H MOAP - 1 deficiency promotes activation of Nrf2 signaling . Transcript of downstream targets of Nrf2 ( i . e . HMOX - 1 , NQO1 , SLC7A11 , GCLC G6PD ) in WT MOAP - 1 KO LO2 were determined by RT - PCR normalized to GAPDH .

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H MOAP-1 deficiency promotes activation of Nrf2 signaling. Transcript levels of downstream targets of Nrf2 (i.e. HMOX-1, NQO1, SLC7A11, GCLC and G6PD) in WT and MOAP-1 KO LO2 cells were determined by RT-PCR and normalized to GAPDH.


I Elevated transcriptional activity of Nrf2 in MOAP - 1 deficient is mediated through p62 . Transcript of Nrf2 target genes ( HMOX - 1 NQO1 ) in WT , MOAP - 1 KO MOAP - 1 / p62 DKO LO2 were determined by RT - PCR normalized to GAPDH .

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I Elevated transcriptional activity of Nrf2 in the MOAP-1 deficient cells is mediated through p62. Transcript levels of Nrf2 target genes (HMOX-1 and NQO1) in WT, MOAP-1 KO and MOAP-1/p62 DKO LO2 cells were determined by RT-PCR and normalized to GAPDH.


J Re - expression of MOAP - 1 MOAP - 1 - L120E , but not p62 binding defective mutant of MOAP - 1 , MOAP - 1 - M3 , inhibits p62 / Keap1 interaction in MOAP - 1 deficient . MOAP - 1 KO LO2 were transfected with plasmid encoding Myc - tagged MOAP - 1 , L120E or M3 . transfected were then subjected to co - IP with anti - p62 antibody .

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J Re-expression of MOAP-1 and MOAP-1-L120E, but not the p62 binding defective mutant of MOAP-1, MOAP-1-M3, inhibits p62/Keap1 interaction in the MOAP-1 deficient cells. MOAP-1 KO LO2 cells were transfected with plasmid encoding Myc-tagged MOAP-1, L120E or M3. The transfected cells were then subjected to co-IP assay with anti-p62 antibody.


K Re - introduction of MOAP - 1 MOAP - 1 - L120E , but not p62 binding defective MOAP - 1 - M3 , reduces Nrf2 transcriptional activity in MOAP - 1 KO . MOAP - 1 KO LO2 were transfected with Myc - tagged MOAP - 1 , MOAP - 1 - L120E or MOAP - 1 - M3 ARE - Nrf2 - firely renilla luciferase reporter constructs for overnight subjected to luciferase .

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K Re-introduction of MOAP-1 and MOAP-1-L120E, but not the p62 binding defective MOAP-1-M3, reduces Nrf2 transcriptional activity in the MOAP-1 KO cells. MOAP-1 KO LO2 cells were transfected with Myc-tagged MOAP-1, MOAP-1-L120E or MOAP-1-M3 and ARE-Nrf2-firely and renilla luciferase reporter constructs for overnight and subjected to luciferase assay.


A High level of p62 bodies in liver of MOAP - 1 KO mice subjected to acute diethylnitrosamine ( DEN ) treatment . Eight - week - old WT MOAP - 1 KO mice were injected with an acute dose of DEN ( 100 µg / g ) . Mice were sacrificed at 48 hours post - injection . Livers were harvested for immunohistochemistry ( ) with anti - p62 antibody ( in red ) . Nuclei were counterstained with DAPI ( in blue ) . Insets in upper panels are enlarged in lower panels . Scale bar : 20 µm .

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A High level of p62 bodies in the liver of the MOAP-1 KO mice subjected to acute diethylnitrosamine (DEN) treatment. Eight-week-old male WT and MOAP-1 KO mice were injected with an acute dose of DEN (100 µg/g body weight). Mice were sacrificed at 48 hours post-injection. Livers were harvested for immunohistochemistry (IHC) analysis with anti-p62 antibody (in red). Nuclei were counterstained with DAPI (in blue). Insets in the upper panels are enlarged in the lower panels. Scale bar: 20 µm.


B Loss of MOAP - 1 results in higher of Nrf2 its downstream target , HMOX - 1 , in liver upon acute DEN treatment . Lysates of livers harvested from mice subjected to acute DEN treatment as described in ( A ) were prepared in RIPA lysis buffer containing 1 % SDS analyzed by Western blotting . Actin as loading control .

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B Loss of MOAP-1 results in higher levels of Nrf2 and its downstream target protein, HMOX-1, in liver upon acute DEN treatment. Lysates of the livers harvested from mice subjected to acute DEN treatment as described in (A) were prepared in RIPA lysis buffer containing 1% SDS and analyzed by Western blotting. Actin as loading control.


C MOAP - 1 deficiency elevates of p62 in detergent - insoluble fractions of liver lysates from mice subjected to acute DEN treatment . Livers from mice injected as described in ( A ) were lysed in 1 % Triton - X lysis buffer , separated into detergent - soluble ( S ) insoluble ( I ) fractions analyzed by Western blotting .

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C MOAP-1 deficiency elevates levels of p62 protein in the detergent-insoluble fractions of the liver lysates from mice subjected to acute DEN treatment. Livers from mice injected as described in (A) were lysed in 1% Triton-X lysis buffer, separated into detergent-soluble (S) and insoluble (I) fractions and analyzed by Western blotting.


D High of p62 bodies detected in tumors of MOAP - 1 KO mice . sections from livers dissected from 8 . 5 - month - old WT MOAP - 1 KO mice injected with a single dose of DEN ( 25 µg / g ) at 15 - day - old were subjected to with anti - p62 antibody ( in red ) . were stained with DAPI ( in blue ) . Insets were enlarged in bottom right . Scale bar : 20 µm . E of p62 bodies in WT MOAP - 1 KO tumors . ( Left panel ) number of p62 bodies per . n = 7 tumors from livers of WT MOAP - 1 KO mice . ( Right panel ) of p62 bodies . n = 456 1211 bodies detected in WT MOAP - 1 KO tumors , respectively , from three independent samples . Error bar represents S . E . M . * P < 0 . 05 , * * * P < 0 . 001 , student ' s t - .

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D High levels of p62 bodies detected in the tumors of the MOAP-1 KO mice. Tumor sections from livers dissected from the 8.5-month-old male WT and MOAP-1 KO mice injected with a single dose of DEN (25 µg/g body weight) at 15-day-old were subjected to IHC analysis with anti-p62 antibody (in red). Nuclear were stained with DAPI (in blue). Insets were enlarged in the bottom right. Scale bar: 20 µm. E Quantification of the p62 bodies in the WT and MOAP-1 KO tumors. (Left panel) number of p62 bodies per cell. n = 7 tumors from the livers of WT and MOAP-1 KO mice. (Right panel) size of p62 bodies. n = 456 and 1211 bodies detected in the WT and MOAP-1 KO tumors, respectively, from three independent samples. Error bar represents S.E.M. *P<0.05, ***P<0.001, student's t-test.


D High of p62 bodies detected in tumors of MOAP - 1 KO mice . sections from livers dissected from 8 . 5 - month - old WT MOAP - 1 KO mice injected with a single dose of DEN ( 25 µg / g ) at 15 - day - old were subjected to with anti - p62 antibody ( in red ) . were stained with DAPI ( in blue ) . Insets were enlarged in bottom right . Scale bar : 20 µm .

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D High levels of p62 bodies detected in the tumors of the MOAP-1 KO mice. Tumor sections from livers dissected from the 8.5-month-old male WT and MOAP-1 KO mice injected with a single dose of DEN (25 µg/g body weight) at 15-day-old were subjected to IHC analysis with anti-p62 antibody (in red). Nuclear were stained with DAPI (in blue). Insets were enlarged in the bottom right. Scale bar: 20 µm.


F Increased of Nrf2 its downstream target , HMOX - 1 , in MOAP - 1 deficient tumors . While p62 is upregulated in tumors compared to adjacent non - tissues in both WT MOAP - 1 KO livers , no significant difference is observed in p62 between WT MOAP - 1 KO tumors . Lysates of tumors non - parts of livers from mice injected with DEN as described in ( D ) were prepared in RIPA lysis buffer containing 1 % SDS subjected to Western blotting . Actin as loading control . P , PBS injected control ; N , non - ; T , .

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F Increased levels of Nrf2 and its downstream target protein, HMOX-1, in the MOAP-1 deficient tumors. While p62 protein is upregulated in the tumors compared to the adjacent non-tumor tissues in both WT and MOAP-1 KO livers, no significant difference is observed in the p62 protein levels between the WT and MOAP-1 KO tumors. Lysates of the tumors and non-tumor parts of the livers from mice injected with DEN as described in (D) were prepared in RIPA lysis buffer containing 1% SDS and subjected to Western blotting. Actin as loading control. P, PBS injected control; N, non-tumor; T, tumor.


G Upregulation of mRNA of HMOX - 1 NQO1 in MOAP - 1 deficient tumors . Livers harvested from mice injected with PBS or DEN as described in ( D ) were subjected to RT - PCR . n = 3 WT 3 MOAP - 1 KO mice injected with PBS control , 8 WT 9 MOAP - 1 KO mice injected with DEN . Error bar represents S . E . M . * * P < 0 . 01 , student ' s t - .

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G Upregulation of mRNA levels of HMOX-1 and NQO1 in the MOAP-1 deficient tumors. Livers harvested from the mice injected with PBS or DEN as described in (D) were subjected to RT-PCR analysis. n = 3 WT and 3 MOAP-1 KO mice injected with the PBS control, and 8 WT and 9 MOAP-1 KO mice injected with DEN. Error bar represents S.E.M. **P<0.01, student's t-test.


H MOAP - 1 deficient mice bear higher burden in DEN - mediated liver cancer model . Representative of liver tumors dissected from 8 . 5 - month - old WT MOAP - 1 KO mice injected with a single dose of DEN ( 25 µg / g ) at 15 - day - old . Scale bar : 5 mm . I Measurements of number , maximum diameter of nodules total liver from WT MOAP - 1 KO mice ( n = 13 14 respectively ) injected with DEN as described in ( H ) . Error bar represents S . E . M . * * P < 0 . 01 , * * * P < 0 . 001 , student ' s t - .

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H MOAP-1 deficient mice bear higher tumor burden in the DEN-mediated liver cancer model. Representative images of liver tumors dissected from the 8.5-month-old male WT and MOAP-1 KO mice injected with a single dose of DEN (25 µg/g body weight) at 15-day-old. Scale bar: 5 mm. I Measurements of the number, maximum diameter of the tumor nodules and total liver weight from WT and MOAP-1 KO mice (n=13 and 14 respectively) injected with DEN as described in (H). Error bar represents S.E.M. **P<0.01, ***P<0.001, student's t-test.


H MOAP - 1 deficient mice bear higher burden in DEN - mediated liver cancer model . Representative of liver tumors dissected from 8 . 5 - month - old WT MOAP - 1 KO mice injected with a single dose of DEN ( 25 µg / g ) at 15 - day - old . Scale bar : 5 mm .

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H MOAP-1 deficient mice bear higher tumor burden in the DEN-mediated liver cancer model. Representative images of liver tumors dissected from the 8.5-month-old male WT and MOAP-1 KO mice injected with a single dose of DEN (25 µg/g body weight) at 15-day-old. Scale bar: 5 mm.


MOAP - 1 deficiency promotes initiation . measurements of number , diameter of largest nodules total liver of 4 . 5 - month - old WT MOAP - 1 KO mice ( n = 18 23 respectively ) injected with a single dose of DEN ( 25 µg / g ) at 15 - day - old .

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MOAP-1 deficiency promotes tumor initiation. measurements of the number, diameter of the largest nodules and total liver weight of the 4.5-month-old WT and MOAP-1 KO mice (n=18 and 23 respectively) injected with a single dose of DEN (25 µg/g body weight) at 15-day-old.


( B ) Confocal of HeLa transfected with EGFP - ORP5 or EGFP - ORP8 showing variable expression . Arrows indicate ORP5 ORP8 localization to cortical ER ( cER ) . Scale bar , 10 μm .

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(B) Confocal images of HeLa cells transfected with EGFP-ORP5 or EGFP-ORP8 showing variable expression levels. Arrows indicate ORP5 and ORP8 localization to cortical ER (cER). Scale bar, 10 μm.


( C - D ) Electron microscopy of ultrathin cryosections of HeLa transfected with EGFP - ORP5 or EGFP - ORP8 immunogold stained with anti - GFP ( 15 nm gold ) to detect ORP5 or ORP8 anti - PDI ( 10 nm gold ) to label ER lumen . ORP5 ORP8 localize at ER - PM ( arrows ) ER - mitochondria contact sites ( arrowheads ) . m , mitochondria ; cER , cortical ER . Scale bar 200nm . ( E - F ) of IEM labelling for EGFP - ORP5 , EGFP - ORP8 ( E ) GFP - Sec61β ( F ) in transfected HeLa . Results are presented as of total number of gold particles ( 800 per condition , n = 35 - 45 ) for ORP5 , ORP8 Sec61β in indicated compartments ( non - cER , non - cortical ER ; ER - PM , ER - PM contact sites ; ER - mito , ER - mitochondria contact sites . % gold particles ± . Data are mean of three independent replicates . * P < 0 , 001 compared to non - cER ER - mito [ EGFP - ORP5 ] to ER - PM ER - mito [ EGFP - ORP8 GFP - Sec61β ] , # P < 0 , 001 compared to ER - PM ER - mito [ GFP - Sec61β ] ) .

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(C-D) Electron microscopy of ultrathin cryosections of HeLa cells transfected with EGFP-ORP5 or EGFP-ORP8 and immunogold stained with anti-GFP (15 nm gold) to detect ORP5 or ORP8 and anti-PDI (10 nm gold) to label the ER lumen. ORP5 and ORP8 localize at ER-PM (arrows) and ER-mitochondria contact sites (arrowheads). m, mitochondria; cER, cortical ER. Scale bar 200nm. (E-F) Quantification of the IEM labelling for EGFP-ORP5, EGFP-ORP8 (E) and GFP-Sec61β (F) in transfected HeLa cells. Results are presented as the percentage of the total number of gold particles (800 per condition, n=35-45 cells) for ORP5, ORP8 and Sec61β in the indicated compartments (non-cER, non-cortical ER; ER-PM, ER-PM contact sites; ER-mito, ER-mitochondria contact sites. % gold particles ±SEM. Data are the mean of three independent replicates. *P<0,001 compared to non-cER and ER-mito [EGFP-ORP5] and to ER-PM and ER-mito [EGFP-ORP8 and GFP-Sec61β], #P<0,001 compared to ER-PM and ER-mito [GFP-Sec61β]).


( G ) Representative electron microscopy of mitochondria ( H ) of of ER - mitochondria contact sites ( ER - mito contact ) per mitochondria of HeLa expressing EGFP - ORP5 , EGFP - ORP8 together with HRP - KDEL , to visualize ER . Control ( Ctrl ) consisted of HeLa overexpressing only HRP - KDEL .

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(G) Representative electron microscopy of mitochondria and (H) quantification of the percentage of ER-mitochondria contact sites (ER-mito contact) per mitochondria of HeLa cells expressing EGFP-ORP5, EGFP-ORP8 together with HRP-KDEL, to visualize the ER. Control (Ctrl) consisted of HeLa cells overexpressing only HRP-KDEL.


( I ) Crude mitochondria , mitochondria MAM fractions were purified from HeLa equal amount of ( 25g ) from each fraction were loaded on a 4 - 20 % gradient SDS - PAGE gel immunoblotted using anti - ORP5 , anti - ORP8 anti - IP3R - 3 ( MAM ) , anti - PDI ( ER ) anti - cytochrome c ( mitochondrial ) . TC , Total ; MAM , mitochondria - associated ER .

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(I) Crude mitochondria, mitochondria and MAM fractions were purified from HeLa cells and equal amount of protein (25g) from each fraction were loaded on a 4-20% gradient SDS-PAGE gel and immunoblotted using anti-ORP5, anti-ORP8 and anti-IP3R-3 (MAM protein), anti-PDI (ER protein) and anti- cytochrome c (mitochondrial protein). TC,Total cell; MAM, mitochondria-associated ER membrane .


( A ) Confocal of HeLa transfected with EGFP - ORP5 , EGFP - ORP8 , or GFP - Sec61β together with PTPIP51 - HA immunostained using HA antibody to detect PTPIP51 . Scale bar , 10 μm .

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(A) Confocal images of HeLa cells transfected with EGFP-ORP5, EGFP-ORP8, or GFP-Sec61β together with PTPIP51-HA and immunostained using HA antibody to detect PTPIP51. Scale bar, 10 μm.


( B ) Electron microscopy of ultrathin cryosections of HeLa co - transfected with EGFP - ORP5 PTPIP51 - HA immunogold stained with anti - GFP ( 15 nm gold ) anti - HA ( 10 nm gold ) showing EGFP - ORP5 localization at ER - mitochondria contacts ( arrows ) . Arrowheads indicate highly expanded ER juxtaposed to mitochondria . Scale bar 200 nm . ( C ) of IEM labelling for EGFP - ORP5 , EGFP - ORP8 GFP - Sec61β in HeLa co - transfected with PTPIP51 - HA . Results are presented as of total number of gold particles ( 800 per condition , n = 30 - 35 ) for ORP5 , ORP8 Sec61β in indicated compartments ( ER , reticular ER ; ER - mito MCS , ER - mitochondria contact sites ; ER - PM MCS , ER - PM contact sites ) . % gold particles ± . Data are representative of three independent replicates . * * P < 0 , 001 .

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(B) Electron microscopy of ultrathin cryosections of HeLa cells co-transfected with EGFP-ORP5 and PTPIP51-HA and immunogold stained with anti-GFP (15 nm gold) and anti-HA (10 nm gold) showing EGFP-ORP5 localization at ER-mitochondria contacts (arrows). Arrowheads indicate the highly expanded ER membrane juxtaposed to mitochondria. Scale bar 200 nm. (C) Quantification of the IEM labelling for EGFP-ORP5, EGFP-ORP8 and GFP-Sec61β in HeLa cells co-transfected with PTPIP51-HA. Results are presented as the percentage of the total number of gold particles (800 per condition, n=30-35 cells) for ORP5, ORP8 and Sec61β in the indicated compartments (ER, reticular ER; ER-mito MCS, ER-mitochondria membrane contact sites; ER-PM MCS, ER-PM membrane contact sites). % gold particles ±SEM. Data are representative of three independent replicates. **P<0,001.


( D ) Confocal of HeLa transfected with EGFP - ORP5 or EGFP - ORP8 together with PTPIP51 - HA RFP - Sec61β immunostained using HA antibody to detect PTPIP51 . Scale bar , 10 μm .

(D) Confocal images of HeLa cells transfected with EGFP-ORP5 or EGFP-ORP8 together with PTPIP51-HA and RFP-Sec61β and immunostained using HA antibody to detect PTPIP51. Scale bar, 10 μm.


( E ) Representative EM of ER - mitochondria contacts in indicated overexpression conditions . ( F ) of of ER - mitochondria contact sites ( ER - mito contact ) per mitochondria of HeLa transfected with HRP - KDEL ( Ctrl ) or co - transfected with PTPIP51 - HA EGFP or PTPIP51 - HA EGFP - ORP5 or PTPIP51 - HA EGFP - ORP8 ( n = 30 1082 - 1223 mitochondria ) . % ER - mitochondria contact sites ± . Data are representative of three independent replicates . * * P < 0 , 001 , * P < 0 , 05 .

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(E) Representative EM images of ER-mitochondria contacts in the indicated overexpression conditions. (F) Quantification of the percentage of ER-mitochondria contact sites (ER-mito contact) per mitochondria of HeLa cells transfected with HRP-KDEL (Ctrl) or co-transfected with PTPIP51-HA and EGFP or PTPIP51-HA and EGFP-ORP5 or PTPIP51-HA and EGFP-ORP8 (n=30 cells and 1082-1223 mitochondria). % ER-mitochondria contact sites ±SEM. Data are representative of three independent replicates. **P<0,001, *P<0,05.


( G ) EGFP , EGFP - ORP5 or EGFP - ORP8 were immuno - precipitated from lysates of HeLa co - expressing PTPIP51 - HA treated with control ( siCtrl ) or VAPA VAPB ( SiVAPA / B ) siRNAs analyzed by western blot using antibodies against GFP ( ORPs ) , HA ( PTPIP51 ) , VAPA , VAPB tubulin .

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(G) EGFP, EGFP-ORP5 or EGFP-ORP8 were immuno-precipitated from lysates of HeLa cells co-expressing PTPIP51-HA and treated with control (siCtrl) or VAPA and VAPB (SiVAPA/B) siRNAs and analyzed by western blot using antibodies against GFP (ORPs), HA (PTPIP51), VAPA, VAPB and tubulin.


( A ) Confocal of HeLa transfected with EGFP - ORP5 , EGFP - ORP5PH or EGFP - ORP5ORD immunostained using anti - TOM20 antibody to visualize mitochondria . Scale bar , 10 μm .

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(A) Confocal images of HeLa cells transfected with EGFP-ORP5, EGFP-ORP5PH or EGFP-ORP5ORD and immunostained using anti-TOM20 antibody to visualize mitochondria. Scale bar, 10 μm.


( B ) Confocal of HeLa transfected with EGFP - tagged ORD domain of ORP5 ( EGFP - ORD ORP5 ) immunostained using anti - TOM20 antibody ( upper panels ) or co - transfected with PTPIP51 - HA immunostained using HA antibody to detect PTPIP51 . Scale bar , 10 μm .

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(B) Confocal images of HeLa cells transfected with the EGFP-tagged ORD domain of ORP5 (EGFP-ORD ORP5) and immunostained using anti-TOM20 antibody (upper panels) or co-transfected with PTPIP51-HA and immunostained using HA antibody to detect PTPIP51. Scale bar, 10 μm.


( C - D ) Confocal of HeLa transfected with EGFP - ORP5PH or EGFP - ORP5ORD ( C ) together with PTPIP51 - HA immunostained using HA antibody to detect PTPIP51 . Scale bar , 10 μm .

(C-D) Confocal images of HeLa cells transfected with EGFP-ORP5PH or EGFP-ORP5ORD (C) together with PTPIP51-HA and immunostained using HA antibody to detect PTPIP51. Scale bar, 10 μm.


( C - D ) Confocal of HeLa transfected with EGFP - ORP5 , EGFP - ORP5L389D or EGFP - ORP8L425D ( D ) together with PTPIP51 - HA immunostained using HA antibody to detect PTPIP51 . Scale bar , 10 μm .

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(C-D) Confocal images of HeLa cells transfected with EGFP-ORP5, EGFP-ORP5L389D or EGFP-ORP8L425D (D) together with PTPIP51-HA and immunostained using HA antibody to detect PTPIP51. Scale bar, 10 μm.


( E ) EGFP - ORP5 , EGFP - ORP8 , EGFP - ORP5L389D , EGFP - ORP8L425D , GFP - Sec61β or EGFP alone were immunoprecipitated from lysates of HeLa co - expressing PTPIP51 - HA analyzed by western blot with antibodies against GFP HA .

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(E) EGFP-ORP5, EGFP-ORP8, EGFP-ORP5L389D, EGFP-ORP8L425D, GFP-Sec61β or EGFP alone were immunoprecipitated from lysates of HeLa cells co-expressing PTPIP51-HA and analyzed by western blot with antibodies against GFP and HA.


( A ) ORP5 , ORP8 actin of HRP - KDEL - expressing HeLa treated with Ctrl siRNAs ( siCtrl ) or siRNAs against ORP5 ( siORP5 ) or ORP8 ( siORP8 ) .

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(A) ORP5, ORP8 and actin levels of HRP-KDEL-expressing HeLa cells treated with Ctrl siRNAs (siCtrl) or siRNAs against ORP5 (siORP5) or ORP8 (siORP8).


( B ) Electron microscopy of HRP - KDEL - expressing HeLa treated with Ctrl siRNAs or siRNAs against ORP5 or ORP8 . Red arrows indicate ER - PM contact sites . Scale bar , 2 μm . Insets show representative mitochondria red arrows in insets indicate ER - mitochondria contacts . Inset scale bar , 500nm .

(B) Electron microscopy of HRP-KDEL-expressing HeLa cells treated with Ctrl siRNAs or siRNAs against ORP5 or ORP8. Red arrows indicate ER-PM contact sites. Scale bar, 2 μm. Insets show representative mitochondria and red arrows in the insets indicate ER-mitochondria contacts. Inset scale bar, 500nm.


( C ) Representative EM showing of mitochondria in siCtrl , siORP5 siORP8 - treated . Scale bar , 1 μm . ( D ) Quantifications of number of mitochondria with altered in indicated siRNA conditions . % altered mitochondria ± , n = 25 632 - 710 mitochondria . * P < 0 , 001 compared to siCtrl .

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(C) Representative EM micrographs showing the morphology of mitochondria in siCtrl, siORP5 and siORP8-treated cells. Scale bar, 1 μm. (D) Quantifications of the number of mitochondria with altered morphology in the indicated siRNA conditions. % altered mitochondria ±SEM, n=25 cells and 632-710 mitochondria. *P<0,001 compared to siCtrl.


( E ) Mitochondrial oxygen consumption rate ( OCR ) measured in Ctrl ORP5 siRNA transfected . OCR trace was obtained by sequential measurement of basal OCR ( OCRBAS ) , OCR after addition of oligomycin ( OM ) OCR after addition of antimycin A ( AA ) . Note reduced basal OCR ( OCRBAS ) compared to ctrl siRNA . Error bars denote ± . Data are mean of 4 independent repeats ( n = 4 ) , * P < 0 , 05 .

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(E) Mitochondrial oxygen consumption rate (OCR) measured in Ctrl and ORP5 siRNA transfected cells. OCR trace was obtained by sequential measurement of basal OCR (OCRBAS), OCR after the addition of oligomycin (OM) and OCR after addition of antimycin A (AA). Note the reduced basal OCR (OCRBAS) compared to ctrl siRNA cells. Error bars denote ±SEM. Data are the mean of 4 independent repeats (n=4), *P<0,05.


( C ) Representative traces obtained with ADP • BeFx . upper FRET trace was calculated from middle traces obtained by exciting donor fluorophore measuring both donor ( green ) acceptor ( red ) . lowest trace was obtained by exciting acceptor fluorophore directly . arrow indicates a bleaching event . ( D ) Distribution of FRET values determined from 97 traces as in ( C ) fit with a Gaussian model ( black curve ) .

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(C) Representative traces obtained with ADP•BeFx. The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor (green) and acceptor (red) fluorescence. The lowest trace was obtained by exciting the acceptor fluorophore directly. The arrow indicates a bleaching event. (D) Distribution of FRET values determined from 97 traces as in (C) fit with a Gaussian model (black curve).


( E ) As in ( C ) , but in presence of ATP . Periods in which a fluorescently labeled SecA molecule is bound are indicated by grey shading . ( F ) As in ( D ) , but with ATP ( 257 traces ) .

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(E) As in (C), but in the presence of ATP. Periods in which a fluorescently labeled SecA molecule is bound are indicated by grey shading. (F) As in (D), but with ATP (257 traces).


( A ) A representative FRET trace ( blue ) was obtained as in Fig 1 , fit with a hidden Markov model ( black dashed ) .

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(A) A representative FRET trace (blue line) was obtained as in Fig 1, and fit with a hidden Markov model (black dashed line).


( B ) Traces as in ( A ) obtained in presence of different nucleotides , were used to determine number of states best fit by Markov model . ( C ) Transition density plot of idealized ATP FRET states obtained in ( B ) .

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(B) Traces as in (A) obtained in the presence of different nucleotides, were used to determine the number of states best fit by the Markov model. (C) Transition density plot of idealized ATP FRET states obtained in (B).


( D ) distributions of dwell times of low FRET states observed in ATP were fit with a single exponential ( 1500 low FRET states ) . inset shows average dwell time error , defined as standard error on number of traces . ( E ) As in ( D ) , but with high FRET ( 1656 high FRET states ) .

(D) The distributions of dwell times of the low FRET states observed in ATP were fit with a single exponential (1500 low FRET states). The inset shows average dwell time and error, defined as the standard error based on the number of traces. (E) As in (D), but with high FRET (1656 high FRET states).


( F ) Representative traces obtained with ATPγS . upper FRET trace was calculated from middle traces obtained by exciting donor fluorophore measuring both donor ( green ) acceptor ( red ) . lowest trace was obtained by exciting acceptor fluorophore directly . arrow indicates a bleaching event . ( G ) Distribution of FRET values determined from 168 traces as in ( D ) fit with a Gaussian model ( black curve ) .

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(F) Representative traces obtained with ATPγS. The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor (green) and acceptor (red) fluorescence. The lowest trace was obtained by exciting the acceptor fluorophore directly. The arrow indicates a bleaching event. (G) Distribution of FRET values determined from 168 traces as in (D) fit with a Gaussian model (black curve).


( C ) Representative traces obtained with ADP • BeFx . upper FRET trace was calculated from middle traces obtained by exciting donor fluorophore measuring both donor ( green ) acceptor ( red ) . lowest trace was obtained by exciting acceptor fluorophore directly . arrow indicates a bleaching event . ( D ) Distribution of FRET values determined from 197 traces as in ( B ) fit with a Gaussian model ( black curve ) .

(C) Representative traces obtained with ADP•BeFx. The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor (green) and acceptor (red) fluorescence. The lowest trace was obtained by exciting the acceptor fluorophore directly. The arrow indicates a bleaching event. (D) Distribution of FRET values determined from 197 traces as in (B) fit with a Gaussian model (black curve).


( E ) As in ( C ) , but with ATP . Periods in which a fluorescently labeled SecA molecule is bound are indicated by grey shading . ( F ) As in ( D ) , but with ATP ( 228 traces ) .

(E) As in (C), but with ATP. Periods in which a fluorescently labeled SecA molecule is bound are indicated by grey shading. (F) As in (D), but with ATP (228 traces).


( A ) FRET traces of clamp movements in presence of different nucleotides were used to determine number of states best fit by a Markov model . ( B ) Transition density plot of idealized ATP FRET states obtained in ( A ) .

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(A) FRET traces of clamp movements in the presence of different nucleotides were used to determine the number of states best fit by a Markov model. (B) Transition density plot of idealized ATP FRET states obtained in (A).


( C ) Representative traces obtained with ATPγS . upper FRET trace was calculated from middle traces obtained by exciting donor fluorophore measuring both donor ( green ) acceptor ( red ) . lowest trace was obtained by exciting acceptor fluorophore directly . arrow indicates a bleaching event . ( D ) Distribution of FRET values determined from 315 traces as in ( C ) fit with a Gaussian model ( black curve ) .

(C) Representative traces obtained with ATPγS. The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor (green) and acceptor (red) fluorescence. The lowest trace was obtained by exciting the acceptor fluorophore directly. The arrow indicates a bleaching event. (D) Distribution of FRET values determined from 315 traces as in (C) fit with a Gaussian model (black curve).


( E ) Comparison of high low FRET state occupancy in ADP • Pi ADP • Vi for clamp THF .

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(E) Comparison of high and low FRET state occupancy in ADP•Pi and ADP•Vi for the clamp and THF.


( F ) distributions of dwell times of low FRET states observed in ATP were fit with a single exponential ( 1539 low FRET states ) . inset shows average dwell time error , defined as standard error on number of traces . ( G ) As in ( F ) , but with high FRET ( 1773 high FRET states ) .

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(F) The distributions of dwell times of the low FRET states observed in ATP were fit with a single exponential (1539 low FRET states). The inset shows average dwell time and error, defined as the standard error based on the number of traces. (G) As in (F), but with high FRET (1773 high FRET states).


( H ) Comparison of dwell times of high FRET states for two - helix finger ( THF ) clamp for different fluorophore positions . Errors as in ( G ) with significance on two sample Kolmogorov - Smirnov with a 1 % threshold . n . s . P = 0 . 012 ( left ) , n . s . P = 0 . 681 ( right ) ; * P < 1 * 10 - 35 ( between each pair of THF / clamp mutants ) . 809C / 394C : 1656 high FRET states ; 809C / 103C : 1349 states ; 233C / 103C : 1773 states ; 233C / 336C : 1778 states .

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(H) Comparison of dwell times of the high FRET states for the two-helix finger (THF) and clamp for different fluorophore positions. Errors as in (G) with significance based on two sample Kolmogorov-Smirnov tests with a 1% threshold. n.s.P = 0.012 (left), n.s.P = 0.681 (right); *P < 1*10-35 (between each pair of THF/clamp mutants). 809C/394C: 1656 high FRET states; 809C/103C: 1349 states; 233C/103C: 1773 states; 233C/336C: 1778 states.


A - B : of pDCs ( A ) mDCs ( B ) from total PBMCs collected at D1 was quantified by flow cytometry grouped to self - reported duration of symptoms prior to D1 ( A - B ; 0 - 4 n = 22 ; 5 - 8 n = 39 ; 9 - 12 n = 39 ; ≥ 13 n = 16 ) . Data information : Each dot represents a , lines with error bars show median values with interquartile ranges Statistical significance was determined using Kruskal - Wallis * < p0 . 05 , * * < p0 . 01 * * * < p0 . 001 , ns = not significant .

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A-B: Percentage of pDCs (A) and mDCs (B) from total PBMCs collected at D1 was quantified by flow cytometry and grouped to self-reported duration of symptoms prior to D1 (A-B; 0-4 n= 22; 5-8 n=39; 9-12 n= 39; ≥13 n=16). Data information: Each dot represents a patient, lines with error bars show the median values with interquartile ranges Statistical significance was determined using the Kruskal-Wallis test *<p0.05, **<p0.01 ***<p0.001, ns= not significant.


PBMCs plasma samples were collected from SARS - CoV - 2 infected individuals at time of hospitalization ( D1 ) after 5 days ( D5 ) . C - D : of pDCs ( C ) mDCs ( D ) at D1 D5 matched samples from independent of symptom duration ( n = 93 ) . Data information : Each dot represents a , lines with error bars show median values with interquartile ranges Statistical significance was determined using Wilcoxon matched pairs signed rank * < p0 . 05 , * * < p0 . 01 * * * < p0 . 001 , ns = not significant .

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PBMCs and plasma samples were collected from SARS-CoV-2 infected individuals at time of hospitalization (D1) and after 5 days (D5). C-D: Percentage of pDCs (C) and mDCs (D) at D1 and D5 matched samples from all patients independent of symptom duration (n=93). Data information: Each dot represents a patient, lines with error bars show the median values with interquartile ranges Statistical significance was determined using the Wilcoxon matched pairs signed rank test *<p0.05, **<p0.01 ***<p0.001, ns= not significant.


PBMCs plasma samples were collected from SARS - CoV - 2 infected individuals at time of hospitalization ( D1 ) after 5 days ( D5 ) . E - J : Matched D1 D5 plasma samples were available from 55 donors were determined for IFNα2a ( E , n = 30 ) , IL - 3 ( F , n = 38 ) , IL - 8 ( G , n = 44 ) , IL - 6 ( H , n = 51 ) , TNFα ( I , n = 42 ) CXCL10 ( J , n = 46 ) , note that some samples could not be quantified were excluded from . Data information : Each dot represents a , lines with error bars show median values with interquartile ranges Statistical significance was determined using Wilcoxon matched pairs signed rank * < p0 . 05 , * * < p0 . 01 * * * < p0 . 001 , ns = not significant .

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PBMCs and plasma samples were collected from SARS-CoV-2 infected individuals at time of hospitalization (D1) and after 5 days (D5). E-J: Matched D1 and D5 plasma samples were available from 55 donors and protein levels were determined for IFNα2a (E, n=30), IL-3 (F, n=38), IL-8 (G, n=44), IL-6 (H, n=51), TNFα (I, n=42) and CXCL10 (J, n=46), note that some samples could not be quantified and were excluded from the analysis. Data information: Each dot represents a patient, lines with error bars show the median values with interquartile ranges Statistical significance was determined using the Wilcoxon matched pairs signed rank test *<p0.05, **<p0.01 ***<p0.001, ns= not significant.


K - L : of pDCs grouped by COVID - 19 severity , as determined by oxygen supplementation ( no oxygen n = 43 , nasal oxygen n = 65 high flow oxygen n = 5 ) . of pDCs ( K ) was correlated to COVID - 19 disease severity ( L ) using simple linear regression . Data information : Each dot represents a , lines with error bars show median values with interquartile ranges mean values with standard error of mean Statistical significance was determined using Kruskal - Wallis simple linear regression * < p0 . 05 , * * < p0 . 01 * * * < p0 . 001 , ns = not significant .

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K-L: Percentage of pDCs grouped by COVID-19 severity, as determined by oxygen supplementation (no oxygen n=43, nasal oxygen n=65 and high flow oxygen n=5). Percentage of pDCs (K) was correlated to COVID-19 disease severity (L) using simple linear regression. Data information: Each dot represents a patient, lines with error bars show the median values with interquartile ranges mean values with the standard error of the mean Statistical significance was determined using the Kruskal-Wallis test and simple linear regression *<p0.05, **<p0.01 ***<p0.001, ns= not significant.


C - J : FR2020 was used in subsequent experiments where pDC were either mock treated ( mock , grey ) , exposed to SARS - CoV - 2 at 1 MOI ( SARS - 2 , purple ) , TLR7 ( 2 . 5 μg / mL R837 , blue ) or TLR3 agonist ( 800 ng / mL poly ( I : C ) , pink ) . Supernatants were collected after 24 hrs analyzed for type I IFNα ( C ) , IFNβ ( D ) , type II IFNγ ( E ) , type III IFNλ1 ( F ) , IL - 6 ( G ) , IL - 8 ( H ) , CXCL10 ( I ) TNFα ( J ) expression by ELISA . Data information : Bars represent mean values with standard error of mean symbols represent pDC donors ( n = 3 - 4 ) . Equal symbols represent equal donors Statistical significance was determined using paired student T compared treated condition with time point - matched mock condition * < p0 . 05 , * * < p0 . 01 * * * < p0 . 001 .

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C- J: The FR2020 strain was used in subsequent experiments where pDC were either mock treated (mock, grey), exposed to SARS-CoV-2 at 1 MOI (SARS-2, purple), TLR7 (2.5 μg/mL R837, blue) or TLR3 agonist (800 ng/mL poly(I:C), pink). Supernatants were collected after 24 hrs and analyzed for type I IFNα (C), IFNβ (D), type II IFNγ (E), type III IFNλ1 (F), IL-6 (G), IL-8 (H), CXCL10 (I) and TNFα (J) expression by ELISA. Data information: Bars represent mean values with standard error of the mean and symbols represent individual pDC donors (n=3-4). Equal symbols represent equal donors Statistical significance was determined using the ratio paired student T test and compared the treated condition with the time point-matched mock condition *<p0.05, **<p0.01 ***<p0.001.


Using CRISPR / Cas9 , MyD88 knock - out ( KO ) AAVS1KO ( control ) pDCs were generated . MyD88 in KO control pDCs were analyzed by western

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Using CRISPR/Cas9, MyD88 knock-out (KO) and AAVS1KO (control) pDCs were generated. MyD88 protein levels in KO and control pDCs were analyzed by western blotting


C - D : MyD88KO control pDCs were either mock treated ( mock ) or exposed to SARS - CoV - 2 ( SARS - 2 , 1 MOI ) , supernatants were collected at indicated time points analyzed for type I IFNα ( C ) CXCL10 ( D ) . Done with n = 4 Data information : Bars represent mean values equal symbols represent equal donors . Statistical significance was determined using Mann - Whitney one - T in ( C ) as MyD88 - KO data had no variance values of 0 . For ( D ) a Mann - Whitney two - T was used for comparison between conditions of mock MyD88 KO donors .

C-D: MyD88KO and control pDCs were either mock treated (mock) or exposed to SARS-CoV-2 (SARS-2, 1 MOI), supernatants were collected at indicated time points and analyzed for type I IFNα (C) and CXCL10 (D). Done with n=4 Data information: Bars represent mean values and equal symbols represent equal donors. Statistical significance was determined using Mann-Whitney one-tailed T test in (C) as MyD88-KO data had no variance and values of 0. For (D) a Mann-Whitney two-tailed T test was used for comparison between conditions of mock and MyD88 KO donors.


E - F : Type I IFNα production was then determined in culture supernatant from SARS - CoV - 2 exposed TRIFKO or TRIG + MyD88KO ( E ) RIG - IKO or RIG - I + MyD88KO ( F ) pDCs . Done with n =

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E-F: Type I IFNα production was then determined in cell culture supernatant from SARS-CoV-2 exposed TRIFKO or TRIG+MyD88KO (E) and RIG-IKO or RIG-I+MyD88KO (F) pDCs. Done with n=2


( A ) Surface plasmon resonance ( SPR ) KAP1 - KRAB binding . MBP - KRAB was immobilized on chip . WT or R311E KAP1 RBCC were flowed over chip . Data points are shown in red or blue ; fits are shown as red or blue lines .

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(A) Surface plasmon resonance (SPR) KAP1-KRAB binding assay. MBP-KRAB was immobilized on the chip. WT or R311E KAP1 RBCC were flowed over the chip. Data points are shown in dark red or blue; fits are shown as light red or blue lines.


( C ) - 1 reporter repression with single point mutants a previously described KRAB - binding deficient KAP1 variant containing four mutations in CC domain ( CCmut ; V293S / K296A / M297A / L300S ) .

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(C) LINE-1 reporter repression with single point mutants and a previously described KRAB-binding deficient KAP1 variant containing four mutations in the CC domain (CCmut; V293S/K296A/M297A/L300S).


( D ) SVA reporter repression with same set of mutants as in ( B ) . Data were normalized to KAP1 KO transfected with an empty vector ( EV ) .

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(D) SVA reporter repression with the same set of mutants as in (B). Data were normalized to KAP1 KO cells transfected with an empty vector (EV).


( A ) BMDMs were infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis ( MOI = 10 ) for 4 h ( as described in Materials Methods ) , then incubated for 24 h ( left ) or indicated periods of time ( right ) . were fixed , stained with DAPI to visualize nuclei ( blue ) , immunolabeled with an anti - LC3 antibody . antibody was detected using an Alexa Fluor 488 - conjugated goat anti - rabbit IgG ( green ) . Left : representative immunofluorescence of LC3 ; right : of data ( LC3 - punctated were counted manually ) . * * * p < 0 . 001 , vs . Mtb - WT - infected condition . Scale bars , 5 µm . (

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(A) BMDMs were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis (MOI = 10) for 4 h (as described in the Materials and Methods), and then incubated for 24 h (left) or the indicated periods of time (right). Cells were fixed, stained with DAPI to visualize nuclei (blue), and immunolabeled with an anti-LC3 antibody. Primary antibody was detected using an Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). Left: representative immunofluorescence images of LC3 punctae; right: quantification of data (LC3-punctated cells were counted manually). ***p<0.001, vs. Mtb-WT-infected condition. Scale bars, 5 µm. (


( B ) BMDMs were infected with Mtb - Δeis in absence or presence of 3 - methyladenine ( 3 - MA ; 10 mM ) subjected to confocal as described in Figure 1A . LC3 - punctated were counted manually . Each condition was assayed in triplicate , at least 250 were counted in each well . * * * p < 0 . 001 , vs . SC .

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(B) BMDMs were infected with Mtb-Δeis in the absence or presence of 3-methyladenine (3-MA; 10 mM) and subjected to confocal analysis as described in Figure 1A. LC3-punctated cells were counted manually. Each condition was assayed in triplicate, and at least 250 cells were counted in each well. ***p<0.001, vs. SC.


( C ) Immunoblot analyses performed using Abs raised to LC3 or β - actin . Experimental conditions were identical to those outlined in panel A . Gel representative of three experiments are shown .

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(C) Immunoblot analyses performed using Abs raised to LC3 or β-actin. Experimental conditions were identical to those outlined in panel A. Gel images representative of three experiments are shown.


( D ) Electron of Mtb - Δeis - infected BMDMs under low ( left ) high ( right ) magnification show accumulation of autophagic vesicles ( black arrow , initial autophagic vacuoles ; arrow , degradative autophagic vacuoles ) . Scale bars : 2 µm ( left ) , 0 . 5 µm ( right ) .

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(D) Electron micrographs of Mtb-Δeis-infected BMDMs under low (left) and high (right) magnification show the accumulation of autophagic vesicles (black arrow, initial autophagic vacuoles; white arrow, degradative autophagic vacuoles). Scale bars: 2 µm (left), 0.5 µm (right).


( E ) Immunoblot analyses performed using Abs raised to LC3 or β - actin . BMDMs were infected with Mtb - Δeis in presence or absence of 3 - MA ( 10 mM ) or bafilomycin A1 ( Baf - A1 ; 100 nM ) . Gel representative of three experiments are shown . of intensities of LC3 - II / LC3 - I β - actin bands is indicated below each lane ( C E ) . UI , uninfected ; SC , solvent control ( 0 . 1 % distilled water ( B ) , 0 . 1 % DMSO ( E ) ) .

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(E) Immunoblot analyses performed using Abs raised to LC3 or β-actin. BMDMs were infected with Mtb-Δeis in the presence or absence of 3-MA (10 mM) or bafilomycin A1 (Baf-A1; 100 nM). Gel images representative of three experiments are shown. The ratio of the intensities of the LC3-II/LC3-I and β-actin bands is indicated below each lane (C and E). UI, uninfected; SC, solvent control (0.1% distilled water (B), 0.1% DMSO (E)).


( A B ) BMDMs were infected with Mtb - WT , Mtb - Δeis or Mtb - c - eis at different MOIs ( 0 . 1 , 1 10 ) for 18 h ( A ) or for indicated periods of time ( B ; MOI = 10 ) . Supernatants were assessed by ELISA for of TNF - α IL - 6 . Data ( A B ) are presented as mean ± SD of five experiments .

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(A and B) BMDMs were infected with Mtb-WT, Mtb-Δeis or Mtb-c-eis at different MOIs (0.1, 1 and 10) for 18 h (A) or for the indicated periods of time (B; MOI = 10). Supernatants were assessed by ELISA for levels of TNF-α and IL-6. Data (A and B) are presented as the mean±SD of five experiments.


( C D ) BMDMs were stimulated with Mtb - WT , Mtb - Δeis , or Mtb - c - eis for 30 min . were then incubated with 10 µM DHE or 5 µM DCFH - DA for 15 min , washed thoroughly , immediately analyzed for superoxide or H2O2 production by flow cytometry ( C , Left ) . were labeled with MitoSOX for 30 min analyzed for mitochondrial ROS by flow cytometry ( D , top ) . Quantitative of ROS generation ( C , right ; D , bottom ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , vs . Mtb - WT - infected condition . UI , uninfected .

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(C and D) BMDMs were stimulated with Mtb-WT, Mtb-Δeis, or Mtb-c-eis for 30 min. Cells were then incubated with 10 µM DHE or 5 µM DCFH-DA for 15 min, washed thoroughly, and immediately analyzed for superoxide or H2O2 production by flow cytometry (C, Left). Cells were labeled with MitoSOX for 30 min and analyzed for mitochondrial ROS levels by flow cytometry (D, top). Quantitative analysis of ROS generation (C, right; D, bottom). *p<0.05, **p<0.01, ***p<0.001, vs. Mtb-WT-infected condition. UI, uninfected.


( A - C ) BMDMs were infected with Mtb - Δeis ( MOI = 10 ) for 18 h in presence or absence of DPI ( 10 µM ) , NAC ( 20 mM ) , catalase ( Cat , 1 mU / mL ) , or tiron ( 5 mM ) . ( A ) Representative immunofluorescence ( top ) ; of endogenous LC3 - punctated ( bottom )

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(A-C) BMDMs were infected with Mtb-Δeis (MOI = 10) for 18 h in the presence or absence of DPI (10 µM), NAC (20 mM), catalase (Cat, 1 mU/mL), or tiron (5 mM). (A) Representative immunofluorescence images (top); percentage of endogenous LC3-punctated cells (bottom)


( B ) Immunoblot analyses of BMDMs with antibodies raised to LC3 or β - actin . Gel are representative of three experiments . of intensities of LC3 - II / LC3 - I β - actin bands is indicated below each lane .

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(B) Immunoblot analyses of BMDMs with antibodies raised to LC3 or β-actin. Gel images are representative of three experiments. The ratio of the intensities of the LC3-II/LC3-I and β-actin bands is indicated below each lane.


( C ) Experimental conditions were identical to those outlined in Figure 3A . Supernatants collected 18 h after infection were assessed for cytokine by ELISA . Data represent mean ± SD of five experiments .

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(C) Experimental conditions were identical to those outlined in Figure 3A. Supernatants collected 18 h after infection were assessed for cytokine levels by ELISA. Data represent the mean±SD of five experiments.


( D - F ) BMDMs from WT NOX2 KO mice were infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis for 18 h . ( D ) Numbers of LC3 - punctated ( counted manually ) are shown ( at least 250 were counted in each well ) .

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(D-F) BMDMs from WT and NOX2 KO mice were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis for 18 h. (D) Numbers of LC3-punctated cells (counted manually) are shown (at least 250 cells were counted in each well).


( E ) Immunoblot analyses performed using Abs raised to LC3 or β - actin . BMDMs from WT NOX2 KO mice were infected with Mtb - Δeis for 18 h . Gel representative of three experiments are shown .

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(E) Immunoblot analyses performed using Abs raised to LC3 or β-actin. BMDMs from WT and NOX2 KO mice were infected with Mtb-Δeis for 18 h. Gel images representative of three experiments are shown.


( F ) Supernatants collected 18 h after infection were assessed for cytokine by ELISA . Data are presented as mean ± SD of at least three separate experiments , each performed in triplicate . * * * p < 0 . 001 , vs . SC ( A C ) ; WT mice ( D F ) . UI , uninfected ; SC , solvent control ( 0 . 1 % DMSO ) .

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(F) Supernatants collected 18 h after infection were assessed for cytokine levels by ELISA. Data are presented as the mean±SD of at least three separate experiments, each performed in triplicate. ***p<0.001, vs. SC (A and C); WT mice (D and F). UI, uninfected; SC, solvent control (0.1% DMSO).


( A ) BMDMs were infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis ( MOI = 10 ) for indicated periods of time , washed to remove unbound mycobacteria , then incubated in complete DMEM at 37 ° C in 5 % CO2 . viability was assessed by PI then examined by microscopy . ( B C ) Experimental conditions were identical to those outlined in panel A . BMDMs were infected with three strains of mycobacteria for 36 h .

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(A) BMDMs were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis (MOI = 10) for the indicated periods of time, washed to remove unbound mycobacteria, and then incubated in complete DMEM at 37°C in 5% CO2. Cell viability was assessed by PI staining and then examined by fluorescence microscopy. (B and C) Experimental conditions were identical to those outlined in panel A. BMDMs were infected with the three strains of mycobacteria for 36 h.


B ) Apoptosis was assessed using a TUNEL / apoptosis detection kit , according to manufacturer ' s protocol . were then examined under a - scanning confocal microscope ( LSM 510 ; Zeiss ) . Percentages of TUNEL - positive , PI - positive , TUNEL - / PI - double - positive were calculated . Data are representative of three separate experiments .

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B) Apoptosis was assessed using a TUNEL/apoptosis detection kit, according to the manufacturer's protocol. Cells were then examined under a laser-scanning confocal microscope (LSM 510; Zeiss). Percentages of TUNEL-positive, PI-positive, and TUNEL-/PI-double-positive cells were calculated. Data are representative of three separate experiments.


( C ) Morphological changes in BMDMs infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis at MOIs of 1 10 . Representative are shown . Scale bars : 50 µm ( low magnification ) , 20 µm ( high magnification ) .

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(C) Morphological changes in BMDMs infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis at MOIs of 1 and 10. Representative images are shown. Scale bars: 50 µm (low magnification), 20 µm (high magnification).


( D ) Macrophages were infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis in presence or absence of zVAD - fmk ( 20 µM ) or 3 - MA ( 10 mM ) . Staurosporine ( STS ; 500 nM ) was used as a positive control . After 36 h , were stained with PI then examined by microscopy . Data are presented as mean ± SD of at least three separate experiments , each performed in duplicate . * p < 0 . 05 , * * * p < 0 . 001 , vs . Mtb - WT - infected condition ( A B ) ; Mtb - Δeis - infected condition without inhibitors ( D ) . UI , uninfected .

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(D) Macrophages were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis in the presence or absence of zVAD-fmk (20 µM) or 3-MA (10 mM). Staurosporine (STS; 500 nM) was used as a positive control. After 36 h, cells were stained with PI and then examined by fluorescence microscopy. Data are presented as the mean±SD of at least three separate experiments, each performed in duplicate. *p<0.05, ***p<0.001, vs. Mtb-WT-infected condition (A and B); Mtb-Δeis-infected condition without inhibitors (D). UI, uninfected.


( A B ) BMDMs were infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis ( MOI = 1 ) for indicated periods of time , then subjected to Western blot using Abs raised to p - ERK1 / 2 , p - p38 , p - JNK , β - actin . Data shown are representative of three independent experiments that yielded similar results ( A ) . Expression of phospho - MAPK / β - actin in extracts of BMDMs was quantified densitometrically ( B ) .

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(A and B) BMDMs were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis (MOI = 1) for the indicated periods of time, and then subjected to Western blot analysis using Abs raised to p-ERK1/2, p-p38, p-JNK, and β-actin. Data shown are representative of three independent experiments that all yielded similar results (A). Expression of phospho-MAPK/β-actin protein in cytoplasmic extracts of BMDMs was quantified densitometrically (B).


( C D ) BMDMs were pretreated with U0126 ( 5 , 10 , 20 µM ) , SB203580 ( SB ; 1 , 5 , 10 µM ) , or SP600125 ( SP ; 5 , 10 , 20 µM ) for 45 min , then infected with Mtb - Δeis for 30 min ( C ) or 36 h ( D ) . ( C ) were then incubated with DHE for 15 min , washed rapidly thoroughly , analyzed immediately for superoxide by flow cytometry . Quantitative DHE data represent mean ± SD of four experiments . ( D ) after 36 h was assessed by PI then examined by microscopy . (

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(C and D) BMDMs were pretreated with U0126 (5, 10, 20 µM), SB203580 (SB; 1, 5, 10 µM), or SP600125 (SP; 5, 10, 20 µM) for 45 min, and then infected with Mtb-Δeis for 30 min (C) or 36 h (D). (C) Cells were then incubated with DHE for 15 min, washed rapidly and thoroughly, and analyzed immediately for superoxide levels by flow cytometry. Quantitative DHE fluorescence data represent the mean±SD of four experiments. (D) Cell death after 36 h was assessed by PI staining and then examined by fluorescence microscopy. (


( E ) Raw264 . 7 were transfected with siRNA specific for JNK1 ( siJNK ) or a non - specific control siRNA ( siNS ) . At 24 h after transfection , were infected with Mtb - Δeis for 36 h . was then assessed by PI , then examined by flow cytometry . Transfection efficiency was assessed by RT - PCR ( inset ) . Data represent mean ± SD of five random fields are representative of three independent experiments ( D E ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , vs . Mtb - WT - infected condition ( B ) ; SC ( C D ) ; siNS ( E ) . UI , uninfected ; SC , solvent control ( 0 . 1 % DMSO ) .

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(E) Raw264.7 cells were transfected with siRNA specific for JNK1 (siJNK) or a non-specific control siRNA (siNS). At 24 h after transfection, cells were infected with Mtb-Δeis for 36 h. Cell death was then assessed by PI staining, and then examined by flow cytometry. Transfection efficiency was assessed by RT-PCR (inset). Data represent the mean±SD of five random fields and are representative of three independent experiments (D and E). *p<0.05, **p<0.01, ***p<0.001, vs. Mtb-WT-infected condition (B); SC (C and D); siNS (E). UI, uninfected; SC, solvent control (0.1% DMSO).


C57BL / 6 mice were challenged , by aerosol , with 10 - 30 CFU Mtb - WT , Mtb - Δeis , or Mtb - c - eis sacrificed 4 post - infection . ( A ) High - low - magnification electron of lung tissue sections from mice infected with Mtb - Δeis show accumulation of autophagic vesicles ( black arrow , in autophagic vacuoles ; arrow , degradative autophagic vacuoles ) . Scale bars : 2 µm ( left upper ) , 0 . 5 µm ( right ) . Numbers of autophagic vacuoles per in each section ( left lower ) ( mean ± SD ; n = 50 ) .

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C57BL/6 mice were challenged, by aerosol, with 10-30 CFU Mtb-WT, Mtb-Δeis, or Mtb-c-eis and sacrificed 4 weeks post-infection. (A) High- and low-magnification electron micrographs of lung tissue sections from mice infected with Mtb-Δeis show accumulation of autophagic vesicles (black arrow, bacteria in autophagic vacuoles; white arrow, degradative autophagic vacuoles). Scale bars: 2 µm (left upper), 0.5 µm (right). Numbers of autophagic vacuoles per cell in each TEM section (left lower) (mean±SD; n = 50).


( B ) Quantitative RT - PCR of lung tissue from Mtb - WT - , Mtb - Δeis - , Mtb - c - eis - infected mice . Total was extracted from paraffin - embedded lung tissue sections , as described in Materials Methods .

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(B) Quantitative RT-PCR analysis of lung tissue from Mtb-WT-, Mtb-Δeis-, and Mtb-c-eis-infected mice. Total RNA was extracted from paraffin-embedded lung tissue sections, as described in the Materials and Methods.


( C ) To assess in vivo , bronchoalveolar lavage fluid from Mtb - WT - , Mtb - Δeis - , Mtb - c - eis - infected mice were subjected to PI , analyzed by flow cytometry . Data are presented as mean ± ( n = 4 ) .

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(C) To assess in vivo cell death, bronchoalveolar lavage fluid cells from Mtb-WT-, Mtb-Δeis-, and Mtb-c-eis-infected mice were subjected to PI staining, and analyzed by flow cytometry. Data are presented as the mean±SEM (n = 4).


( D ) Numbers of CFUs in lung spleen 4 after infection with Mtb - WT , Mtb - Δeis , or Mtb - c - eis . Data are presented as log10 CFU ± ( n = 4 ) .

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(D) Numbers of CFUs in lung and spleen 4 weeks after infection with Mtb-WT, Mtb-Δeis, or Mtb-c-eis. Data are presented as log10 CFU±SEM (n = 4).


( E ) BMDMs were infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis then analyzed by CFU . CFU data represent mean ± SD of four experiments . * * p < 0 . 01 , * * * p < 0 . 001 , vs . Mtb - WT - infected condition . UI , uninfected .

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(E) BMDMs were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis and then analyzed by CFU assay. CFU data represent the mean±SD of four individual experiments. **p<0.01, ***p<0.001, vs. Mtb-WT-infected condition. UI, uninfected.


( A ) Intracellular superoxide production was analyzed by flow cytometric . BMDMs were infected with Mtb - Δeis ( MOI = 10 ) in presence or absence of recombinant Eis ( rEis ; 5 , 10 , 20 µg / mL ) or 30 kDa Mtb antigen ( 30 k ; 5 , 10 , 20 µg / mL ) . Upper , representative flow cytometric ; lower , quantitation of superoxide generation .

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(A) Intracellular superoxide production was analyzed by flow cytometric analysis. BMDMs were infected with Mtb-Δeis (MOI = 10) in the presence or absence of recombinant Eis protein (rEis; 5, 10, 20 µg/mL) or 30 kDa Mtb antigen (30 k; 5, 10, 20 µg/mL). Upper, representative flow cytometric analysis; lower, quantitation of superoxide generation.


( B ) THP - 1 were transfected with mock , Eis - WT , or Eis - ΔAT constructs , infected with Mtb - Δeis for 30 min . were stained with DHE ( for superoxide ) or DCFH - DA ( for H2O2 ) subjected to flow cytometric . Inset , transfection efficiency .

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(B) THP-1 cells were transfected with mock, Eis-WT, or Eis-ΔAT constructs, and infected with Mtb-Δeis for 30 min. Cells were stained with DHE (for superoxide) or DCFH-DA (for H2O2) and subjected to flow cytometric analysis. Inset, transfection efficiency.


( C D ) Experimental conditions were identical to those outlined in panels A B , respectively . Supernatants were collected 18 h after infection assessed by ELISA for of TNF - α IL - 6 . Data are presented as mean ± SD of five experiments .

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(C and D) Experimental conditions were identical to those outlined in panels A and B, respectively. Supernatants were collected 18 h after infection and assessed by ELISA for levels of TNF-α and IL-6. Data are presented as the mean±SD of five experiments.


( E F ) THP - 1 transfected with mock , Eis - WT , or Eis - ΔAT constructs were pretreated with SP600125 ( SP ; 20 µM ) or SB203580 ( SB ; 5 µM ) for 45 min before infection with Mtb - Δeis for 30 min ( E ) or 18 h ( F ) . E , Intracellular superoxide production was analyzed by flow cytometric . F , ELISA for TNF - α IL - 6 . Data are presented as mean ± SD of three experiments . * * p < 0 . 01 , * * * p < 0 . 001 , vs . SC ( A , C , E , F ) ; mock control ( B D ) . UI , uninfected ; SC , solvent control ( 0 . 1 % DMSO ) .

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(E and F) THP-1 cells transfected with mock, Eis-WT, or Eis-ΔAT constructs were pretreated with SP600125 (SP; 20 µM) or SB203580 (SB; 5 µM) for 45 min before infection with Mtb-Δeis for 30 min (E) or 18 h (F). E, Intracellular superoxide production was analyzed by flow cytometric analysis. F, ELISA analysis for TNF-α and IL-6 levels. Data are presented as the mean±SD of three experiments. **p<0.01, ***p<0.001, vs. SC (A, C, E, and F); mock control (B and D). UI, uninfected; SC, solvent control (0.1% DMSO).


DFL inhibits HMGB1 - induced , but not fMLP - induced migration . Mouse 3T3 fibroblasts were subjected to chemotaxis assays in Boyden chambers , 1 nM HMGB1 or no chemoattractant was added in lower chamber , together with indicated concentrations of DFL . Data points with average ± standard deviation ( avg ± sd ; n = 3 , each point represents a biological replicate ) in a representative experiment .

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DFL inhibits HMGB1-induced, but not fMLP-induced cell migration. Mouse 3T3 fibroblasts were subjected to chemotaxis assays in Boyden chambers, 1 nM HMGB1 or no chemoattractant was added in the lower chamber, together with the indicated concentrations of DFL. Data points with average ± standard deviation (avg ± sd; n=3, each point represents a biological replicate) in a representative experiment.


Histogram showing residue - specific CSPs of 15N - labelled HMGB1 ( ~ 0 . 1 mM ) upon addition of ten - fold excess of DFL ( helices are schematically represented on top ) . Missing residues are prolines or are absent because of exchange with solvent . Box A Box B residues with CSP > avg + sd are represented in magenta blue , respectively .

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Histogram showing residue-specific CSPs of 15N-labelled HMGB1 (~0.1 mM) upon addition of ten-fold excess of DFL (helices are schematically represented on the top). Missing residues are prolines or are absent because of exchange with the solvent. Box A and Box B residues with CSP > avg + sd are represented in magenta and blue, respectively.


R23A / R109A HMGB1 double mutant has chemotactic activity but is not inhibited by DFL ( experiment was carried out as described in panel B ) . data points represent three biological replicates in one experiment ( of 2 performed in different days ) ; avg ± sd is shown .

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The R23A/R109A HMGB1 double mutant has chemotactic activity but is not inhibited by DFL (the experiment was carried out as described in panel B). The data points represent three biological replicates in one experiment (of 2 performed in different days); the avg ± sd is shown.


Histogram showing CSPs of 15N - labelled CXCL12 amides ( ~ 0 . 1 mM ) upon addition of ten - fold excess of DFL . Missing residues are prolines . Elements of secondary structure are depicted on top .

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Histogram showing the CSPs of 15N-labelled CXCL12 amides (~0.1 mM) upon addition of ten-fold excess of DFL. Missing residues are prolines. Elements of secondary structure are depicted on top.


Top : 1H spectrum of DFL , where numbered peaks correspond to DFL protons ( left ) . Middle : superimposition of STD spectra obtained for 0 . 5 mM DFL with 0 . 05 mM CXCL12 ( black ) upon addition of 0 . 2 mM of CXCR41 - 38sY21 ( red ) . Bottom : waterLOGSY spectra obtained for 0 . 5 mM DFL with 0 . 05 mM CXCL12 ( black ) upon addition of 0 . 2 mM of CXCR41 - 38sY21 ( red ) .

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Top: 1H spectrum of DFL, where the numbered peaks correspond to DFL protons (left). Middle: superimposition of STD spectra obtained for 0.5 mM DFL with 0.05 mM CXCL12 (black line) and upon addition of 0.2 mM of CXCR41-38sY21 (red line). Bottom: waterLOGSY spectra obtained for 0.5 mM DFL with 0.05 mM CXCL12 (black line) upon addition of 0.2 mM of CXCR41-38sY21 (red line).


DFL does not inhibit CXCL12 induced chemotaxis . Mouse 3T3 fibroblasts were subjected to chemotaxis assays in Boyden chambers ; 1 nM CXCL12 or no chemoattractant was added in lower chamber , together with indicated concentrations of DFL . Data points ( n = 3 ) with avg ± sd in one representative experiment ( of 2 performed in different days ) ; migration in presence of increasing concentrations of

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 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

DFL does not inhibit CXCL12 induced chemotaxis. Mouse 3T3 fibroblasts were subjected to chemotaxis assays in Boyden chambers; 1 nM CXCL12 or no chemoattractant was added in the lower chamber, together with the indicated concentrations of DFL. Data points (n=3) with avg ± sd in one representative experiment (of 2 performed in different days); the migration in the presence of increasing concentrations of DFL


DFL inhibits chemotaxis toward HMGB1 / CXCL12 heterocomplex . Data points ( n = 3 ) with avg ± sd in one representative experiment ( of 3 performed in different days ) . Migration in presence or absence of DFL is significantly different ( p = 0 . 0037 , one - way ANOVA plus Dunnett ' s post ; * * p < 0 . 01 relative to no DFL addition ) .

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 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

DFL inhibits chemotaxis toward the HMGB1/CXCL12 heterocomplex. Data points (n=3) with avg ± sd in one representative experiment (of 3 performed in different days). Migration in the presence or absence of DFL is significantly different (p=0.0037, one-way ANOVA plus Dunnett's post test; ** p<0.01 relative to no DFL addition).


1H-15N HSQC HMGB1 (0.1 mM) spectrum


1H-15N HSQC HMGB1 (0.1 mM) spectrum with 0.2 mM CXCL12.


MST measurements of CXCL12 titrated into 50 nM fluorescently labelled HMGB1 ; signal increases from ∼ 930 ( free ) to ∼ 950 a . u . ( bound ) yielding an apparent Kd of 4 ± 0 . 4 μM . n = 3 ; error bars represent avg ± sd .

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

MST measurements of CXCL12 titrated into 50 nM fluorescently labelled HMGB1; the signal increases from ∼930 (free) to ∼950 a.u. (bound) yielding an apparent Kd of 4 ± 0.4 μM. n=3; error bars represent avg ± sd.


H - 15N HSQC of HMGB1 ( 0 . 1 mM ) with CXCL12 ( 0 . 2 mM ) upon addition of ( D ) 0 . 2 mM ( E ) 1 mM DFL .

O O O O O O O O O O O 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

H-15N HSQC of HMGB1 (0.1 mM) with CXCL12 (0.2 mM) upon addition of (D) 0.2 mM and (E) 1 mM DFL.


MST measurements of heterocomplex ( preformed using 50 nM HMGB1 50 μM CXCL12 ) in presence of increasing concentrations of DFL . Decreasing MST signal ( from ∼ 950 to ∼ 930 a . u . ) upon DFL titration indicates that CXCL12 has been displaced from HMGB1 . n = 3 ; error bars represent avg ± sd .

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

MST measurements of the heterocomplex (preformed using 50 nM HMGB1 and 50 μM CXCL12) in the presence of increasing concentrations of DFL. Decreasing MST signal (from ∼950 to ∼930 a.u.) upon DFL titration indicates that CXCL12 has been displaced from HMGB1. n=3; error bars represent avg ± sd.


DFL does not affect cytokine - inducing activities of disulfide HMGB1 ( ds - HMGB1 ) . Mouse Bone Marrow Derived Macrophages were activated or not for 3 h with 3 µg / ml ds - HMGB1 ( ~ 100 nM ) or 10 ng / ml LPS , in presence of indicated concentrations of DFL . data points represent n = 3 biological replicates , with avg ± sd , in one representative experiment ( of 2 performed in different days ) . of TNFα mRNA are not significantly different in presence of increasing concentrations of DFL ( one - way ANOVA plus post ) .

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

DFL does not affect the cytokine-inducing activities of disulfide HMGB1 (ds-HMGB1). Mouse Bone Marrow Derived Macrophages were activated or not for 3 h with 3 µg/ml ds-HMGB1 (~100 nM) or 10 ng/ml LPS, in the presence of the indicated concentrations of DFL. The data points represent n=3 biological replicates, with avg ± sd, in one representative experiment (of 2 performed in different days). The levels of TNFα mRNA are not significantly different in the presence of increasing concentrations of DFL (one-way ANOVA plus post tests).


DFL does not inhibit chemotaxis towards 3S . Mouse 3T3 fibroblasts were subjected to chemotaxis assays in Boyden chambers ; 1 nM fully reduced HMGB1 ( fr - HMGB1 ) ( green ) or 3S ( red ) , or no chemoattractant ( black ) , were added in lower chamber , together with 1 µg / ml of anti - CXCL12 monoclonal antibody or 30 nM DFL . Data points ( n = 4 ) with avg ± sd in one representative experiment ( of two performed in different days ) . Statistics : one - way ANOVA ( p < 0 . 0001 ) , followed by Dunnett ' s post - . * * * , p < 0 . 001 relative to migration towards HMGB1 , migrations towards 3S are not significantly different among each other .

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 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

DFL does not inhibit chemotaxis towards 3S. Mouse 3T3 fibroblasts were subjected to chemotaxis assays in Boyden chambers; 1 nM fully reduced HMGB1 (fr-HMGB1) (green) or 3S (red), or no chemoattractant (black), were added in the lower chamber, together with 1 µg/ml of anti-CXCL12 monoclonal antibody or 30 nM DFL. Data points (n=4) with avg ± sd in one representative experiment (of two performed in different days). Statistics: one-way ANOVA (p<0.0001), followed by Dunnett's post-test. ***, p<0.001 relative to migration towards HMGB1, the migrations towards 3S are not significantly different among each other.


of CD45 + isolated with immunobeads from injured 6 hours after damage . Before damage mice were treated either with Phosphate Buffered Saline vehicle , Glycyrrhizin or DFL . Two independent experiments . Each point represents a different mouse , avg ± sd are shown ( statistics : Kruskal - Willis plus post - ) .

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

Quantification of CD45+ cells isolated with immunobeads from injured muscles 6 hours after damage. Before damage mice were treated either with Phosphate Buffered Saline vehicle, Glycyrrhizin or DFL. Two independent experiments. Each point represents a different mouse, avg ± sd are shown (statistics: Kruskal-Willis plus post-tests).


( A ) Schematic representation of study design Western blot for TIP30 GAPDH in hearts from TIP30 wild - type ( WT ) , heterozygous ( Het ) homozygous knock - out ( KO ) mice under basal conditions .

(A) Schematic representation of the study design and Western blot analysis for TIP30 and GAPDH in hearts from TIP30 wild-type (WT), heterozygous (Het) and homozygous knock-out (KO) mice under basal conditions.


of heart ( HW ) / tibia ( TL ) ( B ) N = 4 to 18 mice / group , 6 after TAC or sham surgery .

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

Quantification of heart weight (HW)/tibia length (TL) ratio (B) N=4 to 18 mice/group, all 6 weeks after TAC or sham surgery.


of lung ( LuW / TL ) ( C ) N = 4 to 18 mice / group , 6 after TAC or sham surgery .

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

Quantification of lung weight (LuW/TL) ratio (C) N=4 to 18 mice/group, all 6 weeks after TAC or sham surgery.


of , echocardiographic fractional change ( D ) N = 4 to 18 mice / group , 6 after TAC or sham surgery .

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

Quantification of , echocardiographic fractional area change (D) N=4 to 18 mice/group, all 6 weeks after TAC or sham surgery.


of left ventricular enddiastolic ( LVEDA ; E ) N = 4 to 18 mice / group , 6 after TAC or sham surgery .

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

Quantification of left ventricular enddiastolic area (LVEDA; E) N=4 to 18 mice/group, all 6 weeks after TAC or sham surgery.


of dP / dt max dP / dt min ( Millar catheter ; F , G ) N = 4 to 18 mice / group , 6 after TAC or sham surgery .

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

Quantification of dP/dt max and dP/dt min (Millar catheter; F, G) N=4 to 18 mice/group, all 6 weeks after TAC or sham surgery.


of α - MHC transcript abundance N = 4 to 18 mice / group , 6 after TAC or sham surgery .

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

Quantification of α-MHC transcript abundance N=4 to 18 mice/group, all 6 weeks after TAC or sham surgery.


of β - MHC transcript abundance N = 4 to 18 mice / group , 6 after TAC or sham surgery .

Quantification of β-MHC transcript abundance N=4 to 18 mice/group, all 6 weeks after TAC or sham surgery.


Representative of Sirius - red - stained heart sections ( scale bar : 1mm ) ( J )

Representative images of Sirius-red-stained heart sections (scale bar: 1mm) (J)


fibrosis ( K ) of indicated mice 6 after TAC or sham surgery .

fibrosis quantification (K) of indicated mice 6 weeks after TAC or sham surgery.


of cardiomyocyte of isolated adult cardiac myocytes ( N = 4 to 11 mice / group ) of indicated mice 6 after TAC or sham surgery . * P < 0 . 05 . One - way ANOVA with Sidak ´ s multiple comparisons .

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

Quantification of cardiomyocyte area of isolated adult cardiac myocytes (N=4 to 11 mice/group) of indicated mice 6 weeks after TAC or sham surgery. *P<0.05. One-way ANOVA with Sidak´s multiple comparisons test.


Microscopy of heart sections of indicated mice 6 after TAC surgery stained for Isolectin B4 ( green ) WGA ( red , M )

Microscopy images of heart sections of indicated mice 6 weeks after TAC surgery stained for Isolectin B4 (green) and WGA (red, M)


of capillaries per myocyte ( N ) . ( N = 5 to 7 mice / group , scale bar : 50µm ) . * * P < 0 . 01 . One - way ANOVA with Sidak ´ s multiple comparisons .

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

quantification of capillaries per myocyte (N). (N=5 to 7 mice/group, scale bar: 50µm). **P<0.01. One-way ANOVA with Sidak´s multiple comparisons test.


( P ) Western blot for TIP30 GAPDH in hearts from TIP30 wild - type ( WT ) heterozygous ( Het ) after AAV - TropT - TIP30 or AAV - Control ( AAV - Con ) injection followed by 6 of TAC surgery .

(P) Western blot analysis for TIP30 and GAPDH in hearts from TIP30 wild-type (WT) and heterozygous (Het) after AAV-TropT-TIP30 or AAV-Control (AAV-Con) injection followed by 6 weeks of TAC surgery.


of HW / TL ( Q ) in AAV - Con or AAV - TropT - TIP30 treated Tip30 heterozygous ( Het ) or WT mice 6 after TAC or sham surgery ( N = 5 to 11 mice / group ) .

Quantification of HW/TL ratio (Q) ratio in AAV-Con or AAV-TropT-TIP30 treated Tip30 heterozygous (Het) or WT mice 6 weeks after TAC or sham surgery (N=5 to 11 mice/group).


of LuW / TL ( R ) in AAV - Con or AAV - TropT - TIP30 treated Tip30 heterozygous ( Het ) or WT mice 6 after TAC or sham surgery ( N = 5 to 11 mice / group ) .

Quantification of LuW/TL (R) ratio in AAV-Con or AAV-TropT-TIP30 treated Tip30 heterozygous (Het) or WT mice 6 weeks after TAC or sham surgery (N=5 to 11 mice/group).


( A ) Western blot for TIP30 GAPDH in neonatal rat cardiomyocytes ( NRCM ) after adenoviral transduction with Ad . Control ( Ad . Con ) or Ad . TIP30 .

O O O O O O O O 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) Western blot for TIP30 and GAPDH in neonatal rat cardiomyocytes (NRCM) after adenoviral transduction with Ad.Control (Ad.Con) or Ad.TIP30.


of cardiomyocyte ( B ) , N = 6 to 8 samples / group in NRCM transduced with Ad . Con or Ad . TIP30 stimulated as indicated . ET - 1 : endothelin - 1 , FBS : bovine serum , PE : phenylephrine .

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

Quantification of cardiomyocyte area (B), N=6 to 8 samples/group in NRCM transduced with Ad.Con or Ad.TIP30 and stimulated as indicated. ET-1: endothelin-1, FBS: fetal bovine serum, PE: phenylephrine.


of / ( C ) , N = 9 samples / group in NRCM transduced with Ad . Con or Ad . TIP30 stimulated as indicated . PE : phenylephrine .

O O O 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 B-CONTROLLED_VAR O

Quantification of protein/DNA ratio (C), N=9 samples/group in NRCM transduced with Ad.Con or Ad.TIP30 and stimulated as indicated. PE: phenylephrine.


of Acta1 mRNA transcript abundance ( D ) , N = 3 samples / group in NRCM transduced with Ad . Con or Ad . TIP30 stimulated as indicated . ET - 1 : endothelin - 1 PE : phenylephrine .

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

Quantification of Acta1 mRNA transcript abundance (D), N=3 samples/group in NRCM transduced with Ad.Con or Ad.TIP30 and stimulated as indicated. ET-1: endothelin-1 PE: phenylephrine.


of with a 7 - AAD ( E ) , N = 7 samples / group in NRCM transduced with Ad . Con or Ad . TIP30 stimulated as indicated . PE : phenylephrine .

O O O O O O O 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 B-CONTROLLED_VAR O

Quantification of cell death with a 7-AAD assay (E), N=7 samples/group in NRCM transduced with Ad.Con or Ad.TIP30 and stimulated as indicated. PE: phenylephrine.


( G ) Western blot for TIP30 Actin in mouse hearts with AAV9 mediated overexpression of TIP30 ( AAV - TIP30 ) or from mice treated with a control AAV9 construct ( AAV - Con ) followed by 2 of TAC surgery .

(G) Western blot for TIP30 and Actin in mouse hearts with AAV9 mediated overexpression of TIP30 (AAV-TIP30) or from mice treated with a control AAV9 construct (AAV-Con) followed by 2 weeks of TAC surgery.


of HW / TL ( H ) , N = 8 to 13 mice / group 2 after sham or TAC surgery in AAV - Con or AAV - TIP30 treated C57BL / 6 WT mice . *

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

Quantification of HW/TL ratio (H), N=8 to 13 mice/group 2 weeks after sham or TAC surgery in AAV-Con or AAV-TIP30 treated C57BL/6 WT mice. *


of cardiomyocyte ( I ) , N = 4 to 5 mice / group 2 after sham or TAC surgery in AAV - Con or AAV - TIP30 treated C57BL / 6 WT mice .

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

Quantification of cardiomyocyte area (I), N=4 to 5 mice/group 2 weeks after sham or TAC surgery in AAV-Con or AAV-TIP30 treated C57BL/6 WT mice.


( J ) Serial echocardiography with of echocardiographic fractional change 2 , 4 6 after sham or TAC surgery in AAV - Con or AAV - TIP30 treated C57BL / 6 WT mice ( N = 10 to 14 mice / group time point ) .

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

(J) Serial echocardiography with quantification of echocardiographic fractional area change 2, 4 and 6 weeks after sham or TAC surgery in AAV-Con or AAV-TIP30 treated C57BL/6 WT mice (N=10 to 14 mice/group and time point).


( A ) Western blot of GST - pulldown assays with GST - TIP30 fragments of indicated ( as : from N - terminus ) with Myc - tagged binding partners .

(A) Western blot analysis of GST-pulldown assays with GST-TIP30 fragments of the indicated length (as: amino acids from N-terminus) with Myc-tagged binding partners.


( B ) Western blot of co - immunoprecipitation ( IP ) from HEK transfected with GST - TIP30 eEF1A1 - Myc .

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

(B) Western blot analysis of co-immunoprecipitation (IP) from HEK cells transfected with GST-TIP30 and eEF1A1-Myc.


( C ) Confocal microscopy of neonatal rat cardiomyocytes ( NRCM ) stained with antibodies for endogenous TIP30 ( green ) endogenous eEF1A1 ( red ) . DAPI : blue . ( Scale bar : 20µm ) .

(C) Confocal microscopy images of neonatal rat cardiomyocytes (NRCM) stained with antibodies for endogenous TIP30 (green) and endogenous eEF1A1 (red). DAPI: blue. (Scale bar: 20µm).


( D ) Microscopy of isolated neonatal rat cardiomyocytes subsequent Proximity Ligation ( PLA ) . Red : endogenous TIP30 / eEF1A1 interaction ; Blue : DAPI . Control were stained for TIP30 / Myc - Tag interaction ( scale bar : 50µm ) .

(D) Microscopy images of isolated neonatal rat cardiomyocytes and subsequent Proximity Ligation Assay (PLA). Red: endogenous TIP30/eEF1A1 interaction; Blue: DAPI. Control cells were stained for TIP30/Myc-Tag interaction (scale bar: 50µm).


( E ) Western blot of co - immunoprecipitation ( IP ) for endogenous TIP30 eEF1A1 in NRCM after stimulation with phenylephrine for 24 h . IgG HC - High chain of IgG molecule .

(E) Western blot analysis of co-immunoprecipitation (IP) for endogenous TIP30 and eEF1A1 protein in NRCM after stimulation with phenylephrine for 24 h. IgG HC - High chain of IgG molecule.


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

Western blot analysis of GST-pulldown assays with GST or GST-tagged eEF1A1 (GST-eEF1A1) and TIP30-His full length protein (TIP30-His) and TIP30 deletion mutants ΔN25 (TIP30-ΔN25-His), ΔN52 (TIP30-ΔN52-His), ΔC15 (TIP30-ΔC15-His) and Δ102-107 (TIP30-ΔN102-104-His, G)


Western blot of GST - tagged eEF1A1 GST - Control ( H ) .

Western blot analysis of GST-tagged eEF1A1 and GST-Control (H).


( J ) Quantitative Real - Time PCR of eEF1A1 eEF1A2 mRNA abundance in hearts from neonatal mice , adult wild - type mice ( Adult ) adult wild - type mice 2 after TAC surgery ( Adult TAC , N = 3 to 4 mice / group ) . * P < 0 . 05 , * * * P < 0 . 001 . One - way ANOVA with Sidak ´ s multiple comparisons .

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

(J) Quantitative Real-Time PCR analysis of eEF1A1 and eEF1A2 mRNA abundance in hearts from neonatal mice, adult wild-type mice (Adult) and adult wild-type mice 2 weeks after TAC surgery (Adult TAC, N=3 to 4 mice/group). *P<0.05, ***P<0.001. One-way ANOVA with Sidak´s multiple comparisons test.


( K ) Western blot for eEF1A1 , eEF1A2 GAPDH in hearts from wild - type mice at of 7 days ( 7d ) 8 ( 8w ) as well as 2 ( TAC 2w ) after TAC surgery .

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

(K) Western blot analysis for eEF1A1, eEF1A2 and GAPDH in hearts from wild-type mice at the age of 7 days (7d) and 8 weeks (8w) as well as 2 weeks (TAC 2w) after TAC surgery.


( L ) Western blot of co - immunoprecipitation ( IP ) with GST - TIP30 eEF1A2 - His .

(L) Western blot analysis of co-immunoprecipitation (IP) with GST-TIP30 and eEF1A2-His.


( A ) Western blot of GST - pulldown assays with GST or GST - eEF1A1 purified eEF1B2 - His . Purified TIP30 - His was added in indicated amounts .

O O O O O O O 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) Western blot analysis of GST-pulldown assays with GST or GST-eEF1A1 and purified eEF1B2-His. Purified TIP30-His was added in indicated amounts.


( B ) Western blot of anti - Myc immunoprecipitation ( IP ) in NRCM co - transduced with Ad . eEF1A1 - Myc either control ( Ad . con ) or Ad . TIP30 . Endogenous eEF1B2 was detected . IP input for eEF1B2 , eEF1A1 - myc TIP30 is shown below . ( C ) of eEF1B2 abundance after eEF1A1 - myc IP under conditions shown in ( B )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of anti-Myc immunoprecipitation (IP) in NRCM co-transduced with Ad.eEF1A1-Myc and either control virus (Ad.con) or Ad.TIP30. Endogenous eEF1B2 was detected. The IP input for eEF1B2, eEF1A1-myc and TIP30 is shown below. (C) Quantification of eEF1B2 abundance after eEF1A1-myc IP under conditions shown in (B)


( D ) Microscopy of NRCM after adenoviral transduction with Ad . TIP30 or control ( Ad . Con ) stimulation with phenylephrine ( PE ) subsequent Proximity Ligation ( PLA ) . Red : eEF1A1 - eEF1B2 interaction ; Blue : DAPI . ( scale bar : 50µm ) . ( E ) of eEF1A1 - eEF1B2 interaction in conditions described in ( D )

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 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

(D) Microscopy images of NRCM after adenoviral transduction with Ad.TIP30 or control virus (Ad.Con) and stimulation with phenylephrine (PE) and subsequent Proximity Ligation Assay (PLA). Red: eEF1A1-eEF1B2 interaction; Blue: DAPI. (scale bar: 50µm). (E) Quantification of eEF1A1-eEF1B2 interaction in conditions described in (D)


( F ) Microscopy of adult mouse cardiomyocytes isolated from hearts 3 days ( d ) after TAC or sham surgery subsequent PLA . Red : eEF1A1 - eEF1B2 interaction ; Blue : DAPI ( scale bar : 100µm ) . ( G ) of eEF1A1 - eEF1B2 interaction in adult mouse cardiomyocytes isolated from hearts 3 days after TAC or sham surgery subsequent PLA ( N = 4 to 6 mice / group ) . *

(F) Microscopy images of adult mouse cardiomyocytes isolated from hearts 3 days (d) after TAC or sham surgery and subsequent PLA. Red: eEF1A1-eEF1B2 interaction; Blue: DAPI (scale bar: 100µm). (G) Quantification of eEF1A1-eEF1B2 interaction in adult mouse cardiomyocytes isolated from hearts 3 days after TAC or sham surgery and subsequent PLA (N=4 to 6 mice/group). *


Western blot for TIP30 , eEF1A1 GAPDH in C57BL / 6 WT mice 3 days ( A , B ) , N = 4 mice / group , 2 ( C , D ) , N = 4 to 8 mice / group 6 ( E , F ) , N = 4 to 8 mice / group after TAC or sham surgery their . KO denotes TIP30 homozygous knock - out .

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

Western blot analysis for TIP30, eEF1A1 and GAPDH in C57BL/6 WT mice 3 days (A, B), N=4 mice/group, 2 weeks (C, D), N=4 to 8 mice/group and 6 weeks (E, F), N=4 to 8 mice/group after TAC or sham surgery and their quantification. KO denotes TIP30 homozygous knock-out.


( G , H ) Western blot for TIP30 , eEF1A1 Actin in TIP30 Het mice 6 after TAC surgery their ( N = 4 mice / group ) .

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

(G, H) Western blot for TIP30, eEF1A1 and Actin in TIP30 Het mice 6 after TAC surgery and their quantification (N=4 mice/group).