physical activity of 10 - 12 week old WT KO mice on normal chow were measured using CLAM metabolic cage system ( , n = 6 ) . Bar graphs show average value of metabolic measurements made within 60 minutes flanking 6AM or 6PM . Data information : values shown are average ± SD . * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons ) .

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physical activity of 10-12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system (males, n=6). Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM. Data information: values shown are average ±SD. *** = p < 0.001 (two-way ANOVA with multiple comparisons).


Transcript for Rcan1 . 1 Rcan1 . 4 during differentiation of 3T3 - L1 adipocytes , measured at 0 , 1 , 2 , 3 , 4 , 6 24 hours following addition MDI as well as at 3 , 7 10 days as indicated ( n = 3 ) . * = p < 0 . 05 ; * * = p < 0 . 01 , student ' s t - , unpaired , vs 0 time point . Data information : Transcript were normalized to 18S . values shown are mean ±

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Transcript levels for Rcan1.1 and Rcan1.4 during differentiation of 3T3-L1 adipocytes, measured at 0, 1, 2, 3, 4, 6 and 24 hours following the addition MDI as well as at 3, 7 and 10 days as indicated (n=3). * = p < 0.05; ** = p < 0.01, student's t-test, unpaired, vs 0 time point. Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD


B Western blot for RCAN1 . 1 , RCAN1 . 4 tubulin ( TUB ) in whole extracts of 3T3 - L1 adipocytes transfected with a control si - or ones targeting either Rcan1 . 4 or Rcan1 . 1 ( 20 μg per lane ) .

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B Western blot for RCAN1.1, RCAN1.4 and tubulin (TUB) in whole cell extracts of 3T3-L1 adipocytes transfected with a control si-RNA or ones targeting either Rcan1.4 or Rcan1.1 (20 μg per lane).


C Representative of 3T3 - L1 adipocytes transfected with indicated si - RNAs prior to differentiation stained with Oil Red O on day 10 . Scale bar = 100 μm .

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C Representative images of 3T3-L1 adipocytes transfected with the indicated si-RNAs prior to differentiation and stained with Oil Red O on day 10. Scale bar = 100 μm.


D Western blot for adiponectin ( ADPOQ ) released into media during differentiation by 3T3 - L1 adipocytes transfected with indicated si - RNAs . Ponceau stained image provides loading control .

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D Western blot for adiponectin (ADPOQ) released into the media during differentiation by 3T3-L1 adipocytes transfected with the indicated si-RNAs. Ponceau stained image provides loading control.


E Transcript for Pgc1 - α , in 3T3 - L1 adipocytes transfected with indicated si - RNAs assayed over 10 - days of differentiation ( n = 3 ) . Data information : Transcript were normalized to 18S . values shown are mean ±

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E Transcript levels for Pgc1-α, in 3T3-L1 adipocytes transfected with the indicated si-RNAs and assayed over 10-days of differentiation (n=3). Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD


Representative bright - field showing lipid accumulation at 10 - days differentiation of Ing - svf treated with indicated siRNA . Scale bar = 50 μm .

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Representative bright-field images showing lipid accumulation at 10-days differentiation of Ing-svf cells treated with the indicated siRNA. Scale bar = 50 μm.


G Transcript for Adrenoceptor beta 3 ( Adrb3 ) , uncoupling 1 ( Ucp1 ) , PPARγ coactivator 1 alpha ( PGC1α ) , deiodinase type II ( Dio2 ) Ing - svf cultures transfected with control siRNA ( C ) or one targeting Rcan1 ( KD ) then maintained in growth media ( G ) , or differentiated for ten - days ( D ) with without addition of 100 mM isoproterionol for 6 hours prior to harvesting ( D + ISO ) ( n = 2 in triplicate ) . * = p < 0 . 05 ; * * = p < 0 . 01 , two - way ANOVA with multiple comparisons . Data information : Transcript were normalized to 18S . values shown are mean ±

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G Transcript levels for Adrenoceptor beta 3 (Adrb3), uncoupling protein 1 (Ucp1), PPARγ coactivator 1 alpha (PGC1α), and deiodinase type II (Dio2) Ing-svf cultures transfected with control siRNA (C) or one targeting Rcan1 (KD) then maintained in growth media (G), or differentiated for ten-days (D) with and without the addition of 100 mM isoproterionol for 6 hours prior to harvesting (D+ISO) (n=2 in triplicate). * = p < 0.05; ** = p < 0.01, two-way ANOVA with multiple comparisons. Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD


H , I Transcript for indicated panel of genes relevant to adipocyte differentiation in Platelet - derived growth factor receptor alpha - positive ( PDGFRα + ) lineage - negative ( Lin - ) stromal vascular isolated by FACS sorting from sWAT of WT ( black bars ) KO ( red bars ) . In H , isolated were maintained in growth media . In I , cultures were treated with MDI then differentiated for 10 days . * = p < 0 . 05 ; * * = p < 0 . 01 , student ' s t - , unpaired , WT vs . KO . Data information : Transcript were normalized to 18S . values shown are mean ±

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H, I Transcript levels for the indicated panel of genes relevant to adipocyte differentiation in Platelet-derived growth factor receptor alpha-positive (PDGFRα+) and lineage-negative (Lin-) stromal vascular cells isolated by FACS sorting from sWAT of WT (black bars) and KO (red bars). In H, isolated cells were maintained in growth media. In I, cultures were treated with MDI then differentiated for 10 days. * = p < 0.05; ** = p < 0.01, student's t-test, unpaired, WT vs. KO. Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD


A Representative H & E histological sections of sWAT from WT KO mice probed with anti - UCP1 antibodies . Mice were either maintained at 24 ° C ( RT ) or housed at 6 ° C for 5 days . Scale bar = 200 μm .

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A Representative H&E histological sections of sWAT from WT and KO mice probed with anti-UCP1 antibodies. Mice were either maintained at 24°C (RT) or housed at 6°C for 5 days. Scale bar = 200 μm.


Transcript of Pgc1α Ucp1 in sWAT of WT KO mice after 24 hours at RT or 4 ° C ( n = Data information : Transcript were normalized to 18S . values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 . ( two - way ANOVA with multiple

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Transcript levels of Pgc1α and Ucp1 in sWAT of male and female WT and KO mice after 24 hours at RT or 4°C (n= Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD. * = p < 0.05; ** = p < 0.01. (two-way ANOVA with multiple comparisons


C Transcript of Ucp1 , Pgc1α , Adrb3 , retinoblastoma 1 ( Rb1 ) , zinc finger 423 ( Zfp423 ) , PR domain containing 16 ( Prdm16 ) were quantified in sWAT of WT KO mice after 25 on a HFD ( n = 4 - 5 ) . Data information : Transcript were normalized to 18S . values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 . ; unpaired student ' s t -

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C Transcript levels of Ucp1, Pgc1α, Adrb3, retinoblastoma 1 (Rb1), zinc finger protein 423 (Zfp423), and PR domain containing 16 (Prdm16) were quantified in sWAT of WT and KO mice after 25 weeks on a HFD (n=4-5). Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD. * = p < 0.05; ** = p < 0.01. ; unpaired student's t-test


D Transcript of Pgc1α - 1b Pgc1α - 1a isoforms in sWAT of WT KO 8 week old maintained at 24 ° C ( RT ) or housed at 4 ° C for 24 hours ( n = 5 ) . Data information : Transcript were normalized to 18S . values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 . ( two - way ANOVA with multiple

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D Transcript levels of Pgc1α-1b and Pgc1α-1a isoforms in sWAT of WT and KO 8 week old females maintained at 24°C (RT) or housed at 4°C for 24 hours (n=5). Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD. * = p < 0.05; ** = p < 0.01. (two-way ANOVA with multiple comparisons


E . Western blot for RCAN1 in various skeletal muscle groups . Each lane was loaded with 20 μg of total from plantaris ( PL ) , soleus ( SOL ) , extensor digitorum longus ( EDL ) , tibialis anterior ( TA ) , red gastrocnemius ( RG ) , gastrocnemius ( WG ) , vastus lateralis ( WV ) , heart ventricle ( HT ) , atria , diaphragm ( Dia ) , Tong . lane containing brain extract was loaded with 10 μg of . Antibody specificity was validated by running 20 μg of from heart of an Rcan1 - KO ( HT - KO ) . asterisks * indicate location of putative proteolytic fragments of RCAN1 . 1 .

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E. Western blot for RCAN1 in various skeletal muscle groups. Each lane was loaded with 20 μg of total protein from plantaris (PL), soleus (SOL), extensor digitorum longus (EDL), tibialis anterior (TA), red gastrocnemius (RG), white gastrocnemius (WG), white vastus lateralis (WV), heart ventricle (HT), atria, diaphragm (Dia), and Tong. The lane containing brain extract was loaded with 10 μg of protein. Antibody specificity was validated by running 20 μg of protein from the heart of an Rcan1-KO animal (HT-KO). The asterisks * indicate the location of putative proteolytic fragments of RCAN1.1.


F Transcript of Sln in skeletal muscle ( tibialis anterior ) of 8 - 10 week old WT KO mice housed at RT . ( n = 4 - 5 each gender ) . G Transcript of Sln in skeletal muscle ( tibialis anterior ) of 8 - 10 week old WT KO housed at RT . ( n = 5 ) . Data information : Transcript were normalized to 18S . values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 . ( two - way ANOVA with multiple comparisons F , ; unpaired student ' s t - in G .

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F Transcript levels of Sln in skeletal muscle (tibialis anterior) of 8-10 week old WT and KO mice housed at RT. (n=4-5 each gender). G Transcript levels of Sln in skeletal muscle (tibialis anterior ) of 8-10 week old male WT and KO animals housed at RT. (n=5).Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD. * = p < 0.05; ** = p < 0.01. (two-way ANOVA with multiple comparisons F, ; unpaired student's t-test in G.


H Representative western blot showing SLN in soleus muscle of 8 - 10 week old WT KO housed at RT . GAPDH was used as a loading control .

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H Representative western blot showing SLN protein in soleus muscle of 8-10 week old WT and KO animals housed at RT. GAPDH was used as a loading control.


J Activity of Sln - Luc reporter in C2C12 myoblasts transiently transfected with an empty control vector or ones expressing constitutively active calcineurin ( CnA * ) Rcan1 . Luciferase activity was normalized to beta - galactosidase activity from a co - transfected vector . ( n = 3 , assayed in duplicate ) . Data information : Transcript were normalized to 18S . values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 . ( two - way ANOVA with multiple

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J Activity of the Sln-Luc reporter in C2C12 myoblasts transiently transfected with an empty control vector or ones expressing constitutively active calcineurin (CnA*) and Rcan1. Luciferase activity was normalized to beta-galactosidase activity from a co-transfected vector. (n=3, assayed in duplicate). Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD. * = p < 0.05; ** = p < 0.01. (two-way ANOVA with multiple comparisons


A Correlation of Rcan1 expression in liver gonadal adipose tissue with HOMA - IR , using mice from over 100 strains from hybrid mouse diversity panel . Regression ( red ) , r = biweight midcorrelation , p = p - value .

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A Correlation of Rcan1 expression in liver and gonadal adipose tissue with HOMA-IR, using male mice from over 100 strains from the hybrid mouse diversity panel. Regression line (red), r = biweight midcorrelation, p = p-value.


B , C Correlation of Rcan1 expression in gonadal adipose liver with , plasma insulin plasma triglycerides , in a data set comparing backgrounds susceptible ( BTBR background ) or resistant ( C57BL / 6 background ) to diabetes when carrying leptinob / ob ( ob ) mutation . Measures are reported as of mean log10 ( ml ) . Regression ( black ) , r = linear regression , p = p - value .

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B, C Correlation of Rcan1 expression in gonadal adipose and liver with body weight, plasma insulin and plasma triglycerides, in a data set comparing backgrounds susceptible (BTBR background) or resistant (C57BL/6 background) to diabetes when carrying the leptinob/ob (ob) mutation. Measures are reported as the ratio of the mean log10 intensity (ml ratio). Regression line (black), r = linear regression, p = p-value.


A ) Live confocal imaging of liver stage P . berghei expressing UIS4 ‐ mCherry GFP reveals different morphologies of LS ‐ TVN : elongated clusters ( left ) , vesicles in host cytoplasm ( center ) , a thin tubule protruding from PVM ( right ) . Live imaging was performed 20 h after infection of hepatoma . Features are marked with arrowheads .

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A) Live confocal imaging of liver stage P. berghei expressing UIS4‐mCherry and cytoplasmic GFP reveals different morphologies of the LS‐TVN: elongated membrane clusters (left), vesicles in the host cell cytoplasm (center), and a thin tubule protruding from the PVM (right). Live imaging was performed 20 h after infection of hepatoma cells. Features are marked with white arrowheads.


B ) UIS4 ‐ IBIS1 ‐ positive tubules rapidly extend from retract to PVM . Confocal time ‐ lapse imaging of a UIS4 ‐ mCherry ‐ expressing at 22 h ( top ) an IBIS1 ‐ mCherry ‐ expressing at 26 h ( bottom ) after infection of hepatoma . Tubules are indicated with a yellow arrow , time is shown in minutes seconds .

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B) The UIS4‐ and IBIS1‐positive tubules rapidly extend from and retract to the PVM. Confocal time‐lapse imaging of a UIS4‐mCherry‐expressing parasite at 22 h (top) and an IBIS1‐mCherry‐expressing parasite at 26 h (bottom) after infection of hepatoma cells. Tubules are indicated with a yellow arrow, and the time is shown in minutes and seconds.


C ) Imaging of IBIS1 ‐ mCherry ‐ expressing P . berghei reveals similar features of LS ‐ TVN , i . e . elongated cluster ( left ) , vesicles in host cytoplasm ( center ) , a tubule ( right ) . Infected were fixed 26 h post ‐ infection imaged by epifluorescence microscopy . Features are indicated with a yellow arrowhead . Scale bars , 10 µm .

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C) Imaging of IBIS1‐mCherry‐expressing P. berghei reveals similar features of the LS‐TVN, i.e. elongated membrane cluster (left), vesicles in the host cell cytoplasm (center), and a membrane tubule (right). Infected cells were fixed 26 h post‐infection and imaged by epifluorescence microscopy. Features are indicated with a yellow arrowhead. Scale bars, 10 µm.


D ) of distinct UIS4 ‐ IBIS1 ‐ positive membranous structures in fixed infected hepatoma at three different time points : 6 h after infection ( period of sporozoite transformation into exo ‐ erythrocytic forms ) , 24 h after infection ( onset of replication growth ) , 48 h after infection ( mature liver stages ) . Values are shown as mean ( ± SD ) from three separate wells ( n = 100 per well ) of one experiment are representative of two experimental repetitions .

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D) Quantification of the distinct UIS4‐ and IBIS1‐positive membranous structures in fixed infected hepatoma cells at three different time points: 6 h after infection (the period of sporozoite transformation into exo‐erythrocytic forms), 24 h after infection (onset of parasite replication and growth), and 48 h after infection (mature liver stages). Values are shown as mean (± SD) from three separate wells (n = 100 per well) of one experiment and are representative of two experimental repetitions.


Hepatoma infected with Plasmodium berghei ANKA were fixed stained with anti - UIS4 anti - HSP70 A ) Some UIS4 ‐ positive vesicles are tethered to PVM as ' beads ‐ ona ‐ string ' . Infected fixed 24 h post ‐ infection .

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Hepatoma cells infected with Plasmodium berghei ANKA parasites were fixed and stained with anti-UIS4 and anti-HSP70 A) Some UIS4‐positive vesicles are tethered to the PVM as 'beads‐ona‐string'. Infected cell fixed 24 h post‐infection.


B ) UIS4 ‐ mCherry features we detect in live imaging of UIS4 ‐ mCherry ‐ expressing P . berghei , i . e . elongated clusters ( left ) , vesicles ( center ) tubules ( right ) are also apparent in fixed wildtype stained with anti ‐ UIS4 antibody . Infected 24 h post ‐ infection . Scale bars , 10 µm .

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B) The UIS4‐mCherry features we detect in live imaging of UIS4‐mCherry‐expressing P. berghei, i.e. elongated membrane clusters (left), vesicles (center) and tubules (right) are also apparent in fixed wildtype parasites stained with anti‐UIS4 antibody. Infected cells 24 h post‐infection. Scale bars, 10 µm.


Hepatoma infected with Plasmodium berghei ANKA were fixed stained with anti - UIS4 . C ) Often LS ‐ TVN is a composite of vesicles , tubules clusters . Infected 22 h post ‐ infection .

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Hepatoma cells infected with Plasmodium berghei ANKA parasites were fixed and stained with anti-UIS4. C) Often the LS‐TVN is a composite of vesicles, tubules and membrane clusters. Infected cell 22 h post‐infection.


D ) LS ‐ TVN detectable in hepatocytes infected with UIS4 ‐ mCherry ‐ expressing P . berghei also consists of elongated clusters ( yellow arrowhead ) , vesicles ( arrow ) tubules ( yellow arrow ) . Infected 18 h post ‐ infection .

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D) The LS‐TVN detectable in primary hepatocytes infected with UIS4‐mCherry‐expressing P. berghei also consists of elongated membrane clusters (yellow arrowhead), vesicles (white arrow) and tubules (yellow arrow). Infected cell 18 h post‐infection.


E ) Dynamic tubules ( yellow arrow ) are detectable with UIS4 ‐ mCherry in infected hepatocytes 24 h post ‐ infection . Location of is marked with a yellow asterisk . Scale bars , 10 µm .

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E) Dynamic tubules (yellow arrow) are detectable with UIS4‐mCherry in infected primary hepatocytes 24 h post‐infection. Location of parasite is marked with a yellow asterisk. Scale bars, 10 µm.


A ) Confocal microscopy show a tubule ( upper panel ) labeled with UIS4 IBIS1 ( arrowheads ) , an elongated cluster ( middle panel ) in which both are present , vesicles ( bottom panel ) that are often labeled with both UIS4 IBIS1 ( yellow arrow ) frequently labeled with only one ( yellow arrowheads ) .

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A) Confocal microscopy images show a tubule (upper panel) labeled with UIS4 and IBIS1 (white arrowheads), an elongated membrane cluster (middle panel) in which both proteins are present, and vesicles (bottom panel) that are often labeled with both UIS4 and IBIS1 (yellow arrow) and frequently labeled with only one protein (yellow arrowheads).


B ) Epifluorescence microscopy of IBIS1 UIS4 reveals that while most tubules are labeled with both as above ( arrowhead ) , some are labeled exclusively with UIS4 ( upper panel ) or IBIS1 ( bottom panel ) . Single ‐ labeled tubules were highlighted with arrows their frequency in infected with tubules is indicated in ( ± SD ) , which was calculated from two separate wells 30 h post ‐ infection ( n > 150 infected per well ) .

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B) Epifluorescence microscopy of IBIS1 and UIS4 reveals that while most tubules are labeled with both proteins as above (white arrowhead), some are labeled exclusively with UIS4 (upper panel) or IBIS1 (bottom panel). Single‐labeled tubules were highlighted with white arrows and their frequency in infected cells with tubules is indicated in percent (± SD), which was calculated from two separate wells 30 h post‐infection (n > 150 infected cells per well).


A ‐ C ) Time ‐ lapse imaging of hepatoma infected with UIS4 ‐ mCherry ‐ expressing Plasmodium berghei was used to determine velocities of UIS4 ‐ positive features , e . g . , a tubule ( A ) , elongated cluster ( B ) vesicle ( C ) were tracked over time . Measured speeds of dynamic features indicated with arrows are depicted graphically to right . Scale bars , 10 µm .

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A‐C) Time‐lapse imaging of hepatoma cells infected with UIS4‐mCherry‐expressing Plasmodium berghei was used to determine the velocities of the UIS4‐positive features, e.g., a tubule (A), elongated membrane cluster (B) and vesicle (C) were tracked over time. Measured speeds of the dynamic features indicated with white arrows are depicted graphically to the right. Scale bars, 10 µm.


D ) velocities of several UIS4 ‐ or IBIS1 ‐ positive features [ tubules ( left ) , elongated clusters ( center ) vesicles ( right ) ] were measured over time , peak velocity of each was plotted . Plotted data was derived from multiple imaged infected : For IBIS1 ‐ positive tubules data is derived from three infected for UIS4 ‐ positive tubules from 13 infected . Elongated cluster speeds were analyzed in at least five infected for both UIS4 IBIS1 , vesicle speeds were analyzed in at least 12 different for both markers .

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D) The velocities of several UIS4‐ or IBIS1‐positive features [tubules (left), elongated membrane clusters (center) and vesicles (right)] were measured over time, and the peak velocity of each was plotted. Plotted data was derived from multiple imaged infected cells: For IBIS1‐positive tubules data is derived from three infected cells and for UIS4‐positive tubules from 13 infected cells. Elongated membrane cluster speeds were analyzed in at least five infected cells for both UIS4 and IBIS1, and vesicle speeds were analyzed in at least 12 different cells for both protein markers.


B ) infected with UIS4 ‐ mCherry ‐ expressing were treated with nocodazole for 1 . 5 h ( top ) or cytochalasin D for 30 min ( bottom ) imaged by confocal microscopy . Imaging occurred 29 24 h post ‐ infection , respectively . UIS4 ‐ positive tubules , static ( top ) dynamic ( bottom ) , are indicated with arrowheads . Displayed are representative of five imaged from at least two different experiments .

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B) Cells infected with UIS4‐mCherry‐expressing parasites were treated with nocodazole for 1.5 h (top) or cytochalasin D for 30 min (bottom) and imaged by confocal microscopy. Imaging occurred 29 and 24 h post‐infection, respectively. UIS4‐positive tubules, static (top) and dynamic (bottom), are indicated with white arrowheads. Displayed cells are representative of five imaged parasites from at least two different experiments.


C ) No colocalization between host microtubules UIS4 ‐ positive tubules is detectable . Infected were fixed 25 h post ‐ infection stained with antibodies against UIS4 Tubulin . Scale bars , 10 µm .

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C) No colocalization between host microtubules and UIS4‐positive tubules is detectable. Infected cells were fixed 25 h post‐infection and stained with antibodies against UIS4 and Tubulin. Scale bars, 10 µm.


A B ) bleach was applied to elongated clusters of LS ‐ TVN approximately 27 h post ‐ infection . UIS4 ‐ mCherry signal typically did not recover in bleached ( A ) , although it was found to recover in some elongated clusters ( B ) .

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A and B) The bleach was applied to elongated membrane clusters of the LS‐TVN approximately 27 h post‐infection. The UIS4‐mCherry signal typically did not recover in the bleached area (A), although it was found to recover in some elongated membrane clusters (B).


C ) When bleach was applied to PVM at 25 h post ‐ infection , signal recovered rapidly in bleached region . Upon application of additional bleach pulses in same , bleached ( region # 1 measured graphically ) repeatedly recovered , remaining PVM ‐ associated UIS4 ‐ mCherry signal depleted ( region # 2 ) although LS ‐ TVN ‐ associated signal remained ( region # 3 ) . When a region of LS ‐ TVN was subsequently bleached ( yellow arrow , region # 4 ) , no recovery was observed . times are shown in minutes seconds . Scale bars , 10 µm .

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C) When the bleach was applied to the PVM at 25 h post‐infection, the signal recovered rapidly in the bleached region. Upon the application of additional bleach pulses in the same area, the bleached area (region #1 measured graphically) repeatedly recovered, and the remaining PVM‐associated UIS4‐mCherry signal depleted (region #2) although the LS‐TVN‐associated signal remained (region #3). When a region of the LS‐TVN was subsequently bleached (yellow arrow, region #4), no recovery was observed. The times are shown in minutes and seconds. Scale bars, 10 µm.


A B ) A phase ‐ contrast image containing infected of interest ( A ) was used for comparison to EM grid after fixation preparation of for ( B ) . Green outlining of in phase ‐ contrast image was used as a tool for comparison alignment . insets defined in are shown in right panels merged with image of UIS4 ‐ mCherry signal acquired immediately prior to fixation . Scale bars , 10 µm .

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A and B) A phase‐contrast image containing the infected cell of interest (A) was used for comparison to the EM grid after fixation and preparation of the cells for TEM (B). Green outlining of the cells in the phase‐contrast image was used as a tool for comparison and alignment. The insets defined in white are shown in the right panels merged with the fluorescent image of the UIS4‐mCherry signal acquired immediately prior to fixation. Scale bars, 10 µm.


C ) Detection of UIS4 ‐ mCherry signal by widefield microscopy in of interest immediately prior to fixation . location of is marked with an asterisk , UIS4 ‐ positive features are highlighted with arrowheads .

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C) Detection of the UIS4‐mCherry signal by widefield fluorescence microscopy in the cell of interest immediately prior to fixation. The location of the parasite is marked with an asterisk, and UIS4‐positive features are highlighted with arrowheads.


D ) Thirty ‐ six sections of this sample were prepared imaged ( Movies S4 S5 ) . Features corresponding to UIS4 ‐ positive tubule ( arrowhead ) were found in sections 10 - 16 to UIS4 ‐ positive signal adjacent to PVM ( yellow arrowhead ) were found in Sections 16 - 29 . Sections 11 ( top ) 20 ( bottom ) are displayed to highlight these two features . Scale bars , 1 µm .

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D) Thirty‐six TEM sections of this sample were prepared and imaged (Movies S4 and S5). Features corresponding to the UIS4‐positive tubule (white arrowhead) were found in sections 10-16 and to the UIS4‐positive signal adjacent to the PVM (yellow arrowhead) were found in Sections 16-29. Sections 11 (top) and 20 (bottom) are displayed to highlight these two features. Scale bars, 1 µm.


A ) Hepatoma infected with UIS4 − mCherry − expressing Plasmodium berghei were fixed 22 h post − infection stained with antibodies against host markers of distinct organelles : anti − EEA1 to label early endosomes , anti − CD63 anti − LAMP1 to label late endosomes lysosomes , anti − Calnexin to label endoplasmic reticulum , anti − Golgin97 to label Golgi . In samples , either anti − mCherry or anti − UIS4 was used to amplify UIS4 − mCherry signal . Imaging was carried out by epifluorescence microscopy . In right panels , is merged , selected regions with areas of interest are shown enlarged . At least 20 infected possessing LS − TVN features were visually examined for each condition , representative of UIS4 − positive elongated clusters ( left ) UIS4 − positive tubules ( right , indicated with yellow arrows ) are displayed . Scale bars , 10 µm .

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A) Hepatoma cells infected with UIS4−mCherry−expressing Plasmodium berghei parasites were fixed 22 h post−infection and stained with antibodies against host cell protein markers of distinct organelles: anti−EEA1 to label early endosomes, anti−CD63 and anti−LAMP1 to label late endosomes and lysosomes, anti−Calnexin to label the endoplasmic reticulum, and anti−Golgin97 to label the Golgi. In all samples, either anti−mCherry or anti−UIS4 was used to amplify the UIS4−mCherry signal. Imaging was carried out by epifluorescence microscopy. In the right panels, fluorescence is merged, and selected regions with areas of interest are shown enlarged. At least 20 infected cells possessing LS−TVN features were visually examined for each condition, and representative images of UIS4−positive elongated membrane clusters (left) and UIS4−positive tubules (right, and indicated with yellow arrows) are displayed. Scale bars, 10 µm.


B ) Profiling of red ( UIS4 ) green ( CD63 , left or LAMP ‐ 1 , right ) signals along indicated within insets from in ( A ) above .

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B) Profiling of the red (UIS4) and green (CD63, left or LAMP‐1, right) signals along the indicated line within the insets from the images in (A) above.


LC3 colocalizes with UIS4 on Plasmodium liver stage PVM TVNHepatoma infected with Plasmodium berghei expressing UIS4 ‐ mCherry ( top ) or IBIS1 ‐ mCherry ( bottom ) were fixed 22 h or 48 h post ‐ infection , respectively , stained with anti ‐ LC3 to label autophagic membranes . In right panels , is merged , selected areas of interest are shown enlarged . Scale bars , 10 µm .

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LC3 colocalizes with UIS4 on the Plasmodium liver stage PVM and TVNHepatoma cells infected with Plasmodium berghei expressing UIS4‐mCherry (top) or IBIS1‐mCherry (bottom) were fixed 22 h or 48 h post‐infection, respectively, and stained with anti‐LC3 to label autophagic membranes. In the right panels, fluorescence is merged, and selected areas of interest are shown enlarged. Scale bars, 10 µm.


( A ) of AURKB transcripts in non - IPF lung derived resident - fibroblasts treated with indicated mitogens for 16hrs . * P < 0 . 05 , * * * P < 0 . 0005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 4 ) .

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(A) Quantification of AURKB transcripts in non-IPF lung derived resident-fibroblasts treated with indicated mitogens for 16hrs. *P < 0.05, ***P < 0.0005, and ****P < 0.00005, 1-way ANOVA, (n=4).


( B ) of AURKB gene transcripts in isolated fibroblasts from non - IPF IPF lung stromal cultures . * * P < 0 . 005 , unpaired t - , ( n = 6 ) .

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(B) Quantification of AURKB gene transcripts in isolated fibroblasts from non-IPF and IPF lung stromal cell cultures. **P < 0.005, unpaired t-test, (n=6).


( C ) IPF non - IPF lung sections were immunostained using AURKB antibody ( n = 4 ) . Representative were obtained at 40X magnification . Scale bar : 40µm . Dotted : black ; myofibroblastic , blue ; active fibrotic front . Arrows indicate AURKB positive .

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(C) IPF and non-IPF lung sections were immunostained using AURKB antibody (n=4). Representative images were obtained at 40X magnification. Scale bar: 40µm. Dotted area: black; myofibroblastic core, blue; active fibrotic front. Arrows indicate AURKB positive cells.


( D ) Immunoblots of Aurkb Aurka in total lung lysates of normal ( CCSP / - ) fibrotic ( CCSP / TGFα ) mice fed with Dox for 6wks . was performed using phosphor imager software normalization was done using loading control GAPDH . * P < 0 . 05 , unpaired t - , ( n = 4 ) .

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(D) Immunoblots of Aurkb and Aurka in total lung lysates of normal (CCSP/-) and fibrotic (CCSP/TGFα) mice fed with Dox for 6wks. Quantification was performed using phosphor imager software and normalization was done using loading control GAPDH. *P < 0.05, unpaired t-test, (n=4).


( E ) Immunostaining was performed using AURKB antibody in lung sections of control ( CCSP / - ) TGFα ( CCSP / TGFα ) mice on Dox for 6wks ( n = 6 ) . Representative were obtained at 20X ( low ; Scale bar : 100µm ) 63X ( high ; Scale bar : 30µm ) magnification . Dashed box indicates of section showing in high magnification .

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(E) Immunostaining was performed using AURKB antibody in lung sections of control (CCSP/-) and TGFα (CCSP/TGFα) mice on Dox for 6wks (n=6). Representative images were obtained at 20X (low; Scale bar: 100µm) and 63X (high; Scale bar: 30µm) magnification. Dashed box indicates area of the section showing in high magnification.


( F ) Western blot of Aurkb in total lung lysates from saline bleomycin treated mice . was performed using phosphor imager software normalization was done using loading control Gapdh . * * P < 0 . 005 , unpaired t - , ( n = 4 - 5 ) .

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(F) Western blot analysis of Aurkb in total lung lysates from saline and bleomycin treated mice. Quantification was performed using phosphor imager software and normalization was done using loading control Gapdh. **P < 0.005, unpaired t-test, (n=4-5).


( A ) Human IPF lung fibroblasts were transiently transfected with control or WT1 siRNA for 72hrs AURKB transcripts were quantified . * * * * P < 0 . 00005 , unpaired t - , ( n = 4 ) .

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(A) Human IPF lung fibroblasts were transiently transfected with control or WT1 siRNA for 72hrs and AURKB transcripts were quantified. ****P < 0.00005, unpaired t-test, (n=4).


( B ) Lung - resident fibroblasts from TGFα mice on Dox for 4 wks were transiently transfected with control or WT1 siRNA for 72hrs AURKB transcripts were quantified . * * * * P < 0 . 00005 , unpaired t - , ( n = 4 ) .

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(B) Lung-resident fibroblasts from TGFα mice on Dox for 4 wks were transiently transfected with control or WT1 siRNA for 72hrs and AURKB transcripts were quantified. ****P < 0.00005, unpaired t-test, (n=4).


( C ) Immunoblot of AURKB WT1 in lysates of non - IPF fibroblasts transduced with control or WT1 - adenoviral particles for 72hrs . * * P < 0 . 005 , unpaired t - , ( n = 3 ) .

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(C) Immunoblot analysis of AURKB and WT1 in the lysates of non-IPF fibroblasts transduced with control or WT1-adenoviral particles for 72hrs. **P < 0.005, unpaired t-test, (n=3).


( D ) of AURKB transcripts in fibroblasts from IPF lung treated with either control or WT1 siRNA stimulated with media or TGFα ( 100ng / mL ) for 16hrs . * * * * P < 0 . 00005 , unpaired t - , ( n = 4 ) .

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(D) Quantification of AURKB transcripts in primary fibroblasts from IPF lung treated with either control or WT1 siRNA and stimulated with media or TGFα (100ng/mL) for 16hrs. ****P < 0.00005, unpaired t-test, (n=4).


( E ) lung - resident fibroblasts were isolated from stromal cultures of TGFα mice placed on Dox for 8 wks . lysates were prepared , ChIP was performed with anti - WT1 antibody or normal rabbit IgG as a negative control using AURKB gene promoter - specific PCR primers . Non - immunoprecipitated is represented as input ( product , 104 bp ) . * * * P < 0 . 0005 , unpaired t - , ( n = 2 ) .

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(E) Primary lung-resident fibroblasts were isolated from stromal cultures of TGFα mice placed on Dox for 8 wks. Cell lysates were prepared, and the ChIP assay was performed with anti-WT1 antibody or normal rabbit IgG as a negative control using AURKB gene promoter-specific PCR primers. Non-immunoprecipitated DNA is represented as input DNA (product size, 104 bp). ***P < 0.0005, unpaired t-test, (n=2).


( F ) AURKB - promoter luciferase activity was measured in HEK293 transfected with control or WT1 overexpressing ( OE ) vector . * * * * P < 0 . 00005 , one - way - ANOVA , ( n = 6 ) .

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(F) AURKB-promoter luciferase activity was measured in HEK293 cells transfected with control or WT1 overexpressing (OE) vector. ****P < 0.00005, one-way-ANOVA, (n=6).


( C ) Lung sections from control TGFα mice fed with Dox food for 4 wks were stained for AURKB ( green ) Ki - 67 ( red ) . Merged image shows that co - express AURKB Ki - 67 ( yellow ) .

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(C) Lung sections from control and TGFα mice fed with Dox food for 4 wks were stained for AURKB (green) and Ki-67(red). Merged image shows cells that co-express AURKB and Ki-67 (yellow).


( D ) of Ki - 67 + AURKB + double positive was performed using five confocal per mice in control TGFα mice . were obtained at 40X magnification . Scale bar : 50µm . * * * * P < 0 . 00005 , unpaired t - , ( n = 4 ) .

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(D) Quantification of Ki-67+ and AURKB+ double positive cells was performed using five confocal images per mice in control and TGFα mice. Images were obtained at 40X magnification. Scale bar: 50µm. ****P < 0.00005, unpaired t-test, (n=4).


( E ) Proliferation was measured in lung resident fibroblast isolated from either IPF lung or TGFα mice treated with either control or AURKB siRNA . * * * * P < 0 . 00005 , unpaired t - , ( n = 9 - 11 ) .

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(E) Proliferation was measured in primary lung resident fibroblast isolated from either IPF lung or TGFα mice and treated with either control or AURKB siRNA. ****P < 0.00005, unpaired t-test, (n=9-11).


( F ) Proliferation was measured in lung - resident fibroblasts isolated from stromal cultures of TGFα mice on Dox for 2 wks transiently transfected with control or AURKB siRNA stimulated with TGFα ( 20ng / mL ) for 24hrs . * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 9 - 11 ) .

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(F) Proliferation was measured in primary lung-resident fibroblasts isolated from stromal cultures of TGFα mice on Dox for 2 wks and transiently transfected with control or AURKB siRNA and stimulated with TGFα (20ng/mL) for 24hrs. ****P < 0.00005, 1-way ANOVA, (n=9-11).


( G ) of CCNA2 PLK1 transcripts in human IPF fibroblasts transiently transfected with control or AURKB siRNA for 72hrs . * * P < 0 . 005 , * * * P < 0 . 0005 , unpaired t - , ( n = 3 ) .

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(G) Quantification of CCNA2 and PLK1 transcripts in human IPF fibroblasts transiently transfected with control or AURKB siRNA for 72hrs. **P < 0.005, ***P < 0.0005, unpaired t-test, (n=3).


( H ) Lung sections from control TGFα mice fed with Dox food for 6 wks were stained for AURKB ( green ) αSMA ( red ) . were obtained at 40X magnification . Scale bar : 50µm . ( n = 4 ) .

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(H) Lung sections from control and TGFα mice fed with Dox food for 6 wks were stained for AURKB (green) and αSMA (red). Images were obtained at 40X magnification. Scale bar: 50µm. (n=4).


( I ) of apoptotic using Incucyte ZOOM ( Caspase - 3 / 7 - positive ) in lung - resident fibroblasts isolated from IPF TGFα mice on Dox for 6 wks treated with control or AURKB siRNA for 72h . * * P < 0 . 005 , * * * * P < 0 . 00005 , 2 - way ANOVA , ( n = 4 ) .

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(I) Quantification of apoptotic cells using Incucyte ZOOM (Caspase-3/7- positive cells) in lung-resident fibroblasts isolated from IPF and TGFα mice on Dox for 6 wks and treated with control or AURKB siRNA for 72h. **P < 0.005, ****P < 0.00005, 2-way ANOVA, (n=4).


( J ) of BAK , BAX FAS gene transcripts in human IPF lung resident fibroblasts treated with either control or AURKB siRNA for 72hrs . * * P < 0 . 005 , * * * P < 0 . 0005 , unpaired t - , ( n = 3 ) .

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(J) Quantification of BAK, BAX and FAS gene transcripts in human IPF lung resident fibroblasts treated with either control or AURKB siRNA for 72hrs. **P < 0.005, ***P < 0.0005, unpaired t-test, (n=3).


( K ) of Bak , Bax Fas gene transcripts in lung resident fibroblasts isolated from TGFα mice treated with either control or AURKB siRNA for 72hrs . * P < 0 . 005 , * * P < 0 . 005 , unpaired t - , ( n = 3 ) .

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(K) Quantification of Bak, Bax and Fas gene transcripts in lung resident fibroblasts isolated from TGFα mice and treated with either control or AURKB siRNA for 72hrs. *P < 0.005, **P < 0.005, unpaired t-test, (n=3).


( A ) Proliferation was assessed using BrdU incorporation in human IPF fibroblasts treated with indicated doses of barasertib for total of 48hrs . * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 9 - 11 ) .

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(A) Proliferation was assessed using BrdU incorporation assay in human IPF fibroblasts treated with indicated doses of barasertib for total of 48hrs. ****P < 0.00005, 1-way ANOVA, (n=9-11).


( B ) Proliferation was assessed using BrdU incorporation in fibroblasts isolated from TGFα mice lung treated with indicated doses of barasertib for total of 48hrs . * * * P < 0 . 0005 , 1 - way ANOVA , ( n = 9 - 11 ) .

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(B) Proliferation was assessed using BrdU incorporation assay in fibroblasts isolated from TGFα mice lung and treated with indicated doses of barasertib for total of 48hrs. ***P < 0.0005, 1-way ANOVA, (n=9-11).


( C ) lung resident fibroblasts isolated from TGFα mice on Dox for 4 wks were treated with vehicle or 5µM barasertib for 48hrs immunostained using PCNA antibody . were obtained at 40X magnification . Scale bar : 50µm . number of PCNA - positive total DAPI - positive were quantified using MetaMorph image software . Proliferation is indicated as of proliferating in total DAPI - positive . * * * * P < 0 . 00005 , unpaired t - , ( n = 3 - 4 ) .

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(C) Primary lung resident fibroblasts isolated from TGFα mice on Dox for 4 wks were treated with vehicle or 5µM barasertib for 48hrs and immunostained using PCNA antibody. Images were obtained at 40X magnification. Scale bar: 50µm. The number of PCNA-positive cells and total DAPI-positive cells were quantified using MetaMorph image analysis software. Proliferation is indicated as the percentage of proliferating cells in total DAPI-positive cells. ****P < 0.00005, unpaired t-test, (n=3-4).


( D ) Proliferation was measured in fibroblasts treated with TGFα ( 20ng / mL ) indicated doses of barasertib for 48hrs . . * * P < 0 . 005 , 1 - way ANOVA , ( n = 9 - 11 ) .

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(D) Proliferation was measured in primary fibroblasts treated with TGFα (20ng/mL) and indicated doses of barasertib for 48hrs.. **P<0.005, 1-way ANOVA, (n=9-11).


( E ) of apoptotic using Incucyte ZOOM ( Caspase - 3 / 7 - positive ) in resident fibroblasts isolated from IPF lung stromal cultures treated with vehicle or 5µM Barasertib . * * * * P < 0 . 00005 , 2 - way ANOVA , ( n = 4 ) .

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(E) Quantification of apoptotic cells using Incucyte ZOOM (Caspase-3/7- positive cells) in resident fibroblasts isolated from IPF lung stromal cultures and treated with vehicle or 5µM Barasertib. ****P < 0.00005, 2-way ANOVA, (n=4).


( B ) Representative of Masson ' s trichrome - stained lung sections from vehicle barasertib treated mice . were obtained at 10X magnification . Scale bar : 200μm .

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(B) Representative images of Masson's trichrome-stained lung sections from the vehicle and barasertib treated mice. Images were obtained at 10X magnification. Scale bar: 200μm.


( C ) of right lung of mice treated with vehicle or barasertib . * * * P < 0 . 0005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 8 - 10 mice / group ) .

(C) Quantification of right lung weight of mice treated with vehicle or barasertib. ***P< 0.0005, ****P < 0.00005, 1-way ANOVA, (n=8-10 mice/group).


( D ) of total lung hydroxyproline in mice treated with vehicle or barasertib . * P < 0 . 05 , * * * P < 0 . 0005 , 1 - way ANOVA , ( n = 8 - 10 mice / group ) .

(D) Quantification of total lung hydroxyproline levels in mice treated with vehicle or barasertib. *P< 0.05, ***P < 0.0005, 1-way ANOVA, (n=8-10 mice/group).


( E ) of Col1α , Col5α Fn1 gene transcripts in total lung of mice treated with vehicle or barasertib . * * * P < 0 . 0005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 8 mice / group ) .

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(E) Quantification of Col1α, Col5α and Fn1 gene transcripts in total lung of mice treated with vehicle or barasertib.***P< 0.0005, ****P < 0.00005, 1-way ANOVA, (n=8 mice/group).


( A ) Lung sections from vehicle barasertib treated mice were immunostained using Ki - 67 antibody . Representative were obtained at 20X magnification . Scale bar : 50μm .

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(A) Lung sections from vehicle and barasertib treated mice were immunostained using Ki-67 antibody. Representative images were obtained at 20X magnification. Scale bar: 50μm.


( B ) Immunoblot in lung lysates from vehicle barasertib treated mice using PCNA antibody . Gapdh is used as loading control . * * P < 0 . 005 , 1 - way ANOVA , ( n = 4 - 5 mice / group ) .

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(B) Immunoblot analysis in lung lysates from vehicle and barasertib treated mice using PCNA antibody. Gapdh is used as loading control. **P< 0.005, 1-way ANOVA, (n=4-5 mice/group).


( C ) of Aurkb , Plk1 CcnA2 gene transcripts in total lungs of mice treated with vehicle or barasertib . * * P < 0 . 005 , * * * P < 0 . 0005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 8 mice / group ) .

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(C) Quantification of Aurkb, Plk1 and CcnA2 gene transcripts in total lungs of mice treated with vehicle or barasertib. **P< 0.005, ***P<0.0005, ****P < 0.00005, 1-way ANOVA, (n=8 mice/group).


( B ) of right lung of mice treated with vehicle barasertib . * * P < 0 . 005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 9 - 10 mice / group ) .

(B) Quantification of right lung weight of mice treated with vehicle and barasertib. **P< 0.005, ****P < 0.00005, 1-way ANOVA, (n=9-10 mice/group).


( C ) Western blot in lung lysates from vehicle barasertib treated mice using Col1α Fn1 antibodies . Gapdh is used as loading control . * P < 0 . 05 , * * P < 0 . 005 , * * * P < 0 . 0005 , 1 - way ANOVA , ( n = 4 - 5 mice / group ) .

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(C) Western blot analysis in lung lysates from vehicle and barasertib treated mice using Col1α and Fn1 antibodies. Gapdh is used as loading control. *P< 0.05, **P<0.005, ***P < 0.0005, 1-way ANOVA, (n=4-5 mice/group).


( D ) Representative of Masson ' s trichrome stained lung section from mice treated with vehicle barasertib . were obtained at 10X magnification . Scale bar : 200μm .

(D) Representative images of Masson's trichrome stained lung section from mice treated with vehicle and barasertib. Images were obtained at 10X magnification. Scale bar: 200μm.


( E ) of lung mechanics in mice treated with vehicle barasertib . * P < 0 . 05 , * * P < 0 . 005 , * * * P < 0 . 0005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 9 - 10 mice / group ) .

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(E) Quantification of lung mechanics in mice treated with vehicle and barasertib. *P<0.05, **P< 0.005, ***P<0.0005, ****P < 0.00005, 1-way ANOVA, (n=9-10 mice/group).


( B ) Representative of Masson ' s trichrome stained lung section from mice treated with vehicle barasertib . were obtained at 10X magnification . Scale bar : 200μm .

(B) Representative images of Masson's trichrome stained lung section from mice treated with vehicle and barasertib. Images were obtained at 10X magnification. Scale bar: 200μm.


( C ) of total lung hydroxyproline in mice treated with vehicle barasertib . * P < 0 . 05 , * * * P < 0 . 0005 , 1 - way ANOVA , ( n = 7 - 8 mice / group ) .

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(C) Quantification of total lung hydroxyproline in mice treated with vehicle and barasertib. *P< 0.05, ***P < 0.0005, 1-way ANOVA, (n=7-8 mice/group).


( D ) of Col1α1 , Col3α , Col5α , Col14 , Col15 , αSma , Ccna2 , Fas gene transcripts in total lungs of mice treated with vehicle or barasertib . * P < 0 . 05 , * * P < 0 . 005 * * * P < 0 . 0005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 6 mice / group ) .

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(D) Quantification of Col1α1, Col3α, Col5α, Col14, Col15, αSma, Ccna2, and Fas gene transcripts in total lungs of mice treated with vehicle or barasertib. *P< 0.05, **P<0.005 ***P<0.0005, ****P < 0.00005, 1-way ANOVA, (n=6 mice/group).


( E ) of αSma in lung lysates from vehicle barasertib treated mice . Gapdh is used as loading control . Data are presented as mean ± . * * P < 0 . 005 , unpaired t - ( n = 5mice / group ) .

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(E) Quantification of αSma protein levels in lung lysates from vehicle and barasertib treated mice. Gapdh is used as loading control. Data are presented as mean ± SEM. **P<0.005, unpaired t-test (n=5mice /group).


A CD4 + CD127 - CD25hiFoxP3 + CTLA4 + Treg induction from PBMCs incubated with CVF , CLys CSN ( 10µg / ml ) after 4h , 24h , 48h , 72h ( plots graphs shown ) , 120h 168h .

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A CD4+CD127-CD25hiFoxP3+CTLA4+ Treg induction from PBMCs incubated with CVF, CLys and CSN (10µg/ml) after 4h, 24h, 48h, 72h (plots and graphs shown), 120h and 168h.


B Trafficking receptors ( CXCR3 , CCR4 , CCR6 CCR7 ) evaluation on induced Treg ( CD25hiCD127 - ) non - Treg ( CD127 + CD25 - ) .

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B Trafficking receptors (CXCR3, CCR4, CCR6 and CCR7) evaluation on induced Treg (CD25hiCD127-) and non-Treg cells (CD127+CD25-).


C Induced Treg proliferation with Ki67 Treg expression of IL - 10 IFNγ .

C Induced Treg proliferation with Ki67 staining and Treg expression of IL-10 and IFNγ.


A of pro - anti - inflammatory cytokines in PBMC culture supernatants with CVF , CLys CSN ( 10µg / ml ) after 72h determined using a LUMINEX - technology .

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A Levels of pro- and anti-inflammatory cytokines in PBMC culture supernatants with CVF, CLys and CSN (10µg/ml) after 72h determined using a LUMINEX-based technology.


B Composition of bioactive lipid mediators ( LMs ) , including prostaglandins , leukotrienes , lipoxins , thromboxanes , their precursors in culture with CVF , CLys CSN quantified by LC - MS / MS - profiling . Heat maps show relative amounts of LMs detected by LC - MS / MS in supernatants . Asterisks show statistical differences analyzed using two - way ANOVA followed by Turkey ' s multiple comparison .

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B Composition and levels of bioactive lipid mediators (LMs), including prostaglandins, leukotrienes, lipoxins, thromboxanes, and their precursors in culture with CVF, CLys and CSN quantified by LC-MS/MS-based profiling. Heat maps show relative amounts of LMs detected by LC-MS/MS in supernatants. Asterisks show statistical differences analyzed using two-way ANOVA followed by Turkey's multiple comparison test.


C Dynamic of PGE2 release correlated with Treg induction by CLys ( upper panel ) CSN ( lower panel ) using a Pearson correlation .

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C Dynamic of PGE2 release correlated with Treg induction by CLys (upper panel) and CSN (lower panel) using a Pearson correlation.


D Treg induction by CLys or CSN following PBMC pre - incubation with 10µM mPGES1 inhibitor 934 ( or DMSO as a control ) .

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D Treg induction by CLys or CSN following PBMC pre-incubation with 10µM mPGES1 inhibitor 934 (or DMSO as a control).


E Induction of Tregs with PGE2 analog ( DINP ) ( 5µM ) / or recombinant human IL - 10 ( rhIL - 10 ) ( 10ng / ml ) as compared to CLys .

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E Induction of Tregs with PGE2 analog (DINP) (5µM) and/or recombinant human IL-10 (rhIL-10) (10ng/ml) as compared to CLys.


A of bioactive LMs IL - 10 released from monocyte non - monocyte fractions .

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A Levels of bioactive LMs and IL-10 released from monocyte and non-monocyte fractions.


B Non - monocyte fractions were pre - incubated with IL - 10R inhibitor PGE2 receptor ( EP2 EP4 ) antagonists ( or DMSO as a control ) incubated with CLys ( left upper panel ) or CSN ( 10µg / ml ) ( left lower panel ) in presence or absence of monocytes Treg induction was analysed after 72h . On right panel , PGE2 receptors were first blocked on monocytes ( 2 ) or non - monocytes ( 3 ) or both ( 4 ) before addition of non - monocytes ( 2 ) or monocytes ( 3 ) incubation with CSN for 72h of Treg induction compared to control condition ( 1 ) .

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B Non-monocyte fractions were pre-incubated with IL-10R inhibitor and PGE2 receptor (EP2 and EP4) antagonists (or DMSO as a control) and incubated with CLys (left upper panel) or CSN (10µg/ml) (left lower panel) in presence or absence of monocytes and Treg induction was analysed after 72h. On the right panel, PGE2 receptors were first blocked on monocytes (2) or non-monocytes (3) or both (4) before addition of non-monocytes (2) or monocytes (3) and incubation with CSN for 72h and analysis of Treg induction compared to control condition (1).


C Treg induction by CLys CSN in presence of monocyte / non - monocyte fractions naïve CD4 + CD45RA + CD45RO - T .

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C Treg induction by CLys and CSN in presence of monocyte/non-monocyte fractions and naïve CD4+CD45RA+CD45RO- T cells.


D Monocyte phenotyping in presence of CVF , CLys CSN by FACS using CD68 , CD80 , CD163 CD206 surface expression .

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D Monocyte phenotyping in presence of CVF, CLys and CSN by FACS using CD68, CD80, CD163 and CD206 surface expression.


E of LMs PGE2 released by macrophages in presence of CVF , CLys CSN Additionally , macrophages were lysed gene expression of PTGS2 , PTGES , ALOX5 , ALOX15 MaR1 quantified by RT - PCR .

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E Levels of LMs and PGE2 released by macrophages in presence of CVF, CLys and CSN Additionally, macrophages were lysed and gene expression of PTGS2, PTGES, ALOX5, ALOX15 and MaR1 quantified by RT-PCR.


F Treg induction following naïve CD4 + CD45RA + CD45RO - T incubation with CLys - , CSN - monocyte conditioned media .

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F Treg induction following naïve CD4+CD45RA+CD45RO- T cells incubation with CLys-, CSN-monocyte conditioned media.


A Induction of Treg following PBMC treatment with either native or proteinase K - treated CLys or CSN ( 10µg / ml ) for 72h .

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A Induction of Treg following PBMC treatment with either native or proteinase K-treated CLys or CSN (10µg/ml) for 72h.


C PBMCs were treated with GDH inhibitor bithionol ( BT ) ( 20µM ) or IDH inhibitor IDH - 305 ( 50nM ) alone or in combination before incubation with CLys or CSN . of Treg was determined PGE2 IL - 10 were evaluated .

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C PBMCs were treated with GDH inhibitor bithionol (BT) (20µM) or IDH inhibitor IDH-305 (50nM) alone or in combination before incubation with CLys or CSN. Percentage of Treg was determined and PGE2 and IL-10 levels were evaluated.


D Treg induction from PBMC by CLys , CSN TsGDH ( 5µg / ml ) of PGE2 IL - 10 in culture supernatants .

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D Treg induction from PBMC by CLys, CSN and TsGDH (5µg/ml) and levels of PGE2 and IL-10 in culture supernatants.


E Treg induction from monocyte - naïve CD4 + - TsGDH culture of PGE2 IL - 10 in monocyte - CD4 + - TsGDH monocyte - TsGDH culture supernatants .

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E Treg induction from monocyte-naïve CD4+-TsGDH culture and levels of PGE2 and IL-10 in monocyte-CD4+-TsGDH and monocyte-TsGDH culture supernatants.


F Treg induction from PBMC by CLys , CSN rhGDH ( 10µg / ml ) .

F Treg induction from PBMC by CLys, CSN and rhGDH (10µg/ml).


( A ) In vitro Methyltransferase activity . Recombinant Nsp10 Nsp16 wt or Nsp16mut were combined with cap0 Adenosyl - L - methionine ( [ methyl - 3H ] SAM ) for 90 min at 37 ° C . Methylation of cap0 was quantified using a scintillation counter ( Beckmann ) . ( - ) control did not contain any recombinant . Results are plotted as mean of triplicate reactions with error bars representing SD . One out of three independent biological repeats is shown . Data information : Statistical was done using a one - way ANOVA followed by Tukey ' s multiple comparisons .

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(A) In vitro Methyltransferase activity. Recombinant proteins Nsp10 and Nsp16 wt or Nsp16mut were combined with cap0 RNA and Adenosyl-L-methionine ([methyl-3H] SAM) for 90 min at 37°C. Methylation of cap0 RNA was quantified using a scintillation counter (Beckmann).(-) control did not contain any recombinant protein. Results are plotted as mean of triplicate reactions with error bars representing SD. One out of three independent biological repeats is shown. Data information: Statistical analysis was done using a one-way ANOVA followed by Tukey's multiple comparisons test.


( B ) MTase activity of Nsp16 upon viral infection . mRNA was purified 24 h postinfection with SARS - CoV - 2 wt or Nsp16mut ( triplicate infections , biological replicates ) . Purified mRNA was incubated with 100U of recombinant MTase VACV VP39 ( NEB ) [ methyl - 3H ] SAM for 90 min at 37 ° C . Methylation of mRNA from SARS - CoV - 2 infected was quantified using a scintillation counter ( DPM3H ; Beckmann ) . ( - ) control did not contain . Results are plotted as mean of triplicate infections with error bars representing SD . One out of three independent experiments is shown ( n = 3 ) . Data information : Statistical was done using a one - way ANOVA followed by Tukey ' s multiple comparisons .

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(B) MTase activity of Nsp16 upon viral infection. Cellular mRNA was purified 24 h postinfection with SARS-CoV-2 wt or Nsp16mut (triplicate infections, biological replicates). Purified mRNA was incubated with 100U of the recombinant MTase VACV VP39 (NEB) and [methyl-3H] SAM for 90 min at 37 °C. Methylation of mRNA from SARS-CoV-2 infected cells was quantified using a scintillation counter (DPM3H; Beckmann). (-) control did not contain cellular RNA. Results are plotted as mean of triplicate infections with error bars representing SD. One out of three independent experiments is shown (n=3). Data information: Statistical analysis was done using a one-way ANOVA followed by Tukey's multiple comparisons test.


( A ) Calu - 3 were incubated with indicated amounts of type I IFN 24 h prior to infection with normalized amounts of SARS - CoV - 2 wt ( WT ) or Nsp16mut at 0 . 015 copies / . Viral load in supernatant was quantified at indicated time points by RT - qPCR targeting viral polymerase RdRp . Results are plotted as mean of triplicate infections with error bars representing SD ( biological replicates ) . One out of three independent experiments is shown ( n = 3 ) . Three days postinfection , expression of IFIT1 was analyzed by immunoblot to control for type I IFN treatment . Membranes were probed with antibodies targeting IFIT1 housekeeping gene HSP90 . Data information : Statistical was done using two - way ANOVA followed by Bonferroni ' s multiple comparisons . ns = not significant ( p > 0 . 05 ) .

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(A) Calu-3 cells were incubated with the indicated amounts of type I IFN 24 h prior to infection with normalized amounts of SARS-CoV-2 wt (WT) or Nsp16mut at 0.015 RNA copies/cell. Viral load in the supernatant was quantified at the indicated time points by RT-qPCR targeting viral polymerase RdRp. Results are plotted as mean of triplicate infections with error bars representing the SD (biological replicates). One out of three independent experiments is shown (n=3). Three days postinfection, protein expression of IFIT1 was analyzed by immunoblot to control for type I IFN treatment. Membranes were probed with antibodies targeting IFIT1 and the housekeeping gene HSP90. Data information: Statistical analysis was done using two-way ANOVA followed by Bonferroni's multiple comparisons test. ns = not significant (p>0.05).


( C ) Calu - 3 lacking MDA5 ( MDA5 KO ) , RIG - I ( RIG - I KO ) , or control KO ( Ctrl ) were seeded into 96well plates infected with increasing amounts of SARS - CoV - 2 wt ( WT ) or Nsp16mut in triplicates ( biological replicates ) . Type I IFN release was quantified after 48 h by incubating supernatants with HEK - Blue IFN - α / β reporter . SEAP activity in supernatant of reporter is shown as mean of quadruplicates with error bars representing SD . ( D ) Calu - 3 ( Ctrl ) were seeded into 96 well plates infected with 10 copies / of SARS - CoV - 2 wt ( WT ) or Nsp16mut in presence of increasing amounts of Remdesivir ( RDV ) . Type I IFN release was quantified after 48 h by IFN bioassay on HEK - Blue IFN - α / β is depicted as mean of quadruplicates with error bars representing SD . In parallel , infectivity of SARS - CoV - 2 wt Nsp16 in presence of increasing amounts of RDV was determined at indicated time points by RT - qPCR targeting viral polymerase RdRp transcripts . Data information : Statistical was performed using two - way ANOVA followed by Bonferroni ' s multiple comparisons . ns = not significant ( p > 0 . 05 ) .

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

(C) Calu-3 cells lacking MDA5 (MDA5 KO), RIG-I (RIG-I KO), or control KO cells (Ctrl) were seeded into 96well plates and infected with increasing amounts of SARS-CoV-2 wt (WT) or Nsp16mut in triplicates (biological replicates). Type I IFN release was quantified after 48 h by incubating supernatants with HEK-Blue IFN-α/β reporter cells. SEAP activity in the supernatant of the reporter cells is shown as mean of quadruplicates with error bars representing SD. (D) Calu-3 cells (Ctrl) were seeded into 96 well plates and infected with 10 RNA copies/cell of SARS-CoV-2 wt (WT) or Nsp16mut in the presence of increasing amounts of Remdesivir (RDV). Type I IFN release was quantified after 48 h by IFN bioassay on HEK-Blue IFN-α/β and is depicted as mean of quadruplicates with error bars representing SD. In parallel, infectivity of SARS-CoV-2 wt and Nsp16 in the presence of increasing amounts of RDV was determined at the indicated time points by RT-qPCR targeting viral polymerase RdRp transcripts. Data information: Statistical analysis was performed using two-way ANOVA followed by Bonferroni's multiple comparisons test. ns = not significant (p>0.05).


( B ) Three days after SARS - CoV - 2 Nsp16mut infection of A549 described in ( A ) , expression of IFIT1 viral nucleocapsid N was analyzed by immunoblot . Membranes were probed with antibodies targeting IFIT1 , SARS - CoV - 2 N , housekeeping gene HSP90 .

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(B) Three days after SARS-CoV-2 Nsp16mut infection of A549 cells described in (A), expression of IFIT1 and viral nucleocapsid protein N was analyzed by immunoblot. Membranes were probed with antibodies targeting IFIT1, SARS-CoV-2 N protein, and the housekeeping gene HSP90.


( D ) IFIT1 expression in Calu - 3 Ctrl , IFIT1 KO # 1 # 2 lines treated with type I IFN ( 24 h ; 100 U / ml ) were analyzed by immunoblot . Membranes were probed with antibodies targeting IFIT1 or housekeeping gene HSP90 .

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

(D) IFIT1 expression levels in Calu-3 Ctrl, IFIT1 KO #1 and #2 lines treated with type I IFN (24 h; 100 U/ml) were analyzed by immunoblot. Membranes were probed with antibodies targeting IFIT1 or the housekeeping gene HSP90.


( F ) Three days postinfection , A549 described in ( E ) were analyzed by immunoblot . Membranes were probed with antibodies targeting IFIT1 , SARS - CoV - 2 N , or housekeeping gene HSP90 .

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 B-MEASURED_VAR O O O O O O

(F) Three days postinfection, A549 cells described in (E) were analyzed by immunoblot. Membranes were probed with antibodies targeting IFIT1, SARS-CoV-2 N protein, or the housekeeping gene HSP90.


( H ) IFIT1 expression was analyzed in mock treated , SARS - CoV - 2 wt ( WT ) or Nsp16mut infected at 48 hpi by immunoblot . Membranes were probed with antibodies targeting IFIT1 housekeeping gene HSP90 .

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

(H) IFIT1 expression was analyzed in mock treated, SARS-CoV-2 wt (WT) or Nsp16mut infected cells at 48 hpi by immunoblot. Membranes were probed with antibodies targeting IFIT1 and the housekeeping gene HSP90.


. MEFs from δWD littermate control mice were incubated with HBSS , chloroquine or monensin for 2 hours as indicated . were fixed permeabilized stained for LC3 . Arrows indicate LC3 vacuoles where a ring of LC3 signal surrounds vacuoles ranging between 2 8 μm diameter . B . Numbers of LC3 LC3 vacuoles in each were quantified by microscopy . LC3 were identified using spot function software to locate ranging from 0 . 5 - 1 . 0mm diameter . LC3 vacuoles were identified by eye as rings of ranging between 2 8 μm diameter .

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

. MEFs from δWD and littermate control mice were incubated with HBSS, chloroquine or monensin for 2 hours as indicated. Cells were fixed and permeabilized and stained for LC3. Arrows indicate LC3 vacuoles where a ring of LC3 signal surrounds vacuoles ranging between 2 and 8 μm diameter. B. Numbers of fluorescent LC3 puncta and LC3 vacuoles in each cell were quantified by fluorescence microscopy. LC3 puncta were identified using spot function software to locate puncta ranging from 0.5-1.0mm diameter. LC3 vacuoles were identified by eye as rings of fluorescence ranging between 2 and 8 μm diameter.


MEFs from δWD littermate control mice were incubated with HBSS , chloroquine or monensin for 2 h as indicated lysates analysed by western blot for ATG16L1 , δWD LC3 as indicated . Control MEFS express α β isoforms of ATG16L1 at 70 kDa , MEFs from δWD mice express a truncated ATG16L1 at 30kDa . ( D ) shows level of conversion of LC3 to LC3II estimated by densitometry from a mean ( ± SD ) of three replicate blots .

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

MEFs from δWD and littermate control mice were incubated with HBSS, chloroquine or monensin for 2 h as indicated and cell lysates analysed by western blot for ATG16L1, δWD and LC3 as indicated. Control MEFS express α and β isoforms of ATG16L1 at 70 kDa, and MEFs from δWD mice express a truncated ATG16L1 at 30kDa. (D) shows the level of conversion of LC3 to LC3II estimated by densitometry from a mean (±SD) of three replicate blots.


E ) of phagosomes following engulfment of Zymosan ( red ) by bone marrow - derived macrophages ( BMDM ) from control δWD mice . indicates track used for profile to compare LC3 signal on limiting of phagosome with center . F shows examples of profile G , H . ( G ) shows of LC3 positive phagosomes per ( H ) shows profile of data from 10 , bars represent mean ± SD were compared by Student ' s t - ( * * * * P < 0 . 0001 ) .

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

E) fluorescence images of phagosomes following engulfment of Zymosan (red) by bone marrow-derived macrophages (BMDM) from control and δWD mice. White line indicates track used for line profile analysis to compare the LC3 signal on the limiting membrane of the phagosome with the center. F shows examples of line profile analysis G, H. (G) shows the percentage of LC3 positive phagosomes per cell and (H) shows line profile analysis of data from 10 cells, bars represent the mean ± SD and were compared by Student's t-test (****P<0.0001).


Littermate control δWD mice were challenged intranasally with IAV X31 ( 103 pfu ) . ( A ) Mice were monitored for loss at indicated time - points . ( n = 8 ) .

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

Littermate control and δWD mice were challenged intranasally with IAV strain X31 (103 pfu). (A) Mice were monitored for weight loss at indicated time-points. (n = 8).


Littermate control δWD mice were challenged intranasally with IAV X31 ( 103 pfu ) . ( B ) Survival was assessed at indicated time points ( n = 15 ) .

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

Littermate control and δWD mice were challenged intranasally with IAV strain X31 (103 pfu). (B) Survival was assessed at indicated time points (n = 15).


Littermate control δWD mice were challenged intranasally with IAV X31 ( 103 pfu ) . ( C ) IAV titre in lungs was determined by plaque at 5 d . p . i . ( n = 6 ) .

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

Littermate control and δWD mice were challenged intranasally with IAV strain X31 (103 pfu). (C) IAV titre in lungs was determined by plaque assay at 5 d.p.i. (n = 6).


Littermate control δWD mice were challenged intranasally with IAV X31 ( 103 pfu ) . ( D ) presence of IAV antigen was assessed by IH at 7 d . p . i . ( representative from n = 6 ) .

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Littermate control and δWD mice were challenged intranasally with IAV strain X31 (103 pfu). (D) The presence of IAV antigen was assessed by IH at 7 d.p.i. (representative images from n = 6).


Littermate control δWD mice ( n = 5 ) were challenged with IAV X31 ( 103 pfu ) . ( A ) At indicated time points , cytokine mRNA transcripts in lung tissue ( n = 5 ) were evaluated by qPCR .

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

Littermate control and δWD mice (n = 5) were challenged with IAV X31 (103 pfu). (A) At the indicated time points, cytokine mRNA transcripts in lung tissue (n = 5) were evaluated by qPCR.


Littermate control δWD mice ( n = 5 ) were challenged with IAV X31 ( 103 pfu ) . ( B ) Representative lung sections from ( n = 6 ) taken at 3 d . p . i . were stained by IH for neutrophils ( Ly6G ) or neutrophil extracellular traps ( NET ; anti - H3 ) . Sections at 7 d . p . i . were stained for macrophages ( Iba - 1 ) .

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Littermate control and δWD mice (n = 5) were challenged with IAV X31 (103 pfu). (B) Representative lung sections from animals (n = 6) taken at 3 d.p.i. were stained by IH for neutrophils (Ly6G) or neutrophil extracellular traps (NET; anti-H3). Sections at 7 d.p.i. were stained for macrophages (Iba-1).


Littermate control δWD mice ( n = 5 ) were challenged with IAV X31 ( 103 pfu ) . C ) BAL ( n = 5 ) was taken at 2 d . p . i evaluated by flow cytometry , with pre - gating on CD45 + . of neutrophils ( CD11b + , Ly6G + ) is shown ± were compared using Mann - Whitney U ( * P < 0 . 05 ) .

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Littermate control and δWD mice (n = 5) were challenged with IAV X31 (103 pfu). C) BAL (n = 5) was taken at 2 d.p.i and evaluated by flow cytometry, with pre-gating on CD45+. The percentage of neutrophils (CD11b+, Ly6G+) cells is shown ±SEM and were compared using Mann-Whitney U test (*P<0.05).


Littermate control δWD mice ( n = 5 ) were challenged with IAV X31 ( 103 pfu ) . ( D ) Single suspensions were prepared from lungs taken at 5 d . p . i evaluated by flow cytometry , with pre - gating on CD45 + . of macrophage / monocytes ( CD11b + , F4 / 80 + ) is shown ± were compared using Mann - Whitney U ( * P < 0 . 05 ) .

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

Littermate control and δWD mice (n = 5) were challenged with IAV X31 (103 pfu). (D) Single cell suspensions were prepared from lungs taken at 5 d.p.i and evaluated by flow cytometry, with pre-gating on CD45+. The percentage of macrophage/monocytes (CD11b+, F4/80+) cells is shown ±SEM and were compared using Mann-Whitney U test (*P<0.05).


( E ) δWD mice were treated with either anti - PCDA - 1 ( to deplete plasmacytoid DC ) or an isotype - matched control . Litter - mate control mice were used as comparator . loss was measured at indicated days p . i . ( n = 5 ) .

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

(E) δWD mice were treated with either anti-PCDA-1 (to deplete plasmacytoid DC) or an isotype-matched control. Litter-mate control mice were used as comparator. Weight loss was measured at the indicated days p.i. (n = 5).


A - D . Bone marrow from wild - type ( Atg16L1 + / + ) was used to reconstitute irradiated littermate control mice ( B6 WT → control [ ● ] ) or δWD mice ( B6 WT → δWD [ ○ ] ) . Bone marrow from δWD mice was used to reconstitute irradiated δWD mice ( δWD → δWD [ ▲ ] ) . After 12 , mice ( n = 5 per group ) were challenged with IAV X31 ( 103 pfu ) . ( A ) Mice were monitored for loss at indicated time - points .

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

A-D. Bone marrow from wild-type (Atg16L1+/+) was used to reconstitute irradiated littermate control mice (B6 WT → control [●] ) or δWD mice (B6 WT → δWD [○] ). Bone marrow from δWD mice was used to reconstitute irradiated δWD mice (δWD → δWD [▲] ). After 12 weeks, mice (n = 5 per group) were challenged with IAV X31 (103 pfu). (A) Mice were monitored for weight loss at indicated time-points.


Bone marrow from wild - type ( Atg16L1 + / + ) was used to reconstitute irradiated littermate control mice ( B6 WT → control [ ● ] ) or δWD mice ( B6 WT → δWD [ ○ ] ) . Bone marrow from δWD mice was used to reconstitute irradiated δWD mice ( δWD → δWD [ ▲ ] ) . After 12 , mice ( n = 5 per group ) were challenged with IAV X31 ( 103 pfu ) . ( B ) Survival was assessed at indicated time points .

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

Bone marrow from wild-type (Atg16L1+/+) was used to reconstitute irradiated littermate control mice (B6 WT → control [●] ) or δWD mice (B6 WT → δWD [○] ). Bone marrow from δWD mice was used to reconstitute irradiated δWD mice (δWD → δWD [▲] ). After 12 weeks, mice (n = 5 per group) were challenged with IAV X31 (103 pfu). (B) Survival was assessed at indicated time points.


Bone marrow from wild - type ( Atg16L1 + / + ) was used to reconstitute irradiated littermate control mice ( B6 WT → control [ ● ] ) or δWD mice ( B6 WT → δWD [ ○ ] ) . Bone marrow from δWD mice was used to reconstitute irradiated δWD mice ( δWD → δWD [ ▲ ] ) . After 12 , mice ( n = 5 per group ) were challenged with IAV X31 ( 103 pfu ) . ( C ) IAV titre in lungs was determined by plaque at 5 d . p . i . ( n = 6 ) .

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

Bone marrow from wild-type (Atg16L1+/+) was used to reconstitute irradiated littermate control mice (B6 WT → control [●] ) or δWD mice (B6 WT → δWD [○] ). Bone marrow from δWD mice was used to reconstitute irradiated δWD mice (δWD → δWD [▲] ). After 12 weeks, mice (n = 5 per group) were challenged with IAV X31 (103 pfu). (C) IAV titre in lungs was determined by plaque assay at 5 d.p.i. (n = 6).


Bone marrow from wild - type ( Atg16L1 + / + ) was used to reconstitute irradiated littermate control mice ( B6 WT → control [ ● ] ) or δWD mice ( B6 WT → δWD [ ○ ] ) . Bone marrow from δWD mice was used to reconstitute irradiated δWD mice ( δWD → δWD [ ▲ ] ) . After 12 , mice ( n = 5 per group ) were challenged with IAV X31 ( 103 pfu ) . ( D ) Lungs taken at 5 d . p . i . were analysed for neutrophils ( Ly6G ) , neutrophil extracellular traps ( NET ; anti - H3 ) macrophages ( Iba - 1 ) .

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

Bone marrow from wild-type (Atg16L1+/+) was used to reconstitute irradiated littermate control mice (B6 WT → control [●] ) or δWD mice (B6 WT → δWD [○] ). Bone marrow from δWD mice was used to reconstitute irradiated δWD mice (δWD → δWD [▲] ). After 12 weeks, mice (n = 5 per group) were challenged with IAV X31 (103 pfu). (D) Lungs taken at 5 d.p.i. were analysed for neutrophils (Ly6G), neutrophil extracellular traps (NET; anti-H3) and macrophages (Iba-1) .


δWDphag mice lack non - canonical autophagy in myeloid ( LysMcre ) Offspring negative for LysMcre were used as littermate controls . Mice ( n = 6 per group ) were challenged intranasally with IAV X31 ( 103 pfu ) . ( A ) Mice were monitored for loss at indicated time - points .

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

δWDphag mice lack non-canonical autophagy in myeloid (LysMcre) cells Offspring negative for LysMcre were used as littermate controls. Mice (n = 6 per group) were challenged intranasally with IAV X31 (103 pfu). (A) Mice were monitored for weight loss at indicated time-points.


δWDphag mice lack non - canonical autophagy in myeloid ( LysMcre ) Offspring negative for LysMcre were used as littermate controls . Mice ( n = 6 per group ) were challenged intranasally with IAV X31 ( 103 pfu ) . ( B ) IAV titre in lungs was determined by plaque at 5 d . p . i . ( n = 6 ) .

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

δWDphag mice lack non-canonical autophagy in myeloid (LysMcre) cells Offspring negative for LysMcre were used as littermate controls. Mice (n = 6 per group) were challenged intranasally with IAV X31 (103 pfu). (B) IAV titre in lungs was determined by plaque assay at 5 d.p.i. (n = 6).


δWDphag mice lack non - canonical autophagy in myeloid ( LysMcre ) Offspring negative for LysMcre were used as littermate controls . Mice ( n = 6 per group ) were challenged intranasally with IAV X31 ( 103 pfu ) . ( C ) IL - 1β mRNA transcripts in lung at 5 d . p . i . were determined by qPCR .

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

δWDphag mice lack non-canonical autophagy in myeloid (LysMcre) cells Offspring negative for LysMcre were used as littermate controls. Mice (n = 6 per group) were challenged intranasally with IAV X31 (103 pfu). (C) IL-1β mRNA transcripts in lung at 5 d.p.i. were determined by qPCR.


δWDphag mice lack non - canonical autophagy in myeloid ( LysMcre ) Offspring negative for LysMcre were used as littermate controls . Mice ( n = 6 per group ) were challenged intranasally with IAV X31 ( 103 pfu ) . ( D ) Precision - cut lung slices from control δWD mice were infected with IAV . titres were determined at indicated time points .

δWDphag mice lack non-canonical autophagy in myeloid (LysMcre) cells Offspring negative for LysMcre were used as littermate controls. Mice (n = 6 per group) were challenged intranasally with IAV X31 (103 pfu). (D) Precision-cut lung slices from control and δWD mice were infected with IAV. Virus titres were determined at indicated time points.


A ) . WD domain of ATG16L1 reduces IAV replication in MEFs . were infected with IAV titres were estimated at 8 hours post infection by plaque .

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

A). The WD domain of ATG16L1 reduces IAV replication in MEFs. Cells were infected with IAV and virus titres were estimated at 8 hours post infection by plaque assay.


B ) . IAV binding is similar between control δWD MEFs . were bound with IAV X31 labeled with AF488 on ice for 1 h , fixed immediately . were stained with anti - HA ( H3SKE , red ) without permeabilisation to visualise extracellular IAV particles , counter stained with Hoechst ( nuclei ) WGA - AF647 to label . were imaged by automated confocal microscopy using a 40x objective , maximum projection were analysed to quantify number of bound IAV particles per . results means ( horizontal ) of n = 3 independent experiments ( 50 - 100 per experiment ) are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). IAV binding is similar between control and δWD MEFs. Cells were bound with IAV X31 labeled with AF488 on ice for 1 h, and fixed immediately. The cells were stained with anti-HA (H3SKE, red) without permeabilisation to visualise extracellular IAV particles, counter stained with Hoechst (nuclei) and WGA-AF647 to label the cell membrane. Cells were imaged by automated confocal microscopy using a 40x objective, and the maximum intensity projection images were analysed to quantify the number of bound IAV particles per cell. The results and means (horizontal line) of n = 3 independent experiments (50-100 cells per experiment) are shown.


C ) IAV infection following acid bypass is comparable between control δWD MEFs . IAV was bound on ice for 1 h , warmed at either neutral pH ( 6 . 8 ) or low pH ( pH 5 . 0 ) for 2 min , followed by incubation in STOP medium containing NH4Cl for 18 hours . were fixed stained for NP Hoechst , imaged by automated confocal microscopy maximum projection analysed to quantify viral infectivity . results from N = 3 independent experiments ( 5 , 000 - 10 , 000 quantified per experiment ) , means are shown . Scale bar ; 500 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) IAV infection following acid bypass is comparable between control and δWD MEFs. IAV was bound on ice for 1 h, and warmed at either neutral pH (6.8) or low pH (pH 5.0) for 2 min, followed by incubation in STOP medium containing NH4Cl for 18 hours. Cells were fixed and stained for NP and Hoechst, imaged by automated confocal microscopy and maximum intensity projection images analysed to quantify viral infectivity. The results from N=3 independent experiments (5,000-10,000 cells quantified per experiment), and the means are shown. Scale bar; 500 µm.


D ) . IAV fusion with endosomes is increased in δWD MEFs . were infected with dual labeled ( R18 / SP - DiOC18 ) IAV for 1 h , fixed , counterstained with Hoechst ( nuclei blue ) WGA - AF647 ( perimeter in ) . were imaged by automated confocal microscopy number of fusion events per ( represented by number of SP - DiOC18 ) was quantified . Fused non - fused viral particles are shown as green red spots respectively . results means ( horizontal lines ) of n = 3 independent experiments ( 30 - 60 per experiment ) are shown . Representative overlayed with boundary segmented from WGA are shown on right .

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D). IAV fusion with endosomes is increased in δWD MEFs. Cells were infected with dual labeled (R18/SP-DiOC18) IAV for 1 h, fixed, counterstained with Hoechst (nuclei blue) and WGA-AF647 (cell perimeter in white). Cells were imaged by automated confocal microscopy and the number of fusion events per cell (represented by number of SP-DiOC18 puncta) was quantified. Fused and non-fused viral particles are shown as green and red spots respectively. The results and means (horizontal lines) of n = 3 independent experiments (30-60 cells per experiment) are shown. Representative cells overlayed with the cell boundary segmented from the WGA staining are shown on the right.