was performed by two-way ANOVA. ***P < 0.0001. Representative images of xenograft tumors are shown, and mice tails represent complete regression of tumors.


( C ) The IC50 values of thioparib in six acquired PARPi - resistant cell lines . Numbers 1 - 6 represent the PARPi - resistant Capan - 1 / OP , Capan - 1 / TP , MDA - MB - 436 / OP , MDA - MB - 436 / TP , U251 / OP , and U251 / TP cells , respectively . Cells were treated with the indicated PARP inhibitors for 7 days and then subjected to SRB assay . The IC50 values are expressed as the mean ± SD from three separate experiments .

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(C) The IC50 values of thioparib in six acquired PARPi-resistant cell lines. Numbers 1-6 represent the PARPi-resistant Capan-1/OP, Capan-1/TP, MDA-MB-436/OP, MDA-MB-436/TP, U251/OP, and U251/TP cells, respectively. Cells were treated with the indicated PARP inhibitors for 7 days and then subjected to SRB assay. The IC50 values are expressed as the mean ± SD from three separate experiments.


( E ) Efficacy of thioparib in the PDX model BR - 05 - 0028 ( BRCA1 - deficient ) . Mice bearing BR - 05 - 0028 tumors derived from a patient with breast cancer ( n = 6 ) were treated with 10 or 30 mg / kg thioparib or 100 mg / kg olaparib for 6 weeks . Data are depicted as mean ± SEM . * * * P < 0 . 0001 .

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(E) Efficacy of thioparib in the PDX model BR-05-0028 (BRCA1-deficient). Mice bearing BR-05-0028 tumors derived from a patient with breast cancer (n = 6) were treated with 10 or 30 mg/kg thioparib or 100 mg/kg olaparib for 6 weeks. Data are depicted as mean ± SEM. ***P < 0.0001.


( B ) Concentration - dependent effects of thioparib on the viability of malignant hematologic cells . Cells were treated with thioparib or other PARP inhibitors for 72 h and then subjected to CCK8 assay . Data from three independent experiments were presented as mean ± SEM .

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(B) Concentration-dependent effects of thioparib on the viability of malignant hematologic cells. Cells were treated with thioparib or other PARP inhibitors for 72 h and then subjected to CCK8 assay. Data from three independent experiments were presented as mean ± SEM.


( D ) Kaplan - Meier survival curve of M - NSG mice transplanted with JeKo - 1 cells treated with 10 mg / kg thioparib , 0 . 3 mg / kg talazoparib , or vehicle ( n = 10 ) . Statistical analysis was performed by log - rank ( Mantel - Cox ) test . * * * P < 0 . 0001 , ns : P = 0 . 3526 .

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(D) Kaplan-Meier survival curve of M-NSG mice transplanted with JeKo-1 cells treated with 10 mg/kg thioparib, 0.3 mg/kg talazoparib, or vehicle (n = 10). Statistical analysis was performed by log-rank (Mantel-Cox) test. ***P < 0.0001, ns: P = 0.3526.


( B ) Western blotting of γH2AX and phosphor - RPA32 in cells exposed to thioparib , talazoparib , or olaparib for 12 h .

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(B) Western blotting of γH2AX and phosphor-RPA32 in cells exposed to thioparib, talazoparib, or olaparib for 12 h.


( E ) and ( F ) . Thioparib caused changes in apoptosis - related proteins in JeKo - 1 or THP - 1 cells , as determined by western blotting . Cells were exposed to thioparib , talazoparib , olaparib , or Cpd 391 for 24 h and then subjected to western blotting .

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(E) and (F). Thioparib caused changes in apoptosis-related proteins in JeKo-1 or THP-1 cells, as determined by western blotting. Cells were exposed to thioparib, talazoparib, olaparib, or Cpd 391 for 24 h and then subjected to western blotting.


( G ) and ( H ) . Depletion of PARP1 in HT - 29 and Capan - 1 / TP cells resulted in a decrease in DNA damage and thioparib resistance . The upper panel shows the changes in γH2AX and phospho - RPA32 in HT - 29 PARP1 knockout ( KO ) clones ( G ) or Capan - 1 / TP PARP1 KO clones ( H ) and their parental cells after exposure to thioparib for 24 h . The lower panel shows the IC50 values of thioparib in these PARP1 KO clones and parental cells . Cells were treated with thioparib for 5 or 7 d and the IC50 values were determined by SRB assay from three separate experiments . Data are presented as the mean ± SD . ThP : Thioparib , OP : Olaparib , TP : Talazoparib , 391 : Cpd 391 .

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(G) and (H). Depletion of PARP1 in HT-29 and Capan-1/TP cells resulted in a decrease in DNA damage and thioparib resistance. The upper panel shows the changes in γH2AX and phospho-RPA32 in HT-29 PARP1 knockout (KO) clones (G) or Capan-1/TP PARP1 KO clones (H) and their parental cells after exposure to thioparib for 24 h. The lower panel shows the IC50 values of thioparib in these PARP1 KO clones and parental cells. Cells were treated with thioparib for 5 or 7 d and the IC50 values were determined by SRB assay from three separate experiments. Data are presented as the mean ± SD. ThP: Thioparib, OP: Olaparib, TP: Talazoparib, 391: Cpd 391.


( A ) HR repair assays in U2OS - DR - GFP cells treated with increasing concentrations of thioparib . Cells were incubated with the indicated drugs at the time of I - SceI introduction for 48 h . GFP - positive cells were analyzed after I - SceI transfection for 72 h by flow cytometry . The ATR inhibitor VE - 821 was used as a positive control . Three independent experiments were performed and data are expressed as mean ± SD . Statistical analysis was performed by one - way ANOVA , and all groups were compared with control . * * * P < 0 . 0001 , * P = 0 . 0205 , 0 . 0101 ( from left to right ) .

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(A) HR repair assays in U2OS-DR-GFP cells treated with increasing concentrations of thioparib. Cells were incubated with the indicated drugs at the time of I-SceI introduction for 48 h. GFP-positive cells were analyzed after I-SceI transfection for 72 h by flow cytometry. The ATR inhibitor VE-821 was used as a positive control. Three independent experiments were performed and data are expressed as mean ± SD. Statistical analysis was performed by one-way ANOVA, and all groups were compared with control. ***P < 0.0001, *P = 0.0205, 0.0101 (from left to right).


( C ) Representative images of RAD51 foci in U2OS - DR - GFP cells with or without radiation . Graphs ( right panel ) show quantification of cells with ≥ 5 RAD51 foci in vehicle - and drug - treated cells . Scale bar : 5 μm . Data are obtained from three biological replicates and presented as mean ± SD . Statistical analysis was performed by one - way ANOVA . Left to right : P < 0 . 0001 , = 0 . 0033 , < 0 . 0001 , = 0 . 0006 , = 0 . 0031 , = 0 . 0015 .

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(C) Representative images of RAD51 foci in U2OS-DR-GFP cells with or without radiation. Graphs (right panel) show quantification of cells with ≥ 5 RAD51 foci in vehicle- and drug-treated cells. Scale bar: 5 μm. Data are obtained from three biological replicates and presented as mean ± SD. Statistical analysis was performed by one-way ANOVA. Left to right: P < 0.0001, = 0.0033, < 0.0001, = 0.0006, = 0.0031, = 0.0015.


( H ) HR repair assay . U2OS - DR - GFP ( WT ) and PARP1 KO cells ( KO1 and KO2 ) were treated with the indicated drugs for 48 h and collected after 72 h of I - SceI transfection as in ( A ) . VE - 821 served as a positive control . PARP1 knockout efficiency was evaluated by western blotting . Data from three biological replicates are presented as mean ± SD . Statistical analysis was performed by one - way ANOVA , and each group was compared with the control . * * * P < 0 . 0001 , * P = 0 . 0143 , 0 . 0483 , ns : P = 0 . 3061 , 0 . 8269 , 0 . 9997 , 0 . 3906 ( from left to right ) .

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(H) HR repair assay. U2OS-DR-GFP (WT) and PARP1 KO cells (KO1 and KO2) were treated with the indicated drugs for 48 h and collected after 72 h of I-SceI transfection as in (A). VE-821 served as a positive control. PARP1 knockout efficiency was evaluated by western blotting. Data from three biological replicates are presented as mean ± SD. Statistical analysis was performed by one-way ANOVA, and each group was compared with the control. ***P < 0.0001, *P = 0.0143, 0.0483, ns: P = 0.3061, 0.8269, 0.9997, 0.3906 (from left to right).


( C ) Effects of thioparib on CXCL9 , CXCL10 , and IL15 mRNA in JeKo - 1 cells . Data from three independent experiments are shown as the mean ± SD . Statistical analysis was performed by two - way ANOVA . Left panel : * P = 0 . 0367 , * * P = 0 . 0011 ; median panel : * * * P = 0 . 0002 , < 0 . 0001 , < 0 . 0001 ; right panel : * * * P = 0 . 0005 , 0 . 0009 , 0 . 0001 ( from left to right ) .

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(C) Effects of thioparib on CXCL9, CXCL10, and IL15 mRNA in JeKo-1 cells. Data from three independent experiments are shown as the mean ± SD. Statistical analysis was performed by two-way ANOVA. Left panel: *P = 0.0367, **P = 0.0011; median panel: ***P = 0.0002, < 0.0001, < 0.0001; right panel: ***P = 0.0005, 0.0009, 0.0001 (from left to right).


( D ) The protein levels of p - STAT1 , p - TBK1 , γH2AX , and phospho - RPA32 in HT - 29 parent , PARP1 − / − ( # KO1 ) , and PARP7 − / − cell lines after 24 or 48 h thioparib treatment .

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(D) The protein levels of p-STAT1, p-TBK1, γH2AX, and phospho-RPA32 in HT-29 parent, PARP1−/− (#KO1), and PARP7−/− cell lines after 24 or 48 h thioparib treatment.


( F ) Western blot analysis of p - STAT1 , p - TBK1 , and γH2AX in HT - 29 parent , STING − / − , and TBK1 − / − cells after 24 or 48 h thioparib treatment .

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(F) Western blot analysis of p-STAT1, p-TBK1, and γH2AX in HT-29 parent, STING−/−, and TBK1−/−cells after 24 or 48 h thioparib treatment.


( H ) Immunohistochemistry staining of CD4 , CD8 , and CD45 expression in MC38 tumor tissue sections . Quantification of cells positive of CD4 , CD8 , CD45 were shown . Scale bar : 25 μm . Data from ten technical replicates from each of the two mice are presented as mean ± SD . Statistical analysis was performed by unpaired t - test . * * * P < 0 . 0001 , ns : P = 0 . 1958 .

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(H) Immunohistochemistry staining of CD4, CD8, and CD45 expression in MC38 tumor tissue sections. Quantification of cells positive of CD4, CD8, CD45 were shown. Scale bar: 25 μm. Data from ten technical replicates from each of the two mice are presented as mean ± SD. Statistical analysis was performed by unpaired t-test. ***P < 0.0001, ns: P = 0.1958.


Western blot analysis of soluble and insoluble fractions ( Red - and yellow rectangles , highlights CTF - 35 and CTF - 25 respectively in the insoluble fraction ) .

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Western blot analysis of soluble and insoluble fractions (Red- and yellow rectangles, highlights CTF-35 and CTF-25 respectively in the insoluble fraction).


Immunofluorescent analysis showing fluorescence intensity of phosphorylated TDP - 43 ( pTDP - 43 ) . Scale bar : 20µm . Presented immunofluorescent images of control ( C2 ) and mutant TDP - 43 ( P2 ) . I represents its corresponding profile intensity plot of the selected ( square ) region . Scale bar : 10µm , J quantification pooled control ( C0 = 10 , C1 = 10 and C2 = 10 ) n = 30 and pooled mutant TDP - 43 ( P1 = 10 , P2 = 10 and P3 = 10 ) n = 30 cells from each differentiation . Unpaired Mann - Whitney - test .

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Immunofluorescent analysis showing fluorescence intensity of phosphorylated TDP-43 (pTDP-43). Scale bar: 20µm. Presented immunofluorescent images of control (C2) and mutant TDP-43 (P2). I represents its corresponding profile intensity plot of the selected (square) region. Scale bar: 10µm, J quantification pooled control (C0=10, C1=10 and C2=10) n=30 and pooled mutant TDP-43 (P1=10, P2=10 and P3=10) n=30 cells from each differentiation. Unpaired Mann-Whitney-test.


Western blots showing nucleo - cytoplasmic fractionation of WT - mCherry and MUT - mCherry iPSC - derived motor neurons with F ( left panel ) quantification of total mCherry and total untagged TDP - 43 on α - tubulin and ( right panel ) relative cytoplasmic mCherry and untagged TDP - 43 on total mCherry and total untagged TDP - 43

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Western blots showing nucleo-cytoplasmic fractionation of WT-mCherry and MUT-mCherry iPSC-derived motor neurons with F (left panel) quantification of total mCherry and total untagged TDP-43 on α-tubulin and (right panel) relative cytoplasmic mCherry and untagged TDP-43 on total mCherry and total untagged TDP-43 respectively


Representative kymographs from control and mutant TDP - 43 iPSC - derived motor neurons . The x - axis represent distance in µm while on the y - axis time in seconds is revealed . Stationary mitochondria are visible as straight vertical lines , while moving mitochondria are depicted as skewed lines . B Quantification of percentage of moving mitochondria .

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Representative kymographs from control and mutant TDP-43 iPSC-derived motor neurons. The x-axis represent distance in µm while on the y-axis time in seconds is revealed. Stationary mitochondria are visible as straight vertical lines, while moving mitochondria are depicted as skewed lines. B Quantification of percentage of moving mitochondria.


Immunofluorescent analysis showing fluorescence intensity of pTDP - 43 in motor neurons . Scale bar : 10µm and its perspective intensity plot in F . G Quantification of E , each dot represents one analysed cell , unpaired Mann - Whitney - test .

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Immunofluorescent analysis showing fluorescence intensity of pTDP-43 in motor neurons. Scale bar: 10µm and its perspective intensity plot in F. G Quantification of E, each dot represents one analysed cell, unpaired Mann-Whitney-test.


axonal transport analysis . Quantification of percentage of moving mitochondria ( J ) , number of moving mitochondria ( K ) and number of stationary mitochondria ( L ) normalized to neurite length 100µm .

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axonal transport analysis. Quantification of percentage of moving mitochondria (J), number of moving mitochondria (K) and number of stationary mitochondria (L) normalized to neurite length 100µm.


Total cell lysates were fractionated into soluble and insoluble fractions and analysed by Western blot , representative Western blots of soluble and insoluble fraction without C and with D HDAC6 inhibitor treatment .

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Total cell lysates were fractionated into soluble and insoluble fractions and analysed by Western blot, representative Western blots of soluble and insoluble fraction without C and with D HDAC6 inhibitor treatment.