Z - projection of HeLa cells expressing GFP - centrin1 pre - treated with DMSO or 0 . 2mM CK666 during prometaphase arrest and forced mitotic exit and stained with phalloidin for F - actin .

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Z-projection of HeLa cells expressing GFP-centrin1 pre-treated with DMSO or 0.2mM CK666 during prometaphase arrest and forced mitotic exit and stained with phalloidin for F-actin.


Quantification of the level of actin around the centrosome from ( C ) , which shows the reduction in actin accumulation around centrosomes following CK666 pre - treatment . DMSO - STLC = 1 ± 0 . 02768 , n = 99 , CK666 STLC = 0 . 776 ± 0 . 02186 , n = 87 , DMSO - RO - 3306 = 1 . 339 ± 0 . 03048 , n = 127 , CK666 - RO - 3306 = 0 . 7699 ± 0 . 02246 , n = 70 , one - way ANOVA , p < 0 . 0001 .

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Quantification of the level of actin around the centrosome from (C), which shows the reduction in actin accumulation around centrosomes following CK666 pre-treatment. DMSO-STLC= 1 ± 0.02768, n=99, CK666 STLC= 0.776 ± 0.02186, n=87, DMSO-RO-3306= 1.339 ± 0.03048, n=127, CK666-RO-3306= 0.7699 ± 0.02246, n=70, one-way ANOVA, p<0.0001.


Quantification of tubulin around centrosomes for cells treated with DMSO or 0 . 2mM CK666 , showing the failure to reduce tubulin density around centrosomes following CK666 treatment during bipolar exit . DMSO - metaphase - 1 ± 0 . 1968 , n = 54 , DMSO - anaphase - 0 . 867 ± 0 . 2345 , n = 78 , CK666 - metaphase - 0 . 7995 ± 0 . 1275 , n = 43 , CK666 - anaphase - 0 . 7407 ± 0 . 1859 , n = 74 ; one - way ANOVA , p < 0 . 0001 . Data pooled from 3 independent experiments . Error bars indicated standard deviation .

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Quantification of tubulin around centrosomes for cells treated with DMSO or 0.2mM CK666, showing the failure to reduce tubulin density around centrosomes following CK666 treatment during bipolar exit. DMSO-metaphase-1±0.1968, n=54, DMSO-anaphase-0.867±0.2345, n=78, CK666-metaphase-0.7995±0.1275, n=43, CK666-anaphase-0.7407±0.1859, n=74; one-way ANOVA, p<0.0001. Data pooled from 3 independent experiments. Error bars indicated standard deviation.


Quantification of tubulin around centrosomes for cells treated with DMSO or 0 . 2mM CK666 , during monopolar exit , showing the failure to reduce tubulin density around centrosomes following CK666 treatment . Data from three independent experiments . Prometaphase arrest ( p . a . ) 1 ± 0 . 34 , n = 27 ; forced mitotic exit ( f . m . e ) 0 . 78 ± 0 . 30 , n = 26 ; p . a . plus CK666 0 . 49 ± 0 . 34 , n = 30 ; f . m . e plus CK666 0 . 92 ± 0 . 37 , n = 30 . One - way ANOVA with Sidak ' s multiple comparisons test .

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Quantification of tubulin around centrosomes for cells treated with DMSO or 0.2mM CK666, during monopolar exit, showing the failure to reduce tubulin density around centrosomes following CK666 treatment. Data from three independent experiments. Prometaphase arrest (p.a.) 1±0.34 , n=27; forced mitotic exit (f.m.e) 0.78±0.30, n=26; p.a. plus CK666 0.49±0.34, n=30 ; f.m.e plus CK666 0.92±0.37, n=30. One-way ANOVA with Sidak's multiple comparisons test.


Z - projection of HeLa cells expressing GFP - centrin1 immunostained for p34 - Arc ( sub - domain Arp2 / 3 ) following prometaphase arrest and forced mitotic exit for 5min . N = 3 independent experiments . Scale bar = 10µm and for zoom 2µm .

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Z-projection of HeLa cells expressing GFP-centrin1 immunostained for p34-Arc (sub-domain Arp2/3) following prometaphase arrest and forced mitotic exit for 5min. N=3 independent experiments. Scale bar = 10µm and for zoom 2µm.


Levels of p34 - Arc around the centrosome ( 3um area ) were quantified relative to metaphase and increase during forced mitotic exit . STLC arrested = 1 ± 0 . 01576 , n = 153 , STLC + RO - 3306 = 1 . 268 ± 0 . 01748 , n = 191 , * * * * p < 0 . 0001 , Unpaired t - test with Welch correction . Error bars represent standard deviation .

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Levels of p34-Arc around the centrosome (3um area) were quantified relative to metaphase and increase during forced mitotic exit. STLC arrested = 1 ± 0.01576, n=153, STLC+RO-3306= 1.268 ± 0.01748, n=191, ****p<0.0001, Unpaired t-test with Welch correction. Error bars represent standard deviation.


Z - projection of centrosomes isolated from cells expressing GFP - centrin1 during prometaphase arrest and forced exit , which were immunostained with p34 - Arc . N = 2 independent experiments . Scale bar = 2µm .

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Z-projection of centrosomes isolated from cells expressing GFP-centrin1 during prometaphase arrest and forced exit, which were immunostained with p34-Arc. N=2 independent experiments. Scale bar = 2µm.


Quantification of p34 - Arc levels around the centrosome in ( C ) , relative to metaphase , showing its increase during forced exit . STLC arrested = 1 ± 0 . 00779 , n = 93 , STLC + RO - 3306 = 1 . 249 ± 0 . 02446 , n = 134 , * * * * p < 0 . 0001 , Unpaired t - test with Welch correction . Error bars represent standard deviation .

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Quantification of p34-Arc levels around the centrosome in (C), relative to metaphase, showing its increase during forced exit. STLC arrested = 1 ± 0.00779, n=93, STLC+RO-3306= 1.249 ± 0.02446, n=134, ****p<0.0001, Unpaired t-test with Welch correction. Error bars represent standard deviation.


Time - lapse of in vitro assay on centrosomes isolated from prometaphase arrested and cells forced to exit mitosis showing centrosomal actin nucleation over time . Scale bar = 10µm Quantification of actin nucleation from isolated centrosomes as in ( E ) over an area of 2 μm , ( white circle ) , showing an increase in actin nucleation around centrosomes isolated from cells undergoing forced mitotic exit . N = 2 isolations , 5 independent experiments , n = 16 STLC and n = 19 STLC RO .

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Time-lapse of in vitro assay on centrosomes isolated from prometaphase arrested and cells forced to exit mitosis showing centrosomal actin nucleation over time. Scale bar = 10µm Quantification of actin nucleation from isolated centrosomes as in (E) over an area of 2 μm, (white circle), showing an increase in actin nucleation around centrosomes isolated from cells undergoing forced mitotic exit. N=2 isolations, 5 independent experiments, n=16 STLC and n=19 STLC RO.


Color switch experiment using green - and red - labelled actin . Red actin was used first , followed by Capping protein , then green - labelled actin . The accumulation of green in the centre indicates that actin is nucleated at the centrosome . Scale bar = 10µm . Quantification of ( G ) over the time . N = 2 isolations , 2 independent experiments , n = 19 STLC and n = 14 STLC RO . Error bars represent standard deviation .

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Color switch experiment using green- and red-labelled actin. Red actin was used first, followed by Capping protein, then green-labelled actin. The accumulation of green in the centre indicates that actin is nucleated at the centrosome. Scale bar = 10µm. Quantification of (G) over the time. N=2 isolations, 2 independent experiments, n=19 STLC and n=14 STLC RO. Error bars represent standard deviation.


Time - lapse showing cytoplasmic actin filament formation at centrosomes isolated from cells undergoing forced exit when pre - treated with DMSO or CK666 . Scale bar = 10µm . Quantification of ( I ) , showing that centrosomal actin nucleation fails when centrosomes isolated from cells undergoing exit are pre - treated with CK666 . N = 2 isolations , 2 independent experiments , n = 17 DMSO , 26 Ro CK666 24 STLC DMSO , 23 STLC CK666 . Error bars represent standard deviation .

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Time-lapse showing cytoplasmic actin filament formation at centrosomes isolated from cells undergoing forced exit when pre-treated with DMSO or CK666. Scale bar = 10µm. Quantification of (I), showing that centrosomal actin nucleation fails when centrosomes isolated from cells undergoing exit are pre-treated with CK666. N=2 isolations, 2 independent experiments, n = 17 DMSO, 26 Ro CK666 24 STLC DMSO, 23 STLC CK666. Error bars represent standard deviation.


Phos - tag Western blot of MCF7 - Her2 - 18 cells synchronised into distinct phases of the cell cycle showing the phospho - shift in WASH1 during mitosis .

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Phos-tag Western blot of MCF7-Her2-18 cells synchronised into distinct phases of the cell cycle showing the phospho-shift in WASH1 during mitosis.


Blue native PAGE Western blot showing shift in the size of WASH1 protein complex isolated from STLC treated prometaphase arrested cells when compared to cells forced to exit mitosis following Ro3306 treatment .

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Blue native PAGE Western blot showing shift in the size of WASH1 protein complex isolated from STLC treated prometaphase arrested cells when compared to cells forced to exit mitosis following Ro3306 treatment.


Time - lapse of HeLa cells expressing GFP - alpha tubulin and RFP - lifeact treated with control siRNA or siRNA against WASH1 for 48 hours arrested at prometaphase and forced to exit mitosis with Ro3306 addition ( t = 0 ) . Scale bar = 5µm .

Time-lapse of HeLa cells expressing GFP-alpha tubulin and RFP-lifeact treated with control siRNA or siRNA against WASH1 for 48 hours arrested at prometaphase and forced to exit mitosis with Ro3306 addition (t=0). Scale bar = 5µm.


Quantification of actin around the centrosome in ( A ) , showing the failure to accumulate actin around the centrosome in siWASH . N = 4 experiments . Error bars represent standard deviation .

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Quantification of actin around the centrosome in (A), showing the failure to accumulate actin around the centrosome in siWASH. N = 4 experiments. Error bars represent standard deviation.


Western blots of lysates from cells treated with siRNA control and siRNA against WASH1 probed with WASH1 antibody , showing the reduction in WASH levels following treatment of cells with siRNA against WASH . Scale bar = 5µm and 2µm in zoom .

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Western blots of lysates from cells treated with siRNA control and siRNA against WASH1 probed with WASH1 antibody, showing the reduction in WASH levels following treatment of cells with siRNA against WASH. Scale bar = 5µm and 2µm in zoom.


Z - projection of HeLa cells expressing GFP - centrin1 treated with control siRNA or siRNA against WASH , arrested at prometaphase or forced to exit mitosis and immunostained with phalloidin . Scale bar - 5µm Quantification of actin around the centrosome in ( F ) , showing the failure to accumulated actin around the centrosome during forced exit in cells treated with siRNA against WASH . siControl - STLC = 1 ± 0 . 0189 , n = 165 , siWASH - STLC = 0 . 9138 ± 0 . 0298 , n = 118 , siControl - STLC + RO - 3306 = 1 . 911 ± 0 . 06221 , n = 147 , siWASH - STLC + RO - 3306 = 1 . 103 ± 0 . 0301 , n = 140 . Error bars represent standard deviation . P < 0 . 0001 , one - way ANOVA .

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Z-projection of HeLa cells expressing GFP-centrin1 treated with control siRNA or siRNA against WASH, arrested at prometaphase or forced to exit mitosis and immunostained with phalloidin. Scale bar-5µm Quantification of actin around the centrosome in (F), showing the failure to accumulated actin around the centrosome during forced exit in cells treated with siRNA against WASH. siControl-STLC= 1 ± 0.0189, n=165, siWASH-STLC=0.9138 ± 0.0298, n=118, siControl-STLC+RO-3306=1.911 ± 0.06221, n=147, siWASH-STLC+RO-3306=1.103 ± 0.0301, n=140. Error bars represent standard deviation. P<0.0001, one-way ANOVA.


Time - lapse from in vitro assay with centrosomes isolated from cells undergoing forced exit were pre - treated with either no antibody or anti - WASH1 antibody for 1 hour . Scale bar - 10µm Quantification of in vitro assay from ( F ) , showing the reduction in actin nucleation around centrosomes when they are pre - treated with anti - WASH1 . Error bars represent standard deviation . 2 independent experiments .

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Time-lapse from in vitro assay with centrosomes isolated from cells undergoing forced exit were pre-treated with either no antibody or anti-WASH1 antibody for 1 hour. Scale bar- 10µm Quantification of in vitro assay from (F), showing the reduction in actin nucleation around centrosomes when they are pre-treated with anti-WASH1. Error bars represent standard deviation. 2 independent experiments.


HeLa cells overexpressing the HA - tagged MICU1 WT , MICU1 S124D or MICU1 S124A mutants were stained for HA or HSP60 ( mitochondrial marker ) . Merged images are indicated ( merge ) . Scale bar 10

HeLa cells overexpressing the HA-tagged MICU1 WT, MICU1 S124D or MICU1 S124A mutants were stained for HA or HSP60 (mitochondrial marker). Merged images are indicated (merge). Scale bar 10 μm


Representative images of the 2mt - GCaMP6m 474 / 410 ratio of ShMICU1 HeLa stable cells expressing an empty vector ( ctrl ) or the MICU1 WT , MICU1 SD , and MICU1 SA . Scale bar 10

Representative images of the 2mt-GCaMP6m 474/410 ratio of ShMICU1 HeLa stable cells expressing an empty vector (ctrl) or the MICU1 WT, MICU1 SD, and MICU1 SA. Scale bar 10 μm


Resting mitochondrial calcium levels , evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m , in ShRNA control ( plko ) or ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs ( n = 5 independent experiments ; 55 - 67cells )

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Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m, in ShRNA control (plko) or ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs (n=5 independent experiments; 55-67cells)


Representative kinetics ( E ) and analysis ( F ) of aequorin - based [ Ca2 + ] m measurements in ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs and challenged with 20 μM 2 , 5 - di - tert - butylhydroquinone ( TBHQ ) in the absence of extracellular Ca2 + ( n = 3 independent experiments )

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Representative kinetics (E) and analysis (F) of aequorin-based [Ca2+]m measurements in ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs and challenged with 20 μM 2,5-di-tert-butylhydroquinone (TBHQ) in the absence of extracellular Ca2+ (n=3 independent experiments)


Representative kinetics ( G ) and analysis ( H ) of aequorin - based [ Ca2 + ] m measurements in intact ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs and challenged with 10 μM cyclopiazonic acid ( CPA ) in the presence of 100 μM EGTA ( n = 3 independent experiments )

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Representative kinetics (G) and analysis (H) of aequorin-based [Ca2+]m measurements in intact ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs and challenged with 10 μM cyclopiazonic acid (CPA) in the presence of 100 μM EGTA (n=3 independent experiments)


Western blot analysis for the presence of MICU1 in clones arising from single cells generated by CRISPR / Cas9 - mediated genome editing . The results for 12 of the 36 clones that were examined are

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Western blot analysis for the presence of MICU1 in clones arising from single cells generated by CRISPR/Cas9-mediated genome editing. The results for 12 of the 36 clones that were examined are shown


Resting mitochondrial calcium levels , evaluated through ratiometric imaging of the mitochondrial - targeted GCaMP6m , in MICU1 - KO cells generated using the CRISPR / Cas9 technique and transfected with the indicated constructs ( n = 3 independent experiments ; 30 - 56 cells )

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Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial-targeted GCaMP6m, in MICU1-KO cells generated using the CRISPR/Cas9 technique and transfected with the indicated constructs (n=3 independent experiments; 30-56 cells)


Representative kinetics ( K ) and analysis ( L ) of aequorin - based [ Ca2 + ] m measurements in intact MICU1 - KO cells generated using the CRISPR / Cas9 technique , transfected with the indicated constructs and challenged with 20 μM TBHQ in the absence of extracellular Ca2 + ( n = 3 independent experiments . )

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Representative kinetics (K) and analysis (L) of aequorin-based [Ca2+]m measurements in intact MICU1-KO cells generated using the CRISPR/Cas9 technique, transfected with the indicated constructs and challenged with 20 μM TBHQ in the absence of extracellular Ca2+ (n=3 independent experiments.)


Representative kinetics ( M ) and analysis ( N ) of aequorin - based [ Ca2 + ] m measurements in intact MICU1 - KO cells generated using the CRISPR / Cas9 technique , transfected with the indicated constructs and challenged with 10 μM CPA in the presence of 100 μM EGTA ( n = 3 independent experiments ) .

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Representative kinetics (M) and analysis (N) of aequorin-based [Ca2+]m measurements in intact MICU1-KO cells generated using the CRISPR/Cas9 technique, transfected with the indicated constructs and challenged with 10 μM CPA in the presence of 100 μM EGTA (n=3 independent experiments).


HeLa cells treated with vehicle or 1 μM rapamycin for 4 h were stained for phosphorylated ( S473 ) Akt ( p - Akt ) or HSP60 ( mitochondrial marker ) . Merged images are indicated ( merge ) . Scale bar 10

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HeLa cells treated with vehicle or 1 μM rapamycin for 4 h were stained for phosphorylated (S473) Akt (p-Akt) or HSP60 (mitochondrial marker). Merged images are indicated (merge). Scale bar 10 μm


Analysis of the number of cells , expressed as a percentage , showing obvious mitochondrial staining of activated ( S473 phosphorylated ) Akt ( p - Akt ) ( n = 3 independent experiments ; 350 - 355 cells )

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Analysis of the number of cells, expressed as a percentage, showing obvious mitochondrial staining of activated (S473 phosphorylated) Akt (p-Akt) (n=3 independent experiments; 350-355 cells)


Hek293T cells treated with vehicle or 1 μM rapamycin ( Rapa . ) for 4 h were fractionated into Cytosol ( Cyt . ) or mitochondrial ( Mito . ) extracts and analyzed by western blotting . E - cadherin : plasma membrane marker ; β - tubulin : cytosolic marker ; VDAC : mitochondrial marker ; Homo : cell

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Hek293T cells treated with vehicle or 1 μM rapamycin (Rapa.) for 4 h were fractionated into Cytosol (Cyt.) or mitochondrial (Mito.) extracts and analyzed by western blotting. E-cadherin: plasma membrane marker; β-tubulin: cytosolic marker; VDAC: mitochondrial marker; Homo: cell homogenate


Mouse livers treated with vehicle or 1 μM rapamycin ( Rapa . ) for 16 h by an intraperitoneal injection were fractionated into cytosol ( Cyt . ) or mitochondrial ( Mito . ) extracts and analyzed by western blotting . Phosphorylated ( S2468 ) mTOR ( p - mTOR ) was used to assess the rapamycin activity . E - cadherin : plasma membrane marker ; β - tubulin : cytosolic marker ; VDAC : mitochondrial marker ; Homo : cell

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Mouse livers treated with vehicle or 1 μM rapamycin (Rapa.) for 16 h by an intraperitoneal injection were fractionated into cytosol (Cyt.) or mitochondrial (Mito.) extracts and analyzed by western blotting. Phosphorylated (S2468) mTOR (p-mTOR) was used to assess the rapamycin activity. E-cadherin: plasma membrane marker; β-tubulin: cytosolic marker; VDAC: mitochondrial marker; Homo: cell homogenate


Mitochondria isolated from Hek293T cells were subjected to the indicated treatments and analyzed by western blotting against Akt , phosphorylated ( S473 ) Akt ( p - Akt ) , and mitochondrial proteins with known localizations . Osmotic swelling through the removal of sucrose from the buffer was used to induce OMM rupture . OMM : outer mitochondrial membrane ; IMS : intermembrane

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Mitochondria isolated from Hek293T cells were subjected to the indicated treatments and analyzed by western blotting against Akt, phosphorylated (S473) Akt (p-Akt), and mitochondrial proteins with known localizations. Osmotic swelling through the removal of sucrose from the buffer was used to induce OMM rupture. OMM: outer mitochondrial membrane; IMS: intermembrane space


Western blot analysis of the supernatant ( S ) and insoluble pellet ( P ) fractions of vehicle - or rapamycin ( Rapa . , 1 μM for 4 h ) - treated Hek293T cell mitochondria following carbonate extraction at pH 11 . 5 . Akt , phosphorylated ( S473 ) Akt ( p - Akt ) , MICU1 , the established integral membrane proteins TIM23 and MCU , and the soluble protein cytochrome c ( Cyt . c ) was

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Western blot analysis of the supernatant (S) and insoluble pellet (P) fractions of vehicle- or rapamycin (Rapa., 1 μM for 4 h)-treated Hek293T cell mitochondria following carbonate extraction at pH 11.5. Akt, phosphorylated (S473) Akt (p-Akt), MICU1, the established integral membrane proteins TIM23 and MCU, and the soluble protein cytochrome c (Cyt. c) was analyzed


Hek293T cells were transfected with the GFP - tagged wild - type ( WT ) MICU1 and then treated with 1 μM rapamycin ( Rapa . ) alone for 4 h or in combination with the Akt inhibitor triciribine ( 10 μM ) . GFP - MICU1 immunocomplexes were precipitated with a GFP antibody and analyzed with PAS ( phospho - Akt substrate ) and phosphorylated ( S473 ) Akt ( p - Akt ) antibodies by western

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Hek293T cells were transfected with the GFP-tagged wild-type (WT) MICU1 and then treated with 1 μM rapamycin (Rapa.) alone for 4 h or in combination with the Akt inhibitor triciribine (10 μM). GFP-MICU1 immunocomplexes were precipitated with a GFP antibody and analyzed with PAS (phospho-Akt substrate) and phosphorylated (S473) Akt (p-Akt) antibodies by western blotting


Hek293T cells were transfected with either the GFP - tagged wild - type ( WT ) MICU1 or MICU1 S124A - GFP mutant . MICU1 - GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS ( phospho - Akt substrate ) antibody by western blotting .

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Hek293T cells were transfected with either the GFP-tagged wild-type (WT) MICU1 or MICU1 S124A-GFP mutant. MICU1-GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS (phospho-Akt substrate) antibody by western blotting.


Mitochondrial preparations of both Hek293T ( left ) and HeLa ( right ) cells transiently transfected with an HA - tagged constitutively active Akt T308D / S473D ( Akt D / D ) construct were analyzed by western blotting . GAPDH : cytosolic marker ; TIM23 : mitochondrial

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Mitochondrial preparations of both Hek293T (left) and HeLa (right) cells transiently transfected with an HA-tagged constitutively active Akt T308D/S473D (Akt D/D) construct were analyzed by western blotting. GAPDH: cytosolic marker; TIM23: mitochondrial marker


Resting mitochondrial calcium levels in the control ( ctrl ) and Akt D / D - expressing HeLa cells evaluated by ratiometric imaging of the mitochondrial - targeted GCaMP6m ( n = 4 independent experiments ; 44 - 49 cells )

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Resting mitochondrial calcium levels in the control (ctrl) and Akt D/D-expressing HeLa cells evaluated by ratiometric imaging of the mitochondrial-targeted GCaMP6m (n=4 independent experiments; 44-49 cells)


Schematic model of the HA - tagged constitutively active Akt form targeted to the mitochondrial intermembrane space ( mt - Akt D / D ) . Both the expression and the mitochondrial localization of the chimera were assessed by immunofluorescence . TOM20 was used as a mitochondrial marker . Merged images are indicated ( merge ) . Scale bar 10

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Schematic model of the HA-tagged constitutively active Akt form targeted to the mitochondrial intermembrane space (mt-Akt D/D). Both the expression and the mitochondrial localization of the chimera were assessed by immunofluorescence. TOM20 was used as a mitochondrial marker. Merged images are indicated (merge). Scale bar 10 μm


Hek293T cells were transfected with the indicated constructs . The GFP - tagged MICU1 was immunoprecipitated from the whole - cell lysate , and the precipitated proteins were immunoblotted with the HA and phosphorylated ( S473 ) Akt ( p - Akt )

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Hek293T cells were transfected with the indicated constructs. The GFP-tagged MICU1 was immunoprecipitated from the whole-cell lysate, and the precipitated proteins were immunoblotted with the HA and phosphorylated (S473) Akt (p-Akt) antibodies


Hek293T cells were transfected with the indicated constructs . MICU1 - GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS ( phospho - Akt substrate ) antibody by western

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Hek293T cells were transfected with the indicated constructs. MICU1-GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS (phospho-Akt substrate) antibody by western blotting


Resting mitochondrial calcium levels in control ( ctrl ) and mitochondrial - targeted Akt D / D ( mt - Akt D / D ) - expressing HeLa cells , evaluated through ratiometric imaging of the mitochondrial - targeted GCaMP6m ( n = 4 independent experiments ; 43 - 47 cells )

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Resting mitochondrial calcium levels in control (ctrl) and mitochondrial-targeted Akt D/D (mt-Akt D/D)-expressing HeLa cells, evaluated through ratiometric imaging of the mitochondrial-targeted GCaMP6m (n=4 independent experiments; 43-47 cells)


Resting mitochondrial calcium levels in ShRNA MICU1 HeLa stable cells transfected with the indicated constructs and evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m ( n = 3 independent experiments ; 38 - 50 cells )

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Resting mitochondrial calcium levels in ShRNA MICU1 HeLa stable cells transfected with the indicated constructs and evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m (n=3 independent experiments; 38-50 cells)


Representative kinetics ( H ) and analysis ( I ) of aequorin - based [ Ca2 + ] m measurements in permeabilized ShRNA MICU1 HeLa stable cells transfected with the indicated constructs and challenged with 400 nM buffered [ Ca2 + ] ( n = 3 independent experiments ) .

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Representative kinetics (H) and analysis (I) of aequorin-based [Ca2+]m measurements in permeabilized ShRNA MICU1 HeLa stable cells transfected with the indicated constructs and challenged with 400 nM buffered [Ca2+] (n=3 independent experiments).


Western blot analysis of ShRNA MICU1 HeLa stable cells transfected with the indicated constructs . pre - MICU1 : precursor form of MICU1 ; m - MICU1 : mature form of

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Western blot analysis of ShRNA MICU1 HeLa stable cells transfected with the indicated constructs. pre-MICU1: precursor form of MICU1; m-MICU1: mature form of MICU1


Western blot analysis of ShRNA MICU1 HeLa stable cells transfected with the indicated constructs . Where indicated , the cells were treated with 5 μM MG132 for 4 h . pre : precursor ; int : intermediate ; m : mature

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Western blot analysis of ShRNA MICU1 HeLa stable cells transfected with the indicated constructs. Where indicated, the cells were treated with 5 μM MG132 for 4 h. pre: precursor; int: intermediate; m: mature form


Western blot analysis of the supernatant ( S ) and insoluble pellet ( P ) fractions of ShRNA MICU1 HeLa cell mitochondria transfected with the indicated constructs , following carbonate extraction at pH 10 . The established integral membrane protein TIM23 and the soluble protein cytochrome c ( Cyt . c ) were analyzed as markers . The cells were treated with 5 μM MG132 for 4 h before

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

Western blot analysis of the supernatant (S) and insoluble pellet (P) fractions of ShRNA MICU1 HeLa cell mitochondria transfected with the indicated constructs, following carbonate extraction at pH 10. The established integral membrane protein TIM23 and the soluble protein cytochrome c (Cyt. c) were analyzed as markers. The cells were treated with 5 μM MG132 for 4 h before fractionation


Western blot analysis of the supernatant ( S ) and insoluble pellet ( P ) fractions of ShRNA MICU1 HeLa cell mitochondria transfected with MICU1 WT - HA or MICU1 SD - HA , together with MCU - flag and EMRE constructs , following carbonate extraction at pH 7 . 4 , pH 10 . 5 , pH 11 . 5 or pH 12 . 5 . The cells were treated with 5 μM MG132 for 4 h before

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 the supernatant (S) and insoluble pellet (P) fractions of ShRNA MICU1 HeLa cell mitochondria transfected with MICU1 WT-HA or MICU1 SD-HA, together with MCU-flag and EMRE constructs, following carbonate extraction at pH 7.4, pH 10.5, pH 11.5 or pH 12.5. The cells were treated with 5 μM MG132 for 4 h before fractionation


ShRNA MICU1 HeLa stable cells were transfected with the indicated constructs . Cells were incubated with 50 μg / ml cycloheximide ( CHX ) for the indicated times , collected and subjected to western blotting analysis as indicated . In the graph , the amount of HA ( MICU1 ) , both WT and mutants , is represented relative to the amount at time 0 . ( n = 3 independent experiments )

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ShRNA MICU1 HeLa stable cells were transfected with the indicated constructs. Cells were incubated with 50 μg/ml cycloheximide (CHX) for the indicated times, collected and subjected to western blotting analysis as indicated. In the graph, the amount of HA (MICU1), both WT and mutants, is represented relative to the amount at time 0. (n=3 independent experiments)


ShRNA MICU1 HeLa stable cells were transfected with the indicated constructs . Cells were harvested , and total protein was extracted and subjected to western blotting analysis with the indicated antibodies . Immunoblotting was performed under reducing ( with the presence of DTT ) or non - reducing ( non - red ) conditions ( without DTT ) . Cells were treated with 5 μM MG132 for 4 h before

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ShRNA MICU1 HeLa stable cells were transfected with the indicated constructs. Cells were harvested, and total protein was extracted and subjected to western blotting analysis with the indicated antibodies. Immunoblotting was performed under reducing (with the presence of DTT) or non-reducing (non-red) conditions (without DTT). Cells were treated with 5 μM MG132 for 4 h before lysis


Quantification of HA levels in non - reducing condition showed in ( E ) . ( n = 3 independent experiments )

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Quantification of HA levels in non-reducing condition showed in (E). (n=3 independent experiments)


Resting mitochondrial calcium levels , evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m , in ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs ( n = 3 independent experiments ; 46 - 54 cells ) .

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Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m, in ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs (n=3 independent experiments; 46-54 cells).


Western blot analysis of endogenous MICU1 levels in HeLa cells upon silencing of IMMP1L and / or IMMP2L . int : intermediate ; m : mature

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Western blot analysis of endogenous MICU1 levels in HeLa cells upon silencing of IMMP1L and/or IMMP2L. int: intermediate; m: mature form


Western blot analysis of HeLa cells transfected with the indicated constructs and silenced with IMMP1L and / or IMMP2L . int : intermediate ; m : mature form . Cells were treated with 5 μM MG132 for 4 h before

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Western blot analysis of HeLa cells transfected with the indicated constructs and silenced with IMMP1L and/or IMMP2L. int: intermediate; m: mature form. Cells were treated with 5 μM MG132 for 4 h before lysis


Western blot analysis of the supernatant ( S ) and insoluble pellet ( P ) fractions of control or IMMP1L - silenced HeLa cell mitochondria following carbonate extraction at pH 10 . The established integral membrane protein TIM23 and the soluble protein cytochrome c ( Cyt . c ) were analyzed as

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 the supernatant (S) and insoluble pellet (P) fractions of control or IMMP1L-silenced HeLa cell mitochondria following carbonate extraction at pH 10. The established integral membrane protein TIM23 and the soluble protein cytochrome c (Cyt. c) were analyzed as markers


Western blot analysis of the supernatant ( S ) and insoluble pellet ( P ) fractions of control or IMMP1L - silenced HeLa cell mitochondria transfected with HA - tagged MICU1 WT following carbonate extraction at pH 10 . The established integral membrane protein TIM23 and the soluble protein cytochrome c ( Cyt . c ) were analyzed as markers . pre : precursor ; int : intermediate ; m : mature form . Cells were treated with 5 μM MG132 for 4 h before

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 the supernatant (S) and insoluble pellet (P) fractions of control or IMMP1L-silenced HeLa cell mitochondria transfected with HA-tagged MICU1 WT following carbonate extraction at pH 10. The established integral membrane protein TIM23 and the soluble protein cytochrome c (Cyt. c) were analyzed as markers. pre: precursor; int: intermediate; m: mature form. Cells were treated with 5 μM MG132 for 4 h before lysis


Control or IMMP1L - silenced HeLa cells were transfected with HA - tagged MICU1 WT . Cells were incubated with 50 μg / ml cycloheximide ( CHX ) for the indicated times , collected and subjected to western blotting analysis as indicated . In the graph , the amount of HA ( MICU1 ) is represented relative to the amount at time 0 . ( n = 3 independent experiments )

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Control or IMMP1L-silenced HeLa cells were transfected with HA-tagged MICU1 WT. Cells were incubated with 50 μg/ml cycloheximide (CHX) for the indicated times, collected and subjected to western blotting analysis as indicated. In the graph, the amount of HA (MICU1) is represented relative to the amount at time 0. (n=3 independent experiments)


Representative kinetics ( F ) and analysis ( G ) of aequorin - based [ Ca2 + ] m measurements in control or IMMP1L - silenced HeLa cells challenged with 10 μM cyclopiazonic acid ( CPA ) in the presence of 100 μM EGTA ( n = 3 independent experiments )

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Representative kinetics (F) and analysis (G) of aequorin-based [Ca2+]m measurements in control or IMMP1L-silenced HeLa cells challenged with 10 μM cyclopiazonic acid (CPA) in the presence of 100 μM EGTA (n=3 independent experiments)


Resting mitochondrial calcium levels , evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m , in control or IMMP1L - silenced HeLa cells ( n = 3 independent experiments ; 37 - 55 cells ) .

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Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m, in control or IMMP1L-silenced HeLa cells (n=3 independent experiments; 37-55 cells).


Western blot analysis of MICU1 and MICU2 levels in lung cancer cells with low or high Akt activity


Lung cancer cells were transfected with the GFP - tagged WT MICU1 . MICU1 - GFP immunocomplexes were precipitated with a GFP antibody and analyzed with a PAS ( phospho - Akt substrate ) antibody by western

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

Lung cancer cells were transfected with the GFP-tagged WT MICU1. MICU1-GFP immunocomplexes were precipitated with a GFP antibody and analyzed with a PAS (phospho-Akt substrate) antibody by western blotting


Western blot analysis of MICU1 and MICU2 levels in melanoma cells with low or high Akt activity


Melanoma cells were transfected with the GFP - tagged WT MICU1 . MICU1 - GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS ( phospho - Akt substrate ) antibody by western

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Melanoma cells were transfected with the GFP-tagged WT MICU1. MICU1-GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS (phospho-Akt substrate) antibody by western blotting


Western blot analysis of MICU1 and MICU2 levels in glioblastoma cells with low or high Akt activity


Glioblastoma cells were transfected with the GFP - tagged WT MICU1 . MICU1 - GFP immunocomplexes were precipitated with GFP antibody and analyzed with PAS ( phospho - Akt substrate ) antibody by western

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Glioblastoma cells were transfected with the GFP-tagged WT MICU1. MICU1-GFP immunocomplexes were precipitated with GFP antibody and analyzed with PAS (phospho-Akt substrate) antibody by western blotting


Number of soft agar colonies ( n = 3 independent experiments formed by Rat2 cells stably expressing the indicated

Number of soft agar colonies (n=3 independent experiments formed by Rat2 cells stably expressing the indicated constructs


xenograft tumor volumes ( H ) and tumor weights at day 21 ( n = 8 mice for each group ) ( I ) formed by Rat2 cells stably expressing the indicated

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xenograft tumor volumes (H) and tumor weights at day 21 (n=8 mice for each group) (I) formed by Rat2 cells stably expressing the indicated constructs


Representative images ( J ) and analysis ( K ) of MitoSOX - based ROS measurements in ShRNA MICU1 HeLa stable cells stably expressing either MICU1 S124D ( SD ) or MICU1 S124A ( SA ) ( n = 3 independent experiments ) . Scale bar 10 μm . f . a . u . : fluorescence arbitrary

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Representative images (J) and analysis (K) of MitoSOX-based ROS measurements in ShRNA MICU1 HeLa stable cells stably expressing either MICU1 S124D (SD) or MICU1 S124A (SA) (n=3 independent experiments). Scale bar 10 μm. f.a.u.: fluorescence arbitrary unit


ShRNA MICU1 HeLa stable cells stably expressing either MICU1 S124D ( SD ) or MICU1 S124A ( SA ) were treated with 500 μM H2O2 alone or in combination with the ROS scavenger Tempol and analyzed by western blotting as indicated . l . e . : long exposure .

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ShRNA MICU1 HeLa stable cells stably expressing either MICU1 S124D (SD) or MICU1 S124A (SA) were treated with 500 μM H2O2 alone or in combination with the ROS scavenger Tempol and analyzed by western blotting as indicated. l. e.: long exposure.


B Plot of the whole - cell current ( Vhold - 65 mV ) in control conditions ( ctrl ) and after application of NASPM ( NSP , p < 0 . 0001 , n = 18 ) .

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B Plot of the whole-cell current (Vhold -65 mV) in control conditions (ctrl) and after application of NASPM (NSP, p<0.0001, n=18).


B Current - voltage relationship of the tonic Ba2 + - sensitive ( 100 µM ) outward GIRK current demonstrating reversal near expected EK and inward rectification ( n = 6 biological replicates ) .

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B Current-voltage relationship of the tonic Ba2+-sensitive (100 µM) outward GIRK current demonstrating reversal near expected EK and inward rectification (n=6 biological replicates).


A With GDPβS - containing internal solution , noradrenaline - induced GluD1R current ( INA ) was diminished by > 20 minutes post - dialysis , shown in a representative trace .

A With GDPβS-containing internal solution, noradrenaline-induced GluD1R current (INA) was diminished by >20 minutes post-dialysis, shown in a representative trace.


C Plot of the magnitude of tonic GluD1R current measured after dialysis with GTP + LiCl - , GDPβS - Li3 - , and GTP - containing internal solution , displaying no significant difference between the groups ( p = 0 . 46 , n = 9 , 13 , 8 biological replicates respectively ) .

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C Plot of the magnitude of tonic GluD1R current measured after dialysis with GTP + LiCl-, GDPβS-Li3-, and GTP-containing internal solution, displaying no significant difference between the groups (p=0.46, n=9, 13, 8 biological replicates respectively).


B - C Plot of the amplitude of the α1 - AR - EPSC in control conditions ( ctrl ) and in glycine ( 1 mM , p = 0 . 43 , n = 10 biological replicates , B ) , or D - serine ( 1 mM , p = 0 . 13 , n = 9 biological replicates , C ) .

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B-C Plot of the amplitude of the α1-AR-EPSC in control conditions (ctrl) and in glycine (1 mM, p=0.43, n=10 biological replicates, B), or D-serine (1 mM, p=0.13, n=9 biological replicates, C).


E Glycine had no significant effect on the amplitude of the α1 - AR - EPSC ( arrow ) or whole - cell current , shown in a representative trace .

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E Glycine had no significant effect on the amplitude of the α1-AR-EPSC (arrow) or whole-cell current, shown in a representative trace.


A Increasing extracellular calcium to 4 . 8 mM reduced the amplitude of the α1 - AR - EPSC , shown in a time - course plot ( n = 18 and 26 biological replicates for 2 . 4 and 4 . 8 mM respectively ) .

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A Increasing extracellular calcium to 4.8 mM reduced the amplitude of the α1-AR-EPSC, shown in a time-course plot (n=18 and 26 biological replicates for 2.4 and 4.8 mM respectively).


F Plot of the magnitude of tonic GluD1R current measured by application of NASPM in control conditions ( 1 . 2 mM ) , 2 . 4 mM and 4 . 8 mM calcium . The magnitude of tonic GluD1R current in 2 . 4 and 4 . 8 mM calcium was reduced relative to control conditions ( 1 . 2 mM : n = 18 ; 2 . 4 mM : p = 0 . 03 , n = 17 ; 4 . 8 mM : p = 0 . 005 , n = 22 biological replicates ) .

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F Plot of the magnitude of tonic GluD1R current measured by application of NASPM in control conditions (1.2 mM), 2.4 mM and 4.8 mM calcium. The magnitude of tonic GluD1R current in 2.4 and 4.8 mM calcium was reduced relative to control conditions (1.2 mM: n=18; 2.4 mM: p=0.03, n=17; 4.8 mM: p=0.005, n=22 biological replicates).


J Elevating extracellular calcium to 4 . 8 mM reduces the change in whole - cell current to NASPM and accelerates recovery of the α1 - AR - EPSC from NASPM - block in control conditions ( 1 . 2 mM extracellular calcium ) , shown in a representative whole - cell voltage - clamp recording .

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J Elevating extracellular calcium to 4.8 mM reduces the change in whole-cell current to NASPM and accelerates recovery of the α1-AR-EPSC from NASPM-block in control conditions (1.2 mM extracellular calcium), shown in a representative whole-cell voltage-clamp recording.


A Representative traces of whole - cell current clamp recordings of membrane potential and AP firing evoked by current injection ( 1 . 5 s ) demonstrating hyperpolarization by NASPM . Dashed line is at - 80 mV .

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A Representative traces of whole-cell current clamp recordings of membrane potential and AP firing evoked by current injection (1.5 s) demonstrating hyperpolarization by NASPM. Dashed line is at -80 mV.


C Plot of the membrane potential ( Vm ) versus injected current relative to rheobase ( rb ) in control and NASPM , demonstrating that NASPM produced a hyperpolarization at subthreshold potentials ( n = 10 biological replicates ) .

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C Plot of the membrane potential (Vm) versus injected current relative to rheobase (rb) in control and NASPM, demonstrating that NASPM produced a hyperpolarization at subthreshold potentials (n=10 biological replicates).


( A ) The T cell clone 20 - 4 / A4 recognizes an epitope derived from the NeoR protein in the context of HLA ‐ DP3 . MHC class II expression on RCC1 . 24 ‐ NeoR cells following IFN ‐ γ treatment confers recognition by 20 - 4 / A4 . This recognition is almost completely blocked by the HLA ‐ DP ‐ specific antibody B7 / 21 . EBV ‐ B cells transfected with pINCO ‐ NeoR are able to present endogenous NeoR on MHC class II . As exemplified by the cell lines EBV ‐ B1 . 11 ( HLA ‐ DP3 ) and EBV ‐ B1 . 25 ( HLA ‐ DP4 ) , recognition by 20 - 4 / A4 is dependent on HLA ‐ DP3 expression . Bacterially expressed NeoR protein and a synthetic peptide encompassing amino acids NeoR216 - 229 served as controls .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The T cell clone 20-4/A4 recognizes an epitope derived from the NeoR protein in the context of HLA‐DP3. MHC class II expression on RCC1.24‐NeoR cells following IFN‐γ treatment confers recognition by 20-4/A4. This recognition is almost completely blocked by the HLA‐DP‐specific antibody B7/21. EBV‐B cells transfected with pINCO‐NeoR are able to present endogenous NeoR on MHC class II. As exemplified by the cell lines EBV‐B1.11 (HLA‐DP3) and EBV‐B1.25 (HLA‐DP4), recognition by 20-4/A4 is dependent on HLA‐DP3 expression. Bacterially expressed NeoR protein and a synthetic peptide encompassing amino acids NeoR216-229 served as controls.


( B ) Cross - presentation experiments to exclude release of NeoR and re ‐ uptake as exogenous protein . EBV ‐ B1 . 25 cells transfected with the neoR expression plasmid pINCO ‐ NeoR , were co ‐ cultured for 24 h with the same number of untransfected EBV ‐ B1 . 11 cells , or vice versa . Then , T cells were added and co ‐ cultured with the cells for additional 24 h . To test for NeoR release by neoR ‐ transfected EBV ‐ B1 . 11 cells , total supernatant ( 200 μl ) of 1 × 105 EBV ‐ B1 . 11 cells was harvested 48 h post transfection by centrifugation , and was used as culture media for 1 × 105 untransfected EBV ‐ B1 . 11 cells . After 48 h , 1 × 105 20 - 4 / A4 T cells were added and GM ‐ CSF concentration determined after additional 24 h .

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(B) Cross-presentation experiments to exclude release of NeoR and re‐uptake as exogenous protein. EBV‐B1.25 cells transfected with the neoR expression plasmid pINCO‐NeoR, were co‐cultured for 24 h with the same number of untransfected EBV‐B1.11 cells, or vice versa. Then, T cells were added and co‐cultured with the cells for additional 24 h. To test for NeoR release by neoR‐transfected EBV‐B1.11 cells, total supernatant (200 μl) of 1×105 EBV‐B1.11 cells was harvested 48 h post transfection by centrifugation, and was used as culture media for 1×105 untransfected EBV‐B1.11 cells. After 48 h, 1×105 20-4/A4 T cells were added and GM‐CSF concentration determined after additional 24 h.


( A ) , pINCO ‐ NeoR transfected EBV ‐ B1 . 11 cells ( B ) , and as control pINCO ‐ Tyrosinase ‐ transfected EBV ‐ B1 . 11 cells ( C ) , were either left untreated or treated with 5 μM lactacystin or 50 μM AAF ‐ CMK for 20 h . Following fixation with 0 . 5 % paraformaldehyde , cells were incubated with T cells , and 24 h later GM ‐ CSF release was determined by ELISA .

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(A), pINCO‐NeoR transfected EBV‐B1.11 cells (B), and as control pINCO‐Tyrosinase‐transfected EBV‐B1.11 cells (C), were either left untreated or treated with 5 μM lactacystin or 50 μM AAF‐CMK for 20 h. Following fixation with 0.5% paraformaldehyde, cells were incubated with T cells, and 24 h later GM‐CSF release was determined by ELISA.


( D ) FACS analysis of EBV ‐ B1 . 11 cells ( open histograms ) and EBV ‐ B1 . 11 cells stably transfected with the ICP47 gene ( shaded histograms ) . Inhibition of TAP by ICP47 down ‐ regulates HLA class I but not HLA ‐ DP expression . An isotype ‐ matched antibody was used as control ( thin ‐ lined histogram ) . EBV ‐ B1 . 11 ‐ ICP47 cells were transfected with pINCO ‐ NeoR ( E ) , pINCO ‐ Tyrosinase ( F ) , or mock transfected ( pINCO ) and MHC ‐ restricted presentation monitored with T cells .

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(D) FACS analysis of EBV‐B1.11 cells (open histograms) and EBV‐B1.11 cells stably transfected with the ICP47 gene (shaded histograms). Inhibition of TAP by ICP47 down‐regulates HLA class I but not HLA‐DP expression. An isotype‐matched antibody was used as control (thin‐lined histogram). EBV‐B1.11‐ICP47 cells were transfected with pINCO‐NeoR (E), pINCO‐Tyrosinase (F), or mock transfected (pINCO) and MHC‐restricted presentation monitored with T cells.


( A ) RCC1 . 24 ‐ NeoR incubated with IFN ‐ γ were either left untreated ( w / o ) or treated with chloroquine or leupeptin for 24 h to block lysosomal processing . Subsequently cells were fixed with 0 . 5 % paraformaldehyde and tested with 20 - 4 / A4 in a GM ‐ CSF release assay . EBV ‐ B1 . 11 cells transfected with pINCO ‐ NeoR ( B ) or pINCO ‐ Tyrosinase ( C ) were tested likewise . Both substances abrogated recognition of neoR ‐ transfected cells , while HLA ‐ A2 ‐ restricted presentation of the tyrosinase peptide was not

O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RCC1.24‐NeoR incubated with IFN‐γ were either left untreated (w/o) or treated with chloroquine or leupeptin for 24 h to block lysosomal processing. Subsequently cells were fixed with 0.5% paraformaldehyde and tested with 20-4/A4 in a GM‐CSF release assay. EBV‐B1.11 cells transfected with pINCO‐NeoR (B) or pINCO‐Tyrosinase (C) were tested likewise. Both substances abrogated recognition of neoR‐transfected cells, while HLA‐A2‐restricted presentation of the tyrosinase peptide was not affected


( D ) ) EBV ‐ B1 . 11 cells were incubated with 200 ng / ml of recombinant NeoR protein and treated with leupeptin or chloroquine as described in ( A ) . Presentation of exogenous NeoR on HLA ‐ DP3 is blocked by both substances as well , indicating that NeoR presentation is dependent on lysosomal processing .

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(D) ) EBV‐B1.11 cells were incubated with 200 ng/ml of recombinant NeoR protein and treated with leupeptin or chloroquine as described in (A). Presentation of exogenous NeoR on HLA‐DP3 is blocked by both substances as well, indicating that NeoR presentation is dependent on lysosomal processing.


( A ) 293T cells transfected with pINCO ‐ NeoR were harvested 6 and 96 h post transfection and NeoR localization within various cell fractions determined by Western blot . In addition , supernatant of NeoR ‐ pINCO transfected 293T cells ( 96 h post transfection ) was included to test for release of NeoR into the culture supernatant . At 6 h post transfection , NeoR protein becomes detectable in the cytoplasm , while at 96 h post transfection a substantial amount of NeoR protein is also localized in the endosomal / lysosomal fraction . For further characterization of the various fractions , blots were stripped and re ‐ probed with antibodies specific for the cytoplasmic protein LDH , the nuclear protein c ‐ myc , early endosomes ( Rab ‐ 5 ) , late endosomes and lysosomes ( Lamp ‐ 1 ) and mitochondria ( cytochrome c ) , which co ‐ purify with endosomes / lysosomes .

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(A) 293T cells transfected with pINCO‐NeoR were harvested 6 and 96 h post transfection and NeoR localization within various cell fractions determined by Western blot. In addition, supernatant of NeoR‐pINCO transfected 293T cells (96 h post transfection) was included to test for release of NeoR into the culture supernatant. At 6 h post transfection, NeoR protein becomes detectable in the cytoplasm, while at 96 h post transfection a substantial amount of NeoR protein is also localized in the endosomal/lysosomal fraction. For further characterization of the various fractions, blots were stripped and re‐probed with antibodies specific for the cytoplasmic protein LDH, the nuclear protein c‐myc, early endosomes (Rab‐5), late endosomes and lysosomes (Lamp‐1) and mitochondria (cytochrome c), which co‐purify with endosomes/lysosomes.


( B ) Endosomal / lysosomal fractions were prepared from pINCO ‐ NeoR ‐ transfected cells 96 h post transfection as in ( A ) . To prevent lysosomal degradation of NeoR , half of the cells were treated with leupeptin for the last 48 h . These fractions were separated on a 27 % Percoll gradient and eight different fractions collected . Successful separation into fractions with increasing density was verified by Western blot analysis with antibodies directed against Lamp ‐ 1 , Rab ‐ 5 and cytochrome c . Inhibition of NeoR degradation by leupeptin causes accumulation of NeoR protein in the late endosomal / lysosomal fractions 7 and 8 .

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(B) Endosomal/lysosomal fractions were prepared from pINCO‐NeoR‐transfected cells 96 h post transfection as in (A). To prevent lysosomal degradation of NeoR, half of the cells were treated with leupeptin for the last 48 h. These fractions were separated on a 27% Percoll gradient and eight different fractions collected. Successful separation into fractions with increasing density was verified by Western blot analysis with antibodies directed against Lamp‐1, Rab‐5 and cytochrome c. Inhibition of NeoR degradation by leupeptin causes accumulation of NeoR protein in the late endosomal/lysosomal fractions 7 and 8.


( C ) To determine where NeoR gains access to the vacuolar system , 293T cells were transfected with pINCO ‐ NeoR , treated with leupeptin to prevent NeoR degradation , and the endosomal / lysosomal fractions prepared at various time points post transfection and separated by Percoll gradient centrifugation . Starting at 16 h post transfection , NeoR becomes detectable in the Rab ‐ 5 ‐ positive fraction 5 , and then spreads into late endosomal / lysosomal fractions . At 26 h post transfection an almost identical NeoR distribution pattern is observed in cells treated with or without BfA .

O 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 O O O O O

(C) To determine where NeoR gains access to the vacuolar system, 293T cells were transfected with pINCO‐NeoR, treated with leupeptin to prevent NeoR degradation, and the endosomal/lysosomal fractions prepared at various time points post transfection and separated by Percoll gradient centrifugation. Starting at 16 h post transfection, NeoR becomes detectable in the Rab‐5‐positive fraction 5, and then spreads into late endosomal/lysosomal fractions. At 26 h post transfection an almost identical NeoR distribution pattern is observed in cells treated with or without BfA.


( A ) RCC were transfected with the NeoR ‐ GFP expression construct and subcellular localization of the fusion protein monitored by UV fluorescence . As compared to untreated cells , cells treated with 3 ‐ MA show a much brighter UV fluorescence .

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(A) RCC were transfected with the NeoR‐GFP expression construct and subcellular localization of the fusion protein monitored by UV fluorescence. As compared to untreated cells, cells treated with 3‐MA show a much brighter UV fluorescence.


( B ) Semiquantitative analysis of fluorescence intensity of cells transfected with the NeoR ‐ GFP fusion construct . Compared to untreated cells , cells treated for 24 h with 7 . 5 mM 3 ‐ MA show an about threefold increase in mean fluorescence intensity .

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(B) Semiquantitative analysis of fluorescence intensity of cells transfected with the NeoR‐GFP fusion construct. Compared to untreated cells, cells treated for 24 h with 7.5 mM 3‐MA show an about threefold increase in mean fluorescence intensity.


( C ) Western blot analysis of neoR ‐ transfected cells . Treatment of cells with 7 . 5 mM 3 ‐ MA causes a dramatic increase in NeoR protein levels in only 12 h . The ER ‐ resident protein BiP was used as gel loading control .

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(C) Western blot analysis of neoR‐transfected cells. Treatment of cells with 7.5 mM 3‐MA causes a dramatic increase in NeoR protein levels in only 12 h. The ER‐resident protein BiP was used as gel loading control.


( D ) This increase in total NeoR protein in 3 ‐ MA ‐ treated cells is associated with an increase in cytoplasmic , and a decrease in endosomal / lysosomal NeoR protein levels .

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(D) This increase in total NeoR protein in 3‐MA‐treated cells is associated with an increase in cytoplasmic, and a decrease in endosomal/lysosomal NeoR protein levels.


( E ) Western blot analysis of whole cell lysates prepared from neoR ‐ transfected cells . Inhibition of protein synthesis by cycloheximide treatment leads to a rapid decrease in NeoR protein levels . Simultaneous treatment of cells with 3 ‐ MA prevents NeoR degradation . BiP was again used as a gel loading control .

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(E) Western blot analysis of whole cell lysates prepared from neoR‐transfected cells. Inhibition of protein synthesis by cycloheximide treatment leads to a rapid decrease in NeoR protein levels. Simultaneous treatment of cells with 3‐MA prevents NeoR degradation. BiP was again used as a gel loading control.


( A ) RCC1 . 24 ‐ NeoR ( endogenous ) , and RCC1 . 24 cells to which NeoR protein had been added ( exogenous ) , were treated with different concentrations of 3 ‐ MA and cell surface presentation of the NeoR ‐ derived peptide on HLA ‐ DP3 monitored with the T cell clone 20 - 4 / A4 ‐ specific T cell clone 20 - 4 / A4 . Inhibition of autophagy by 3 ‐ MA causes an almost complete inhibition of endogenous antigen presentation , while presentation of exogenous NeoR is not affected .

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(A) RCC1.24‐NeoR (endogenous), and RCC1.24 cells to which NeoR protein had been added (exogenous), were treated with different concentrations of 3‐MA and cell surface presentation of the NeoR‐derived peptide on HLA‐DP3 monitored with the T cell clone 20-4/A4‐specific T cell clone 20-4/A4. Inhibition of autophagy by 3‐MA causes an almost complete inhibition of endogenous antigen presentation, while presentation of exogenous NeoR is not affected.


( B ) EBV ‐ B1 . 11 cells transfected with pINCO ‐ NeoR ( endogenous ) , or incubated with recombinant NeoR protein ( exogenous ) , were treated with the inhibitors of autophagy , wortmannin and 3 ‐ MA , and HLA ‐ DP3 ‐ restricted presentation of the NeoR ‐ derived peptide determined . Inhibition of antigen presentation by leupeptin served as control .

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(B) EBV‐B1.11 cells transfected with pINCO‐NeoR (endogenous), or incubated with recombinant NeoR protein (exogenous), were treated with the inhibitors of autophagy, wortmannin and 3‐MA, and HLA‐DP3‐restricted presentation of the NeoR‐derived peptide determined. Inhibition of antigen presentation by leupeptin served as control.


( C ) Presentation of the HLA ‐ A2 ‐ restricted tyrosinase peptide in EBV ‐ B1 . 11 cells transfected with pINCO ‐ Tyrosinase is not affected by these inhibitors of autophagy .

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(C) Presentation of the HLA‐A2‐restricted tyrosinase peptide in EBV‐B1.11 cells transfected with pINCO‐Tyrosinase is not affected by these inhibitors of autophagy.


FACS analysis of HLA ‐ class I and HLA ‐ DP expression on RCC1 . 24 ( D ) and EBV ‐ B1 . 11 cells ( E ) As compared to untreated cells ( thick ‐ lined histogram ) , treatment of cells with 7 . 5 mM 3 ‐ MA ( shaded histogram ) does not cause down ‐ regulation of HLA ‐ class I or HLA ‐ DP . Isotype control is shown as thin ‐ lined histogram .

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FACS analysis of HLA‐class I and HLA‐DP expression on RCC1.24 (D) and EBV‐B1.11 cells (E) As compared to untreated cells (thick‐lined histogram), treatment of cells with 7.5 mM 3‐MA (shaded histogram) does not cause down‐regulation of HLA‐class I or HLA‐DP. Isotype control is shown as thin‐lined histogram.


Human monocytic ( THP - 1 ) cells were induced to acquire an M2 - like phenotype ( see Methods ) and treated with different concentrations of TLR7 - 54 or FA - TLR7 - 54 for either 48h In the latter case , after the 2h incubation , culture medium was replaced with drug - free medium and incubation was continued for 46h . All treatment groups were then analyzed by qPCR for gene expression ( A ) Changes in mRNA levels of indicated profibrotic macrophage markers by induced by different concentrations of TLR7 - 54 and FA - TLR7 - 54 ( n = 3 , technical replicates ) .

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Human monocytic (THP-1) cells were induced to acquire an M2-like phenotype (see Methods) and treated with different concentrations of TLR7-54 or FA-TLR7-54 for either 48h In the latter case, after the 2h incubation, culture medium was replaced with drug-free medium and incubation was continued for 46h. All treatment groups were then analyzed by qPCR for gene expression (A) Changes in mRNA levels of indicated profibrotic macrophage markers by induced by different concentrations of TLR7-54 and FA-TLR7-54 (n=3, technical replicates).


Human monocytic ( THP - 1 ) cells were induced to acquire an M2 - like phenotype ( see Methods ) and treated with different concentrations of TLR7 - 54 or FA - TLR7 - 54 for either 48h or 2h In the latter case , after the 2h incubation , culture medium was replaced with drug - free medium and incubation was continued for 46h . cell supernatants were analyzed for secreted cytokines by ELISA . ( B - C ) Changes in CCL18 and IL - 1β in the culture media induced upon treatment with TLR7 - 54 and FA - TLR7 - 54 for the treatment regimens ( n = 3 , technical replicates ) .

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Human monocytic (THP-1) cells were induced to acquire an M2-like phenotype (see Methods) and treated with different concentrations of TLR7-54 or FA-TLR7-54 for either 48h or 2h In the latter case, after the 2h incubation, culture medium was replaced with drug-free medium and incubation was continued for 46h. cell supernatants were analyzed for secreted cytokines by ELISA. (B-C) Changes in CCL18 and IL-1β in the culture media induced upon treatment with TLR7-54 and FA-TLR7-54 for the treatment regimens (n=3, technical replicates).


M2 - induced human monocyte - derived macrophages were treated with 100 nM of the indicated drug either continuously for 48h , or initially for 2h in the presence or absence of FA - glucosamine ( competition ) followed by 46h in the absence of drug ( 2 + 46h ) , as described in Fig 1 . mRNA levels of profibrotic markers , Arg1 ( A ) , CD206 ( B ) and CD163 ( C ) Changes in both sets of cytokines were inhibited by blockade of unoccupied folate receptors with excess FA - glucosamine ( 2 + 46h , competition ) .

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M2-induced human monocyte-derived macrophages were treated with 100 nM of the indicated drug either continuously for 48h, or initially for 2h in the presence or absence of FA-glucosamine (competition) followed by 46h in the absence of drug (2+46h), as described in Fig 1. mRNA levels of profibrotic markers, Arg1 (A), CD206 (B) and CD163 (C) Changes in both sets of cytokines were inhibited by blockade of unoccupied folate receptors with excess FA-glucosamine (2+46h, competition).


M2 - induced human monocyte - derived macrophages were treated with 100 nM of the indicated drug either continuously for 48h , or initially for 2h in the presence or absence of FA - glucosamine ( competition ) followed by 46h in the absence of drug ( 2 + 46h ) , as described in Fig 1 . protein levels of secreted profibrotic CCL18 ( D ) and antifibrotic cytokines , CXCL10 ( E ) and IL - 6 ( F ) ( n = 3 , technical replicates ) were then determined . Changes in both sets of cytokines were inhibited by blockade of unoccupied folate receptors with excess FA - glucosamine ( 2 + 46h , competition ) .

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M2-induced human monocyte-derived macrophages were treated with 100 nM of the indicated drug either continuously for 48h, or initially for 2h in the presence or absence of FA-glucosamine (competition) followed by 46h in the absence of drug (2+46h), as described in Fig 1. protein levels of secreted profibrotic CCL18 (D) and antifibrotic cytokines, CXCL10 (E) and IL-6 (F) (n=3, technical replicates) were then determined. Changes in both sets of cytokines were inhibited by blockade of unoccupied folate receptors with excess FA-glucosamine (2+46h, competition).


( A ) Healthy mice or BLM - induced mice were tail vein injected on day 10 with 10 nmol OTL38 in the absence or presence of 200 - fold excess FA - glucosamine to block all folate receptors . After 2h , mice were euthanized , resected and imaged for fluorescence intensity ( n = 5 ) .

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(A) Healthy mice or BLM-induced mice were tail vein injected on day 10 with 10 nmol OTL38 in the absence or presence of 200-fold excess FA-glucosamine to block all folate receptors. After 2h, mice were euthanized, resected and imaged for fluorescence intensity (n=5).


( B ) Alternatively , lungs from mice injected with 100 nmol OTL38 in the present or absence of 200 - fold excess FA - glucosamine were collagenase digested and stained with 7AAD plus antibodies to CD11b and F4 / 80 prior to FACS analysis ( n = 3 ) . Representative plots showing the gating strategy yielding live macrophages ( 7AAD - CD11b + F4 / 80 + ) and OTL38 positive macrophages are shown .

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(B) Alternatively, lungs from mice injected with 100 nmol OTL38 in the present or absence of 200-fold excess FA-glucosamine were collagenase digested and stained with 7AAD plus antibodies to CD11b and F4/80 prior to FACS analysis (n=3). Representative plots showing the gating strategy yielding live macrophages (7AAD- CD11b+ F4/80+) and OTL38 positive macrophages are shown.


( C ) Percentages of live macrophages in BLM - induced mice ( n = 3 ) .

(C) Percentages of live macrophages in BLM-induced mice (n=3).


( D ) Percentages of lung macrophages that accumulated OTL38 in vivo ( n = 3 ) .

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(D) Percentages of lung macrophages that accumulated OTL38 in vivo (n=3).


Healthy mice or BLM - induced mice were injected intravenously on day 10 with either vehicle ( 3 % DMSO in PBS ) , or 10 nmol TLR7 - 54 or FA - TLR7 - 54 dissolved in vehicle , and 1 or 4h later sacrificed to collect both lungs and bronchioalveolar lavage fluid ( BALF ) . ( A ) Lungs were digested and macrophages were isolated by flow cytometry prior to analysis for expression of the indicated mRNAs by qPCR ( n = 3 ) .

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Healthy mice or BLM-induced mice were injected intravenously on day 10 with either vehicle (3% DMSO in PBS), or 10 nmol TLR7-54 or FA-TLR7-54 dissolved in vehicle, and 1 or 4h later sacrificed to collect both lungs and bronchioalveolar lavage fluid (BALF). (A) Lungs were digested and macrophages were isolated by flow cytometry prior to analysis for expression of the indicated mRNAs by qPCR (n=3).


Healthy mice or BLM - induced mice were injected intravenously on day 10 with either vehicle ( 3 % DMSO in PBS ) , or 10 nmol TLR7 - 54 or FA - TLR7 - 54 dissolved in vehicle , and 1 or 4h later sacrificed to collect both lungs and bronchioalveolar lavage fluid ( BALF ) . ( B ) BALF cells were pelleted and similarly analyzed for the indicated mRNAs ( n = 3 ) .

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Healthy mice or BLM-induced mice were injected intravenously on day 10 with either vehicle (3% DMSO in PBS), or 10 nmol TLR7-54 or FA-TLR7-54 dissolved in vehicle, and 1 or 4h later sacrificed to collect both lungs and bronchioalveolar lavage fluid (BALF). (B) BALF cells were pelleted and similarly analyzed for the indicated mRNAs (n=3).


Healthy mice or BLM - induced mice were injected intravenously on day 10 with either vehicle ( 3 % DMSO in PBS ) , or 10 nmol TLR7 - 54 or FA - TLR7 - 54 dissolved in vehicle , and 1 or 4h later sacrificed to collect both lungs and bronchioalveolar lavage fluid ( BALF ) . ( C ) BALF supernatant was also collected and analyzed by ELISA for IL - 6 , IFNα and TNFα ( n = 3 ) .

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

Healthy mice or BLM-induced mice were injected intravenously on day 10 with either vehicle (3% DMSO in PBS), or 10 nmol TLR7-54 or FA-TLR7-54 dissolved in vehicle, and 1 or 4h later sacrificed to collect both lungs and bronchioalveolar lavage fluid (BALF). (C) BALF supernatant was also collected and analyzed by ELISA for IL-6, IFNα and TNFα (n=3).


( B ) Survival analysis of the treatment groups ( Healthy , n = 5 ; others , n = 10 ) .

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

(B) Survival analysis of the treatment groups (Healthy, n=5; others, n=10).


BALF was collected on day 21 and centrifuged to isolate cell pellets . Cells ( primarily macrophages ) were analyzed by qPCR for Arg1 ( C ) , MMP9 ( D ) , TIMP3 ( E )

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

BALF was collected on day 21 and centrifuged to isolate cell pellets. Cells (primarily macrophages) were analyzed by qPCR for Arg1 (C), MMP9 (D), TIMP3 (E)


BALF was collected on day 21 and centrifuged to isolate cell pellets . Cells ( primarily macrophages ) were analyzed by qPCR for CD86 ( F ) and IRAK4 ( G ) ( n = 5 ) .

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

BALF was collected on day 21 and centrifuged to isolate cell pellets. Cells (primarily macrophages) were analyzed by qPCR for CD86 (F) and IRAK4 (G) (n=5).


BALF supernatant was analyzed by ELISA for IFNγ ( H ) ( n = 5 ) .

BALF supernatant was analyzed by ELISA for IFNγ (H) (n=5).


( I ) Lungs were resected and subjected to hematoxylin - eosin ( H & E ) , trichome ( collagen ) and α - SMA IHC staining ( scale bars , 200 μm ) ,

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

(I) Lungs were resected and subjected to hematoxylin-eosin (H&E), trichome (collagen) and α-SMA IHC staining (scale bars, 200 μm),


Lungs were ( J ) hydrolyzed and analyzed for hydroxyproline content as a measure of collagen content ( Healthy , n = 5 ; others , n = 10 ) ,

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

Lungs were (J) hydrolyzed and analyzed for hydroxyproline content as a measure of collagen content (Healthy, n=5; others, n=10),


( K ) Ashcroft score quantitation of fibrosis ( n = 5 ) .

(K) Ashcroft score quantitation of fibrosis (n=5).


Healthy control ( filled circles ) or BLM - induced mice were treated with vehicle ( filled squares ) , 1 nmol ( empty circles ) , 3 nmol ( empty squares ) , or 10 nmol ( filled triangles ) FA - TLR7 - 54 and then sacrificed on day 21 for analysis . ( A ) Analysis of body weight change versus time ( n = 10 ) .

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 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

Healthy control (filled circles) or BLM-induced mice were treated with vehicle (filled squares), 1 nmol (empty circles), 3 nmol (empty squares), or 10 nmol (filled triangles) FA-TLR7-54 and then sacrificed on day 21 for analysis. (A) Analysis of body weight change versus time (n=10).


Healthy control ( filled circles ) or BLM - induced mice were treated with vehicle ( filled squares ) , 1 nmol ( empty circles ) , 3 nmol ( empty squares ) , or 10 nmol ( filled triangles ) FA - TLR7 - 54 and then sacrificed on day 21 for analysis . ( B ) Analysis of the number of cells per milliliter of BALF ( n = 5 ) .

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 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

Healthy control (filled circles) or BLM-induced mice were treated with vehicle (filled squares), 1 nmol (empty circles), 3 nmol (empty squares), or 10 nmol (filled triangles) FA-TLR7-54 and then sacrificed on day 21 for analysis. (B) Analysis of the number of cells per milliliter of BALF (n=5).


Healthy control ( filled circles ) or BLM - induced mice were treated with vehicle ( filled squares ) , 1 nmol ( empty circles ) , 3 nmol ( empty squares ) , or 10 nmol ( filled triangles ) FA - TLR7 - 54 and then sacrificed on day 21 for analysis . ( C ) Quantitation of total hydroxyproline content per right lung ( Healthy control , n = 5 ; others , n = 7 - 9 ) .

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

Healthy control (filled circles) or BLM-induced mice were treated with vehicle (filled squares), 1 nmol (empty circles), 3 nmol (empty squares), or 10 nmol (filled triangles) FA-TLR7-54 and then sacrificed on day 21 for analysis. (C) Quantitation of total hydroxyproline content per right lung (Healthy control, n=5; others, n=7-9).


Healthy control ( filled circles ) or BLM - induced mice were treated with vehicle ( filled squares ) , 1 nmol ( empty circles ) , 3 nmol ( empty squares ) , or 10 nmol ( filled triangles ) FA - TLR7 - 54 and then sacrificed on day 21 for analysis . ( D ) H & E staining and trichrome staining of lung tissue ( scale bars , 200 μm ) .

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 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

Healthy control (filled circles) or BLM-induced mice were treated with vehicle (filled squares), 1 nmol (empty circles), 3 nmol (empty squares), or 10 nmol (filled triangles) FA-TLR7-54 and then sacrificed on day 21 for analysis. (D) H&E staining and trichrome staining of lung tissue (scale bars, 200 μm).


Healthy mice were tail vein injected with 10 nmol TLR7 - 54 ( circles ) or FA - TLR7 - 54 ( squares ) , and peripheral blood was collected at indicated time points after drug injection . ( A - C ) Measurement of plasma IL - 6 ( A ) , IFNα ( B ) and TNFα ( C ) ( n = 3 ) .

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

Healthy mice were tail vein injected with 10 nmol TLR7-54 (circles) or FA-TLR7-54 (squares), and peripheral blood was collected at indicated time points after drug injection. (A-C) Measurement of plasma IL-6 (A), IFNα (B) and TNFα (C) (n=3).


Healthy mice were tail vein injected with 10 nmol TLR7 - 54 ( circles ) or FA - TLR7 - 54 ( squares ) , and peripheral blood was collected at indicated time points after drug injection . ( D - F ) Effect of drug concentration on plasma levels of IL - 6 ( D ) , IFNα ( E ) , and TNFα ( F ) at 1 . 5h , 1h , or 1h after treatment , respectively ( n = 2 ) .

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

Healthy mice were tail vein injected with 10 nmol TLR7-54 (circles) or FA-TLR7-54 (squares), and peripheral blood was collected at indicated time points after drug injection. (D-F) Effect of drug concentration on plasma levels of IL-6 (D), IFNα (E), and TNFα (F) at 1.5h, 1h, or 1h after treatment, respectively (n=2).


Healthy mice were tail vein injected with 10 nmol TLR7 - 54 ( circles ) or FA - TLR7 - 54 ( squares ) , and peripheral blood was collected at indicated time points after drug injection . ( G ) Change in body weight as a measure of systemic toxicity during alternate day dosing ( n = 2 ) .

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

Healthy mice were tail vein injected with 10 nmol TLR7-54 (circles) or FA-TLR7-54 (squares), and peripheral blood was collected at indicated time points after drug injection. (G) Change in body weight as a measure of systemic toxicity during alternate day dosing (n=2).


( A ) Classical , ( B ) Rolled , and ( C ) Rotated subpopulations of the 80S * HCVIRES complex . Left column shows an overview of the final cryo - EM reconstructions with the HCVIRES ( pink ) , 40S subunit ( yellow ) , 60S subunit ( blue ) and tRNA ( green ) . Middle and right column shows mesh representation of the maps and docked models of the 60S and 40S parts , respectively . 28S - 5S - 5 . 8S rRNA ( blue ) , 60S proteins ( gold ) , 18S rRNA ( yellow ) , 40S proteins ( dark gray ) , HCVIRES ( pink ) , tRNA ( green ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Classical, (B) Rolled, and (C) Rotated subpopulations of the 80S*HCVIRES complex. Left column shows an overview of the final cryo-EM reconstructions with the HCVIRES (pink), 40S subunit (yellow), 60S subunit (blue) and tRNA (green). Middle and right column shows mesh representation of the maps and docked models of the 60S and 40S parts, respectively. 28S-5S-5.8S rRNA (blue), 60S proteins (gold), 18S rRNA (yellow), 40S proteins (dark gray), HCVIRES (pink), tRNA (green).


( D ) Subpopulations of the 80S * HCVIRES complex containing P - site tRNA . The 80S is in the classical configuration and the head tilt is not present . Left column shows an overview of the final cryo - EM reconstructions with the HCVIRES ( pink ) , 40S subunit ( yellow ) , 60S subunit ( blue ) and tRNA ( green ) . Middle and right column shows mesh representation of the maps and docked models of the60S and40S parts , respectively . 28S - 5S - 5 . 8S rRNA ( blue ) , 60S proteins ( gold ) , 18S rRNA ( yellow ) , 40S proteins ( dark gray ) , HCVIRES ( pink ) , tRNA ( green ) .

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

(D) Subpopulations of the 80S*HCVIRES complex containing P-site tRNA. The 80S is in the classical configuration and the head tilt is not present. Left column shows an overview of the final cryo-EM reconstructions with the HCVIRES (pink),40S subunit (yellow),60S subunit (blue) and tRNA (green). Middle and right column shows mesh representation of the maps and docked models of the60S and40S parts, respectively.28S-5S-5.8S rRNA (blue), 60S proteins (gold), 18S rRNA (yellow), 40S proteins (dark gray),HCVIRES (pink), tRNA (green).


( E ) The cryo - EM map was filtered according to resolution . 40S * HCV IRES . Left column shows an overview of the final cryo - EM reconstructions with the HCV IRES ( pink ) , 40S subunit ( yellow ) , 60S subunit ( blue ) and tRNA ( green ) . Middle and right column shows mesh representation of the maps and docked models of the 60S and 40S parts , respectively . 28S - 5S - 5 . 8S rRNA ( blue ) , 60S proteins ( gold ) , 18S rRNA ( yellow ) , 40S proteins ( dark gray ) , HCV IRES ( pink ) , tRNA ( green ) .

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

(E) The cryo-EM map was filtered according to resolution. 40S*HCV IRES. Left column shows an overview of the final cryo-EM reconstructions with the HCV IRES (pink), 40S subunit (yellow), 60S subunit (blue) and tRNA (green). Middle and right column shows mesh representation of the maps and docked models of the 60S and 40S parts, respectively. 28S-5S-5.8S rRNA (blue), 60S proteins (gold), 18S rRNA (yellow), 40S proteins (dark gray), HCV IRES (pink), tRNA (green).


( A ) Comparison of 40S subunit positions in Rolled , Rotated , Classical / no head tilt ( orange ) and Classical 80S * HCVIRES complex ( yellow ) in common 60S alignment . The cryo - EM map of the Classical 80S * HCVIRES was filtered to 6 Å . Arrows indicate the direction of movement during transition between two different states . The distance changes in the 40S subunit positions resulting from the rigid body transformation are color - coded in Å units . ( B ) Comparison of the HCVIRES in Classical 80S * HCVIRES complex ( gray ) and upon P - tRNA binding ( Classical , no head tilt ; purple ) in a common 40S body alignment . The distance of the movement of domain II was indicated with an arrow and Å

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

(A) Comparison of 40S subunit positions in Rolled, Rotated, Classical/no head tilt (orange) and Classical 80S*HCVIRES complex (yellow) in common 60S alignment. The cryo-EM map of the Classical 80S*HCVIRES was filtered to 6 Å. Arrows indicate the direction of movement during transition between two different states. The distance changes in the 40S subunit positions resulting from the rigid body transformation are color-coded in Å units.(B) Comparison of the HCVIRES in Classical 80S*HCVIRES complex (gray) and upon P-tRNA binding (Classical, no head tilt; purple) in a common 40S body alignment. The distance of the movement of domain II was indicated with an arrow and Å units


( A ) Adopted secondary structure of the IRES representing the tertiary fold . ( B ) Atomic model of the HCV IRES RNA . IRES domains are presented as coloured ribbons , overlaid with experimental IRES density ( gray , semi - transparent ) . ( C ) . Molecular model of the HCV IRES ( pink ) with ribosomal interaction sites ( coloured proteins and helices ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Adopted secondary structure of the IRES representing the tertiary fold.(B) Atomic model of the HCV IRES RNA. IRES domains are presented as coloured ribbons, overlaid with experimental IRES density (gray, semi-transparent).(C). Molecular model of the HCV IRES (pink) with ribosomal interaction sites (coloured proteins and helices).


( A ) The secondary structure of the HCVIRES domain III ( hIII1 , hIII2 , IIIe and IIId ) and helix 26 ( h26 ) from the 18S rRNA . Colors of HCVIRES Domains are the same as in Fig 3 . Interaction between the HCVIRES and 18S rRNA are indicated as red lines and a tertiary IRES interaction is indicated as black line . ( B ) The interaction core between IIId , IIIe , hIII1 of HCVIRES ( colored as in ( A ) ) and Expansion segment 7 ( ES7 ) of h26 ( dark gray ) . ( C ) The rearrangement of h26 ( red ) upon binding of HCVIRES . The 80S * HCVIRES complex ( yellow ) is compared to the elongating POST state ( gray ; PDB 5Aj0 ) ( Behrmann et al , 2015 ) . The loop closing the helix is highlighted in red and dark gray .

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

(A) The secondary structure of the HCVIRES domain III (hIII1, hIII2, IIIe and IIId) and helix 26 (h26) from the 18S rRNA. Colors of HCVIRES Domains are the same as in Fig 3. Interaction between the HCVIRES and 18S rRNA are indicated as red lines and a tertiary IRES interaction is indicated as black line.(B) The interaction core between IIId, IIIe, hIII1 of HCVIRES (colored as in (A)) and Expansion segment 7 (ES7) of h26 (dark gray).(C) The rearrangement of h26 (red) upon binding of HCVIRES. The 80S*HCVIRES complex (yellow) is compared to the elongating POST state (gray; PDB 5Aj0) (Behrmann et al, 2015). The loop closing the helix is highlighted in red and dark gray.


( E ) Close up on the interactions between GGG266 - 268 of IIId ( cyan ) and CCC1116 - 1118 of ES7 ( gray ) . ( F ) Close up on the interactions between G295 , A296 and U297 of IIIe ( green ) , A136 and A288 of hIII1 ( yellow ) and U1114 , U1115 of ES7 ( gray ) .

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

(E) Close up on the interactions between GGG266-268 of IIId (cyan) and CCC1116-1118 of ES7 (gray).(F) Close up on the interactions between G295, A296 and U297 of IIIe (green), A136 and A288 of hIII1 (yellow) and U1114, U1115 of ES7 (gray).


( A ) Comparison of the 40S subunit from Classical 80S * HCVIRES complex ( yellow ) and canonical POST state ( gray ; EMD 2875 ) ( Behrmann et al , 2015 ) in common 40S body alignment . The arrow indicates the direction of the head tilt movement . Ribosomal proteins of the 40S head ( uS7 , eS25 and eS28 ) that interact with the HCVIRES domains II and IV ( pink ) on the 40S head are illuminated with blue , green , and purple , respectively . The positions of the proteins in the canonical POST state are illuminated in lighter color . The numbers indicate interaction site of HCVIRES and the 40S subunit . ( B ) Close up view on the interaction site of HCVIRES and the 40S head region . Numbers correspond to red labels in ( A ) . The rearrangement of the uS7 loop ( blue ) compared to the Classical 80S * HCVIRES complex ( gray ) is indicated . The arrow indicates the direction of the rearrangement of the uS7 loop . ( B - 1 and B - 4 ) inter subunit view from 40S subunit . ( B - 2 and - 3 ) inter subunit view from 60S subunit .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Comparison of the 40S subunit from Classical 80S*HCVIRES complex (yellow) and canonical POST state (gray; EMD 2875) (Behrmann et al, 2015) in common 40S body alignment. The arrow indicates the direction of the head tilt movement. Ribosomal proteins of the 40S head (uS7, eS25 and eS28) that interact with the HCVIRES domains II and IV (pink) on the 40S head are illuminated with blue, green, and purple, respectively. The positions of the proteins in the canonical POST state are illuminated in lighter color. The numbers indicate interaction site of HCVIRES and the 40S subunit.(B) Close up view on the interaction site of HCVIRES and the 40S head region. Numbers correspond to red labels in (A). The rearrangement of the uS7 loop (blue) compared to the Classical 80S*HCVIRES complex (gray) is indicated. The arrow indicates the direction of the rearrangement of the uS7 loop. (B-1 and B-4) inter subunit view from 40S subunit. (B-2 and -3) inter subunit view from 60S subunit.