The percentage of IL - 17 - producing cells in CD45 . 1 + Vβ11 + CD4 + T cells in the spinal cords 11 days after transfer Each symbol represents an individual mouse . Small horizontal lines indicate the mean ( error bars , s . d )

The percentage of IL-17-producing cells in CD45.1+Vβ11+ CD4+ T cells in the spinal cords 11 days after transfer Each symbol represents an individual mouse. Small horizontal lines indicate the mean (error bars, s.d)


The absolute number of IL - 17A + CD45 . 1 + Vβ11 + CD4 + T cells in the spinal cords 11 days after transfer . Each symbol represents an individual mouse . Small horizontal lines indicate the mean ( error bars , s . d )

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The absolute number of IL-17A+CD45.1+Vβ11+CD4+ T cells in the spinal cords 11 days after transfer. Each symbol represents an individual mouse. Small horizontal lines indicate the mean (error bars, s.d)


Flow cytometry of Foxp3 staining of splenic cells . Histograms are gated for CD45 . 1 - CD4 + Vβ11 + . Data are representative of two independent experiments with consistent

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

Flow cytometry of Foxp3 staining of splenic cells. Histograms are gated for CD45.1-CD4+Vβ11+. Data are representative of two independent experiments with consistent results


Body weight of RAG - 2 - / - mice after transfer with 5 x 105 CD45 . 1 + CD4 + CD45RBhigh cells alone ( circles ) or with 2 x 105 FACS - sorted CD45 . 1 - CD4 + GFP ( Foxp3 ) + NGFR + cells transduced with control retrovirus ( Min ) ( filled rectangles ) or retrovirus encoding C / EBPβ ( empty rectangles ) and cultured for 2 d in the presence of TGF - β , anti - IFN - γ and anti - IL - 4 Abs . Each time point contains three mice in each group . Error bars represent mean ± s . e .

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Body weight of RAG-2-/- mice after transfer with 5 x 105 CD45.1+CD4+CD45RBhigh cells alone (circles) or with 2 x 105 FACS-sorted CD45.1-CD4+GFP(Foxp3)+NGFR+ cells transduced with control retrovirus (Min)(filled rectangles) or retrovirus encoding C/EBPβ (empty rectangles) and cultured for 2 d in the presence of TGF-β, anti-IFN-γ and anti-IL-4 Abs. Each time point contains three mice in each group. Error bars represent mean ± s.e.m


Hematoxylin and eosin staining of the distal colons of mice ( Scale bars , 200μm )

Hematoxylin and eosin staining of the distal colons of mice (Scale bars, 200μm)


Hematoxylin and eosin staining of the distal colons of mice ( Scale bars , 200μm ) Histological scores . Each symbol represents an individual mouse . Small horizontal lines indicate the mean ( error bars , s . e . m )

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Hematoxylin and eosin staining of the distal colons of mice (Scale bars, 200μm) Histological scores. Each symbol represents an individual mouse. Small horizontal lines indicate the mean (error bars, s.e.m)


The percentage of Foxp3 + cells in CD45 . 1 - CD4 + T cells in colonic lamina propria 11 weeks after

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

The percentage of Foxp3+ cells in CD45.1-CD4+ T cells in colonic lamina propria 11 weeks after transfer


Flow cytometry of intracellular FOXP3 staining ( right ) or isotype control ( left ) in human CD4 + CD25 - CD45RA + naïve T cells transduced with control retrovirus ( Min ) or retrovirus encoding hC / EBPβ and cultured for 2 d under conditions as indicated . Representative experiments are shown in the left panel and pooled data with mean ± s . e . m are shown on the right . Histograms are gated for CD4 + NGFR + . Statistical analysis was performed using unpaired two - tailed t test . ( * p < 0 . 05 ; ns , not significant )

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

Flow cytometry of intracellular FOXP3 staining (right) or isotype control (left) in human CD4+CD25-CD45RA+ naïve T cells transduced with control retrovirus (Min) or retrovirus encoding hC/EBPβ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the left panel and pooled data with mean±s.e.m are shown on the right. Histograms are gated for CD4+NGFR+. Statistical analysis was performed using unpaired two-tailed t test. (*p<0.05; ns, not significant)


Flow cytometry of CFSE - labeled human CD4 + CD25 - T cells ( responder ) stimulated 4 d alone or together with NGFR + - sorted human CD4 + CD25 - CD45RA + naïve T cells ( suppressor ) transduced with control retrovirus ( Min ) or retrovirus encoding hC / EBPβ and cultured for 5 d in the presence of hTGF - β , anti - hIFN - γ and anti - hIL - 4 Abs . Histograms are gated for CFSE + . The ratio of responder to suppressor is 1 : 0 . 2 ( top ) or 1 : 0 . 4 ( bottom ) . Data are representative of two independent experiments with consistent

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

Flow cytometry of CFSE-labeled human CD4+CD25- T cells (responder) stimulated 4 d alone or together with NGFR+-sorted human CD4+CD25-CD45RA+ naïve T cells (suppressor) transduced with control retrovirus (Min) or retrovirus encoding hC/EBPβ and cultured for 5 d in the presence of hTGF-β, anti-hIFN-γ and anti-hIL-4 Abs. Histograms are gated for CFSE+. The ratio of responder to suppressor is 1:0.2 (top) or 1:0.4(bottom). Data are representative of two independent experiments with consistent results


L ) Example images of β - Gal + traced cells post - irradiation and at homeostasis . Scale bar = 100um . M ) The number of fully traced β - Gal + crypt - villus axes / cm in irradiated and non - irradiated mice . Lineage tracing events were quantified from small intestines divided into four equal segments ( S1 - 4 , with S1 being the most proximal ) , stained with X - Gal , embedded in paraffin , cut into 5um thick sections , and counterstained with neutral red . Data are expressed as mean ± SEM . N = 3 mice / group , P - value generated by multiple student ' s t - tests . N ) The number of β - Gal + fully traced crypt - villus axes ( i . e . lineage tracing events ) normalized to the number of pre - injury crypt marked β - Gal + cells ( quantified from d0 control mice ) . S1 + S2 = the two most proximal intestinal segments . Data are expressed as mean ± SEM . N = 4 mice / group , P - value generated by two - tailed student ' s t - test .

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

L) Example images of β-Gal+ traced cells post-irradiation and at homeostasis. Scale bar= 100um. M) The number of fully traced β-Gal+ crypt-villus axes/cm in irradiated and non-irradiated mice. Lineage tracing events were quantified from small intestines divided into four equal segments (S1-4, with S1 being the most proximal), stained with X-Gal, embedded in paraffin, cut into 5um thick sections, and counterstained with neutral red. Data are expressed as mean ± SEM. N=3 mice/group, P-value generated by multiple student's t-tests. N) The number of β-Gal+ fully traced crypt-villus axes (i.e. lineage tracing events) normalized to the number of pre-injury crypt marked β-Gal+ cells (quantified from d0 control mice). S1 + S2 = the two most proximal intestinal segments. Data are expressed as mean ± SEM. N=4 mice/group, P-value generated by two-tailed student's t-test.


D ) Organoid formation efficiency of different aged LRC populations . N = 3 mice / group , N = 3 wells quantified per mouse . Data are expressed as mean ± SEM . N = 3 mice / group ,

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

D) Organoid formation efficiency of different aged LRC populations. N=3 mice/group, N=3 wells quantified per mouse. Data are expressed as mean ± SEM. N=3 mice/group,


E ) Images of organoids quantified in ( D ) .

E) Images of organoids quantified in (D).


C ) Western blot demonstrating LC3 levels from equal numbers of sorted CytoIDlow and CytoIDhigh cells . 200 , 000 cells were loaded per lane .

O O 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) Western blot demonstrating LC3 levels from equal numbers of sorted CytoIDlow and CytoIDhigh cells. 200,000 cells were loaded per lane.


E ) Representative images of organoids formed from sorted CytoIDlow , CytoIDhigh , and Lgr5 - GFPhigh cells . F ) Quantification of the organoid - formation efficiency in ( E ) . N = 4 mice / group , N = 3 wells quantified per mouse . P - Values displayed on graph . Data are expressed as mean ± SEM . P value generated by ratio paired two - tailed t - test .

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E) Representative images of organoids formed from sorted CytoIDlow, CytoIDhigh, and Lgr5-GFPhigh cells. F) Quantification of the organoid-formation efficiency in (E). N=4 mice/group, N=3 wells quantified per mouse. P-Values displayed on graph. Data are expressed as mean ± SEM. P value generated by ratio paired two-tailed t-test.


G ) Effect of bafilomycin on organoid formation efficiency in CytoIDlow and CytoIDhigh cells . Cells were plated in media containing different concentrations of bafilomycin ( 0 , 2 . 5 , 5 , 10 , or 25 ng / mL ) on day 0 , media was changed on day 3 to media that did not contain bafilomycin and organoids were imaged on day 5 after plating . N = 4 mice / group , N = 4 wells quantified per mouse .

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

G) Effect of bafilomycin on organoid formation efficiency in CytoIDlow and CytoIDhigh cells. Cells were plated in media containing different concentrations of bafilomycin (0, 2.5, 5, 10, or 25 ng/mL) on day 0, media was changed on day 3 to media that did not contain bafilomycin and organoids were imaged on day 5 after plating. N=4 mice/group, N=4 wells quantified per mouse.


H ) Representative images of organoids quantified in ( G ) .

H) Representative images of organoids quantified in (G).


A ) Representative plots demonstrating the distribution of cells with and without CytoID puncta within sorted CytoID low , high , Chga + , and c - Kit + cells .

A) Representative plots demonstrating the distribution of cells with and without CytoID puncta within sorted CytoID low, high, Chga+, and c-Kit+ cells.


B ) Quantification of the percentage of cells with and without puncta within CytoID low , high , Chga + , and c - Kit + cells . N = 3 mice / group ( biological replicates ) .

O O O O O O O O O O O O O O O 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) Quantification of the percentage of cells with and without puncta within CytoID low, high, Chga+, and c-Kit+ cells. N=3 mice/group (biological replicates).


C ) Representative images and quantification of organoid - formation efficiency of CytoID low and high cKit + and Chga + cells . Scale bars = 500um . N = 3 mice / group ( biological replicates ) , N = 4 wells quantified per mouse ( technical replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images and quantification of organoid-formation efficiency of CytoID low and high cKit+ and Chga+ cells. Scale bars = 500um. N=3 mice/group (biological replicates), N=4 wells quantified per mouse (technical replicates).


D ) Immunofluorescent images of crypts from CAG - LC3 - GFP - RFP and Lgr5 - GFP mice 6h post - irradiation . Sections were stained for γH2AX , E - cadherin , and either LC3 - RFP or Lgr5 - GFP respectively . E ) Quantification of the number of γH2AX foci in cells with RFP + puncta , without autophagic puncta , or in Lgr5 + CBCs . N = 3 mice / group , 40 - 80 cells quantified / mouse .

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D) Immunofluorescent images of crypts from CAG-LC3-GFP-RFP and Lgr5-GFP mice 6h post-irradiation. Sections were stained for γH2AX, E-cadherin, and either LC3-RFP or Lgr5-GFP respectively. E) Quantification of the number of γH2AX foci in cells with RFP+ puncta, without autophagic puncta, or in Lgr5+ CBCs. N=3 mice/group, 40-80 cells quantified/mouse.


( A ) Serum IgG ELISA reactivity with MTB ( H37Rv ) whole cell lysate , purified cell membrane fraction and secreted culture filtrate proteins ( CFP ) for TB patients and healthy donors ( HD ) . ELISA graphs ( left ; TB : black lines ; n = 25 ; HD : red dotted lines ; n = 2 ) and area under the curve ( AUC ) values for all tested samples ( right ; TB : n = 25 ; HD : n = 17 ) are shown . Median is 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

(A) Serum IgG ELISA reactivity with MTB (H37Rv) whole cell lysate, purified cell membrane fraction and secreted culture filtrate proteins (CFP) for TB patients and healthy donors (HD). ELISA graphs (left; TB: black lines; n=25; HD: red dotted lines; n=2) and area under the curve (AUC) values for all tested samples (right; TB: n=25; HD: n=17) are shown. Median is shown.


( B ) AUC values for the IgA serum ELISA response against MTB whole cell lysate , purified cell membrane fraction and secreted culture filtrate proteins ( CFP ) of TB patients ( n = 25 ) and healthy donors ( HD ; n = 9 ) . Median is 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

(B) AUC values for the IgA serum ELISA response against MTB whole cell lysate, purified cell membrane fraction and secreted culture filtrate proteins (CFP) of TB patients (n=25) and healthy donors (HD; n=9). Median is shown.


( C ) Flow cytometry gating strategy and frequency of circulating plasmablasts ( CD19 + CD27 + + CD38 + ) for one representative TB patient ( TB24 ) and HD ( left ) . Frequency of circulating CD19 + CD27 + + CD38 + plasmablasts of all CD19 + B cells in peripheral blood of TB patients ( n = 24 ) and HD ( n = 8 ) . Dashed line indicates threshold for detectable plasmablast populations ( right ) . Median and SEM 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

(C) Flow cytometry gating strategy and frequency of circulating plasmablasts (CD19+CD27++CD38+) for one representative TB patient (TB24) and HD (left). Frequency of circulating CD19+CD27++CD38+plasmablasts of all CD19+ B cells in peripheral blood of TB patients (n=24) and HD (n=8). Dashed line indicates threshold for detectable plasmablast populations (right). Median and SEM are shown.


( D ) Expression of CD19 and activation markers ( CD86 , CD84 and CD24 ) in plasmablasts and memory B cells as measured by flow cytometry .

O O O O 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) Expression of CD19 and activation markers (CD86, CD84 and CD24) in plasmablasts and memory B cells as measured by flow cytometry.


( E ) Plasmablast response of patient TB7 ( top ) and IgG serum response against MTB cell lysate of patient TB7 , TB19 and TB39 ( bottom ) and one HD at the indicated time points before and after treatment onset .

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(E) Plasmablast response of patient TB7 (top) and IgG serum response against MTB cell lysate of patient TB7, TB19 and TB39 (bottom) and one HD at the indicated time points before and after treatment onset.


( F ) Dots indicate AUC values for the anti - MTB cell lysate IgG serum ELISA response ( y - axis ) versus frequency of circulating plasmablasts ( x - axis ) for individual TB patients .

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

(F) Dots indicate AUC values for the anti-MTB cell lysate IgG serum ELISA response (y-axis) versus frequency of circulating plasmablasts (x-axis) for individual TB patients.


( A ) Gating strategy , phenotype and frequency of circulating plasmablasts ( CD19 + CD27 + + CD38 + ) isolated by flow cytometric cell sorting from 3 TB patients ( TB7 , TB24 , TB33 ) in comparison to one representative HD . Boxes indicate sort gates . The plasmablast frequency is indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Gating strategy, phenotype and frequency of circulating plasmablasts (CD19+CD27++CD38+) isolated by flow cytometric cell sorting from 3 TB patients (TB7, TB24, TB33) in comparison to one representative HD. Boxes indicate sort gates. The plasmablast frequency is indicated.


( B ) Absolute number of somatic hypermutations ( SHM ) in the IGHV , IGKV and IGLV segments of IgA and IgG plasmablast antibody genes sequenced from TB7 , TB24 and TB33 . The absolute number of sequences analyzed is indicated below the graph . Geometric means with SEM are indicated in grey . SHM means of historic data from sorted CD27 + IgA + or CD27 + IgG + cells from peripheral blood of HD are indicated in red for comparison ( Berkowska et al , 2015a ; Tiller et al , 2007 ) .

(B) Absolute number of somatic hypermutations (SHM) in the IGHV, IGKV and IGLV segments of IgA and IgG plasmablast antibody genes sequenced from TB7, TB24 and TB33. The absolute number of sequences analyzed is indicated below the graph. Geometric means with SEM are indicated in grey. SHM means of historic data from sorted CD27+IgA+ or CD27+IgG+ cells from peripheral blood of HD are indicated in red for comparison (Berkowska et al, 2015a; Tiller et al, 2007).


( C ) Isotype distribution of plasmablast and memory B cells from TB7 , TB24 and TB33 . PB , plasmablasts ( CD19 + CD27 + + CD38 + ) ; M , memory B cells ( CD19 + CD27 + ) .

(C) Isotype distribution of plasmablast and memory B cells from TB7, TB24 and TB33. PB, plasmablasts (CD19+CD27++CD38+); M, memory B cells (CD19+CD27+).


( D ) Gating strategy , phenotype and frequency of HBHA - reactive memory B cells ( CD19 + CD27 + HBHA + ) in peripheral blood of one representative TB patient ( TB29 ) , health - care worker ( HCW ) 2 and HD , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Gating strategy, phenotype and frequency of HBHA-reactive memory B cells (CD19+CD27+HBHA+) in peripheral blood of one representative TB patient (TB29), health-care worker (HCW) 2 and HD, respectively.


( E ) Dots indicate frequency of HBHA - reactive memory B cells out of all CD27 + memory B cells in individual TB patients ( n = 23 ) and HCWs ( n = 7 ) . Mean and SEM are indicated . P - value was determined using Wilcoxon - Mann - Whitney test ; * * * * 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 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) Dots indicate frequency of HBHA-reactive memory B cells out of all CD27+ memory B cells in individual TB patients (n=23) and HCWs (n=7). Mean and SEM are indicated. P-value was determined using Wilcoxon-Mann-Whitney test; ****p<0.0001.


( F ) Dots indicate the frequency of resting memory B cells ( CD19 + CD27 + CD10 - ) out of all B cells in peripheral blood of individual TB patients compared to HD .

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

(F) Dots indicate the frequency of resting memory B cells (CD19+CD27+CD10-) out of all B cells in peripheral blood of individual TB patients compared to HD.


( G ) Anti - HBHA serum IgG ELISA reactivity for TB patients and HCW ( black lines ) compared to two representative HD ( red lines ) . Dashed line indicates the threshold OD405nm for positive reactivity ( left ) . Asterisks indicate serum responses of donors selected for single sorting of HBHA - reactive memory B cells . Data are representative of two independent experiments .

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

(G) Anti-HBHA serum IgG ELISA reactivity for TB patients and HCW (black lines) compared to two representative HD (red lines). Dashed line indicates the threshold OD405nm for positive reactivity (left). Asterisks indicate serum responses of donors selected for single sorting of HBHA-reactive memory B cells. Data are representative of two independent experiments.


( H ) Absolute number of somatic hypermutations ( SHM ) in the IGHV ( IGHA , IGHG and IGHM ) , IGKV or IGLV segments of sorted anti - HBHAmemory cells from TB patients and HCWs . Geometric means with SEM are indicated in grey . For comparison , red lines indicate the historic SHM means for randomly sorted CD27 + IgA + , CD27 + IgG + or CD27 + IgM + cells from peripheral blood of HDs ( Berkowska et al , 2015a ; Tiller et al , 2007 ; Tsuiji et al , 2006 ) .

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(H) Absolute number of somatic hypermutations (SHM) in the IGHV (IGHA, IGHG and IGHM), IGKV or IGLV segments of sorted anti-HBHAmemory cells from TB patients and HCWs. Geometric means with SEM are indicated in grey. For comparison, red lines indicate the historic SHM means for randomly sorted CD27+IgA+, CD27+IgG+ or CD27+IgM+ cells from peripheral blood of HDs (Berkowska et al, 2015a; Tiller et al, 2007; Tsuiji et al, 2006).


( I ) Number and size of clonally expanded B cell clusters among HBHA - reactive memory B cells from two TB patients ( TB35 , TB29 ) and HCWs ( HCW1 , HCW 2 ) . Cells in clusters are indicated in grey , single cells are indicated in white . No B cell clusters were shared between donors .

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

(I) Number and size of clonally expanded B cell clusters among HBHA-reactive memory B cells from two TB patients (TB35, TB29) and HCWs (HCW1, HCW 2). Cells in clusters are indicated in grey, single cells are indicated in white. No B cell clusters were shared between donors.


( J ) IgA , IgG and IgM isotype distribution of single - cell - sorted HBHA - reactive memory cells . Mean and SEM are indicated .

(J) IgA, IgG and IgM isotype distribution of single-cell-sorted HBHA-reactive memory cells. Mean and SEM are indicated.


( A , B ) Representative ELISA graphs show reactivity of antibodies from patient TB7 to ( A ) MTB whole cell lysate or ( B ) MTB bacteria ( left ) . Dashed red line indicates the threshold OD405nm for positive reactivity . Green line indicates negative control antibody ( mGO53 ; ( Wardemann et al , 2003 ) ) . AUC ( area under curve ) values indicate the reactivity to ( A ) MTB cell lysate or ( B ) whole MTB bacteria for antibodies from TB7 , TB24 , TB33 . Total numbers of analyzed antibodies are indicated ( right ) .

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

(A, B) Representative ELISA graphs show reactivity of antibodies from patient TB7 to (A) MTB whole cell lysate or (B) MTB bacteria (left). Dashed red line indicates the threshold OD405nm for positive reactivity. Green line indicates negative control antibody (mGO53; (Wardemann et al, 2003)). AUC (area under curve) values indicate the reactivity to (A) MTB cell lysate or (B) whole MTB bacteria for antibodies from TB7, TB24, TB33. Total numbers of analyzed antibodies are indicated (right).


( D ) ELISA graph shows reactivity to the MTB cell membrane fraction for MTB - reactive ( n = 26 ) and non - reactive ( n = 14 ) antibodies .

O O O O O O O O O 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) ELISA graph shows reactivity to the MTB cell membrane fraction for MTB-reactive (n=26) and non-reactive (n=14) antibodies.


( E ) Reactivity to whole BCG bacteria as determined by flow cytometry for three representative MTB cell lysate - reactive antibodies and one non - reactive antibody ( isotype control , mGO53 ) .

O O O O O O O O O O O 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) Reactivity to whole BCG bacteria as determined by flow cytometry for three representative MTB cell lysate-reactive antibodies and one non-reactive antibody (isotype control, mGO53).


( A ) Binding of antibodies TB24PB037 and TB33PB123 to LAM purified from MTB as determined by Western Blot . The negative isotype control ( mGO53 ) and a commercially available anti - LAM antibody ( positive control ) are shown for comparison .

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

(A) Binding of antibodies TB24PB037 and TB33PB123 to LAM purified from MTB as determined by Western Blot. The negative isotype control (mGO53) and a commercially available anti-LAM antibody (positive control) are shown for comparison.


( B ) Anti - BCG specificity of the LAM - reactive antibody TB24PB037 ( black ) compared to E . coli as determined by ELISA with whole bacteria ( left ) and flow cytometry ( right ) .

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(B) Anti-BCG specificity of the LAM-reactive antibody TB24PB037 (black) compared to E.coli as determined by ELISA with whole bacteria (left) and flow cytometry (right).


( C ) Anti - LAMELISA for TB24PB037 or negative control antibody at the indicated concentrations . The mean ± SD of the absorbance was calculated .

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

(C) Anti-LAMELISA for TB24PB037 or negative control antibody at the indicated concentrations. The mean ±SD of the absorbance was calculated.


( D ) Antibody TB24PB037 binds to MTB - LAM but not to LAM from M . smegmatis . ELISA performed as described in ( C ) .

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(D) Antibody TB24PB037 binds to MTB-LAM but not to LAM from M. smegmatis. ELISA performed as described in (C).


( E ) Representative HBHA ELISA for antibodies cloned from HBHA - reactive memory B cells of patient TB35 and HCW1 . Dashed red line indicates the threshold OD405nm for positive reactivity . Green line indicates negative control antibody ( mGO53 ; ( Wardemann et al , 2003 ) ) .

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(E) Representative HBHA ELISA for antibodies cloned from HBHA-reactive memory B cells of patient TB35 and HCW1. Dashed red line indicates the threshold OD405nm for positive reactivity. Green line indicates negative control antibody (mGO53; (Wardemann et al, 2003)).


( F ) Reactivity to HBHA was confirmed by Western Blot for a selected set of antibodies with HBHA ELISA reactivity .

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(F) Reactivity to HBHA was confirmed by Western Blot for a selected set of antibodies with HBHA ELISA reactivity.


( G ) Fluorescence microscopy shows BCG - and MTB - reactivity of representative anti - HBHA antibodies compared to a non - reactive isotype control antibody . Scale bar 10 μm ( top / bottom ) .

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(G) Fluorescence microscopy shows BCG- and MTB-reactivity of representative anti-HBHA antibodies compared to a non-reactive isotype control antibody. Scale bar 10 μm (top/bottom).


Relative bacterial counts ( CFU ) compared to the medium control ( 100 % ) in human lung epithelial A549 cells 1 h after infection with MTB , pre - incubated with recombinant MTB - reactive IgA1 , IgA2 or IgG1 antibodies and non - reactive isotype control as indicated . ( A ) Representative results for individual MTB - reactive plasmablast antibodies . Mean and SEM are indicated .

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Relative bacterial counts (CFU) compared to the medium control (100%) in human lung epithelial A549 cells 1 h after infection with MTB, pre-incubated with recombinant MTB-reactive IgA1, IgA2 or IgG1 antibodies and non-reactive isotype control as indicated. (A) Representative results for individual MTB-reactive plasmablast antibodies. Mean and SEM are indicated.


Relative bacterial counts ( CFU ) compared to the medium control ( 100 % ) in human lung epithelial A549 cells 1 h after infection with MTB , pre - incubated with recombinant MTB - reactive IgA1 , IgA2 or IgG1 antibodies and non - reactive isotype control as indicated . ( B ) Direct comparison of all tested MTB - reactive IgA ( n = 12 ) and IgG ( n = 26 ) antibodies . Mean and SEM are indicated .

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Relative bacterial counts (CFU) compared to the medium control (100%) in human lung epithelial A549 cells 1 h after infection with MTB, pre-incubated with recombinant MTB-reactive IgA1, IgA2 or IgG1 antibodies and non-reactive isotype control as indicated. (B) Direct comparison of all tested MTB-reactive IgA (n=12) and IgG (n=26) antibodies. Mean and SEM are indicated.


Relative bacterial counts ( CFU ) compared to the medium control ( 100 % ) in human lung epithelial A549 cells 1 h after infection with MTB , pre - incubated with recombinant MTB - reactive IgA1 , IgA2 or IgG1 antibodies and non - reactive isotype control as indicated . ( C ) Representative results for individual HBHA - reactive memory B cell antibodies from TB patients and HCW .

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Relative bacterial counts (CFU) compared to the medium control (100%) in human lung epithelial A549 cells 1 h after infection with MTB, pre-incubated with recombinant MTB-reactive IgA1, IgA2 or IgG1 antibodies and non-reactive isotype control as indicated. (C) Representative results for individual HBHA-reactive memory B cell antibodies from TB patients and HCW.


Relative bacterial counts ( CFU ) compared to the medium control ( 100 % ) in human lung epithelial A549 cells 1 h after infection with MTB , pre - incubated with recombinant MTB - reactive IgA1 , IgA2 or IgG1 antibodies and non - reactive isotype control as indicated . ( D ) Same as in ( B ) but with HBHA - reactive antibodies as shown in ( C ) . ( D ) Direct comparison all tested HBHA - reactive IgA ( n = 6 ) and IgG ( n = 7 ) antibodies .

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Relative bacterial counts (CFU) compared to the medium control (100%) in human lung epithelial A549 cells 1 h after infection with MTB, pre-incubated with recombinant MTB-reactive IgA1, IgA2 or IgG1 antibodies and non-reactive isotype control as indicated. (D) Same as in (B) but with HBHA-reactive antibodies as shown in (C). (D) Direct comparison all tested HBHA-reactive IgA (n=6) and IgG (n=7) antibodies.


Relative bacterial counts ( CFU ) compared to the medium control ( 100 % ) in human lung epithelial A549 cells 1 h after infection with MTB , pre - incubated with recombinant MTB - reactive IgA1 , IgA2 or IgG1 antibodies and non - reactive isotype control as indicated . ( E ) Direct comparison of the indicated MTB - reactive antibodies expressed as IgA and IgG . Mean and SEM are indicated . P - values were determined using non - parameric t - test , * p < 0 . 1 ; * * p < 0 . 01 , ns : p > 0 . 5 .

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Relative bacterial counts (CFU) compared to the medium control (100%) in human lung epithelial A549 cells 1 h after infection with MTB, pre-incubated with recombinant MTB-reactive IgA1, IgA2 or IgG1 antibodies and non-reactive isotype control as indicated. (E) Direct comparison of the indicated MTB-reactive antibodies expressed as IgA and IgG. Mean and SEM are indicated. P-values were determined using non-parameric t-test, * p<0.1; **p<0.01, ns: p>0.5.


( A ) Human lung epithelial A549 cells and primary bronchial epithelial cells ( hAECB ) lack FcγR ( CD16 , CD64 and CD32 ) and FcαR ( CD89 ) cell surface expression as analyzed by flow cytometry . Human THP1 cells serve as positive control .

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(A) Human lung epithelial A549 cells and primary bronchial epithelial cells (hAECB) lack FcγR (CD16, CD64 and CD32) and FcαR (CD89) cell surface expression as analyzed by flow cytometry. Human THP1 cells serve as positive control.


( B ) The neonatal FcR ( FcRn ) is expressed in both A549 cells and human THP1 cells as determined by Western Blotting . Data are representative of at least two independent experiments .

(B) The neonatal FcR (FcRn) is expressed in both A549 cells and human THP1 cells as determined by Western Blotting. Data are representative of at least two independent experiments.


( C ) Absolute bacterial counts ( CFUs ) in human THP1 macrophages 1 h after infection with MTB pre - incubated with the LAM - reactive IgA1 ( left ) or IgG1 ( right ) antibody TB24PB037 or the respective isotype control antibody as indicated . Mean and SEM are indicated .

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(C) Absolute bacterial counts (CFUs) in human THP1 macrophages 1 h after infection with MTB pre-incubated with the LAM-reactive IgA1 (left) or IgG1 (right) antibody TB24PB037 or the respective isotype control antibody as indicated. Mean and SEM are indicated.


( A ) Absolute bacterial counts ( CFUs ) in human lung epithelial A549 cells 1 h after infection with MTB pre - incubated with purified serum IgA ( left ) or IgG ( right ) antibodies from TB patients ( TB ) compared to HD as indicated . Mean and SEM are indicated . Data are representative of three independent experiments with three technical replicates each .

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(A) Absolute bacterial counts (CFUs) in human lung epithelial A549 cells 1 h after infection with MTB pre-incubated with purified serum IgA (left) or IgG (right) antibodies from TB patients (TB) compared to HD as indicated. Mean and SEM are indicated. Data are representative of three independent experiments with three technical replicates each.


( B ) Direct comparison of purified serum IgA and IgG antibodies on epithelial cell invasion as measured in ( A ) . P - value was determined using Wilcoxon - Mann - Whitney test ; * * * * p < 0 . 0001 .

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(B) Direct comparison of purified serum IgA and IgG antibodies on epithelial cell invasion as measured in (A). P-value was determined using Wilcoxon-Mann-Whitney test; ****p<0.0001.


Effects of DC and Beva treatment in 786 - O orthoxenograft . Evaluation of tumor progression ( average ± SD ) Box plots indicate median , Q1 / Q3 and max / min value whiskers in Control ( black ) , DC101 ( red ) and Beva ( blue ) treatment groups . 5 - 8 tumors / group were analyzed by Mann - Whitney test , and Mantel - Cox test for survival where * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 and DC * p < 0 . 05 and Beva # p < 0 . 05 vs Control ) .

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

Effects of DC and Beva treatment in 786-O orthoxenograft. Evaluation of tumor progression (average±SD) Box plots indicate median, Q1/Q3 and max/min value whiskers in Control (black), DC101 (red) and Beva (blue) treatment groups. 5-8 tumors/group were analyzed by Mann-Whitney test, and Mantel-Cox test for survival where *p < 0.05, **p < 0.01 and ***p < 0.001 and DC * p < 0.05 and Beva # p < 0.05 vs Control).


Effects of DC and Beva treatment in 786 - O orthoxenograft . Evaluation of ( C ) tumor weight at sacrifice , ( D ) vessel number , Box plots indicate median , Q1 / Q3 and max / min value whiskers in Control ( black ) , DC101 ( red ) and Beva ( blue ) treatment groups . 5 - 8 tumors / group were analyzed by Mann - Whitney test , and Mantel - Cox test for survival where * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 and DC * p < 0 . 05 and Beva # p < 0 . 05 vs Control ) .

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

Effects of DC and Beva treatment in 786-O orthoxenograft. Evaluation of (C) tumor weight at sacrifice, (D) vessel number, Box plots indicate median, Q1/Q3 and max/min value whiskers in Control (black), DC101 (red) and Beva (blue) treatment groups. 5-8 tumors/group were analyzed by Mann-Whitney test, and Mantel-Cox test for survival where *p < 0.05, **p < 0.01 and ***p < 0.001 and DC * p < 0.05 and Beva # p < 0.05 vs Control).


Effects of DC and Beva treatment in 786 - O orthoxenograft . Evaluation of percentage of necrotic area by histology , Box plots indicate median , Q1 / Q3 and max / min value whiskers in Control ( black ) , DC101 ( red ) and Beva ( blue ) treatment groups . 5 - 8 tumors / group were analyzed by Mann - Whitney test , and Mantel - Cox test for survival where * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 and DC * p < 0 . 05 and Beva # p < 0 . 05 vs Control ) .

Effects of DC and Beva treatment in 786-O orthoxenograft. Evaluation of percentage of necrotic area by histology, Box plots indicate median, Q1/Q3 and max/min value whiskers in Control (black), DC101 (red) and Beva (blue) treatment groups. 5-8 tumors/group were analyzed by Mann-Whitney test, and Mantel-Cox test for survival where *p < 0.05, **p < 0.01 and ***p < 0.001 and DC * p < 0.05 and Beva # p < 0.05 vs Control).


Effects of DC and Beva treatment in 786 - O orthoxenograft . Evaluation of survival by Kaplan Meier plot in control and treated animals . Box plots indicate median , Q1 / Q3 and max / min value whiskers in Control ( black ) , DC101 ( red ) and Beva ( blue ) treatment groups . 5 - 8 tumors / group were analyzed by Mann - Whitney test , and Mantel - Cox test for survival where * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 and DC * p < 0 . 05 and Beva # p < 0 . 05 vs Control ) .

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

Effects of DC and Beva treatment in 786-O orthoxenograft. Evaluation of survival by Kaplan Meier plot in control and treated animals. Box plots indicate median, Q1/Q3 and max/min value whiskers in Control (black), DC101 (red) and Beva (blue) treatment groups. 5-8 tumors/group were analyzed by Mann-Whitney test, and Mantel-Cox test for survival where *p < 0.05, **p < 0.01 and ***p < 0.001 and DC * p < 0.05 and Beva # p < 0.05 vs Control).


G ) Representative invasive front profile of 786 - O control and short - term DC and Beva treated tumors in vimentin stained sections ( 4X ) . Front ( red line ) and rear ( white line ) edges of tumor protrusion into kidney determine the depth of invasion ( green arrow ) perpendicularly connecting red and white lines . Box plot shows the quantification of invasion indicating median , Q1 / Q3 and max / min value whiskers of fold - invasion ( ∆ invasion ) in treated vs control tumors ( 7 samples / group ; * p < 0 . 05 by Mann - Whitney test ) .

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G) Representative invasive front profile of 786-O control and short-term DC and Beva treated tumors in vimentin stained sections (4X). Front (red line) and rear (white line) edges of tumor protrusion into kidney determine the depth of invasion (green arrow) perpendicularly connecting red and white lines. Box plot shows the quantification of invasion indicating median, Q1/Q3 and max/min value whiskers of fold-invasion (∆ invasion) in treated vs control tumors (7 samples/group; *p < 0.05 by Mann-Whitney test).


H ) Evaluation of lung micrometastases in survival experiments . Incidence ( pie chart , * p < 0 . 05 by Chi - square ) and proportion of mice with 0 , 1 , 2 or > 3 metastases ( mets ) per lungs ( * p < 0 . 05 by Mann - Whitney test ) are shown .

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H) Evaluation of lung micrometastases in survival experiments. Incidence (pie chart, *p < 0.05 by Chi-square) and proportion of mice with 0, 1, 2 or > 3 metastases (mets) per lungs (*p < 0.05 by Mann-Whitney test) are shown.


Overall mass architecture of paired human specimens and Ren - PDOX tumors observed by HE staining ( 20X ) . TNM staging and Fuhrman grade ( F ) of implanted tumors are reported . For Ren - PDOXs , lung micrometastases in HE and vimentin stained sections are shown ( 40X ) .

Overall mass architecture of paired human specimens and Ren-PDOX tumors observed by HE staining (20X). TNM staging and Fuhrman grade (F) of implanted tumors are reported. For Ren-PDOXs, lung micrometastases in HE and vimentin stained sections are shown (40X).


Human to mouse stroma substitution . CD34 / CD31 staining for respectively human ( h ) and mouse ( m ) blood vessels in original human specimens and paired Ren13 and 28 - PDOX are shown ( 20X ) . Ren - PDOX OT referred to tumor generated from first implantation of human specimen in mice . Insets show human and mouse kidney as positive / negative controls of staining ( 20X ) .

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Human to mouse stroma substitution. CD34/CD31 staining for respectively human (h) and mouse (m) blood vessels in original human specimens and paired Ren13 and 28-PDOX are shown (20X). Ren-PDOX OT referred to tumor generated from first implantation of human specimen in mice. Insets show human and mouse kidney as positive/negative controls of staining (20X).


Frequency histogram of SNV and / or indels found in Ren - PDOX by high - throughput sequencing in Ren13 , Ren28 , Ren36 , Ren86 ( targeted - NGS analysis by Haloplex ) and Ren50 - PDOX ( WES ) . Only non - synonymous / stop gain SNVs and frameshift indels novel or with MAF < 0 . 01 are included .

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Frequency histogram of SNV and/or indels found in Ren-PDOX by high-throughput sequencing in Ren13, Ren28, Ren36, Ren86 (targeted-NGS analysis by Haloplex) and Ren50-PDOX (WES). Only non-synonymous/stop gain SNVs and frameshift indels novel or with MAF<0.01 are included.


Effects of DC and Beva treatments on Ren13 , Ren86 , Ren28 and Ren50 - PDOX . Evaluation of tumor progression average and SD in control ( black ) compared to DC ( red , * p < 0 . 05 ) and Beva ( blue , # p < 0 . 05 ) treatments from 5 - 13 animals per tumor and treatment group by Mann Whitney test .

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Effects of DC and Beva treatments on Ren13, Ren86, Ren28 and Ren50-PDOX. Evaluation of tumor progression average and SD in control (black) compared to DC (red, *p < 0.05) and Beva (blue, # p < 0.05) treatments from 5-13 animals per tumor and treatment group by Mann Whitney test.


Effects of DC and Beva treatments on Ren13 , Ren86 , Ren28 and Ren50 - PDOX . Quantification of tumor weight in control and treated tumors from 4 - 5 samples / tumor / treatment group by Mann Whitney test . Box plots indicate median , Q1 / Q3 and max / min value whiskers .

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Effects of DC and Beva treatments on Ren13, Ren86, Ren28 and Ren50-PDOX. Quantification of tumor weight in control and treated tumors from 4-5 samples/tumor/treatment group by Mann Whitney test. Box plots indicate median, Q1/Q3 and max/min value whiskers.


Effects of DC and Beva treatments on Ren13 , Ren86 , Ren28 and Ren50 - PDOX . Quantification of vessel number in control and treated tumors from 4 - 5 samples / tumor / treatment group by Mann Whitney test . Box plots indicate median , Q1 / Q3 and max / min value whiskers .

Effects of DC and Beva treatments on Ren13, Ren86, Ren28 and Ren50-PDOX. Quantification of vessel number in control and treated tumors from 4-5 samples/tumor/treatment group by Mann Whitney test. Box plots indicate median, Q1/Q3 and max/min value whiskers.


Effects of DC and Beva treatments on Ren13 , Ren86 , Ren28 and Ren50 - PDOX . Quantification of tumor necrosis in control and treated tumors from 4 - 5 samples / tumor / treatment group by Mann Whitney test . Box plots indicate median , Q1 / Q3 and max / min value whiskers .

Effects of DC and Beva treatments on Ren13, Ren86, Ren28 and Ren50-PDOX. Quantification of tumor necrosis in control and treated tumors from 4-5 samples/tumor/treatment group by Mann Whitney test. Box plots indicate median, Q1/Q3 and max/min value whiskers.


Effects of DC and Beva treatments on Ren13 , Ren86 , Ren28 and Ren50 - PDOX . Evaluation of overall survival comparing Control ( black ) , DC101 ( red ) and Beva ( blue ) treated mice from 4 - 13 mice / tumor / treatment group by Mantel - Cox test where * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 .

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Effects of DC and Beva treatments on Ren13, Ren86, Ren28 and Ren50-PDOX. Evaluation of overall survival comparing Control (black), DC101 (red) and Beva (blue) treated mice from 4-13 mice/tumor/treatment group by Mantel-Cox test where *p < 0.05, **p < 0.01 and ***p < 0.001.


Effects of DC and Beva treatment on Ren13 , Ren86 , Ren28 and Ren50 - PDOX on capsular invasion Incidence of Capsular invasion ( stripes ) in each model and treatment . 5 - 8 animals per model and treatment group were analyzed by Chi - square test where * p < 0 . 05 , * * p < 0 . 01 .

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Effects of DC and Beva treatment on Ren13, Ren86, Ren28 and Ren50-PDOX on capsular invasion Incidence of Capsular invasion (stripes) in each model and treatment. 5-8 animals per model and treatment group were analyzed by Chi-square test where *p < 0.05, **p < 0.01.


C ) Effects of DC and Beva treatment on Ren13 , Ren86 , Ren28 and Ren50 - PDOX on tumor front of invasion Representative images of Vimentin staining ( 4X ) , as described in Figure 1 , and box plot quantification of fold - invasion ( ∆ invasion ) of treated vs control tumors indicating median , Q1 / Q3 and max / min value whiskers . 6 - 12 animals per model and treatment group were analyzed by Mann - Whitney test where * p < 0 . 05 , * * p < 0 . 01 .

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C) Effects of DC and Beva treatment on Ren13, Ren86, Ren28 and Ren50-PDOX on tumor front of invasion Representative images of Vimentin staining (4X), as described in Figure 1, and box plot quantification of fold-invasion (∆ invasion) of treated vs control tumors indicating median, Q1/Q3 and max/min value whiskers. 6-12 animals per model and treatment group were analyzed by Mann-Whitney test where *p < 0.05, **p < 0.01.


D ) Effects of sunitinib treatment on Ren13BM and Ren28 on capsular invasion and tumor front invasion . Bar graphs show incidence of capsular invasion ( stripes ) in control and sunitinib treated tumors . 3 - 9 animals per model and treatment group were analyzed by Chi - square test where * p < 0 . 05 , * * p < 0 . 01 . Box plots represent median , Q1 / Q3 and max / min value whiskers of fold - invasion ( ∆ invasion ) of sunitinib - treated vs control tumors . 5 - 8 animals per model and treatment group were analyzed by Mann - Whitney test . * p < 0 . 05 , * * p < 0 . 01 .

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D) Effects of sunitinib treatment on Ren13BM and Ren28 on capsular invasion and tumor front invasion. Bar graphs show incidence of capsular invasion (stripes) in control and sunitinib treated tumors. 3-9 animals per model and treatment group were analyzed by Chi-square test where *p < 0.05, **p < 0.01. Box plots represent median, Q1/Q3 and max/min value whiskers of fold-invasion (∆ invasion) of sunitinib-treated vs control tumors. 5-8 animals per model and treatment group were analyzed by Mann-Whitney test. *p < 0.05, **p < 0.01.


Evaluation of lung micrometastases using HE and vimentin - stained lung sections upon antiangiogenic therapies in Ren13BM Representative images of lung metastasis in Ren13BM upon antiangiogenic therapies .

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Evaluation of lung micrometastases using HE and vimentin-stained lung sections upon antiangiogenic therapies in Ren13BM Representative images of lung metastasis in Ren13BM upon antiangiogenic therapies.


Evaluation of lung micrometastases using HE and vimentin - stained lung sections upon antiangiogenic therapies in Ren13BM and Ren28 . proportion of mice with 0 , 1 , 2 or > 3 metastases ( mets ) per lungs ( * p < 0 . 05 by Mann - Whitney test ) are shown .

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Evaluation of lung micrometastases using HE and vimentin-stained lung sections upon antiangiogenic therapies in Ren13BM and Ren28. proportion of mice with 0, 1, 2 or > 3 metastases (mets) per lungs (*p < 0.05 by Mann-Whitney test) are shown.


Tumor specific ( human ) enriched gene sets in Ren13 and Ren50 Ren - PDOX tumors , after GSEA analysis from RNAseq data . Gene sets most significantly enriched in Ren13 or Ren50 are indicated in the table ( left ) , including their normalized enrichment score ( NES ) , p - value and false discovery rate ( FDR q - value ) . Heatmaps illustrate expression levels of the genes responsible for the enrichment of KRAS signaling up and down in Ren13 and Ren50 , respectively ( right ) .

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Tumor specific (human) enriched gene sets in Ren13 and Ren50 Ren-PDOX tumors, after GSEA analysis from RNAseq data. Gene sets most significantly enriched in Ren13 or Ren50 are indicated in the table (left), including their normalized enrichment score (NES), p-value and false discovery rate (FDR q-value). Heatmaps illustrate expression levels of the genes responsible for the enrichment of KRAS signaling up and down in Ren13 and Ren50, respectively (right).


Validation of candidate gene expression in unique series of 39 RCC patients ( GSE29609 ) , where microvascular invasion ( MVI ) and renal vein involvement ( RV ) are specifically annotated . Progressive invasion relates to the three progressive degrees of invasion toward the vena cava in this series : 1st , no invasion ; 2nd , microvascular invasion ; and 3rd , renal vein involvement . The gene expression is shown for each variable and condition in box plots representing median , Q1 / Q3 and max / min value whiskers , * p < 0 . 05 * * p < 0 . 01 by Mann - Whitney test .

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Validation of candidate gene expression in unique series of 39 RCC patients (GSE29609), where microvascular invasion (MVI) and renal vein involvement (RV) are specifically annotated. Progressive invasion relates to the three progressive degrees of invasion toward the vena cava in this series: 1st, no invasion; 2nd, microvascular invasion; and 3rd, renal vein involvement. The gene expression is shown for each variable and condition in box plots representing median, Q1/Q3 and max/min value whiskers, *p < 0.05 **p < 0.01 by Mann-Whitney test.


Representative images of ALDH1A3 expression on pro - invasive ( Ren13 and Ren86 ) and non - pro - invasive ( Ren28 and Ren50 ) tumors .

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Representative images of ALDH1A3 expression on pro-invasive (Ren13 and Ren86) and non-pro-invasive (Ren28 and Ren50) tumors.


Confirmation of ALDH1A3 mRNA levels of gene expression in pro - invasive ( Ren13 and Ren86 ) and non - pro - invasive ( Ren28 and Ren50 ) tumors . Average ± SD of 4 - 6 independent replicates per group were analyzed by Mann - Whitney test * p < 0 . 05 * * p < 0 . 01 .

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Confirmation of ALDH1A3 mRNA levels of gene expression in pro-invasive (Ren13 and Ren86) and non-pro-invasive (Ren28 and Ren50) tumors. Average ± SD of 4-6 independent replicates per group were analyzed by Mann-Whitney test *p < 0.05 **p < 0.01.


Association of pre - treatment ALDH1A3 protein levels by IHC on a series of Ren - PDOX models treated with sunitinib . ( C ) Representation of ALDH1A3 expression on pro - invasive and non - pro - invasive Ren - PDOX after suntinib treatment ( * * p < 0 . 01 by Chi - Square test ) .

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Association of pre-treatment ALDH1A3 protein levels by IHC on a series of Ren-PDOX models treated with sunitinib. (C) Representation of ALDH1A3 expression on pro-invasive and non-pro-invasive Ren-PDOX after suntinib treatment (**p < 0.01 by Chi-Square test).


D ) Association of pre - treatment ALDH1A3 protein levels by IHC on a series of Ren - PDOX models treated with sunitinib . ( D ) Correlation of pre - treatment ALDH1A3 protein levels by IHC with increased tumor invasion found after sunitinib treatment . n = 7 , Spearman ' s non - parametric correlation , p = 0 . 0095 .

D) Association of pre-treatment ALDH1A3 protein levels by IHC on a series of Ren-PDOX models treated with sunitinib. (D) Correlation of pre-treatment ALDH1A3 protein levels by IHC with increased tumor invasion found after sunitinib treatment. n=7, Spearman's non-parametric correlation, p=0.0095.


F ) Representation of ALDH1A3 score ( 0 = absent , 1 = low , 2 = high expression ) on pre - treatment samples of pro - aggressive and non - pro - aggressive ccRCC patients described above . n = 15 , Chi - square test for independence p = 0 . 015 , and Chi - square test for trend p = 0 . 030 ( * ) .

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F) Representation of ALDH1A3 score (0=absent, 1=low, 2=high expression) on pre-treatment samples of pro-aggressive and non-pro-aggressive ccRCC patients described above. n=15, Chi-square test for independence p=0.015, and Chi-square test for trend p=0.030 (*).


Representative fluorescence microscopy images of total , CD142 - and CD142 + ASPCs isolated with the use of the respective anti - CD142 antibodies : SinoBiological ( SB ) , SinoBiological - LL ( SB - LL ) , BiOrbyt ( BO ) and R & D Systems ( RnD ) ( Appendix Fig . S2B , Appendix Fig . S5A , Methods ) , after in vitro adipogenic differentiation ;

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Representative fluorescence microscopy images of total, CD142- and CD142+ ASPCs isolated with the use of the respective anti-CD142 antibodies: SinoBiological (SB), SinoBiological-LL (SB-LL), BiOrbyt (BO) and R&D Systems (RnD) (Appendix Fig. S2B, Appendix Fig. S5A, Methods), after in vitro adipogenic differentiation;


Fraction of differentiated cells per ASPC type shown in E , as quantified by the " adiposcore " ; marker shapes correspond to different anti - CD142 antibodies used for isolation as indicated , n = 9 - 15 , 3 - 4 biological replicates , 3 - 5 independent wells for each ;

Fraction of differentiated cells per ASPC type shown in E, as quantified by the "adiposcore"; marker shapes correspond to different anti-CD142 antibodies used for isolation as indicated, n=9-15, 3-4 biological replicates, 3-5 independent wells for each;


Representative fluorescence microscopy images of CD142 - , 20 % CD142 + and ( 5 - 7 % ) CD142 + ASPCs isolated following the sorting strategy shown in Appendix Fig . S7A , after in vitro adipogenic differentiation with the indicated white adipogenic differentiation cocktails ( Methods ) ;

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Representative fluorescence microscopy images of CD142-, 20% CD142+ and (5-7%) CD142+ ASPCs isolated following the sorting strategy shown in Appendix Fig. S7A, after in vitro adipogenic differentiation with the indicated white adipogenic differentiation cocktails (Methods);


Fraction of differentiated cells per ASPC type and differentiation cocktail shown in J , as quantified by the " adipo - score " ; marker shapes correspond to different biological replicates , n = 8 - 17 , 3 - 5 biological replicates , 2 - 5 independent wells for each ;

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Fraction of differentiated cells per ASPC type and differentiation cocktail shown in J, as quantified by the "adipo- score"; marker shapes correspond to different biological replicates, n=8-17, 3-5 biological replicates, 2-5 independent wells for each;


Representative fluorescence microscopy images of male - and female - derived total , CD142 - and CD142 + ASPCs , isolated following the sorting strategy shown in Appendix Fig . S8A after in vitro adipogenic differentiation ; Fraction of differentiated cells per ASPC type and per sex shown in L , as quantified by the " adiposcore " ; bar colour shading corresponds to male - and female - derived cells as indicated ; marker shapes correspond to different biological replicates , n = 9 - 10 , 4 biological replicates , 2 - 3 independent wells for each ;

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Representative fluorescence microscopy images of male- and female-derived total, CD142- and CD142+ ASPCs, isolated following the sorting strategy shown in Appendix Fig. S8A after in vitro adipogenic differentiation;   Fraction of differentiated cells per ASPC type and per sex shown in L, as quantified by the "adiposcore"; bar colour shading corresponds to male- and female-derived cells as indicated; marker shapes correspond to different biological replicates, n=9-10, 4 biological replicates, 2-3 independent wells for each;


Representative fluorescence microscopy images of P0 - , P12 - 17 - , 4wo - , 7wo - and 11wo - derived total , CD142 - and CD142 + APSCs after in vitro adipogenic differentiation ; Fraction of differentiated cells per ASPC type shown in D , as quantified by the " adiposcore " ; bar colour shading corresponds to pre - and post - weaning - derived cells as indicated ; marker shapes correspond to different biological replicates , n = 14 - 65 , 12 - 13 biological replicates , 2 - 6 independent wells for each

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Representative fluorescence microscopy images of P0-, P12-17-, 4wo-, 7wo- and 11wo-derived total, CD142- and CD142+ APSCs after in vitro adipogenic differentiation;   Fraction of differentiated cells per ASPC type shown in D, as quantified by the "adiposcore"; bar colour shading corresponds to pre- and post-weaning-derived cells as indicated; marker shapes correspond to different biological replicates, n=14-65, 12-13 biological replicates, 2-6 independent wells for each


Representative fluorescence microscopy images of CD142 - ASPCs co - cultured with an empty transwell ( T - well ) or with wild - type ( WT ) CD142 + ASPCs after in vitro adipogenic differentiation ; Fraction of differentiated CD142 - ASPCs co - cultured with an empty transwell ( T - well ) or with wild - type CD142 + ASPCs ( non - treated or treated with control scrambled siRNA ) after in vitro adipogenic differentiation and as quantified by the " adiposcore " ; marker shapes correspond to different biological replicates , n = 10 , 4 biological replicates , 2 - 3 independent wells for each

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Representative fluorescence microscopy images of CD142- ASPCs co-cultured with an empty transwell (T-well) or with wild-type (WT) CD142+ ASPCs after in vitro adipogenic differentiation;   Fraction of differentiated CD142- ASPCs co-cultured with an empty transwell (T-well) or with wild-type CD142+ ASPCs (non-treated or treated with control scrambled siRNA) after in vitro adipogenic differentiation and as quantified by the "adiposcore"; marker shapes correspond to different biological replicates, n=10, 4 biological replicates, 2-3 independent wells for each


Representative fluorescence microscopy imaged of human - derived stromal vascular fraction ( SVF ) , containing the ASPCs , after in vitro adipogenic differentiation when co - cultured with total , CD142 - or CD142 + mouse ASPCs ; Fraction of differentiated human - derived SVF , containing the ASPCs co - cultured with total , CD142 - or CD142 + mouse ASPCs after in vitro adipogenic differentiation ( Methods ) shown in E ; marker shapes correspond to different biological replicates , n = 9 , 3 biological replicates , 3 independent wells for each

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Representative fluorescence microscopy imaged of human-derived stromal vascular fraction (SVF), containing the ASPCs, after in vitro adipogenic differentiation when co-cultured with total, CD142- or CD142+ mouse ASPCs;   Fraction of differentiated human-derived SVF, containing the ASPCs co-cultured with total, CD142- or CD142+ mouse ASPCs after in vitro adipogenic differentiation (Methods) shown in E; marker shapes correspond to different biological replicates, n=9, 3 biological replicates, 3 independent wells for each


Fraction of differentiated adult - derived CD142 - ASPCs , as quantified by the " adiposcore " , treated with the indicated recombinant proteins shown in J ; marker shapes correspond to different biological replicates , n = 11 - 59 , 2 - 10 biological replicates , 3 - 9 independent wells for

Fraction of differentiated adult-derived CD142- ASPCs, as quantified by the "adiposcore", treated with the indicated recombinant proteins shown in J; marker shapes correspond to different biological replicates, n=11-59, 2-10 biological replicates, 3-9 independent wells for each


Representative fluorescence microscopy images of adult - derived CD142 - ASPCs after in vitro adipogenic differentiation ; the induction cocktail was supplemented with recombinant proteins corresponding to the selected Areg - specific candidates : CD142 , MGP , GDF10 , CLEC11A , CPE and BGN at 100 ng /

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Representative fluorescence microscopy images of adult-derived CD142- ASPCs after in vitro adipogenic differentiation; the induction cocktail was supplemented with recombinant proteins corresponding to the selected Areg-specific candidates: CD142, MGP, GDF10, CLEC11A, CPE and BGN at 100 ng/m


Representative fluorescence microscopy images of CD142 - ASPCs , co - cultured with CD142 + ASPCs ( Aregs ) carrying control ( scr ) or siRNA - mediated knockdowns of selected CD142 + ASPC ( Areg ) - specific candidate genes : F3 , Mgp , and Gdf10 after adipogenic differentiation ( i . e . , after exposure to an adipogenic cocktail , Methods )

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Representative fluorescence microscopy images of CD142- ASPCs, co-cultured with CD142+ ASPCs (Aregs) carrying control (scr) or siRNA-mediated knockdowns of selected CD142+ ASPC (Areg)-specific candidate genes: F3, Mgp, and Gdf10 after adipogenic differentiation (i.e., after exposure to an adipogenic cocktail, Methods)


Fraction of differentiated CD142 - ASPCs , as quantified by the " adiposcore " , co - cultured with CD142 + ASPCs ( Aregs ) subjected to specific knockdowns ( or scrambled ( scr ) siRNA control ) , as indicated in A ; marker shapes correspond to different biological replicates , n = 10 , 4 biological replicates , 2 - 3 independent wells for each ;

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Fraction of differentiated CD142- ASPCs, as quantified by the "adiposcore", co-cultured with CD142+ ASPCs (Aregs) subjected to specific knockdowns (or scrambled (scr) siRNA control), as indicated in A; marker shapes correspond to different biological replicates, n=10, 4 biological replicates, 2-3 independent wells for each;


Representative fluorescence microscopy images of CD142 - ASPCs , co - cultured with CD142 + ASPCs ( Aregs ) ( in the top well ) subjected to the indicated compounds at the indicated concentration ; AGN and BMS are antagonists of RAR , DEAB is an antagonist of RALDH enzymes which convert retinaldehyde to RA , and HX is an antagonist of RXR ; Fraction of differentiated CD142 - ASPCs indicated in G , as quantified by the " adiposcore " ; marker shapes correspond to different biological replicates , n = 4 - 15 , 2 - 5 biological replicates , 2 - 3 independent wells for each ;

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

Representative fluorescence microscopy images of CD142- ASPCs, co-cultured with CD142+ ASPCs (Aregs) (in the top well) subjected to the indicated compounds at the indicated concentration; AGN and BMS are antagonists of RAR, DEAB is an antagonist of RALDH enzymes which convert retinaldehyde to RA, and HX is an antagonist of RXR;   Fraction of differentiated CD142- ASPCs indicated in G, as quantified by the "adiposcore"; marker shapes correspond to different biological replicates, n=4-15, 2-5 biological replicates, 2-3 independent wells for each;


Representative fluorescence microscopy images of CD142 - ASPCs , co - cultured with CD142 + ASPCs ( Aregs ) subjected to control ( scr ) or siRNA - mediated knockdowns of selected RA signalling pathway genes : Rara , Rarb , Rarg , Rbp1 , and Rxrg after adipogenic differentiation ( i . e . , after exposure to an adipogenic cocktail , Methods ) ; Fraction of differentiated CD142 - ASPCs indicated in I , as quantified by the " adiposcore " ; marker shapes correspond to different biological replicates , n = 24 - 29 , 4 biological replicates , 5 - 8 independent wells for each .

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

Representative fluorescence microscopy images of CD142- ASPCs, co-cultured with CD142+ ASPCs (Aregs) subjected to control (scr) or siRNA-mediated knockdowns of selected RA signalling pathway genes: Rara, Rarb, Rarg, Rbp1, and Rxrg after adipogenic differentiation (i.e., after exposure to an adipogenic cocktail, Methods);   Fraction of differentiated CD142- ASPCs indicated in I, as quantified by the "adiposcore"; marker shapes correspond to different biological replicates, n=24-29, 4 biological replicates, 5-8 independent wells for each.


Phenotypic changes of HSCs by citral . Phase - contrast images ( left ) , oil red O - stained images ( middle ) , and autofluorescence images ( right ) are shown for HSCs after passage 1 with or without the treatment of citral ( 100 μM ) for 24 h . Scale bar = 10 μm .

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Phenotypic changes of HSCs by citral. Phase-contrast images (left), oil red O-stained images (middle), and autofluorescence images (right) are shown for HSCs after passage 1 with or without the treatment of citral (100 μM) for 24 h. Scale bar = 10 μm.


Western blot analysis of cell lysates from the citral - treated HSCs in ( A ) . α - tubulin was used as a loading control . The quantitative densitometric data are expressed as the percentage of untreated control and represent the means ( SD ) for three independent experiments . * * P - value = 0 . 002 , paired t - test ( compared to control ) .

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Western blot analysis of cell lysates from the citral-treated HSCs in (A). α-tubulin was used as a loading control. The quantitative densitometric data are expressed as the percentage of untreated control and represent the means (SD) for three independent experiments. **P-value = 0.002, paired t-test (compared to control).


Protein expression of ALDH1A1 and ALDH1A2 during the culture activation of HSCs . Cell lysates were prepared from HSCs at 3 days after plating ( HSCs - d3 ) and from HSCs after passage 1 ( P1 ) , 2 ( P2 ) , and 3 ( P3 ) , and analyzed by Western blotting .

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Protein expression of ALDH1A1 and ALDH1A2 during the culture activation of HSCs. Cell lysates were prepared from HSCs at 3 days after plating (HSCs-d3) and from HSCs after passage 1 (P1), 2 (P2), and 3 (P3), and analyzed by Western blotting.


Real - time PCR analysis for the expression levels of ALDH1A1 and ALDH1A2 during the culture activation of HSCs . The PCR data are expressed as the percentage of HSCs - d3 . * * P - value , paired t - test ( n = 3 ) ( compared to HSCs - d3 ) , ALDH1A1 / HSCs - P3 : 0 . 003 , ALDH1A2 / HSCs - P1 : 0 . 0018 , ALDH1A2 / HSCs - P3 : 0 . 001 .

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Real-time PCR analysis for the expression levels of ALDH1A1 and ALDH1A2 during the culture activation of HSCs. The PCR data are expressed as the percentage of HSCs-d3. **P-value, paired t-test (n = 3) (compared to HSCs-d3), ALDH1A1/HSCs-P3: 0.003, ALDH1A2/HSCs-P1: 0.0018, ALDH1A2/HSCs-P3: 0.001.


Effect of ALDH1A suppression on HSC activation . HSCs - P1 were transfected with non - targeting siRNA or siRNA specific to ALDH1A1 and / or ALDH1A2 , and after 48 h , whole - cell lysates were subjected to Western blotting . * * P = 0 . 0023 , paired t - test ( n = 3 ) ( siRNA - ALDH1A1 / 2 compared to control ) .

Effect of ALDH1A suppression on HSC activation. HSCs-P1 were transfected with non-targeting siRNA or siRNA specific to ALDH1A1 and/or ALDH1A2, and after 48 h, whole-cell lysates were subjected to Western blotting. **P = 0.0023, paired t-test (n = 3) (siRNA-ALDH1A1/2 compared to control).


Reverse - phase HPLC analysis of whole - cell lysates from the siRNA - ALDH1A - treated HSCs in ( E ) . Typical chromatograms of all - trans retinoic acid and 13 - cis retinoic acid . The retention time is 14 min for 13 - cis - RA and 23 min for all - trans RA .

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Reverse-phase HPLC analysis of whole-cell lysates from the siRNA-ALDH1A-treated HSCs in (E). Typical chromatograms of all-trans retinoic acid and 13-cis retinoic acid. The retention time is 14 min for 13-cis-RA and 23 min for all-trans RA.


Phenotypic changes of HSCs by RAR antagonist . HSCs - d3 were incubated with AGN193109 ( 1 μM ) ± all - trans RA ( 10 μM ) for 3 days and subjected to Oil red O staining . Scale bar = 10 μm .

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

Phenotypic changes of HSCs by RAR antagonist. HSCs-d3 were incubated with AGN193109 (1 μM) ± all-trans RA (10 μM) for 3 days and subjected to Oil red O staining. Scale bar = 10 μm.


Western blot analysis of cell lysates from AGN193109 ± RA - treated HSCs in ( G ) . * * P = 0 . 003 , paired t - test ( n = 3 ) ( AGN193109 compared to control ) .

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Western blot analysis of cell lysates from AGN193109 ± RA-treated HSCs in (G). **P = 0.003, paired t-test (n = 3) (AGN193109 compared to control).


Albumin and R - III downregulate RA signaling . HSCs - P1 were either transfected with RARE Cignal reporter and expression plasmid containing vehicle or albumin ( left panel ) or treated with R - III ( 25 - 250 nM ) after transfection with RARE Cignal reporter ( right panel ) . After 36 h , the transfected cells were harvested for luciferase assay . Renilla luciferase activity was normalized to firefly luciferase activity . Ratios were normalized against the cells transfected with vehicle plasmid . * * P - value , two - sample t - test ( n = 9 ) ( compared to control ) , albumin : 0 . 0037 , R - III 125 nM : 0 . 0032 .

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Albumin and R-III downregulate RA signaling. HSCs-P1 were either transfected with RARE Cignal reporter and expression plasmid containing vehicle or albumin (left panel) or treated with R-III (25-250 nM) after transfection with RARE Cignal reporter (right panel). After 36 h, the transfected cells were harvested for luciferase assay. Renilla luciferase activity was normalized to firefly luciferase activity. Ratios were normalized against the cells transfected with vehicle plasmid. **P-value, two-sample t-test (n = 9) (compared to control), albumin: 0.0037, R-III 125 nM: 0.0032.


Phenotypic changes of HSCs by the expression of albumin and / or ALDH1A . HSCs - P1 were transfected with plasmids encoding albumin , ALDH1A1 , and ALDH1A2 , either individually or in combination , and incubated in the presence of all - trans retinoic acid ( 50 nM ) for 18 h . Cells were analyzed by oil red O staining . Scale bar , 20 μm .

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Phenotypic changes of HSCs by the expression of albumin and/or ALDH1A. HSCs-P1 were transfected with plasmids encoding albumin, ALDH1A1, and ALDH1A2, either individually or in combination, and incubated in the presence of all-trans retinoic acid (50 nM) for 18 h. Cells were analyzed by oil red O staining. Scale bar, 20 μm.


Western blot analysis of cell lysates from albumin - and / or ALDH1A - transfected HSCs as in ( B ) . * * P = 0 . 002 , paired t - test ( n = 3 ) ( albumin compared to control ) .

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

Western blot analysis of cell lysates from albumin- and/or ALDH1A-transfected HSCs as in (B). **P = 0.002, paired t-test (n = 3) (albumin compared to control).


Reverse - phase HPLC analysis of whole - cell lysates from albumin - and / or ALDH1A - transfected HSCs as in ( B ) .

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

Reverse-phase HPLC analysis of whole-cell lysates from albumin- and/or ALDH1A-transfected HSCs as in (B).


RAR agonist counteracts R - III action . HSCs - P1 were treated with His - tagged R - III ( final concentration , 0 . 15 μM ) in the presence or absence of RAR agonist ( AGN191183 , 1 μM ) for 18 h and analyzed by Western blotting . * * P = 0 . 0024 , paired t - test ( n = 3 ) ( R - III compared to control ) . The separating lines demarcate bands that come from non - consecutive lanes of the same gel .

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

RAR agonist counteracts R-III action. HSCs-P1 were treated with His-tagged R-III (final concentration, 0.15 μM) in the presence or absence of RAR agonist (AGN191183, 1 μM) for 18 h and analyzed by Western blotting. **P = 0.0024, paired t-test (n = 3) (R-III compared to control). The separating lines demarcate bands that come from non-consecutive lanes of the same gel.


Protein expression of STRA6 during the culture activation of HSCs . Cell lysates were prepared from HSCs at 3 days after plating ( HSCs - 3d ) , and from HSCs after passage 1 ( P1 ) , 2 ( P2 ) , and 3 ( P3 ) , and analyzed by Western blotting . Lysates from NIH3T3 cells were used as a negative control . The separating lines demarcate bands that come from non - consecutive lanes of the same gel .

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Protein expression of STRA6 during the culture activation of HSCs. Cell lysates were prepared from HSCs at 3 days after plating (HSCs-3d), and from HSCs after passage 1 (P1), 2 (P2), and 3 (P3), and analyzed by Western blotting. Lysates from NIH3T3 cells were used as a negative control. The separating lines demarcate bands that come from non-consecutive lanes of the same gel.


Real - time PCR analysis for the expression level of STRA6 during the culture activation of HSCs . The PCR data are expressed as the percentage of HSCs - 3d . * * P - value , paired t - test ( n = 3 ) ( compared to HSCs - d3 ) , HSCs - P2 : 0 . 002 , HSCs - P3 : 0 . 0022 .

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Real-time PCR analysis for the expression level of STRA6 during the culture activation of HSCs. The PCR data are expressed as the percentage of HSCs-3d. **P-value, paired t-test (n = 3) (compared to HSCs-d3), HSCs-P2: 0.002, HSCs-P3: 0.0022.


R - III uptake parallels STRA6 level . HSCs at different stages of activation were incubated with His - tagged R - III ( final concentration , 0 . 15 μM ) for 30 min , washed , and subjected to Western blotting for R - III uptake .

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R-III uptake parallels STRA6 level. HSCs at different stages of activation were incubated with His-tagged R-III (final concentration, 0.15 μM) for 30 min, washed, and subjected to Western blotting for R-III uptake.


STRA6 - mediated uptake of R - III . HSCs - P1 were transfected with non - targeting siRNA or one of the two different STRA6 - specific siRNAs , and after 48 h , cells were incubated with R - III for 30 min and analyzed by Western blotting .

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STRA6-mediated uptake of R-III. HSCs-P1 were transfected with non-targeting siRNA or one of the two different STRA6-specific siRNAs, and after 48 h, cells were incubated with R-III for 30 min and analyzed by Western blotting.


Representative macroscopic pictures of livers from control and CCl4 - , CCl4 / albumin - , CCl4 / RBP - , and CCl4 / R - III - treated mice .

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Representative macroscopic pictures of livers from control and CCl4-, CCl4/albumin-, CCl4/RBP-, and CCl4/R-III-treated mice.


Liver sections were stained with H & amp ; amp ; E and Sirius red , and also subjected to immunohistochemistry for type I collagen . Scale bar , 200 μm .

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Liver sections were stained with H&amp;E and Sirius red, and also subjected to immunohistochemistry for type I collagen. Scale bar, 200 μm.


Hydroxyproline content in the livers from control and CCl4 - , CCl4 / albumin - , and CCl4 / R - III - treated mice ( μg / g liver ) . * P = 0 . 037 , two - sample t - test ( n = 10 ) ( CCl4 + R - III compared to CCl4 - treated mice ) .

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Hydroxyproline content in the livers from control and CCl4-, CCl4/albumin-, and CCl4/R-III-treated mice (μg/g liver). *P = 0.037, two-sample t-test (n = 10) (CCl4 + R-III compared to CCl4-treated mice).


Western blot analysis on liver extracts from the treated mice with antibody against type I collagen .

Western blot analysis on liver extracts from the treated mice with antibody against type I collagen.


Immunohistochemical analysis of α - SMA on representative liver sections from the treated mice . Scale bar , 200 μm .

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Immunohistochemical analysis of α-SMA on representative liver sections from the treated mice. Scale bar, 200 μm.


Liver sections from control and CCl4 - and CCl4 / R - III - treated mice were immunohistochemically stained for TGF - β and F4 / 80 . Scale bar , 50 μm .

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Liver sections from control and CCl4- and CCl4/R-III-treated mice were immunohistochemically stained for TGF-β and F4/80. Scale bar, 50 μm.


Liver sections from treated mice were immunohistochemically stained for desmin , His - tag , and α - SMA . His - positive signals largely overlapped with desmin / α - SMA staining . Scale bar , 50 μm .

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Liver sections from treated mice were immunohistochemically stained for desmin, His-tag, and α-SMA. His-positive signals largely overlapped with desmin/α-SMA staining. Scale bar, 50 μm.


Liver sections from control and CCl4 - and CCl4 / R - III - treated mice were stained with Sirius red . Scale bar , 200 μm .

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Liver sections from control and CCl4- and CCl4/R-III-treated mice were stained with Sirius red. Scale bar, 200 μm.


Hydroxyproline content in the livers ( μg / g liver ) . * P = 0 . 034 , two - sample t - test ( n = 10 ) ( CCl4 + R - III compared to CCl4 - treated mice ) .

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Hydroxyproline content in the livers (μg/g liver). *P = 0.034, two-sample t-test (n = 10) (CCl4 + R-III compared to CCl4-treated mice).


Liver sections from control , BDL , and BDL / R - III - treated mice were stained with Sirius red . Scale bar , 200 μm . .

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Liver sections from control, BDL, and BDL/R-III-treated mice were stained with Sirius red. Scale bar, 200 μm..


Hydroxyproline content in the livers ( μg / g liver ) . * P - value , two - sample t - test ( n = 10 ) ( compared to BDL - treated mice ) ; BDL + R - III ( 10 μg ) : 0 . 031 , BDL + R - III ( 5 μg ) : 0 . 026 .

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Hydroxyproline content in the livers (μg/g liver). *P-value, two-sample t-test (n = 10) (compared to BDL-treated mice); BDL + R-III (10 μg): 0.031, BDL+R-III (5 μg): 0.026.


( A ) Summary of HMCES recovery upon TAP - MS analyses performed with 293T cells stably expressing SFB - PCNA . Cells were treated with CPT : camptothecin ( 1 μg / ml for 6 h ) ; HU : hydroxyurea ( 2 mM for 16 h ) , UV : ultraviolet light irradiation ( 10 mJ / cm2 harvested after 6 h post - irradiation ) or Thymidine ( 100 mM for 6 h ) . Recovered peptide - spectrum matches ( PSMs ) for PCNA and HMCES are indicated for each condition .

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(A) Summary of HMCES recovery upon TAP-MS analyses performed with 293T cells stably expressing SFB-PCNA. Cells were treated with CPT: camptothecin (1 μg/ml for 6 h); HU: hydroxyurea (2 mM for 16 h), UV: ultraviolet light irradiation (10 mJ/cm2 harvested after 6 h post-irradiation) or Thymidine (100 mM for 6 h). Recovered peptide-spectrum matches (PSMs) for PCNA and HMCES are indicated for each condition.


( C ) Immunoprecipitation ( IP ) of SFB - tagged full - length ( FL ) HMCES and indicated HMCES deletion mutants was conducted to decipher the region responsible for PCNA interaction .

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(C) Immunoprecipitation (IP) of SFB-tagged full-length (FL) HMCES and indicated HMCES deletion mutants was conducted to decipher the region responsible for PCNA interaction.


( D ) GST - PCNA pulldown assay was used to evaluate the interaction between PCNA and full - length HMCES or indicated HMCES deletion mutants .

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(D) GST-PCNA pulldown assay was used to evaluate the interaction between PCNA and full-length HMCES or indicated HMCES deletion mutants.


( E ) aniPOND - Western blotting analysis revealed the presence of HMCES on replication fork . Cells were pulsed with EdU for 10 mins , and click - reaction was performed . In thymidine chase samples , cells were incubated with thymidine for another 30 mins prior to click - reaction .

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(E) aniPOND-Western blotting analysis revealed the presence of HMCES on replication fork. Cells were pulsed with EdU for 10 mins, and click-reaction was performed. In thymidine chase samples, cells were incubated with thymidine for another 30 mins prior to click-reaction.


( F ) Analysis of HMCES level throughout the cell cycle . Cells were synchronized by double thymidine block and released for the indicated time points . CYCLIN E and CYCLIN B1 were used as markers for cell cycle phases .

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(F) Analysis of HMCES level throughout the cell cycle. Cells were synchronized by double thymidine block and released for the indicated time points. CYCLIN E and CYCLIN B1 were used as markers for cell cycle phases.


( A ) Western blotting analysis confirmed HMCES knockout in 293A cells ( 293A HMCES KO ) . Blots also included knockout cells reconstituted with WT SFB - HMCES .

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(A) Western blotting analysis confirmed HMCES knockout in 293A cells (293A HMCES KO). Blots also included knockout cells reconstituted with WT SFB- HMCES.


( B , C ) Colony - forming capacity of HMCES KO cells was compared to that of control wild - type 293A cells . ( C ) Box plot showing colony forming efficiencies of 293A wild - type control and HMCES KO cells . Box limits represent 25th and 75th percentile , centerline shows the median and whiskers extend minimum to maximum . Data include more than three independent repeats performed in duplicates . Student ' s t - test were performed for statistical analysis ( * * * * , p < 0 . 0001 ) .

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(B, C) Colony-forming capacity of HMCES KO cells was compared to that of control wild-type 293A cells. (C) Box plot showing colony forming efficiencies of 293A wild-type control and HMCES KO cells. Box limits represent 25th and 75th percentile, centerline shows the median and whiskers extend minimum to maximum. Data include more than three independent repeats performed in duplicates. Student's t-test were performed for statistical analysis (****, p<0.0001).


( D ) Colonogenic survival assay of 293A wild - type control and HMCES KO cells treated with various concentrations of hydroxyurea ( HU ) was shown . Seeding density with respect to control was indicated below each image . The images shown are representative of three biological repeats , each performed in duplicate .

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(D) Colonogenic survival assay of 293A wild-type control and HMCES KO cells treated with various concentrations of hydroxyurea (HU) was shown. Seeding density with respect to control was indicated below each image. The images shown are representative of three biological repeats, each performed in duplicate.


( E ) Quantification showing survival fraction of wild - type control ( WT ) and HMCES KO cells upon HU treatment . Data are presented as mean ± s . e . m ( n = 3 , each performed in duplicates ) and student ' s t - test were performed for statistical analysis ( ns , not significant ; * , p < 0 . 05 ) .

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(E) Quantification showing survival fraction of wild-type control (WT) and HMCES KO cells upon HU treatment. Data are presented as mean±s.e.m (n=3, each performed in duplicates) and student's t-test were performed for statistical analysis (ns, not significant; *, p<0.05).


( F ) Quantification of colonogenic survival assay upon exposure of WT and HMCES KO cells to ionizing radiation ( IR ) . Data are presented as mean ± s . e . m . Quantifications are from three biological repeats performed in duplicates . Student ' s t - test was performed for statistical analysis ( * , p < 0 . 05 ; * * , p < 0 . 01 ) .

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(F) Quantification of colonogenic survival assay upon exposure of WT and HMCES KO cells to ionizing radiation (IR). Data are presented as mean±s.e.m. Quantifications are from three biological repeats performed in duplicates. Student's t-test was performed for statistical analysis (*, p<0.05; **, p<0.01).


( G , H ) Sensitivity assay and quantification of surviving WT control and HMCES KO cells determined by colony - forming assay upon exposure to H2O2 . Experiments were repeated three independent times , each time performed in duplicates and data are presented as mean ± s . e . m . Statistical significance for the corresponding experiment was analyzed by student ' s t - test ( * , p < 0 . 05 ) .

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(G, H) Sensitivity assay and quantification of surviving WT control and HMCES KO cells determined by colony-forming assay upon exposure to H2O2. Experiments were repeated three independent times, each time performed in duplicates and data are presented as mean±s.e.m. Statistical significance for the corresponding experiment was analyzed by student's t-test (*, p<0.05).


( I ) Bar graph showing sensitivity of WT , HMCES KO and stably reconstituted WT cells towards H2O2 treatment . Experiment was repeated two independent times with technical repeats .

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(I) Bar graph showing sensitivity of WT, HMCES KO and stably reconstituted WT cells towards H2O2 treatment. Experiment was repeated two independent times with technical repeats.


( A , B ) Colonogenic assays were performed by adding increasing concentrations of pemetrexed to control wild - type ( WT ) 293A cells and HMCES knockout ( HMCES KO ) cells ( A ) and quantification of results were shown ( B ) . Increasing numbers of cells were seeded with increasing concentrations of the compound to obtain optimal drug - dose response curve . Seeding density with respect to control is indicated below each image . The images shown are representative of three biological repeats , each performed in duplicate . ( B ) Data are presented as mean ± s . e . m . and student ' s t - test was performed for statistical analysis ( * * * * , p < 0 . 0001 ) .

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(A, B) Colonogenic assays were performed by adding increasing concentrations of pemetrexed to control wild-type (WT) 293A cells and HMCES knockout (HMCES KO) cells (A) and quantification of results were shown (B). Increasing numbers of cells were seeded with increasing concentrations of the compound to obtain optimal drug-dose response curve. Seeding density with respect to control is indicated below each image. The images shown are representative of three biological repeats, each performed in duplicate. (B) Data are presented as mean±s.e.m. and student's t-test was performed for statistical analysis (****, p<0.0001).


( C ) Western blot showing APEX2 levels upon 293A cells infected with viruses encoding scrambled or two independent APEX2 shRNAs .

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(C) Western blot showing APEX2 levels upon 293A cells infected with viruses encoding scrambled or two independent APEX2 shRNAs.


( D ) Colony forming assay was performed to indicate colony forming efficiency in 293A and HMCES KO cells upon infection with viruses encoding scrambled or APEX2 shRNAs .

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(D) Colony forming assay was performed to indicate colony forming efficiency in 293A and HMCES KO cells upon infection with viruses encoding scrambled or APEX2 shRNAs.


( E ) Sensitivity assay was conducted with WT and HMCES KO cells upon treatment with AP Endonuclease inhibitor III ( APEi ) . Results are presented from two independent experiments and data are presented as mean ± s . e . m .

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(E) Sensitivity assay was conducted with WT and HMCES KO cells upon treatment with AP Endonuclease inhibitor III (APEi). Results are presented from two independent experiments and data are presented as mean±s.e.m.


( F ) Colony forming assay was performed with WT and HMCES KO cells upon either pemetrexed treatment alone or in combination with APEi . Data are presented as mean ± s . e . m ( n = 2 each with technical repeats ) .

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(F) Colony forming assay was performed with WT and HMCES KO cells upon either pemetrexed treatment alone or in combination with APEi. Data are presented as mean±s.e.m (n=2 each with technical repeats).