( I ) Vascularization revealed by H & E ( n = 7 rats in each group ) . Data information : Values are expressed as mean ± . Statistical : t - ( * P < 0 . 05 vs . respective controls or between indicated groups Scale bar : 20 µm ( I ) .

(I) Vascularization revealed by H&E staining (n = 7 rats in each group). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups Scale bar: 20 µm (I).


( J ) Survival rate of 3 groups ( n = 15 rats in each group ) . Data information : Values are expressed as mean ± . Statistical : t - ( * P < 0 . 05 vs . respective controls or between indicated

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(J) Survival rate of 3 groups (n = 15 rats in each group). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups


A - C 10x106 ZsGreen - luciferase - expressing U - 2932 , RC - K8 RIVA were intravenously injected into six - week - old ( M ) ( F ) MISTRG mice monitored weekly using IVIS for at least four up to six . color scales on right indicate radiance , i . e . sum of photons per second from each pixel inside ROI / number of pixels ( photons / sec / cm2 / sr ) .

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A-C 10x106 ZsGreen- and luciferase-expressing U-2932, RC-K8 and RIVA cells were intravenously injected into six-week-old male (M) and female (F) MISTRG mice and monitored weekly using IVIS for at least four and up to six weeks. The color scales on the right indicate the radiance, i.e. the sum of the photons per second from each pixel inside the ROI/number of pixels (photons/sec/cm2/sr).


10x106 ZsGreen - luciferase - expressing U - 2932 , RC - K8 RIVA were intravenously injected into six - week - old ( M ) ( F ) MISTRG mice monitored weekly using IVIS for at least four up to six . D frequency of involvement of indicated tissues is shown for one cohort of mice is representative of two independently analyzed cohorts per .

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10x106 ZsGreen- and luciferase-expressing U-2932, RC-K8 and RIVA cells were intravenously injected into six-week-old male (M) and female (F) MISTRG mice and monitored weekly using IVIS for at least four and up to six weeks. D The frequency of involvement of the indicated tissues is shown for one cohort of mice and is representative of two independently analyzed cohorts per cell line.


E Gating strategy for identification of U - 2932 residing in spleen bone marrow , among leukocytes in these organs . were stained for human mouse CD45 . Very few hCD45 + U - 2932 were detectable in spleen , whereas their frequencies were much higher in bone marrow ( BM ) . In BM , > 90 % of U - 2932 were positive for ZsGreen . green curve shows U - 2932 recovered from BM , overlayed with unlabelled U - 2932 grown in vitro ( grey curve ) .

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E Gating strategy for the identification of U-2932 cells residing in spleen and bone marrow, among all leukocytes in these organs. Cells were stained for human and mouse CD45. Very few hCD45+ U-2932 cells were detectable in the spleen, whereas their frequencies were much higher in the bone marrow (BM). In BM, >90% of U-2932 cells were positive for ZsGreen. The green curve shows U-2932 cells recovered from BM, overlayed with unlabelled U-2932 cells grown in vitro (grey curve).


F , G Frequencies of human lymphoma , as judged by hCD45 expression , among ( human mouse , h + m ) CD45 + leukocytes in spleen ( S ) , femoral bone marrow ( BM ) jaw bone marrow ( JB ) at study endpoint of mice shown in D ( four post injection for U - 2932 , five p . i . for RIVA six p . i . for RC - K8 ) .

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F, G Frequencies of human lymphoma cells, as judged by hCD45 expression, among all (human and mouse, h+m) CD45+ leukocytes in the spleen (S), femoral bone marrow (BM) and jaw bone marrow (JB) at the study endpoint of the mice shown in D (four weeks post injection for U-2932, five weeks p.i. for RIVA and six weeks p.i. for RC-K8).


MISTRG mice were reconstituted or not at birth with ~ 200 . 000 human CD34 + cord blood HSPCs , subcutaneously transplanted with 10x106 U - 2932 at six of . reconstitution efficiency ( frequency of human among human mouse CD45 + leukocytes ) is shown in

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MISTRG mice were reconstituted or not at birth with ~200.000 human CD34+ cord blood HSPCs, and subcutaneously transplanted with 10x106 U-2932 cells at six weeks of age. The reconstitution efficiency (frequency of human among all human and mouse CD45+ leukocytes) is shown in A


MISTRG mice were reconstituted or not at birth with ~ 200 . 000 human CD34 + cord blood HSPCs , subcutaneously transplanted with 10x106 U - 2932 at six of . average human leukocyte composition in bone marrow of mice is shown in B .

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

MISTRG mice were reconstituted or not at birth with ~200.000 human CD34+ cord blood HSPCs, and subcutaneously transplanted with 10x106 U-2932 cells at six weeks of age. the average human leukocyte composition in the bone marrow of the mice is shown in B.


MISTRG mice were reconstituted or not at birth with ~ 200 . 000 human CD34 + cord blood HSPCs , subcutaneously transplanted with 10x106 U - 2932 at six of . volumes over time are presented in

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

MISTRG mice were reconstituted or not at birth with ~200.000 human CD34+ cord blood HSPCs, and subcutaneously transplanted with 10x106 U-2932 cells at six weeks of age. Tumor volumes over time are presented in C


MISTRG mice were reconstituted or not at birth with ~ 200 . 000 human CD34 + cord blood HSPCs , subcutaneously transplanted with 10x106 U - 2932 at six of . weights volumes at study endpoint in D , where each data point represents one .

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MISTRG mice were reconstituted or not at birth with ~200.000 human CD34+ cord blood HSPCs, and subcutaneously transplanted with 10x106 U-2932 cells at six weeks of age. tumor weights and volumes at the study endpoint in D, where each data point represents one tumor.


MISTRG mice were reconstituted or not at birth with ~ 200 . 000 human CD34 + cord blood HSPCs , subcutaneously transplanted with 10x106 U - 2932 at six of . reconstitution level directly correlates with at endpoint ( E ) .

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

MISTRG mice were reconstituted or not at birth with ~200.000 human CD34+ cord blood HSPCs, and subcutaneously transplanted with 10x106 U-2932 cells at six weeks of age. The reconstitution level directly correlates with the tumor weight at the endpoint (E).


MISTRG mice were reconstituted or not at birth with human CD34 + cord blood HSPCs , intravenously injected with 10x106 U - 2932 or RC - K8 at six of . lymphoma burden at study endpoint was quantified in spleen ( F ) bone marrow ( G ) by for hCD45 ( as well as hCD19 hCD20 for U - 2932 RC - K8 , respectively )

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MISTRG mice were reconstituted or not at birth with human CD34+ cord blood HSPCs, and intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age. The lymphoma burden at the study endpoint was quantified in the spleen (F) and bone marrow (G) by staining for hCD45 (as well as hCD19 and hCD20 for U-2932 and RC-K8, respectively)


MISTRG mice were reconstituted or not at birth with human CD34 + cord blood HSPCs , intravenously injected with RC - K8 at six of . lymphoma burden at study endpoint was quantified by ZsGreen expression in case of RC - K8 , also over time by IVIS ( H ) .

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

MISTRG mice were reconstituted or not at birth with human CD34+ cord blood HSPCs, and intravenously injected with RC-K8 cells at six weeks of age. The lymphoma burden at the study endpoint was quantified by ZsGreen expression and in the case of RC-K8 cells, also over time by IVIS (H).


MISTRG mice were reconstituted or not at birth with human CD34 + cord blood HSPCs , intravenously injected with 10x106 U - 2932 or RC - K8 at six of . frequency of tissue involvement was assessed by ex vivo IVIS macroscopic evaluation ( I ) ,

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

MISTRG mice were reconstituted or not at birth with human CD34+ cord blood HSPCs, and intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age. The frequency of tissue involvement was assessed by ex vivo IVIS and macroscopic evaluation (I),


MISTRG mice were reconstituted or not at birth with human CD34 + cord blood HSPCs , intravenously injected with 10x106 U - 2932 or RC - K8 at six of . occurrence of clinical symptoms ( loss , hind leg paralysis ) was recorded for each mouse ( J ) .

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MISTRG mice were reconstituted or not at birth with human CD34+ cord blood HSPCs, and intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age. the occurrence of clinical symptoms (weight loss, hind leg paralysis) was recorded for each mouse (J).


MISTRG MISTRG6 mice were intravenously injected with 10x106 U - 2932 or RC - K8 at six of monitored weekly by IVIS until study endpoint ( A ) .

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MISTRG and MISTRG6 mice were intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age and monitored weekly by IVIS until the study endpoint (A).


MISTRG MISTRG6 mice were intravenously injected with 10x106 U - 2932 or RC - K8 at six of monitored weekly by IVIS until study endpoint ( A ) . lymphoma burden at study endpoint ( six p . i . for RC - K8 four p . i . for U - 2932 ) was quantified in spleen ( B ) bone marrow ( C ) by flow cytometric for hCD45 by ZsGreen expression .

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MISTRG and MISTRG6 mice were intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age and monitored weekly by IVIS until the study endpoint (A). The lymphoma burden at the study endpoint (six weeks p.i. for RC-K8 and four weeks p.i. for U-2932) was quantified in the spleen (B) and bone marrow (C) by flow cytometric staining for hCD45 and by ZsGreen expression.


MISTRG MISTRG6 mice were intravenously injected with 10x106 U - 2932 or RC - K8 at six of monitored weekly by IVIS until study endpoint involvement of indicated tissues as assessed by ex vivo IVIS is shown in D ( solid bars , MISTRG ; hatched bars , MISTRG6 ) ,

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MISTRG and MISTRG6 mice were intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age and monitored weekly by IVIS until the study endpoint The involvement of the indicated tissues as assessed by ex vivo IVIS is shown in D (solid bars, MISTRG; hatched bars, MISTRG6),


MISTRG MISTRG6 mice were intravenously injected with 10x106 U - 2932 or RC - K8 at six of monitored weekly by IVIS until study endpoint mice that have developed clinical symptoms ( paralysis of hind legs ) are plotted in E .

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MISTRG and MISTRG6 mice were intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age and monitored weekly by IVIS until the study endpoint mice that have developed clinical symptoms (paralysis of hind legs) are plotted in E.


F Surface expression of IL - 6 receptor α - chain ( CD126 ) as assessed by flow cytometry of U - 2932 RC - K8 . Data is representative of two stainings .

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F Surface expression of the IL-6 receptor α-chain (CD126) as assessed by flow cytometry of U-2932 and RC-K8 cells. Data is representative of two stainings.


1x106 DLBCL obtained by passaging through a MISTRG6 mouse were intravenously injected into MISTRG or MISTRG6 recipients . spleen weights ( B , representative pictures in A ) are

1x106 primary DLBCL cells obtained by passaging through a MISTRG6 mouse were intravenously injected into MISTRG or MISTRG6 recipients. The spleen weights (B, representative pictures in A) are shown


1x106 DLBCL obtained by passaging through a MISTRG6 mouse were intravenously injected into MISTRG or MISTRG6 recipients . with representative H & E - stained spleen ( MISTRG6 ) lymph node ( ) sections in

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1x106 primary DLBCL cells obtained by passaging through a MISTRG6 mouse were intravenously injected into MISTRG or MISTRG6 recipients. with representative H&E-stained spleen (MISTRG6) and lymph node (patient) sections in C


1x106 DLBCL obtained by passaging through a MISTRG6 mouse were intravenously injected into MISTRG or MISTRG6 recipients . lymphoma burden at study endpoint eight p . i . , as quantified in spleen bone marrow in D Representative flow cytometry plots are shown in D for bone marrow spleen ( MISTRG , MISTRG6 ) .

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1x106 primary DLBCL cells obtained by passaging through a MISTRG6 mouse were intravenously injected into MISTRG or MISTRG6 recipients. the lymphoma burden at the study endpoint eight weeks p.i., as quantified in the spleen and bone marrow in D Representative flow cytometry plots are shown in D for bone marrow and spleen (MISTRG, MISTRG6).


x106 DLBCL obtained by passaging through a MISTRG6 mouse were intravenously injected into MISTRG or MISTRG6 recipients . lymphoma burden at study endpoint eight p . i . , as quantified in spleen bone

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x106 primary DLBCL cells obtained by passaging through a MISTRG6 mouse were intravenously injected into MISTRG or MISTRG6 recipients. the lymphoma burden at the study endpoint eight weeks p.i., as quantified in the spleen and bone marrow


MISTRG mice were reconstituted or not at birth with human CD34 + cord blood HSPC , intravenously injected with 1x106 DLBCL at six of . lymphoma burden at study endpoint was quantified in bone marrow of reconstituted un - reconstituted by flow cytometric for indicated surface markers .

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MISTRG mice were reconstituted or not at birth with human CD34+ cord blood HSPC, and intravenously injected with 1x106 primary DLBCL cells at six weeks of age. The lymphoma burden at the study endpoint was quantified in the bone marrow of reconstituted and un-reconstituted animals by flow cytometric staining for the indicated surface markers.


lymphoma harvested at study endpoint express IL - 6R α chain ( CD126 , as assessed by flow cytometry , G ) are positive for phosphorylated STAT3 ( G ) .

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Primary lymphoma cells harvested at the study endpoint express the IL-6R α chain (CD126, as assessed by flow cytometry, G) and are positive for phosphorylated STAT3 (G).


lymphoma harvested signaling chain gp130 ( as assessed by immunohistochemistry , H )

Primary lymphoma cells harvested and the signaling chain gp130 (as assessed by immunohistochemistry, H)


expression of IL - 6Rα ( CD126 ) signaling chains ( gp130 ) was assessed by flow cytometry ( A ) on a panel of DLBCL lines . ABC - GCB - DLBCL lines are color - coded in red green ; RC - K8 are depicted in grey , as their subtype is a matter of

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The expression of the IL-6Rα (CD126) and signaling chains (gp130) was assessed by flow cytometry (A) on a panel of DLBCL cell lines. ABC- and GCB-DLBCL cell lines are color-coded in red and green; RC-K8 are depicted in grey, as their subtype is a matter of debate


expression of IL - 6Rα ( CD126 ) signaling chains ( gp130 ) was assessed by SYBR or Taqman qRT - PCR ( B , C ) respectively , on a panel of DLBCL lines . ABC - GCB - DLBCL lines are color - coded in red green ; RC - K8 are depicted in grey , as their subtype is a matter of

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The expression of the IL-6Rα (CD126) and signaling chains (gp130) was assessed by SYBR or Taqman qRT-PCR (B, C) respectively, on a panel of DLBCL cell lines. ABC- and GCB-DLBCL cell lines are color-coded in red and green; RC-K8 are depicted in grey, as their subtype is a matter of debate


D E . expression of gp130 in 114 biopsies of DLBCL was assessed by immunohistochemistry , correlated with ABC / non - GCB vs . GCB subtype , as assessed via Hans classification . Representative are shown in D , alongside of gp130 + cases among samples , among two subtypes , in E . Scale bar , 100 μm .

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D and E. The expression of gp130 in 114 tumor biopsies of DLBCL patients was assessed by immunohistochemistry, and correlated with the ABC/non-GCB vs. GCB subtype, as assessed via Hans classification. Representative micrographs are shown in D, alongside the quantification of gp130+ cases among all samples, and among the two subtypes, in E. Scale bar, 100 μm.


A IL - 6 secretion of indicated lines ( ABC - DLBCL in red , GCB - DLBCL in green ) material , as quantified by ELISA normalized to number ( shown per 1000 ) . n . d . , not detectable .

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A IL-6 secretion of the indicated cell lines (ABC-DLBCL in red, GCB-DLBCL in green) and primary patient material, as quantified by ELISA and normalized to cell number (shown per 1000 cells). n.d., not detectable.


STAT3 phosphorylation as assessed by Western blotting ( B , D ) of indicated ABC - DLBCL GCB - DLBCL lines , with without 1h of exposure to 50ng / ml hIL - 6 ( B , D ) . STAT3 expression are shown as Western blot control .

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STAT3 phosphorylation as assessed by Western blotting (B, D) of the indicated ABC-DLBCL and GCB-DLBCL cell lines, with and without 1h of exposure to 50ng/ml hIL-6 (B,D). STAT3 expression levels are shown as Western blot control.


STAT3 phosphorylation as assessed by flow cytometry ( C ) of indicated ABC - DLBCL GCB - DLBCL lines , Histograms in C represent STAT3 phosphorylation in absence of exogenously added IL - 6 .

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STAT3 phosphorylation as assessed by flow cytometry (C) of the indicated ABC-DLBCL and GCB-DLBCL cell lines, Histograms in C represent STAT3 phosphorylation in the absence of exogenously added IL-6.


E STAT3 phosphorylation as assessed by Western blotting of CD126 - proficient ( WT ) or - deficient ( ko ) RC - K8 , with without 1h of exposure to 50ng / ml hIL - 6 .

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E STAT3 phosphorylation as assessed by Western blotting of CD126-proficient (WT) or -deficient (ko) RC-K8 cells, with and without 1h of exposure to 50ng/ml hIL-6.


F Kinetics of STAT3 phosphorylation as assessed by Western blotting , of IL - 6 - expressing SOCS1wt SOCS1mut lines OCI - Ly3 SU - DHL - 2 , under 1 , 6 24h exposure to an IL - 6 - neutralizing antibody ( α - IL - 6 , anti - IL - 6 neutralizing antibody ) .

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F Kinetics of STAT3 phosphorylation as assessed by Western blotting, of the IL-6-expressing SOCS1wt and SOCS1mut cell lines OCI-Ly3 and SU-DHL-2, under 1, 6 and 24h exposure to an IL-6-neutralizing antibody (α-IL-6, anti-IL-6 neutralizing antibody).


STAT3 phosphorylation gp130 expression of two representative cases ( G ) assessed by immunohistochemistry . specific pSTAT3 gp130 signal is seen as brown ( DAB ) , counterstaining was performed using hematoxylin ( blue nuclei ) . STAT3 phosphorylation is more common in biopsies with gp130 expression ; p - value was calculated by chi - square . Scale bar , 100 μm .

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STAT3 phosphorylation and gp130 expression of two representative cases (G) assessed by immunohistochemistry. The specific pSTAT3 and gp130 signal is seen as brown (DAB staining), and counterstaining was performed using hematoxylin (blue nuclei). STAT3 phosphorylation is more common in biopsies with gp130 expression; the p-value was calculated by chi-square test. Scale bar, 100 μm.


STAT3 phosphorylation gp130 expression of two representative cases ( G ) summarized for entire cohort of 114 ( H ) , as assessed by immunohistochemistry . specific pSTAT3 gp130 signal is seen as brown ( DAB ) , counterstaining was performed using hematoxylin ( blue nuclei ) . STAT3 phosphorylation is more common in biopsies with gp130

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STAT3 phosphorylation and gp130 expression of two representative cases (G) and summarized for the entire cohort of 114 patients (H), as assessed by immunohistochemistry. The specific pSTAT3 and gp130 signal is seen as brown (DAB staining), and counterstaining was performed using hematoxylin (blue nuclei). STAT3 phosphorylation is more common in biopsies with gp130 expression


Disease - specific mortality ( A ) of cohorts of 9 - 12 mice each ( n indicated in figure ) of indicated genotypes .

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Disease-specific mortality (A) of cohorts of 9-12 mice each (n indicated in figure) of the indicated genotypes.


lymphoma burden as determined by lymph node ( B ) , of cohorts of 9 - 12 mice each ( n indicated in figure ) of indicated genotypes .

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lymphoma burden as determined by lymph node weight (B), of cohorts of 9-12 mice each (n indicated in figure) of the indicated genotypes.


Mice of indicated genotypes were immunized once with sheep red blood assessed 10 days later by flow cytometry of splenocytes for their frequencies of GC B - ( C ) , centroblasts ( D ) centrocytes ( E ) .

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Mice of the indicated genotypes were immunized once with sheep red blood cells and assessed 10 days later by flow cytometry of splenocytes for their frequencies of GC B-cells (C), centroblasts (D) and centrocytes (E).


Spleen sections were further stained for Ki67 to reveal GCs ( F ) , which were quantified both in number ( G ) ( H ) . Arrows in F point to GCs ; scale bar , 1000 μm .

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Spleen sections were further stained for Ki67 to reveal GCs (F), which were quantified both in number (G) and area (H). Arrows in F point to GCs; scale bar, 1000 μm.


Frequency of STAT3 phosphorylation ( A ) , of DLBCL stratified according to subtype disease stage Phospho - STAT3 positivity was defined as expression in ≥ 17 % of more common found in ABC - DLBCL tumors , in more advanced ( stage 3 4 , relative to stage 1 2 ) disease ( A ) .

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Frequency of STAT3 phosphorylation (A), of DLBCL patients stratified according to subtype and disease stage Phospho-STAT3 positivity was defined as expression in ≥17% of tumor cells and more common found in ABC-DLBCL tumors, and in more advanced (stage 3 and 4, relative to stage 1 and 2) disease (A).


overall as well as disease - specific survival probability ( B ) of DLBCL pSTAT3 positivity was a strong negative prognostic factor in entire cohort ( B )

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overall as well as disease-specific survival probability (B) of DLBCL patients pSTAT3 positivity was a strong negative prognostic factor in the entire cohort (B)


overall mortality of DLBCL stratified according to subtype disease stage pSTAT3 positivity was associated with higher overall - as well as disease - specific mortality in both ABC - GCB - DLBCL

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overall mortality of DLBCL patients stratified according to subtype and disease stage pSTAT3 positivity was associated with higher overall- as well as disease-specific mortality in both ABC- and GCB-DLBCL subtypes


disease - specific mortality of DLBCL stratified according to subtype disease stage pSTAT3 positivity was associated with higher overall - as well as disease - specific mortality in both ABC - GCB - DLBCL

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

disease-specific mortality of DLBCL patients stratified according to subtype and disease stage pSTAT3 positivity was associated with higher overall- as well as disease-specific mortality in both ABC- and GCB-DLBCL subtypes


Effect of IL - 6R neutralization by twice - weekly injection of 250 μg / dose of tocilizumab or an isotype control antibody , starting two post injection , on ( E F ) spleen weights of MISTRG6 mice transplanted with 1x106 DLBCL ( expanded by one serial passage through MISTRG6 mice ) ,

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Effect of IL-6R neutralization by twice-weekly injection of 250 μg/dose of tocilizumab or an isotype control antibody, starting two weeks post tumor cell injection, on (E and F) the spleen weights of MISTRG6 mice transplanted with 1x106 primary DLBCL cells (expanded by one serial passage through MISTRG6 mice),


Effect of IL - 6R neutralization by twice - weekly injection of 250 μg / dose of tocilizumab or an isotype control antibody , starting two post injection , Representative microscopy in F show STAT3 phosphorylation in spleen - colonizing exposed to either tocilizumab or isotype control antibody ( in brown , counterstained in blue with hematoxylin ) , an H & E - stained section from same mouse . TCZ , tocilizumab . Scale bars , 100 μm .

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Effect of IL-6R neutralization by twice-weekly injection of 250 μg/dose of tocilizumab or an isotype control antibody, starting two weeks post tumor cell injection, Representative microscopy images in F show STAT3 phosphorylation in spleen-colonizing primary cells exposed to either tocilizumab or isotype control antibody (in brown, counterstained in blue with hematoxylin), and an H&E-stained section from the same mouse. TCZ, tocilizumab. Scale bars, 100 μm.


Effect of IL - 6R neutralization by twice - weekly injection of 250 μg / dose of tocilizumab or an isotype control antibody , starting two post injection on bone marrow lymphoma burden of OCI - Ly3 - transplanted MISTRG mice ( G ) assessed at around four post injection .

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Effect of IL-6R neutralization by twice-weekly injection of 250 μg/dose of tocilizumab or an isotype control antibody, starting two weeks post tumor cell injection on the bone marrow lymphoma burden of OCI-Ly3-transplanted MISTRG mice (G) assessed at around four weeks post tumor cell injection.


Effect of IL - 6R neutralization by twice - weekly injection of 250 μg / dose of tocilizumab or an isotype control antibody , starting two post injection on splenic lymphoma burden of RC - K8 - transplanted MISTRG6 mice ( H ) , assessed at around four post injection .

Effect of IL-6R neutralization by twice-weekly injection of 250 μg/dose of tocilizumab or an isotype control antibody, starting two weeks post tumor cell injection on the splenic lymphoma burden of RC-K8-transplanted MISTRG6 mice (H), all assessed at around four weeks post tumor cell injection.


Alveolar macrophages from different donors were purified from BAL fluid by allowing them to attach to culture dish for 2 hours . Subsequently , they were pre - treated overnight with either type I or type III IFNs ( 1 ng / mL IFN - λ1 10 U / mL IFN - α2 , respectively ) before infection with Sendai ( SeV ) influenza A ( IAV , PR8 ) for 6 hours as indicated . Total was harvested subjected to qPCR for of IFN response measured by IFNA2 , IFNB1 , IFNL1 . ISG response measured by ISG15 RSAD2 . Each colour corresponds to a donor ; 9 different donors were analysed . dashed represents detection limit . An ordinary one - way - ANOVA was used for statistical : ns , not significant ; * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ; * * * * , 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 O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-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 O O O O O

Alveolar macrophages from different donors were purified from BAL fluid by allowing them to attach to the culture dish for 2 hours. Subsequently, they were pre-treated overnight with either type I or type III IFNs (1 ng/mL IFN-λ1 and 10 U/mL IFN-α2, respectively) before infection with Sendai virus (SeV) and influenza A virus (IAV, strain PR8) for 6 hours as indicated. Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2, IFNB1, and IFNL1. ISG response measured by ISG15 and RSAD2. Each colour corresponds to a donor; 9 different donors were analysed. The dashed line represents the detection limit. An ordinary one-way-ANOVA test was used for the statistical analysis: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.


Alveolar macrophages from different donors were purified from BAL fluid by allowing them to attach to culture dish for 2 hours . Subsequently , they were pre - treated overnight with either type I or type III IFNs ( 1 ng / mL IFN - λ1 10 U / mL IFN - α2 , respectively ) before infection with Sendai ( SeV ) influenza A ( IAV , PR8 ) for 6 hours as indicated . column labelled c shows control samples collected directly after purification . of presence of SeV or IAV . Each colour corresponds to a donor ; 9 different donors were analysed . dashed represents detection limit . An ordinary one - way - ANOVA was used for statistical : ns , not significant ; * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ; * * * * , 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 O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Alveolar macrophages from different donors were purified from BAL fluid by allowing them to attach to the culture dish for 2 hours. Subsequently, they were pre-treated overnight with either type I or type III IFNs (1 ng/mL IFN-λ1 and 10 U/mL IFN-α2, respectively) before infection with Sendai virus (SeV) and influenza A virus (IAV, strain PR8) for 6 hours as indicated. The column labelled c shows control samples collected directly after purification. Quantification of the presence of SeV or IAV. Each colour corresponds to a donor; 9 different donors were analysed. The dashed line represents the detection limit. An ordinary one-way-ANOVA test was used for the statistical analysis: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.


Alveolar macrophages from different donors were purified from BAL fluid pre - treated with either Poly ( I : C ) or IFN - λ1 ( + ; 1 ng / mL , or + + ; 10 ng / mL ) before infection with SARS - CoV - 2 . ( A ) Total was harvested subjected to qPCR for of IFN response measured by IFNA2 , IFNB1 , IFNL1 ( Donors marked in orange blue were not quantifiable in conditions ) . ( B ) ISG response measured by ISG15 , RSAD2 , IFIT1 .

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

Alveolar macrophages from different donors were purified from BAL fluid and pre-treated with either Poly(I:C) or IFN-λ1 (+; 1 ng/mL, or ++; 10 ng/mL) before infection with SARS-CoV-2. (A) Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2, IFNB1, and IFNL1 (Donors marked in orange and blue were not quantifiable in all test conditions). (B) ISG response measured by ISG15, RSAD2, and IFIT1.


Alveolar macrophages from different donors were purified from BAL fluid pre - treated with either Poly ( I : C ) or IFN - λ1 ( + ; 1 ng / mL , or + + ; 10 ng / mL ) before infection with SARS - CoV - 2 . ( C ) of presence of SARS - CoV - 2 .

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Alveolar macrophages from different donors were purified from BAL fluid and pre-treated with either Poly(I:C) or IFN-λ1 (+; 1 ng/mL, or ++; 10 ng/mL) before infection with SARS-CoV-2. (C) Quantification of the presence of SARS-CoV-2.


Alveolar macrophages from different donors were purified from BAL fluid infected with SARS - CoV - 2 before treatment with either Poly ( I : C ) or IFN - λ1 ( 1 ng / mL ) . ( B ) Total was harvested subjected to qPCR for of IFN response measured by IFNA2 , IFNB1 , IFNL1 . ( C ) ISG response measured by ISG15 , RSAD2 , IFIT1 ( Donors marked in green red were not quantifiable in conditions ) .

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

Alveolar macrophages from different donors were purified from BAL fluid and infected with SARS-CoV-2 before treatment with either Poly(I:C) or IFN-λ1 (1 ng/mL). (B) Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2, IFNB1, and IFNL1. (C) ISG response measured by ISG15, RSAD2, and IFIT1 (Donors marked in green and red were not quantifiable in all test conditions).


Alveolar macrophages from different donors were purified from BAL fluid infected with SARS - CoV - 2 before treatment with either Poly ( I : C ) or IFN - λ1 ( 1 ng / mL ) . ( D ) of presence of SARS - CoV - 2 .

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Alveolar macrophages from different donors were purified from BAL fluid and infected with SARS-CoV-2 before treatment with either Poly(I:C) or IFN-λ1 (1 ng/mL). (D) Quantification of the presence of SARS-CoV-2.


Alveolar macrophages from different donors were purified from BAL fluid infected with SARS - CoV - 2 before treatment with either Poly ( I : C ) or IFN - λ1 ( 1 ng / mL ) . ( E ) Western blots were performed using samples from two donors . ( A - D ) Each colour corresponds to a donor ; 5 different donors were analysed . dashed represents detection limit . An ordinary one - way - ANOVA was used for statistical : ns , not significant ; * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ; * * * * , P ≤ 0 . 0001 .

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Alveolar macrophages from different donors were purified from BAL fluid and infected with SARS-CoV-2 before treatment with either Poly(I:C) or IFN-λ1 (1 ng/mL). (E) Western blots were performed using samples from two donors. (A-D) Each colour corresponds to a donor; 5 different donors were analysed. The dashed line represents the detection limit. An ordinary one-way-ANOVA test was used for statistical analysis: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.


Alveolar macrophages from different donors were purified from BAL treated with SARS - CoV - 2 for either 2 or 24 hours . ( A ) amount of SARS - CoV - 2 nucleocapsid phosphoprotein was quantified using LC - MS / MS . ( B ) After subtraction of background ( unchallenged AMs ) , between amount of SARS - CoV - 2 nucleocapsid phosphoprotein at 24 2 hours was calculated . Each colour corresponds to a donor ; 3 different donors were analyzed . An ordinary one - way - ANOVA was used for statistical : ns , not significant ; * , 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 B-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 B-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

Alveolar macrophages from different donors were purified from BAL and treated with SARS-CoV-2 for either 2 or 24 hours. (A) The amount of SARS-CoV-2 nucleocapsid phosphoprotein was quantified using LC-MS/MS. (B) After subtraction of the background (unchallenged AMs), the ratio between the amount of SARS-CoV-2 nucleocapsid phosphoprotein at 24 and 2 hours was calculated. Each colour corresponds to a donor; 3 different donors were analyzed. An ordinary one-way-ANOVA test was used for statistical analysis: ns, not significant; *, P ≤ 0.05.


( A ) Total was harvested from AMs human airway epithelial ( HAE ) subjected to qPCR for of ACE2 receptor . Each colour corresponds to a donor . dashed represents detection limit .

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(A) Total RNA was harvested from AMs and primary human airway epithelial cells (HAE) and subjected to qPCR for quantification of the ACE2 receptor. Each colour corresponds to a donor. The dashed line represents the detection limit.


( B ) presence of nucleoprotein mRNA of SARS - CoV - 2 in AMs HAE was also quantified after SARS - CoV - 2 treatment . Each colour corresponds to a donor . An unpaired t - was used for statistical : * * * * , P ≤ 0 . 0001 .

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(B) The presence of nucleoprotein mRNA of SARS-CoV-2 in AMs and primary HAE was also quantified after SARS-CoV-2 treatment. Each colour corresponds to a donor. An unpaired t-test test was used for statistical analysis: ****, P≤0.0001.


( C ) Primer efficiency was tested using serial dilutions of positive samples using qPCR . Ct values were plotted on a logarithmic scale along with corresponding dilutions before performing a linear regression in order to calculate slope of trend through data points . This revealed an amplification factor of 1 . 92 , 1 . 91 , 1 . 85 for SARS - CoV - 2 , IAV , SeV primer set , respectively . different colours correspond to different primer sets .

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(C) Primer efficiency was tested using serial dilutions of positive samples using qPCR. The Ct values were plotted on a logarithmic scale along with the corresponding dilutions before performing a linear regression in order to calculate the slope of the trend line through the data points. This analysis revealed an amplification factor of 1.92, 1.91, and 1.85 for the SARS-CoV-2, IAV, and SeV primer set, respectively. The different colours correspond to different primer sets.


( A ) Proportion change of Chinese SL isolates from different phylogenetic clades . Chinese isolates were classified into two groups on their sampling time ( 1993 - 2010 vs 2011 - 2020 ) . of isolates from each clade in each period was compared .

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(A) Proportion change of Chinese SL isolates from different phylogenetic clades. The Chinese isolates were classified into two groups based on their sampling time (1993-2010 vs 2011-2020). The percentage of isolates from each clade in each period was compared.


( A ) resistance to desiccation . Representative isolates that are phylogenetically distinct within Clade - 1 ( n = 2 ) , Clade - 2 ( n = 1 ) , Clade - 4 ( n = 2 ) Clade - 5 ( n = 6 ) were used for desiccation Data information : experiments were triplicated with biological replicates . Data are presented as mean ± SD . P values for ( A ) were calculated using unpaired t - ( two - ) ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) .

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(A) Bacterial resistance to desiccation. Representative isolates that are phylogenetically distinct within Clade-1 (n=2), Clade-2 (n=1), Clade-4 (n=2) and Clade-5 (n=6) were used for desiccation assay Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. The P values for (A) were calculated using unpaired t-test (two-tailed) (*:P<0.05, **:P<0.01, ***:P<0.001).


( B ) survivability under oxidative stress . Representative isolates from C1 ( n = 2 ) , C2 ( n = 1 ) , C4 ( n = 2 ) C5 ( n = 6 ) were treated with hydrogen peroxide . Data information : experiments were triplicated with biological replicates . Data are presented as mean ± SD . P values for ( B ) , were calculated using unpaired t - ( two - ) , ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) .

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(B) Bacterial survivability under oxidative stress. Representative isolates from C1 (n=2), C2 (n=1), C4 (n=2) and C5 (n=6) were treated with hydrogen peroxide. Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. The P values for (B), were calculated using unpaired t-test (two-tailed), (*:P<0.05, **:P<0.01, ***:P<0.001).


( C ) Biofilm formation ability . biofilm of representative isolates from C1 ( n = 2 ) , C2 ( n = 1 ) , C4 ( n = 2 ) C5 ( n = 6 ) were formed at 22 ° C for 48 h . Data information : experiments were triplicated with biological replicates . Data are presented as mean ± SD . P values for ( C ) were calculated using unpaired t - ( two - ) , ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) .

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(C) Biofilm formation ability. The biofilm of the representative isolates from C1 (n=2), C2 (n=1), C4 (n=2) and C5 (n=6) were formed at 22°C for 48 h. Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. The P values for (C) were calculated using unpaired t-test (two-tailed), (*:P<0.05, **:P<0.01, ***:P<0.001).


( D ) Comparison of survival rates of chick after challenging SL C5 C1 isolates . Comparison was on 6 day - post - infection ( dpi ) . Ten were used for each group . Data information : experiments were triplicated with biological replicates . Data are presented as mean ± SD . P values for ( D ) were calculated using Log - rank ( Mantel - Cox , chi - square ) ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) .

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(D) Comparison of survival rates of chick embryo after challenging SL C5 and C1 isolates. Comparison was on 6 day-post-infection (dpi). Ten embryos were used for each group. Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. P values for (D) were calculated using Log-rank (Mantel-Cox, chi-square) test (*:P<0.05, **:P<0.01, ***:P<0.001).


( E ) Survival rates of zebrafish after challenging SL C5 C1 isolates . comparison was on 14 dpi . Ten zebrafish were used for each group . Data information : experiments were triplicated with biological replicates . Data are presented as mean ± SD . P values for ( E ) were calculated using Log - rank ( Mantel - Cox , chi - square ) ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) .

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(E) Survival rates of zebrafish embryo after challenging SL C5 and C1 isolates. The comparison was on 14 dpi. Ten zebrafish embryos were used for each group. Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. P values for (E) were calculated using Log-rank (Mantel-Cox, chi-square) test (*:P<0.05, **:P<0.01, ***:P<0.001).


( F ) loads in distinct organs of mice after challenging SL C5 ( L42 ) C1 ( L1 ) isolates . in liver , spleen , cecum of mice were measured on 3 5 dpi . Ten mice were used per group . Data information : experiments were triplicated with biological replicates . Data are presented as mean ± SD . P values for ( F ) were calculated using unpaired t - ( two - ) , ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) .

(F) Bacterial loads in distinct organs of mice after challenging SL C5 (L42) and C1 (L1) isolates. The bacterial counts in the liver, spleen, and cecum of mice were measured on 3 and 5 dpi. Ten mice were used per group. Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. The P values for (F) were calculated using unpaired t-test (two-tailed), (*:P<0.05, **:P<0.01, ***:P<0.001).


( C ) day 20 ± 2 cerebral organoids derived from controls , EML1 - heKOs stained for neuronal marker MAPT adherens junction marker NCAD , counterstained with DAPI . Ectopic neural rosettes neuronal heterotopia are highlighted with dotted yellow lines . ( D ) of VZ areas with ectopic neural rosettes ( 3 batches , 3 organoids analyzed per batch , significance on Kruskal - Wallis - , P = 0 . 0001 , Dunn ' s post hoc for multiple comparisons performed to define statistical differences between genotypes , single data points are presented colored by batch ) . ( E ) of heterotopic , disorganized or organized cortical areas ( 3 batches , 3 organoids per batch , significance on Kruskal - Wallis - , P = 0 . 0001 for " organized " " heterotopic " ; no significant difference for " disorganized " , Dunn ' s post hoc performed for multiple comparisons to define statistical differences between genotypes ) . Data information : * marks statistical significance in relation to Control 1 , # in relation to Control 2 . P - values : * * * / # # # < 0 . 001 . VZ : ventricular zone ; Data in graphs are represented as means ± SD . Scale bars : 50µm .

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(C) day 20±2 cerebral organoids derived from controls, patients and EML1-heKOs stained for the neuronal marker MAPT and the adherens junction marker NCAD, counterstained with DAPI. Ectopic neural rosettes and neuronal heterotopia are highlighted with dotted yellow lines. (D) Quantification of VZ areas with ectopic neural rosettes (3 batches, 3 organoids analyzed per batch, significance based on Kruskal-Wallis-test, P=0.0001, Dunn's post hoc test for multiple comparisons performed to define statistical differences between genotypes, single data points are presented colored by batch). (E) Quantification of heterotopic, disorganized or organized cortical areas (3 batches, 3 organoids per batch, significance based on Kruskal-Wallis-test, P=0.0001 for "organized" and "heterotopic"; no significant difference for "disorganized", Dunn's post hoc test performed for multiple comparisons to define statistical differences between genotypes). Data information: * marks statistical significance in relation to Control 1, # in relation to Control 2. P-values: ***/###<0.001. VZ: ventricular zone; Data in graphs are represented as means ± SD. Scale bars: 50µm.


( A ) Hybrid organoids generated from controls mixed with control - EGFP + or EML1 - heKO - EGFP + ( day 20 ± 2 ) . Yellow dotted lines indicate VZ areas . ( B ) of control - EGFP + or EML1 - heKO - EGFP + within or at basal side of VZ areas ( 3 batches , 3 organoids each , significance on Mann Whitney , * * * P = 0 . 0001 for " EGFP + at basal side of VZ areas " ) . Data information : Data in graphs are represented as means ± SD . Scale bars : ( A )

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(A) Hybrid organoids generated from controls mixed with control-EGFP+ or EML1-heKO-EGFP+ cells (day 20±2). Yellow dotted lines indicate VZ areas. (B) Quantification of control-EGFP+ or EML1-heKO-EGFP+ cells within or at the basal side of VZ areas (3 batches, 3 organoids each, significance based on Mann Whitney test, ***P= 0.0001 for "EGFP+ cells at the basal side of VZ areas"). Data information: Data in graphs are represented as means ± SD. Scale bars: (A) 50µm


( C ) Representative scheme of cleavage plan orientation . Upper panel : immunostained for pVim ( red ) TPX2 ( green ) , counterstained with DAPI , dotted indicates cleavage plane orientation , yellow dotted VZ - surface . ( D ) of horizontal , vertical oblique plane of divisions in control EML1 - deficient organoids ( day 20 ± 2 ; 3 batches , 3 organoids each ) . Data information : Scale bars :

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(C) Representative images and scheme of cleavage plan orientation. Upper panel: cells immunostained for pVim (red) and TPX2 (green), counterstained with DAPI, white dotted line indicates cleavage plane orientation, yellow dotted line VZ-surface. (D) Quantification of horizontal, vertical and oblique plane of cell divisions in control and EML1-deficient organoids (day 20±2; 3 batches, 3 organoids each). Data information: Scale bars: 5µm


( F ) Electron microscopy of cilia derived from control or EML1 - deficient cortical progenitors in DMSO - control or following EpothiloneD ( EpoD ) exposure ( blue arrow : basal , yellow arrow : appendages , red arrow : ciliary pockets ) . Data information : Scale bars : 0 , 5µm .

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(F) Electron microscopy of primary cilia derived from control or EML1-deficient cortical progenitors in DMSO-control or following EpothiloneD (EpoD) exposure (blue arrow: basal body, yellow arrow: appendages, red arrow: ciliary pockets). Data information: Scale bars: 0,5µm.


( E ) Control EML1 - heKO - derived cerebral organoid stained for COL1A2 MEIS2 . VZ areas are encircled , squares indicate areas enlarged in F . ( F ) Control EML1 - heKO - derived cerebral organoid stained for COL1A2 , MEIS2 MAPT . ( G ) Representative showing EGFP - control EGFP - EML1 - heKO hybrid cerebral organoid stained for EGFP , FAM107A NCAD . Yellow dotted lines indicate of EGFP + ( 4 independent batches each , 3 organoids per batch , at least 30 ectopic rosettes in total ) . Data information : Scale bars : ( E ) 50µm ; ( F ) 10µm ; ( G ) 50µm , enlarged 10µm .

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(E) Control and EML1-heKO-derived cerebral organoid stained for COL1A2 and MEIS2. VZ areas are encircled, squares indicate areas enlarged in F. (F) Control and EML1-heKO-derived cerebral organoid stained for COL1A2, MEIS2 and MAPT. (G) Representative images showing EGFP-control and EGFP-EML1-heKO hybrid cerebral organoid stained for EGFP, FAM107A and NCAD. Yellow dotted lines indicate morphology of EGFP+ cells (4 independent batches each, 3 organoids per batch, at least 30 ectopic rosettes in total). Data information: Scale bars: (E) 50µm; (F) 10µm; (G) 50µm, enlarged 10µm.


( D , E ) Immunofluorescence for ( D ) p - VIM YAP1 or ( E ) Pax6 YAP1 in control EML1 - heKO - derived organoids ( day 20 ± 2 , squares indicate areas enlarged in adjacent part of panel respectively ) . ( F ) of basally located pVim + with YAP1 signal per VZ in control EML1 - heKO - derived organoids ( 3 batches , 3 organoids analyzed per batch ) . ( G ) of PAX6 + with YAP1 signal per VZ in control EML1 - heKO - derived organoids ( 3 batches , 3 organoids analyzed per batch ) . Data information : Data in graphs are represented as means ± SD . Significance on Kruskal - Wallis - Dunn ' s post hoc for multiple comparisons were performed to define statistical differences between genotypes . P - values : * * * < 0 . 001 , * * < 0 . 01 , * < 0 . 05 . Scale bars : ( D E ) 50µm , enlarged

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

(D, E) Immunofluorescence staining for (D) p-VIM and YAP1 or (E) Pax6 and YAP1 in control and EML1-heKO-derived organoids (day 20±2, squares indicate areas enlarged in adjacent part of the panel respectively). (F) Quantification of basally located pVim+ cells with nuclear YAP1 signal per VZ area in control and EML1-heKO-derived organoids (3 batches, 3 organoids analyzed per batch). (G) Quantification of PAX6+ cells with nuclear YAP1 signal per VZ area in control and EML1-heKO-derived organoids (3 batches, 3 organoids analyzed per batch). Data information: Data in graphs are represented as means ± SD. Significance based on Kruskal-Wallis-test Dunn's post hoc test for multiple comparisons were performed to define statistical differences between genotypes. P-values: ***<0.001, **<0.01, *<0.05. Scale bars: (D and E) 50µm, enlarged 10µm


( H ) Immunofluorescence for p - Vim in EML1 - heKO control derived organoids in DMSO , Verteporfin or Fluvastatin treated conditions , counterstained with DAPI . Dotted indicates VZ areas . ( I ) of p - VIM + located at basal side of VZ areas in control or EML1 - heKO - derived organoids under DMSO , Verteporfin or Fluvastatin treated condition ( 3 batches , 3 organoids analyzed per batch ) . Data information : Data in graphs are represented as means ± SD . Significance on two - way ANOVA ( on log10 normalized data ) Tukey post hoc for multiple comparisons were performed to define statistical differences between genotypes . P - values : * * * < 0 . 001 , * * < 0 . 01 , * < 0 . 05 . Scale bars : ( H )

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(H) Immunofluorescence staining for p-Vim in EML1-heKO and control derived organoids in DMSO, Verteporfin or Fluvastatin treated conditions, counterstained with DAPI. Dotted line indicates VZ areas. (I) Quantification of p-VIM+ cells located at the basal side of VZ areas in control or EML1-heKO-derived organoids under DMSO, Verteporfin or Fluvastatin treated condition (3 batches, 3 organoids analyzed per batch). Data information: Data in graphs are represented as means ± SD. Significance based on two-way ANOVA (on log10 normalized data) Tukey post hoc test for multiple comparisons were performed to define statistical differences between genotypes. P-values: ***<0.001, **<0.01, *<0.05. Scale bars: (H) 50µm


expression of SAMHD1 is negatively correlated with EV71 viral replication ability . HEK293T RD were infected with EV71 at a MOI of 0 . 1 , then supernatants were harvested at indicated time points . Immunoblotting ( IB ) of EV71 VP1 in , with tubulin as a loading control . EV71 - VP1 in supernatants was detected after ultracentrifugation .

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The expression of SAMHD1 is negatively correlated with EV71 viral replication ability. HEK293T and RD cells were infected with EV71 at a MOI of 0.1, then the cells and supernatants were harvested at the indicated time points. Immunoblotting (IB) analysis of EV71 VP1 in cells, with tubulin as a loading control. EV71-VP1 protein in the supernatants was detected after ultracentrifugation.


expression of SAMHD1 is negatively correlated with EV71 viral replication ability . HEK293T RD were infected with EV71 at a MOI of 0 . 1 , then supernatants were harvested at indicated time points . EV71 viral in lysates supernatants were detected by RT - qPCR with GAPDH as a control . ( n = 3 , mean ± SD , ns stands for no significance , paired t - )

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The expression of SAMHD1 is negatively correlated with EV71 viral replication ability. HEK293T and RD cells were infected with EV71 at a MOI of 0.1, then the cells and supernatants were harvested at the indicated time points. EV71 viral RNA levels in cell lysates and supernatants were detected by RT-qPCR with GAPDH as a control. (n=3, mean±SD, ns stands for no significance, paired t-test)


expression of SAMHD1 is negatively correlated with EV71 viral replication ability . HEK293T RD were infected with EV71 at a MOI of 0 . 1 , then supernatants were harvested at indicated time points . mRNA of host restriction factors were detected by RT - qPCR in infected or uninfected HEK293T or RD , expression of target genes were normalized to GAPDH . ( n = 3 , mean ± SD , ns stands for no significance , paired t - ) .

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The expression of SAMHD1 is negatively correlated with EV71 viral replication ability. HEK293T and RD cells were infected with EV71 at a MOI of 0.1, then the cells and supernatants were harvested at the indicated time points. The mRNA levels of the host restriction factors were detected by RT-qPCR in infected or uninfected HEK293T or RD cells, and the expression levels of the target genes were normalized to GAPDH. (n=3, mean±SD, ns stands for no significance, paired t-test).


expression of SAMHD1 is negatively correlated with EV71 viral replication ability . HEK293T RD were infected with EV71 at a MOI of 0 . 1 , then supernatants were harvested at indicated time points . IB of SAMHD1 BST - 2 with tubulin as a loading control . densities of bands were analyzed with ImageJ software to calculate values relative to that of tubulin .

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The expression of SAMHD1 is negatively correlated with EV71 viral replication ability. HEK293T and RD cells were infected with EV71 at a MOI of 0.1, then the cells and supernatants were harvested at the indicated time points. IB analysis of SAMHD1 and BST-2 protein levels with tubulin as a loading control. The densities of bands were analyzed with ImageJ software to calculate the values relative to that of tubulin.


SAMHD1 knockdown enhances EV71 replication . stable lines pLKO . 1 sh - SAMHD1 constructed in HEK293T were infected with EV71 at a MOI of 0 . 1 0 . 05 respectively , supernatants were harvested at indicated time points . IB of EV71 VP1 SAMHD1 in was performed with tubulin as a loading control . EV71 - VP1 in supernatants was detected after ultracentrifugation .

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SAMHD1 knockdown enhances EV71 replication. The stable cell lines pLKO.1 and sh-SAMHD1 constructed in HEK293T cells were infected with EV71 at a MOI of 0.1 and 0.05 respectively, and cells and supernatants were harvested at the indicated time points. IB analysis of EV71 VP1 and SAMHD1 in cells was performed with tubulin as a loading control. EV71-VP1 protein in the supernatants was detected after ultracentrifugation.


SAMHD1 knockdown enhances EV71 replication . EV71 viral in lysates was detected by RT - qPCR with GAPDH as a control ( n = 3 , mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , paired t - ) .

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SAMHD1 knockdown enhances EV71 replication. EV71 viral RNA in cell lysates was detected by RT-qPCR with GAPDH as a control (n=3, mean±SD, * P < 0.05, ** P < 0.01, paired t-test).


SAMHD1 knockdown enhances EV71 replication . Viral titers in supernatants were measured by cytopathic effect method .

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SAMHD1 knockdown enhances EV71 replication. Viral titers in the supernatants were measured by the cytopathic effect method.


SAMHD1 knockdown enhances EV71 replication . stable lines pLKO . 1 sh - SAMHD1 constructed in RD ( H ) were infected with EV71 at a MOI of 0 . 1 0 . 05 respectively , supernatants were harvested at indicated time points . IB of EV71 VP1 SAMHD1 in was performed with tubulin as a loading control . EV71 - VP1 in supernatants was detected after ultracentrifugation .

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SAMHD1 knockdown enhances EV71 replication. The stable cell lines pLKO.1 and sh-SAMHD1 constructed in RD (H) cells were infected with EV71 at a MOI of 0.1 and 0.05 respectively, and cells and supernatants were harvested at the indicated time points. IB analysis of EV71 VP1 and SAMHD1 in cells was performed with tubulin as a loading control. EV71-VP1 protein in the supernatants was detected after ultracentrifugation.


HEK293T transfected with VR1012 , which is a eukaryotic expression vector , or Vpx - HA were infected with EV71 at a MOI of 0 . 1 , then supernatants were harvested at indicated time points . IB of EV71 - VP1 , SAMHD1 Vpx - HA in lysates was performed with tubulin as a loading control . EV71 - VP1 was detected in supernatants after ultracentrifugation .

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HEK293T cells transfected with VR1012, which is a eukaryotic expression vector, or Vpx-HA were infected with EV71 at a MOI of 0.1, and then cells and supernatants were harvested at the indicated time points. IB analysis of EV71-VP1, SAMHD1 and Vpx-HA in cell lysates was performed with tubulin as a loading control. EV71-VP1 was detected in the supernatants after ultracentrifugation.


EV71 viral was detected in lysates by RT - qPCR with GAPDH as a control ( n = 3 , mean ± SD , * * P < 0 . 01 , paired t - ) .

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EV71 viral RNA was detected in cell lysates by RT-qPCR with GAPDH as a control (n=3, mean±SD, ** P <0.01, paired t-test).


Viral titres in supernatants were measured by cytopathic effect method . results represent means ± SD from three independent experiments . Statistical significance was analyzed using Student ' s t ( * * P < 0 . 01 ) .

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Viral titres in the supernatants were measured by the cytopathic effect method. The results represent the means ± SD from three independent experiments. Statistical significance was analyzed using Student's t test (**P < 0.01).


Stable SAMHD1 - HA - overexpressing lines were constructed in HEK293T RD detected by IB with pLVX as a negative control .

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Stable SAMHD1-HA-overexpressing cell lines were constructed in HEK293T and RD cells and detected by IB with pLVX as a negative control.


RD SAMHD1 - HA lines were treated with dimethyl sulfoxide ( DMSO ) or 10 μM MG132 for 12 h prior to harvest subjected to IB with tubulin as a loading control .

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RD SAMHD1-HA cell lines were treated with dimethyl sulfoxide (DMSO) or 10 μM MG132 for 12 h prior to harvest and subjected to IB with tubulin as a loading control.


RD SAMHD1 - HA lines were treated as in ( B ) , then HA immunoprecipitation ( IP ) IB were performed . IP elution was analyzed by mass spectrometry .

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RD SAMHD1-HA cell lines were treated as in (B), and then HA immunoprecipitation (IP) and IB were performed. The IP elution was analyzed by mass spectrometry.


TRIM21 induced degradation of SAMHD1 , MG132 rescued TRIM21 - mediated degradation of SAMHD1 . HEK293T were cotransfected with SAMHD1 VR1012 or TRIM21 expression plasmids with or without MG132 treatment for 12 h prior to harvest , then subjected to IB with tubulin as a loading control .

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TRIM21 induced the degradation of SAMHD1, and MG132 rescued the TRIM21-mediated degradation of SAMHD1. HEK293T cells were cotransfected with SAMHD1 and VR1012 or TRIM21 expression plasmids with or without MG132 treatment for 12 h prior to harvest, and then subjected to IB with tubulin as a loading control.


TRIM25 ( E ) induced degradation of SAMHD1 HEK293T were cotransfected with SAMHD1 VR1012 or TRIM25 expression plasmids then subjected to IB with tubulin as a loading control .

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TRIM25 (E) induced the degradation of SAMHD1 HEK293T cells were cotransfected with SAMHD1 and VR1012 or TRIM25 expression plasmids and then subjected to IB with tubulin as a loading control.


Reverse co - IP confirmed interaction between SAMHD1 TRIM21 . SAMHD1 - flag was transfected with TRIM21 - HA into HEK293T , then were treated as in ( B ) subjected to HA IP IB .

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Reverse co-IP confirmed the interaction between SAMHD1 and TRIM21. SAMHD1-flag was transfected with TRIM21-HA into HEK293T cells, and then the cells were treated as in (B) and subjected to HA IP and IB.


Endogenous TRIM21 but not TRIM25 still interacted with SAMHD1 . RD - SAMHD1 - HA treated as in ( B ) were subjected to TRIM21 or TRIM25 IP IB .

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Endogenous TRIM21 but not TRIM25 still interacted with SAMHD1. RD-SAMHD1-HA cells treated as in (B) were subjected to TRIM21 or TRIM25 IP and IB.


mRNA levels of TRIM21 in RD and HEK293T cells.


protein levels of TRIM21 in RD and HEK293T cells.


TRIM21 ( J ) but not TRIM25 ( K ) colocalized with SAMHD1 . were taken under a Zeiss LZM710 confocal microscope , Bars , 10μm .

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TRIM21 (J) but not TRIM25 (K) colocalized with SAMHD1. Images were taken under a Zeiss LZM710 confocal microscope, Bars, 10μm.


TRIM21 Knockout in RD released SAMHD1 inhibition on EV71 replication . Negative control or RD - CRISPR - Cas9 - TRIM21 RD were infected with EV71 at a MOI of 0 . 05 for indicated time harvested for SAMHD1 , TRIM21 EV71 - VP1 detection by IB . Tubulin served as a loading control .

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TRIM21 Knockout in RD cells released SAMHD1 inhibition on EV71 replication. Negative control or RD-CRISPR-Cas9-TRIM21 RD cells were infected with EV71 at a MOI of 0.05 for the indicated time and harvested for SAMHD1, TRIM21 and EV71-VP1 detection by IB. Tubulin served as a loading control.


TRIM21 Knockout in RD released SAMHD1 inhibition on EV71 replication . EV71 in ( B ) were detected by RT - qPCR with GAPDH as a control ( n = 3 , mean ± SD , * * P < 0 . 01 , paired t - ) .

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TRIM21 Knockout in RD cells released SAMHD1 inhibition on EV71 replication. EV71 RNA levels in (B) were detected by RT-qPCR with GAPDH as a control (n=3, mean±SD, ** P < 0.01, paired t-test).


TRIM21 Knockout in RD released SAMHD1 inhibition on EV71 replication . Viral titres in supernatant were measured by cytopathic effect method . results represent means ± SD from three independent experiments . Statistical significance was analyzed using Student ' s t ( * * P < 0 . 01 ) .

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TRIM21 Knockout in RD cells released SAMHD1 inhibition on EV71 replication. Viral titres in the supernatant were measured by the cytopathic effect method. The results represent the means ± SD from three independent experiments. Statistical significance was analyzed using Student's t test (**P < 0.01).


Overexpression of TRIM21 in HEK293T increased EV71 replication by degrading SAMHD1 . HEK293T were transfected with VR1012 or TRIM21 - HA for 24 h then infected with EV71 at a MOI of 0 . 05 . were harvested at indicated time points subjected to IB with tubulin as a loading control .

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Overexpression of TRIM21 in HEK293T cells increased EV71 replication by degrading SAMHD1. HEK293T cells were transfected with VR1012 or TRIM21-HA for 24 h and then infected with EV71 at a MOI of 0.05. Cells were harvested at the indicated time points and subjected to IB with tubulin as a loading control.


Overexpression of TRIM21 in HEK293T increased EV71 replication by degrading SAMHD1 . HEK293T were transfected with VR1012 or TRIM21 - HA for 24 h then infected with EV71 at a MOI of 0 . 05 . EV71 viral in lysates was detected by RT - qPCR with GAPDH as a control ( n = 3 , mean ± SD , * * P < 0 . 01 , paired t - ) ) .

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Overexpression of TRIM21 in HEK293T cells increased EV71 replication by degrading SAMHD1. HEK293T cells were transfected with VR1012 or TRIM21-HA for 24 h and then infected with EV71 at a MOI of 0.05. EV71 viral RNA in cell lysates was detected by RT-qPCR with GAPDH as a control (n=3, mean±SD, ** P < 0.01, paired t-test)).


TRIM21 itself has no effect on EV71 replication . VR1012 or TRIM21 was transfected into pLKO . 1 or sh - SAMHD1 HEK293T for 24 h infected with EV71 . were harvested subjected to IB .

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TRIM21 itself has no effect on EV71 replication. VR1012 or TRIM21 was transfected into pLKO.1 or sh-SAMHD1 HEK293T cells for 24 h and infected with EV71. Cells were harvested and subjected to IB analysis.


TRIM21 itself has no effect on EV71 replication . VR1012 or TRIM21 was transfected into pLKO . 1 or sh - SAMHD1 HEK293T for 24 h infected with EV71 . Viral titers in supernatants were measured by cytopathic effect method . results represent means ± SD from three independent experiments . Statistical significance was analyzed using Student ' s t ( * P < 0 . 05 ) .

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TRIM21 itself has no effect on EV71 replication. VR1012 or TRIM21 was transfected into pLKO.1 or sh-SAMHD1 HEK293T cells for 24 h and infected with EV71. Viral titers in the supernatants were measured by the cytopathic effect method. The results represent the means ± SD from three independent experiments. Statistical significance was analyzed using Student's t test (*P < 0.05).


mRNA of SAMHD1 TRIM21 in different tissues of neonatal mice were detected by RT - qPCR with GAPDH as a control , expression of SAMHD1 TRIM21 in heart was set as 100 % ( n = 3 , mean ± SD , * P < 0 . 05 , paired t - ) .

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The mRNA levels of SAMHD1 and TRIM21 in different tissues of neonatal mice were detected by RT-qPCR with GAPDH as a control, and the expression of SAMHD1 and TRIM21 in the heart was set as 100% (n=3, mean±SD, * P < 0.05, paired t-test).


of SAMHD1 TRIM21 in different tissues of mice were examined by Near Infrared Spectrum ( NIR ) western blot with tubulin as a control .

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The protein levels of SAMHD1 and TRIM21 in different tissues of mice were examined by Near Infrared Spectrum (NIR) western blot with tubulin as a control.


Immunohistochemical ( ) results of SAMHD1 in various tissues of mice . Bars , 100μm .

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Immunohistochemical (IHC) staining results of SAMHD1 in various tissues of mice. Bars, 100μm.


mRNA of EV71 host factors in mock or EV71 infected mice were detected by RT - qPCR with GAPDH as a control . expression of EV71 ( D ) were presented , of corresponding gene in mock infected mice were set as 100 %

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The mRNA levels of EV71 virus and host factors in mock or EV71 infected mice were detected by RT-qPCR with GAPDH as a control. The expression of EV71 (D) were presented, and the levels of corresponding gene in mock infected mice were set as 100%


mRNA of EV71 host factors in mock or EV71 infected mice were detected by RT - qPCR with GAPDH as a control . expression of , IFNα IFNβ ( E ) were presented , of corresponding gene in mock infected mice were set as 100 %

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The mRNA levels of EV71 virus and host factors in mock or EV71 infected mice were detected by RT-qPCR with GAPDH as a control. The expression of , IFNα and IFNβ (E) were presented, and the levels of corresponding gene in mock infected mice were set as 100%


mRNA of EV71 host factors in mock or EV71 infected mice were detected by RT - qPCR with GAPDH as a control . expression of SAMHD1 TRIM21 ( F ) were presented , of corresponding gene in mock infected mice were set as 100 %

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The mRNA levels of EV71 virus and host factors in mock or EV71 infected mice were detected by RT-qPCR with GAPDH as a control. The expression of SAMHD1 and TRIM21 (F) were presented, and the levels of corresponding gene in mock infected mice were set as 100%


IB of THP - 1 treated with scrambled shRNA , TRIM21 - specific shRNA or SAMHD1 - specific shRNA . Tubulin served as loading control . of SAMHD1 TRIM21 in whole in which indicated genes were knocked down by shRNA . Data are normalized to tubulin expressed as fold change over treated with pLKO . 1 without HIV infection ( n = 3 , mean ± SD ) .

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IB analysis of THP-1 cells treated with scrambled shRNA, TRIM21-specific shRNA or SAMHD1-specific shRNA. Tubulin served as loading control. Quantification of SAMHD1 and TRIM21 levels in whole cells in which the indicated genes were knocked down by shRNA. Data are normalized to tubulin and expressed as the fold change over cells treated with pLKO.1 without HIV infection (n=3, mean±SD).


sh - SAMHD1 sh - TRIM21 THP - 1 were treated with PMA for 24 h infected with VSV - G pseudotyped reporter HIV - 1 , then detected 48 h later by flow cytometry ( n = 3 , mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , paired t - ) .

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sh-SAMHD1 and sh-TRIM21 THP-1 cells were treated with PMA for 24 h and infected with the VSV-G pseudotyped reporter HIV-1, then detected 48 h later by flow cytometry analysis (n=3, mean±SD, * P < 0.05, ** P < 0.01, *** P < 0.001, paired t-test).


of SAMHD1 TRIM21 in whole in which indicated genes were knocked down by shRNA . Data are normalized to tubulin expressed as fold change over treated with pLKO . 1 without HIV infection ( n = 3 , mean ± SD ) . IB of MDM from 3 donors transfected with TRIM21 siRNA twice at days 1 3 , respectively , after MDM differentiation .

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Quantification of SAMHD1 and TRIM21 levels in whole cells in which the indicated genes were knocked down by shRNA. Data are normalized to tubulin and expressed as the fold change over cells treated with pLKO.1 without HIV infection (n=3, mean±SD). IB analysis of MDM cells from 3 donors transfected with TRIM21 siRNA twice at days 1 and 3, respectively, after MDM differentiation.


MDM treated with TRIM21 siRNA were infected with VSV - G - pseudotyped HIV - 1 reporter then detected 48 h later by flow cytometry ( n = 3 , mean ± SD , * * P < 0 . 01 , paired t - ) .

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MDM cells treated with TRIM21 siRNA were infected with VSV-G-pseudotyped HIV-1 reporter and then detected 48 h later by flow cytometry analysis (n=3, mean±SD, ** P < 0.01, paired t-test).


of SAMHD1 TRIM21 in whole in which indicated genes were knocked down by shRNA . Data are normalized to tubulin expressed as fold change over treated with pLKO . 1 without HIV infection ( n = 3 , mean ± SD ) . IB of Jurkat KG - 1 treated with scrambled shRNA or TRIM21 - specific shRNA .

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Quantification of SAMHD1 and TRIM21 levels in whole cells in which the indicated genes were knocked down by shRNA. Data are normalized to tubulin and expressed as the fold change over cells treated with pLKO.1 without HIV infection (n=3, mean±SD). IB analysis of Jurkat and KG-1 cells treated with scrambled shRNA or TRIM21-specific shRNA.


Jurkat KG - 1 treated with TRIM21 shRNA were infected with HIV - 1 , then detected 48 h later by flow cytometry ( n = 3 , mean ± SD , ns stands for no significance , paired t - ) .

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Jurkat and KG-1 cells treated with TRIM21 shRNA were infected with HIV-1, then detected 48 h later by flow cytometry analysis (n=3, mean±SD, ns stands for no significance, paired t-test).


TRIM21 interacts with SAMHD1 via PRY SPRY domains . effect of TRIM21 on degradation of SAMHD1 . SAMHD1 - flag was cotransfected with VR1012 , TRIM21 WT or indicated mutant into HEK293T for 48 h , were subjected to IB with tubulin as loading control .

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TRIM21 interacts with SAMHD1 via PRY and SPRY domains. The effect of TRIM21 on the degradation of SAMHD1. SAMHD1-flag was cotransfected with VR1012, TRIM21 WT or the indicated mutant into HEK293T cells for 48 h, and the cells were subjected to IB with tubulin as loading control.


TRIM21 interacts with SAMHD1 via PRY SPRY domains . SAMHD1 - flag was cotransfected with VR1012 or TRIM21 WT or indicated mutant for 24 h , were then treated with 10 μM MG132 for 12 h before harvest subjected to HA IP IB .

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TRIM21 interacts with SAMHD1 via PRY and SPRY domains. SAMHD1-flag was cotransfected with VR1012 or TRIM21 WT or the indicated mutant for 24 h, and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB.


effect of TRIM21 on SAMHD1 WT or its mutants . SAMHD1 - HA or indicated mutant were cotransfected with VR1012 or TRIM21 - flag into HEK293T for 48 h , were subjected to IB with tubulin as a loading control .

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The effect of TRIM21 on SAMHD1 WT or its mutants. SAMHD1-HA or the indicated mutant were cotransfected with VR1012 or TRIM21-flag into HEK293T cells for 48 h, and the cells were subjected to IB with tubulin as a loading control.


SAMHD1 - flag 1 - 547 was cotransfected with VR1012 or TRIM21 - HA for 24 h , were then treated with 10 μM MG132 for 12 h before harvest subjected to HA IP IB .

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SAMHD1-flag 1-547 was cotransfected with VR1012 or TRIM21-HA for 24 h, and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB.


SAMHD1 109 - 626 followed with a His tag or TRIM21 - PRYSPRY followed with a GST tag were expressed in Rosetta ( DE3 ) , pull - down were performed with Ni - Sepharose ( up ) GST Sepharose ( down ) , respectively .

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SAMHD1 109-626 followed with a His tag or TRIM21-PRYSPRY followed with a GST tag were expressed in Rosetta (DE3), and pull-down assay were performed with Ni-Sepharose (up) and GST Sepharose (down), respectively.


FRET indicates interaction between YFP - SAMHD1 CFP - TRIM21 . A representative image of SAMHD1 - YFP ( yellow ) ECFP - TRIM21 ( cyan ) expressing before after photobleaching acceptor fluorophore , YFP . region chosen for photobleaching is marked ( open box ) , Bars , 10μm . quantization of brightness was analyzed by Image J ( n = 3 , mean ± SD , ns stands for no significance , * P < 0 . 05 , paired t - )

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FRET analysis indicates interaction between YFP-SAMHD1 and CFP-TRIM21. A representative image of SAMHD1-YFP (yellow) and ECFP-TRIM21 (cyan) expressing cells before and after photobleaching the acceptor fluorophore, YFP. The region chosen for photobleaching is marked (white open box), Bars, 10μm. The quantization of fluorescence brightness was analyzed by Image J (n=3, mean±SD, ns stands for no significance, * P < 0.05, paired t-test)


microscale thermophoresis curve for interaction between SAMHD1 TRIM21 . Alexa Fluor ® 647 labelled SAMHD1 at a of 20 nM were incubated with two - fold dilution series of unlabeled TRIM21 ( 3 μM to 9 . 16E - 05 μM ) . curve represents signal recorded from three measurements . normalized thermophoretic signals were plotted against of TRIM21 with mean values ± standard deviation as well as a fitting to a 1 : 1 binding model ( NanoTemper ® software , F Norm = F Hot / F Cold ) . Kd of this interaction was determined to be 270 ± 92nM . data was a representive of three independent experiments using different dilution .

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The microscale thermophoresis curve for the interaction between SAMHD1 and TRIM21. Alexa Fluor®647 labelled SAMHD1 at a concentration of 20 nM were incubated with two-fold dilution series of unlabeled TRIM21 (3 μM to 9.16E-05 μM). The curve represents the signal recorded from three measurements. The normalized fluorescence thermophoretic signals were plotted against the concentration of TRIM21 with mean values ± standard deviation as well as a fitting to a 1:1 binding model (NanoTemper® analysis software, F Norm = F Hot / F Cold). The Kd of this interaction was determined to be 270±92nM. The data was a representive of three independent experiments using different dilution.


effect of TRIM21 on SAMHD1 from various species . HEK293T were transfected with VR1012 or TRIM21 indicated SAMHD1 expression vector , then subjected to IB . Tubulin served as a loading control .

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The effect of TRIM21 on SAMHD1 proteins from various species. HEK293T cells were transfected with VR1012 or TRIM21 and the indicated SAMHD1 expression vector, then subjected to IB analysis. Tubulin served as a loading control.


effect of TRIM21 on hSAMHD1 mutants . HEK293T were transfected with VR1012 or TRIM21 SAMHD1 mutants for 48 h then subjected to IB . Tubulin served as a loading control .

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The effect of TRIM21 on hSAMHD1 mutants. HEK293T cells were transfected with VR1012 or TRIM21 and SAMHD1 mutants for 48 h and then subjected to IB analysis. Tubulin served as a loading control.


SAMHD1 - flag WT or G153S or G183R mutant was cotransfected with VR1012 or TRIM21 - HA for 24 h , were then treated with 10 μM MG132 for 12 h before harvest subjected to HA IP IB . Tubulin served as a loading control .

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SAMHD1-flag WT or the G153S or G183R mutant was cotransfected with VR1012 or TRIM21-HA for 24 h, and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB. Tubulin served as a loading control.


SAMHD1 - HA WT or indicated mutants were cotransfected with VR1012 or TRIM21 - flag into HEK293T for 48 h , were subjected to IB with tubulin as a loading control .

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SAMHD1-HA WT or the indicated mutants were cotransfected with VR1012 or TRIM21-flag into HEK293T cells for 48 h, and the cells were subjected to IB with tubulin as a loading control.


SAMHD1 - HA WT VR1012 or TRIM21 were cotransfected with K48 - only K63 - only ubiquitin - flag into HEK293T for 24 h , were then treated with 10 μM MG132 for 12 h before harvest subjected to HA IP IB with tubulin as a loading control .

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SAMHD1-HA WT and VR1012 or TRIM21 were cotransfected with K48-only and K63-only ubiquitin-flag into HEK293T cells for 24 h, and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB with tubulin as a loading control.


K48 - only ubiquitin - flag SAMHD1 - HA WT or mutant K622R were cotransfected with VR1012 or TRIM21 into HEK293T as indicated . were then treated with 10 μM MG132 for 12 h before harvest subjected to HA IP IB with tubulin as a loading control .

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K48-only ubiquitin-flag and SAMHD1-HA WT or mutant K622R were cotransfected with VR1012 or TRIM21 into HEK293T cells as indicated. The cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB with tubulin as a loading control.


VR1012 , SAMHD1 - HA WT or K622R mutant was transfected into HEK293T - shSAMHD1 for 24 h . were then infected with EV71 at a MOI of 0 . 1 harvested at indicated time points . SAMHD1 - HA , TRIM21 EV71 - VP1 were detected by IB with tubulin as a loading control .

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VR1012, SAMHD1-HA WT or the K622R mutant was transfected into HEK293T-shSAMHD1 cells for 24 h. The cells were then infected with EV71 at a MOI of 0.1 and harvested at the indicated time points. SAMHD1-HA, TRIM21 and EV71-VP1 were detected by IB with tubulin as a loading control.


A PTAR1 expression in rat tissues . extracted from various rat tissues ( 15 μg each ) were analyzed by immunoblotting with antibodies against PTAR1 .

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A PTAR1 protein expression in rat tissues. Proteins extracted from various rat tissues (15 μg each) were analyzed by immunoblotting with antibodies against PTAR1.


B Identification of RabGGTβ as a binding of PTAR1 . Anti - FLAG immunoprecipitates from control HeLa S3 or FLAG - PTAR1 - expressing HeLa S3 were analyzed by SDS - PAGE silver . 35 kDa was identified as RabGGTβ by mass spectrometry . asterisk denotes a degradation product of FLAG - PTAR1 .

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B Identification of RabGGTβ as a binding protein of PTAR1. Anti-FLAG immunoprecipitates from control HeLa S3 cells or FLAG-PTAR1-expressing HeLa S3 cells were analyzed by SDS-PAGE and silver staining. The 35 kDa protein was identified as RabGGTβ by mass spectrometry. The asterisk denotes a degradation product of FLAG-PTAR1.


C Co - immunoprecipitation of endogenous PTAR1 RabGGTβ from rat brain thymus cytosol .

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C Co-immunoprecipitation of endogenous PTAR1 and RabGGTβ from rat brain and thymus cytosol.


D Fractionation of rat brain cytosol by Mono Q anion exchange column chromatography . Column fractions were analyzed by immunoblotting with antibodies against RabGGTα , PTAR1 , RabGGTβ . RabGGTβ eluted in two peaks ( peak A peak B ) .

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D Fractionation of rat brain cytosol by Mono Q anion exchange column chromatography. Column fractions were analyzed by immunoblotting with antibodies against RabGGTα, PTAR1, and RabGGTβ. RabGGTβ eluted in two peaks (peak A and peak B).


E Co - immunodepletion of PTAR1 RabGGTβ by anti - PTAR1 IgG . PTAR1 was immunodepleted from Mono Q peak A fraction using anti - PTAR1 IgG - bound A beads , supernatant was analyzed by immunoblotting with antibodies against PTAR1 RabGGTβ . Normal rabbit IgG anti - PTAR1 IgG preabsorbed with antigen peptide were used as controls .

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E Co-immunodepletion of PTAR1 and RabGGTβ by anti-PTAR1 IgG. PTAR1 was immunodepleted from the Mono Q peak A fraction using anti-PTAR1 IgG-bound protein A beads, and the supernatant was analyzed by immunoblotting with antibodies against PTAR1 and RabGGTβ. Normal rabbit IgG and anti-PTAR1 IgG preabsorbed with the antigen peptide were used as controls.


F Fractionation of Mono Q peak A fraction by Superdex 200 gel filtration chromatography . Column fractions were analyzed by immunoblotting with antibodies against PTAR1 RabGGTβ .

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F Fractionation of the Mono Q peak A fraction by Superdex 200 gel filtration chromatography. Column fractions were analyzed by immunoblotting with antibodies against PTAR1 and RabGGTβ.


G Gel filtration chromatography of recombinant PTAR1 - RabGGTβ purified from Sf9 insect on a Superdex 200 column . Inset shows SDS - PAGE Coomassie of purified .

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G Gel filtration chromatography of the recombinant PTAR1-RabGGTβ complex purified from Sf9 insect cells on a Superdex 200 column. Inset shows SDS-PAGE and Coomassie staining analysis of the purified complex.


C Identification of Ykt6 as a biotin - geranylated by GGTase - III . extracted from statin - treated HeLa S3 were applied to GST or GST - GGTase - III affinity columns . Bound were eluted incubated with recombinant GGTase - III BGPP . Reaction products were separated by SDS - PAGE , transferred to a nitrocellulose , probed with horseradish peroxidase ( HRP ) - labeled avidin to detect biotinylated . Mass spectrometry identified 25 kDa as Ykt6 .

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C Identification of Ykt6 as a protein biotin-geranylated by GGTase-III. Cytosolic proteins extracted from statin-treated HeLa S3 cells were applied to GST or GST-GGTase-III affinity columns. Bound proteins were eluted and incubated with recombinant GGTase-III and BGPP. Reaction products were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with horseradish peroxidase (HRP)-labeled avidin to detect biotinylated proteins. Mass spectrometry identified the 25 kDa protein as Ykt6.