a , Sections of thymus muscle of fed starved GFP - LC3 mice were stained with anti - GFP Alexa 488 antibody ( green ) 4 , 6 - diamidino - 2 - phenylindole ( DAPI ; blue ) . are representative of n = 6 .

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a, Sections of the thymus and muscle of fed and starved GFP-LC3 mice were stained with anti-GFP Alexa 488 antibody (green) and 4,6-diamidino-2-phenylindole (DAPI; blue). Images are representative of n = 6.


b , Thymic stromal from GFP - LC3 mice were fixed onto glass slides stained with anti - GFP Alexa 488 DAPI . Typical autophagy - positive cTECs mTEChi are shown . average frequencies ± s . d . of cTECs mTEChi scoring autophagy positive ( that is , ≥ 5 autophagosomes ) in two independent preparations are indicated . Less than 1 % of dendritic ( DCs ) mTEClo were positive .

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

b, Thymic stromal cells from GFP-LC3 mice were fixed onto glass slides and stained with anti-GFP Alexa 488 and DAPI. Typical autophagy-positive cTECs and mTEChi are shown. The average frequencies ± s.d. of cTECs and mTEChi scoring autophagy positive (that is, ≥5 autophagosomes) in two independent preparations are indicated. Less than 1% of dendritic cells (DCs) and mTEClo were positive.


c , Relative distribution of number of autophagosomes in autophagy - positive cTECs mTEChi ; a . u . , arbitrary units .

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c, Relative distribution of the number of autophagosomes in autophagy-positive cTECs and mTEChi; a.u., arbitrary units.


a , Thymi from wild - type ( WT ) or Atg5 - / - were transplanted under kidney capsule of wild - type recipients analysed 6 later . average cellularities ± s . d . are shown ; n = 5 .

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a, Thymi from wild-type (WT) or Atg5-/- embryos were transplanted under the kidney capsule of wild-type recipients and analysed 6 weeks later. The average cellularities ± s.d. are shown; n = 5.


b , Normal compartmentalization of Atg5 - / - grafts in cytokeratin - 8 - positive ( red ; K8 ) cortical cytokeratin - 5 - positive ( green ; K5 ) medullary regions ; n = 8 .

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b, Normal compartmentalization of Atg5-/- grafts in cytokeratin-8-positive (red; K8) cortical and cytokeratin-5-positive (green; K5) medullary regions; n = 8.


c , Expression of MHC class I II on cTECs or mTECs from Atg5 - / - to wild - type ( WT ) grafts ( red ) , or wild - type to wild - type grafts ( blue ) . controls are depicted in grey ; data are representative of three experiments .

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c, Expression of MHC class I and II on cTECs or mTECs from Atg5-/- to wild-type (WT) grafts (red), or wild-type to wild-type grafts (blue). Staining controls are depicted in grey; data are representative of three experiments. d


d , RT - PCR of autoimmune regulator ( Aire ) TRA expression in mTECs isolated from Atg5 - / - to wild - type , or wild - type to wild - type grafts . Expression of β - actin ( Actb ) , intestinal trefoil factor ( Tff3 ) , casein 2 ( Csn2 ) salivary 1 ( Spt1 ) are also shown ( 5 - fold serial dilutions ) . Data are representative of two experiments .

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

d, RT-PCR analysis of autoimmune regulator (Aire) and TRA expression in mTECs isolated from Atg5-/- to wild-type, or wild-type to wild-type grafts. Expression of β-actin (Actb), intestinal trefoil factor (Tff3), casein 2 (Csn2) and salivary protein 1 (Spt1) are also shown (5-fold serial dilutions). Data are representative of two experiments.


a , Polyclonal T - development in Atg5 - / - to wild - type ( WT ) , or wild - type to wild - type grafts . top dot plots show CD4 CD8 ; bottom dot plots show expression of CD25 Foxp3 by CD4 SP thymocytes ; n ≥ 5 .

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a, Polyclonal T-cell development in Atg5-/- to wild-type (WT), or wild-type to wild-type grafts. The top dot plots show CD4 and CD8 staining; the bottom dot plots show expression of CD25 and Foxp3 by CD4 SP thymocytes; n ≥ 5.


b , Atg5 - / - or wild - type lobes were grafted into TCR - HA transgenic recipients . Graft cellularity ± s . d . expression of transgenic TCR on CD4 SP were analysed after 6 . frequency ± s . d . of TCR - HA + among CD4 SP was 32 . 1 % ± 3 . 2 % in wild - type to TCR - HA , 19 . 6 % ± 4 . 3 % in Atg5 - / - to TCR - HAlobes ; P = 1 . 8 × 10 - 8 ( n ≥ 14 ) .

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b, Atg5-/- or wild-type lobes were grafted into TCR-HA transgenic recipients. Graft cellularity ± s.d. and the expression of the transgenic TCR on CD4 SP cells were analysed after 6 weeks. The frequency ± s.d. of TCR-HA+ cells among CD4 SP cells was 32.1% ± 3.2% in wild-type to TCR-HA, and 19.6% ± 4.3% in Atg5-/- to TCR-HAlobes; P = 1.8 × 10-8 (n ≥ 14).


c , Atg5 - / - or wild - type lobes were grafted into transgenic mice . Graft cellularity ± s . d . expression of transgenic TCR ( Vb3 - Va11 ) on CD4 SP were analysed after 6 . frequency ± s . d . of Vb3 - Va11 + CD4 SP was 93 . 5 % ± 0 . 2 % in wild - type to ANDlobes ( n = 3 ) 97 . 9 % ± 1 . 1 % ( n = 6 ) in Atg5 - / - to ANDlobes ( P = 9 × 10 - 4 ) .

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c, Atg5-/- or wild-type lobes were grafted into AND transgenic mice. Graft cellularity ± s.d. and the expression of the transgenic TCR (Vb3-Va11) on CD4 SP cells were analysed after 6 weeks. The frequency ± s.d. of Vb3-Va11+ CD4 SP cells was 93.5% ± 0.2% in wild-type to ANDlobes (n = 3) and 97.9% ± 1.1% (n = 6) in Atg5-/- to ANDlobes (P = 9 × 10-4).


d , Thymi from wild - type or Atg5 - / - were grafted into P14 , OT - I or HY TCR - transgenic recipients . dot plots show expression of transgenic TCR V - segments on CD8 SP . Thymus cellularities ± s . d . are shown in respective bar diagrams . Data are representative of n = 4 ( P14 ) , n ≥ 4 ( OT - I ) n ≥ 2 ( HY ) .

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d, Thymi from wild-type or Atg5-/- embryos were grafted into P14, OT-I or HY TCR-transgenic recipients. The dot plots show the expression of the transgenic TCR V-segments on CD8 SP cells. Thymus cellularities ± s.d. are shown in the respective bar diagrams. Data are representative of n = 4 (P14), n ≥ 4 (OT-I) and n ≥ 2 (HY).


e , Thymi from wild - type or ( BALB / c3C57BL / 6 ) F1 Atg5 - / - were transplanted into ( BALB / c3C57BL / 6 ) F1 recipients . Six after grafting , expression of I - Ea52 - 68 - I - Ab on cTECs was analysed using monoclonal antibody Y - Ae . Atg5 - / - cTECs are shown in red ; wild - type cTECs are shown in blue . Control stainings are shown in orange ( Atg5 - / - ) green ( wild type ) . Data are representative of two independent experiments .

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e, Thymi from wild-type or (BALB/c3C57BL/6)F1 Atg5-/- embryos were transplanted into (BALB/c3C57BL/6)F1 recipients. Six weeks after grafting, expression of the I-Ea52-68-I-Ab complex on cTECs was analysed using the monoclonal antibody Y-Ae. Atg5-/- cTECs are shown in red; wild-type cTECs are shown in blue. Control stainings are shown in orange (Atg5-/-) and green (wild type). Data are representative of two independent experiments.


Atg5 - / - or wild - type ( WT ) lobes were grafted into athymic ( nu / nu ) mice ( nu / nuAtg5 - / - or nu / nuWT , respectively ) . Data are representative of three experiments with n ≥ 4 . a , Expression of activation markers on lymph node CD4 T in nu / nuAtg5 - / - or nu / nuWT chimaeras .

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Atg5-/- or wild-type (WT) lobes were grafted into athymic (nu/nu) mice (nu/nuAtg5-/- or nu/nuWT, respectively). Data are representative of three experiments with n ≥ 4. a, Expression of activation markers on lymph node CD4 T cells in nu/nuAtg5-/- or nu/nuWT chimaeras.


b, The changes in body weight over time are shown.


c , appearance of nu / nuAtg5 - / - or nu / nuWT chimaeras . colon ( arrow ) uterus ( arrowhead ) are highlighted ; note absence of fat pads ( asterisk ) in nu / nuAtg5 - / - chimaeras .

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c, The appearance of nu/nuAtg5-/- or nu/nuWT chimaeras. The colon (arrow) and uterus (arrowhead) are highlighted; note the absence of fat pads (asterisk) in nu/nuAtg5-/- chimaeras.


d , Haematoxylin eosin of organs from nu / nuAtg5 - / - or nu / nuWT chimaeras . Arrows indicate infiltrates .

d, Haematoxylin and eosin staining of organs from nu/nuAtg5-/- or nu/nuWT chimaeras. Arrows indicate infiltrates.


A . Representative of immunofluorescence of α - smooth muscle actin ( α - SMA ) , pan Cytokeratin , Pan - hMENA , hMENA11a E - cadherin expression in CAFs autologous cancer ( Ep - PDAC ) obtained from enzymatically digested PDAC tissue of # 36 . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) . Scale bar : 20 μm .

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

A. Representative images of immunofluorescence of α-smooth muscle actin (α-SMA), pan Cytokeratin, Pan-hMENA, hMENA11a and E-cadherin expression in CAFs and autologous cancer cells (Ep-PDAC) obtained from enzymatically digested primary PDAC tissue of patient #36. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 20 μm.


B . Representative immunoblot ( top ) of hMENA / hMENA ∆ v6 expression ( detected by Pan - hMENA mAb by specific anti - hMENAΔv6 antibody ) in normal fibroblasts derived from transplant donor ( P - NF ) , pancreatic serous cystadenoma # 71 cancer associated fibroblasts ( n = 10 ) obtained from PDAC tissues . Densitometry quantified data ( bottom ) of hMENA ∆ v6 expression . of P # 110 is relative to sample shown in WB on left .

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B. Representative immunoblot (top) of hMENA/hMENA∆v6 expression levels (detected by Pan-hMENA mAb and by the specific anti-hMENAΔv6 antibody) in normal fibroblasts derived from transplant donor (P-NF), pancreatic serous cystadenoma #71 and cancer associated fibroblasts (n=10) obtained from primary PDAC tissues. Densitometry quantified data (bottom) of hMENA∆v6 expression. Quantification of P#110 is relative to the sample shown in the WB on the left.


C . Representative immunoblot ( top ) of hMENA / hMENA ∆ v6 expression ( detected by Pan - hMENA mAb by specific anti - hMENAΔv6 antibody ) in normal lung fibroblasts ( L - NF ) , cancer associated fibroblasts obtained from NSCLC tissues ( n = 4 ) normal dermal fibroblasts ( HNF ) . Densitometry quantified data ( bottom ) of hMENA ∆ v6 expression .

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C. Representative immunoblot (top) of hMENA/hMENA∆v6 expression levels (detected by Pan-hMENA mAb and by the specific anti-hMENAΔv6 antibody) in normal lung fibroblasts (L-NF), cancer associated fibroblasts obtained from NSCLC tissues (n=4) and normal dermal fibroblasts (HNF). Densitometry quantified data (bottom) of hMENA∆v6 expression.


A . Representative of immunofluorescence of Pan - hMENA ( yellow ) α - SMA ( red ) in PDAC tissue of # 138 from whom high hMENA ∆ v6 CAFs were obtained . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) . Scale bar : 50 μm . inset of dashed is provided on right as a zoomed - in cropped image . Scale bar : 20 μm . αSMA ‑ positive CAFs are also positive for Pan - hMENA ( arrow ) .

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A. Representative images of immunofluorescence of Pan-hMENA (yellow) and α-SMA (red) in the primary PDAC tissue of patient #138 from whom high hMENA∆v6 CAFs were obtained. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 50 μm. The inset of the dashed area is provided on the right as a zoomed-in and cropped fluorescence image. Scale bar: 20 μm. αSMA‑positive CAFs are also positive for Pan-hMENA (arrow).


B . Representative of immunofluorescence of Pan - hMENA ( yellow ) α - SMA ( red ) in NSCLC case # 484 from whom high hMENA ∆ v6 CAFs were obtained . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) . Scale bar : 50 μm . inset of dashed is provided on right as a zoomed - in cropped image . Scale bar : 20 μm . As in A , α - SMA signal is evident in stromal which are also positive for Pan - hMENA ( arrow ) .

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B. Representative images of immunofluorescence of Pan-hMENA (yellow) and α-SMA (red) in NSCLC case #484 from whom high hMENA∆v6 CAFs were obtained. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 50 μm. The inset of the dashed area is provided on the right as a zoomed-in and cropped fluorescence image. Scale bar: 20 μm. As in A, α-SMA signal is evident in stromal cells which are also positive for Pan-hMENA (arrow).


C . Boxplots showing mean mRNA expression of ENAH gene ( hMENA ) for 22 in 10 groups of types ( Peng et al . , 2019 ) ( total number of : 38487 , mean of for : 1603 ) . Shown in each boxplot are median value ( horizontal ) , 25th - 75th percentiles ( box outline ) , highest lowest values within 1 . 5x of inter - quartile range ( vertical ) . Expressions from each - type group were compared to other groups by using Mann - Whitney U - ( two - sided ) P - values were adjusted for multiple testing using Benjamini - Hochberg method . Stromal - type groups with significantly up - regulated ENAH expression respect to other stromal groups are : Fibroblast , * * * q = 0 . 0002 ; Stellate , * * * q = 1 . 5e - 07 . Non - stromal - type groups with significantly up - regulated ENAH expression with respect to other non - stromal groups are : 1 , * * q = 0 . 0013 .

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

C. Boxplots showing the mean mRNA expression of ENAH gene (hMENA) for 22 patients in the 10 groups of cell types (Peng et al., 2019) (total number of cells: 38487, mean of cells for patient: 1603). Shown in each boxplot are the median value (horizontal line), 25th-75th percentiles (box outline), and highest and lowest values within 1.5x of the inter-quartile range (vertical line). Expressions from each cell-type group were compared to all other groups by using Mann-Whitney U-test (two-sided) and P-values were adjusted for multiple testing using the Benjamini-Hochberg method. Stromal cell-type groups with significantly up-regulated ENAH expression respect to other stromal groups are: Fibroblast, ***q=0.0002; Stellate, ***q= 1.5e-07. Non-stromal cell-type groups with significantly up-regulated ENAH expression with respect to other non-stromal groups are: Ductal 1, **q=0.0013.


D . Boxplots showing mRNA expression of ENAH gene ( hMENA ) in 7 groups of types from ( Lambrechts et al . , 2018 ) ( total n = 52 ) . Shown in each boxplot are median value ( horizontal ) , 25th - 75th percentiles ( box outline ) , highest lowest values within 1 . 5x of inter - quartile range ( vertical ) . expression from each group were compared to other stromal / not - stromal by using Mann - Whitney U - ( two - sided ) P - values were adjusted for multiple testing using Benjamini - Hochberg method ( Fibroblast group vs other stromal groups , * * q = 0 . 0022 ; Other comparisons , q > 0 .

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D. Boxplots showing the mRNA expression of ENAH gene (hMENA) in the 7 groups of cell types from (Lambrechts et al., 2018) (total n=52). Shown in each boxplot are the median value (horizontal line), 25th-75th percentiles (box outline), and highest and lowest values within 1.5x of the inter-quartile range (vertical line). Cell expression from each group were compared to all other stromal/not-stromal cells by using Mann-Whitney U-test (two-sided) and P-values were adjusted for multiple testing using the Benjamini-Hochberg method (Fibroblast group vs other stromal groups, **q=0.0022; Other comparisons, q>0.05


A . of in vitro matrigel invasion ( bottom ) of P - CAF L - CAF ( P - CAF # 36 , 138 L - CAF # 189 , 484 ) transfected with control siRNA ( CNT ) or hMENA siRNA ( hMENA ( t ) ) indicating that siRNA ‐ mediated knock ‐ down of hMENA / hMENA ∆ v6 reduces invasive ability of CAFs with respect to siCNT CAFs . Number of invading was measured by counting 6 random fields . Data are presented as mean ± SD of two biological replicates , performed in triplicates each . Immunoblot showing hMENA / hMENAΔv6 expression ( detected by Pan - hMENA mAb by specific anti - hMENAΔv6 antibody ) of CAFs employed is reported ( top ) . TUBULIN was used as loading control . P values were calculated by two ‐ sided Student ' s t ‐ . * P < 0 . 05 , * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of in vitro matrigel invasion assay (bottom) of P-CAF and L-CAF (P-CAF # 36, 138 and L-CAF #189, 484) transfected with control siRNA (CNT) or hMENA siRNA (hMENA(t)) indicating that the siRNA‐mediated knock‐down of hMENA/hMENA∆v6 reduces the invasive ability of CAFs with respect to siCNT CAFs. Number of invading cells was measured by counting 6 random fields. Data are presented as the mean ± SD of two biological replicates, performed in triplicates each. Immunoblot showing hMENA/hMENAΔv6 expression (detected by Pan-hMENA mAb and by the specific anti-hMENAΔv6 antibody) of the CAFs employed is reported (top). TUBULIN was used as loading control. P values were calculated by two‐sided Student's t‐test. *P<0.05, ***P<0.001.


B . of in vitro matrigel invasion ( bottom ) of P - NF L - NF P - CAF # 110 L - CAF # 400 transfected with control or hMENA ∆ v6 expressing vectors , demonstrating that overexpression of hMENA ∆ v6 isoform induced invasiveness of P - NFs L - NFs / or P - L - CAFs . Number of invading was measured by counting 6 random fields . Data are presented as mean ± SD of two biological replicates , performed in triplicates each . Immunoblot of hMENAΔv6 expression ( detected by specific anti - hMENAΔv6 antibody ) in fibroblasts employed is reported ( top ) . TUBULIN was used as loading control . P values were calculated by two ‐ sided Student ' s t ‐ . * P < 0 . 05 , * * * P < 0 . 001 .

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

B. Quantification of in vitro matrigel invasion assay (bottom) of P-NF and L-NF and P-CAF#110 and L-CAF#400 transfected with control or hMENA∆v6 expressing vectors, demonstrating that the overexpression of hMENA∆v6 isoform induced the invasiveness of P-NFs and L-NFs and/or P-and L-CAFs. Number of invading cells was measured by counting 6 random fields. Data are presented as the mean ± SD of two biological replicates, performed in triplicates each. Immunoblot of hMENAΔv6 expression (detected by the specific anti-hMENAΔv6 antibody) in fibroblasts employed is reported (top). TUBULIN was used as loading control. P values were calculated by two‐sided Student's t‐test. *P<0.05, ***P<0.001.


A . of in vitro matrigel invasion of PANC - 1 cultured for 48 h with conditioned media ( CM ) of NFs ( P - NFs - CM ) , CAF low # 44 # 110 CAFs high # 36 138 . Histograms show number of invading measured by counting 6 random fields . Data are presented as mean ± SD of three biological replicates , performed at least in duplicate each . Statistical was performed with one - way ANOVA P < 0 . 0001 , followed by Bonferroni ' s multiple comparison . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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A. Quantification of in vitro matrigel invasion assay of PANC-1 cells cultured for 48 h with conditioned media (CM) of NFs (P-NFs-CM), CAF low #44 and #110 and CAFs high #36 and 138. Histograms show the number of invading cells measured by counting 6 random fields. Data are presented as the mean ± SD of three biological replicates, performed at least in duplicate each. Statistical analysis was performed with one-way ANOVA P<0.0001, followed by Bonferroni's multiple comparison test. * P <0.05, **P<0.01, ***P<0.001.


B . of in vitro matrigel invasion of PANC - 1 cultured for 48 h with CM derived from control P - CAFs # 36 ( siCNT - P - CAF - CM # 36 ) hMENA / hMENA ∆ v6 silenced P - CAFs ( sihMENA ( t ) - P - CAF - CM # 36 ) , showing that siRNA ‐ mediated knock ‐ down of hMENA / hMENA ∆ v6 affects PANC - 1 invasive ability mediated by CAF - CM . Culture medium ( DMEM ) was used as control . invading matrigel were counted in 6 random fields . Data are presented as mean ± SD of three biological replicates . Statistical was performed with one - way ANOVA P = 0 . 006 , followed by Bonferroni ' s multiple comparison . * P < 0 . 05 , * * P < 0 . 01 .

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

B. Quantification of in vitro matrigel invasion assay of PANC-1 cultured for 48 h with CM derived from control P-CAFs#36 (siCNT-P-CAF-CM#36) and hMENA/hMENA∆v6 silenced P-CAFs (sihMENA(t)-P-CAF-CM#36), showing that the siRNA‐mediated knock‐down of hMENA/hMENA∆v6 affects PANC-1 invasive ability mediated by CAF-CM. Culture medium (DMEM) was used as control. Cells invading matrigel were counted in 6 random fields. Data are presented as the mean ± SD of three biological replicates. Statistical analysis was performed with one-way ANOVA P=0.006, followed by Bonferroni's multiple comparison test. *P < 0.05, **P<0.01.


C . of in vitro matrigel invasion of H1975 cultured for 48 h with control media ( culture medium ) or conditioned media ( CM ) of L - CAF low # 400 CAFs high # 189 , as described above . Data are presented as mean ± SD of two biological replicates , performed in triplicates each . Statistical was performed with one - way ANOVA P < 0 . 0001 , followed by Bonferroni ' s multiple comparison . * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of in vitro matrigel invasion assay of H1975 cells cultured for 48 h with control media (culture medium) or conditioned media (CM) of L-CAF low #400 and CAFs high #189, as described above. Data are presented as the mean ± SD of two biological replicates, performed in triplicates each. Statistical analysis was performed with one-way ANOVA P<0.0001, followed by Bonferroni's multiple comparison test. ***P<0.001.


D . of in vitro matrigel invasion of H1975 cultured for 48 h with CM derived from control L - CAFs # 484 ( siCNT - L - CAF - CM # 484 ) hMENA / hMENA ∆ v6 silenced L - CAFs ( sihMENA ( t ) - L - CAF - CM # 484 ) , showing that siRNA ‐ mediated knock ‐ down of hMENA / hMENA ∆ v6 affects H1975 invasive ability mediated by CAF - CM . Culture medium ( DMEM ) was used as control . invading matrigel were counted in 6 random fields . Data are presented as mean ± SD of six replicates . Statistical was performed with one - way ANOVA P = 0 . 006 , followed by Bonferroni ' s multiple comparison . * P < 0 . 05 , * * P < 0 . 01 .

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

D. Quantification of in vitro matrigel invasion assay of H1975 cultured for 48 h with CM derived from control L-CAFs#484 (siCNT-L-CAF-CM#484) and hMENA/hMENA∆v6 silenced L-CAFs (sihMENA(t)-L-CAF-CM#484), showing that the siRNA‐mediated knock‐down of hMENA/hMENA∆v6 affects H1975 invasive ability mediated by CAF-CM. Culture medium (DMEM) was used as control. Cells invading matrigel were counted in 6 random fields. Data are presented as the mean ± SD of six replicates. Statistical analysis was performed with one-way ANOVA P=0.006, followed by Bonferroni's multiple comparison test. *P < 0.05, **P<0.01.


E . Representative immunoblot ( top ) densitometry ( bottom ) of hMENA ∆ v6 expression level in P - NFs grown in RPMI control medium ( - ) or PANC - 1 - CM for 24 h ( n = 3 ) . Data are represented as fold increase with respect to control medium ± SD ( set as 1 ) . Data were analyzed using two ‐ sided Student ' s t ‐ . * P < 0 . 05 .

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

E. Representative immunoblot (top) and densitometry quantification (bottom) of hMENA∆v6 expression level in P-NFs grown in RPMI control medium (-) or PANC-1-CM for 24 h (n = 3). Data are represented as fold increase with respect to control medium ± SD (set as 1). Data were analyzed using two‐sided Student's t‐test. *P < 0.05.


F . Representative ( top ) ( bottom ) of collagen gel - contraction ability of CAFs ( monoculture ) or CAFs co - cultured with siCNT ( co - siCNT ) or sihMENA ( t ) PANC - 1 ( co - sihMENA ( t ) ) . Dashed circles illustrate margins of gel . Data are presented as mean ± SD of two biological replicates . Statistical was performed with one - way ANOVA P = 0 . 005 , followed by Bonferroni ' s multiple comparison ANOVA : * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images (top) and quantification (bottom) of collagen gel-contraction ability of CAFs (monoculture) or CAFs co-cultured with siCNT (co-siCNT) or sihMENA(t) PANC-1 cells (co-sihMENA(t)). Dashed white circles illustrate the margins of the gel area. Data are presented as the mean ± SD of two biological replicates. Statistical analysis was performed with one-way ANOVA P=0.005, followed by Bonferroni's multiple comparison test ANOVA: *P<0.05, **P<0.01.


G . Representative immunoblot ( top ) ( bottom ) of hMENA ∆ v6 α - SMA expression in P - CAFs # 110 treated with DMEM medium ( - ) or with conditioned medium ( CM ) derived from siCNT ( siCNT PANC - 1 ) or sihMENA ( t ) PANC - 1 ( ( sihMENA ( t ) PANC - 1 ) ( n = 3 ) . Data are represented as fold increase with respect to DMEM ( set as 1 ± SD ) . Data presented were analyzed using two ‐ sided Student ' s t ‐ : * P < 0 . 05 .

O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative immunoblot (top) and quantification (bottom) of hMENA∆v6 and α-SMA expression in P-CAFs #110 treated with DMEM medium (-) or with conditioned medium (CM) derived from siCNT (siCNT PANC-1) or sihMENA(t) PANC-1 cells ((sihMENA(t) PANC-1) (n = 3). Data are represented as fold increase with respect to DMEM (set as 1± SD). Data presented were analyzed using two‐sided Student's t‐test: *P<0.05.


A . Heat map of identified by LC - MS / MS as differentially secreted in CM of normal fibroblasts ( P - NF # 1 P - NF # 2 ) , CAFs with low level of hMENA ∆ v6 expression ( P - CAFs low # 44 # 49 ) , CAFs with high level of hMENA ∆ v6 expression ( P - CAFs high # 67 # 36 ) . boxed hMENA ∆ v6 associated signature represents exclusively present in CM derived from CAFs high . Color code is shown in upper left corner . Co - variance = 0 . 2 , q value = 0 . 1 ( 10 % false discovery rate ) , adjusted P value = 0 . 0074542 . N = 36 . values of mass spec identified for each were plotted in log2 scale .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Heat map of proteins identified by LC-MS/MS analysis as differentially secreted in CM of normal fibroblasts (P-NF#1 and P-NF#2), CAFs with low level of hMENA∆v6 expression (P-CAFs low#44 and #49), CAFs with high level of hMENA∆v6 expression (P-CAFs high #67 and #36). The boxed hMENA∆v6 associated signature represents proteins exclusively present in the CM derived from CAFs high. Color code is shown in the upper left corner. Co-variance = 0.2, q value = 0.1 (10% false discovery rate), adjusted P value = 0.0074542. N = 36. The values of mass spec identified for each protein were plotted in log2 scale.


B . of GAS6 secretion , as detected by ELISA , in CM of P - NF ( # 1 ) , P - CAFlow ( # 44 # 49 ) P - CAF high ( # 67 # 36 ) . Data are presented as mean ± SD of two biological replicates . Statistical was performed with one - way ANOVA P < 0 . 0001 , followed by Bonferroni ' s multiple comparison . * * * P < 0 . 001 .

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

B. Quantification of GAS6 secretion levels, as detected by ELISA, in the CM of P-NF (#1), P-CAFlow (#44 and #49) and P-CAF high (#67 and #36). Data are presented as the mean ± SD of two biological replicates. Statistical analysis was performed with one-way ANOVA P<0.0001, followed by Bonferroni's multiple comparison test. ***P<0.001.


C . Real time qRT - PCR of relative GAS6 mRNA expression level in NFs ( P - NF # 1 ) , P - CAF low P - CAF high , as described above . Data are presented as mean ± SD of three replicates . Statistical was performed with one - way ANOVA P = 0 . 001 , followed by Bonferroni ' s multiple comparison . * P < 0 . 05 , * * P < 0 . 01 .

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

C. Real time qRT-PCR analysis of the relative GAS6 mRNA expression level in NFs (P-NF#1), P-CAF low and P-CAF high, as described above. Data are presented as the mean ± SD of three replicates. Statistical analysis was performed with one-way ANOVA P=0.001, followed by Bonferroni's multiple comparison test. *P< 0.05, **P<0.01.


D . Real time qRT - PCR of relative GAS6 mRNA expression level in PANC - 1 P - CAF ( n = 3 ) , showing a low GAS6 expression in PANC - 1 . Data are presented as mean ± SD . P values were calculated by two ‐ sided Student ' s t ‐ . * * * P < 0 . 001 .

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

D. Real time qRT-PCR analysis of the relative GAS6 mRNA expression level in PANC-1 and P-CAF (n=3), showing a low GAS6 expression in PANC-1 cells. Data are presented as the mean ± SD. P values were calculated by two‐sided Student's t‐test. ***P<0.001.


E . Boxplots showing mRNA expression of GAS6 in normal lung fibroblasts ( ) ( n = 15 ) versus NSCLC fibroblasts ( blue ) ( n = 15 ) ( GSE22862 data set ) . In each boxplot median value ( horizontal ) , 25th - 75th percentiles ( box outline ) , highest lowest values within 1 . 5x of inter - quartile range ( vertical ) are shown . Statistical significance was calculated by Mann - Whitney U - ( two - sided ) ( P = 0 . 0208 ) .

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

E. Boxplots showing the mRNA expression of GAS6 in normal lung fibroblasts (white) (n=15) versus primary NSCLC fibroblasts (light blue) (n=15) (GSE22862 data set). In each boxplot the median value (horizontal line), 25th-75th percentiles (box outline), and highest and lowest values within 1.5x of the inter-quartile range (vertical line) are shown. Statistical significance was calculated by Mann-Whitney U-test (two-sided) (P=0.0208).


F , G . Real time qRT - PCR of P # 138 CAF ( F ) L # 189 CAF ( G ) transfected with control siRNA ( siCNT ) or hMENA ( t ) siRNA ( sihMENA ( t ) ) as representative cases . siRNA ‐ mediated knock ‐ down of hMENA / hMENA ∆ v6 resulted in a significant reduction of GAS6 mRNA expression compared to siCNT ( set as 100 ) . Data are presented as mean ± SD of three replicates . P values were calculated by two ‐ sided Student ' s t ‐ * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O O 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, G. Real time qRT-PCR analysis of P#138 CAF (F) and L#189 CAF (G) transfected with control siRNA (siCNT) or hMENA(t) siRNA (sihMENA(t)) as representative cases. The siRNA‐mediated knock‐down of hMENA/hMENA∆v6 resulted in a significant reduction of GAS6 mRNA expression levels compared to siCNT cells (set as 100). Data are presented as the mean ± SD of three replicates. P values were calculated by two‐sided Student's t‐test **P<0.01, ***P<0.001.


H . of matrigel invasion of PANC - 1 cultured for 48 h with DMEM ( culture medium ) , or conditioned medium ( CM ) derived from control siRNA P # 106 CAFs ( siCNT P - CAF - CM ) , GAS6 siRNA ( siGAS6 P - CAF - CM ) , hMENA ( t ) siRNA ( sihMENA ( t ) P - CAF - CM ) , hMENA ( t ) siRNA plus rGAS6 ( sihMENA ( t ) P - CAF - CM + rGAS6 ) . Number of invaded PANC - 1 after 48 h of treatment was measured by counting 6 random fields . Data are presented as mean ± SD of three biological replicates . Statistical was performed with one - way ANOVA P = 0 . 004 , followed by Bonferroni ' s multiple comparison . * P < 0 . 05 , * * P < 0 . 01 .

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

H. Quantification of matrigel invasion assay of PANC-1 cultured for 48 h with DMEM (culture medium), or conditioned medium (CM) derived from control siRNA P#106 CAFs (siCNT P-CAF-CM), GAS6 siRNA (siGAS6 P-CAF-CM), hMENA(t) siRNA (sihMENA(t) P-CAF-CM), hMENA(t) siRNA plus rGAS6 (sihMENA(t) P-CAF-CM +rGAS6). Number of invaded PANC-1 cells after 48 h of treatment was measured by counting 6 random fields. Data are presented as the mean ± SD of three biological replicates. Statistical analysis was performed with one-way ANOVA P=0.004, followed by Bonferroni's multiple comparison test. *P< 0.05, **P<0.01.


A . Immunoblot of AXL expression in PANC - 1 A549 cancer upon transfection of control siRNA ( CNT ) or hMENA ( t ) siRNA ( sihMENA ( t ) ) , indicating that knock ‐ down of hMENA isoforms , resulted in a reduction of AXL expression .

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

A. Immunoblot of AXL expression in PANC-1 and A549 cancer cells upon transfection of control siRNA (CNT) or hMENA(t) siRNA (sihMENA(t)), indicating that the knock‐down of hMENA isoforms, resulted in a reduction of AXL protein expression.


B . Real time qRT - PCR of relative AXL mRNA expression in PANC - 1 A549 cancer transfected with control siRNA ( siCNT ) or hMENA ( t ) siRNA ( sihMENA ( t ) ) , indicating that knock ‐ down of hMENA isoforms , resulted in a significant reduction of mRNA AXL expression . Data are presented as mean ± SD of three replicates . P values were calculated by two ‐ sided Student ' s t ‐ . * * P < 0 . 01 , * * * P < 0 . 001 .

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

B. Real time qRT-PCR analysis of the relative AXL mRNA expression in PANC-1 and A549 cancer cells transfected with control siRNA (siCNT) or hMENA(t) siRNA (sihMENA(t)), indicating that the knock‐down of hMENA isoforms, resulted in a significant reduction of mRNA AXL expression. Data are presented as the mean ± SD of three replicates. P values were calculated by two‐sided Student's t‐test. **P<0.01, ***P<0.001.


C . Real time qRT - PCR of relative of mature AXL mRNA or AXL pre - mRNA in PANC - 1 ( left ) A549 ( right ) lines , transfected with control siRNA ( siCNT ) or hMENA ( t ) siRNA , sihMENA ( t ) . Data represent of AXL mRNA or pre - mRNA in hMENA ( t ) silenced relative to siCNT control ( set at 100 ) . Data are presented as mean ± SD of two biological replicates . P values were calculated by two ‐ sided Student ' s t ‐ . * P < 0 . 05 , * * * P < 0 . 001 .

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

C. Real time qRT-PCR analysis of the relative levels of mature AXL mRNA or AXL pre-mRNA in PANC-1 (left) and A549 (right) cell lines, transfected with control siRNA (siCNT) or hMENA(t) siRNA, sihMENA(t). Data represent percent of AXL mRNA or pre-mRNA levels in hMENA(t) silenced cells relative to siCNT control cells (set at 100). Data are presented as the mean ± SD of two biological replicates. P values were calculated by two‐sided Student's t‐test. *P< 0.05, ***P<0.001.


D . Immunoblot with indicated antibodies of PANC - 1 transfected with control siRNA ( CNT ) or hMENA ( t ) siRNA , showing that knock ‐ down of total hMENA isoforms , ( hMENA ( t ) ) inhibits GAS6 - mediated pAXL pAKT expression . were serum starved overnight subsequently stimulated with DMSO ( 0 . 02 % ) in control culture medium ( - ) or rGAS6 ( 200 ng / mL ) , for 30 60 min . fold change of pAXL or pAKT expression respect to siCNT untreated is reported .

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 B-CONTROLLED_VAR O O 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 O O O O O O O O O O O O O O O O O O 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. Immunoblot analysis with the indicated antibodies of PANC-1 cells transfected with control siRNA (CNT) or hMENA(t) siRNA, showing that the knock‐down of total hMENA isoforms, (hMENA(t)) inhibits GAS6-mediated pAXL and pAKT expression. Cells were serum starved overnight and subsequently stimulated with DMSO (0.02%) in control culture medium (-) or rGAS6 (200 ng/mL), for 30 and 60 min. The fold change of pAXL or pAKT expression respect to siCNT untreated cells is reported.


E . of in vitro matrigel invasion of PANC - 1 siCNT ( siCNT ) hMENA / hMENA ∆ v6 silenced ( sihMENA ( t ) ) toward rGAS6 as chemo - attractant ( siCNT + rGAS6 sihMENA ( t ) + rGAS6 ) , showing that knock ‐ down of hMENA ( t ) reduced cancer invasion toward GAS6 . number of invading were counted in 6 random fields . Data are presented as mean ± SD of three biological replicates . Statistical was performed with one - way ANOVA P < 0 . 0001 , followed by Bonferroni ' s multiple comparison . * * P < 0 . 01 .

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

E. Quantification of in vitro matrigel invasion assay of PANC-1 siCNT cells (siCNT) and hMENA/hMENA∆v6 silenced cells (sihMENA(t)) toward rGAS6 as chemo-attractant (siCNT + rGAS6 and sihMENA(t) + rGAS6), showing that the knock‐down of hMENA(t) reduced cancer cell invasion toward GAS6. The number of invading cells were counted in 6 random fields. Data are presented as the mean ± SD of three biological replicates. Statistical analysis was performed with one-way ANOVA P<0.0001, followed by Bonferroni's multiple comparison test. **P<0.01.


F . of in vitro matrigel invasion of PANC - 1 siCNT ( siCNT ) hMENA / hMENA ∆ v6 silenced ( sihMENA ( t ) ) , untreated ( - ) or treated with conditioned media derived from CAFs ( P - CAF # 36 - CM ) for 48 h . number of invaded were counted in 6 random fields . Data are presented as mean ± SD of three biological replicates . Statistical was performed with one - way ANOVA P < 0 . 0001 , followed by Bonferroni ' s multiple comparison . * * * P < 0 . 001 .

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

F. Quantification of in vitro matrigel invasion assay of PANC-1 siCNT cells (siCNT) and hMENA/hMENA∆v6 silenced cells (sihMENA(t)), untreated (-) or treated with conditioned media derived from CAFs (P-CAF#36-CM) for 48 h. The number of invaded cells were counted in 6 random fields. Data are presented as the mean ± SD of three biological replicates. Statistical analysis was performed with one-way ANOVA P<0.0001, followed by Bonferroni's multiple comparison test. *** P< 0.001.


A . Overall survival ( OS ) curves in Pancreatic Adenocarcinoma ( PDAC ) ( n = 172 ) from Cancer Genome Atlas ( TCGA ) , showed that combined expression of AXL , GAS6 ENAH ( hMENA ) , is a prognostic signature in PDAC .

O O O O O O O O O O O O O O O O O O O O O O O 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. Overall survival (OS) curves in Pancreatic Adenocarcinoma patients (PDAC) (n=172) from The Cancer Genome Atlas (TCGA), showed that combined expression of AXL, GAS6 and ENAH (hMENA), is a prognostic signature in PDAC.


B . Overall survival ( OS ) curves in Lung Squamous cancer ( LUSC ) ( n = 501 ) from Cancer Genome Atlas ( TCGA ) , showed that combined expression of AXL , GAS6 ENAH ( hMENA ) , is a prognostic signature in LUSC .

O O O O O O O O O O O O O O O O O O O O O O O O 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. Overall survival (OS) curves in Lung Squamous cancer patients (LUSC) (n=501) from The Cancer Genome Atlas (TCGA), showed that combined expression of AXL, GAS6 and ENAH (hMENA), is a prognostic signature in LUSC.


of maximum projections of forebrain sections from 3 mo old GLASTCreERT2 / eGFP mice 21 days after tamoxifen treatment Double - positive are indicated by arrowheads . CTX : cerebral cortex ; DIE : diencephalon ; HC : hippocampus . Scale bars : 300 µm ( A ) , 20 µm ( B ) , 75 µm ( C ) , 30 µm ( D ) , Histograms showing quantifications ( B , n = 6 ; 5 region of interest ( ROI ) covering diencephalon on a total of 3 slides analyzed per Data information : data are presented as mean ± . Each data point represents one .

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Micrographs of maximum intensity projections of forebrain sections from 3 mo old GLASTCreERT2/eGFP mice 21 days after tamoxifen treatment Double-positive cells are indicated by arrowheads. CTX: cerebral cortex; DIE: diencephalon; HC: hippocampus. Scale bars: 300 µm (A), 20 µm (B), 75 µm (C), 30 µm (D), Histograms showing quantifications (B, n = 6 animals; 5 region of interest (ROI) covering the diencephalon on a total of 3 slides analyzed per animal Data information: data are presented as mean ± SEM. Each data point represents one animal.


of maximum projections of forebrain sections from 3 mo old C57BL / 6J mice after EdU application for 4 in drinking water ( F ) immunostained as indicated on top of panels . Double - positive are indicated by arrowheads . CTX : cerebral cortex ; DIE : diencephalon ; HC : hippocampus . Scale bars : 10 µm ( F ) .

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Micrographs of maximum intensity projections of forebrain sections from 3 mo old C57BL/6J mice after EdU application for 4 weeks in drinking water (F) immunostained as indicated on top of the panels. Double-positive cells are indicated by arrowheads. CTX: cerebral cortex; DIE: diencephalon; HC: hippocampus. Scale bars: 10 µm (F).


G ) Histograms showing quantifications for n = 5 ; 5 ROI covering diencephalon on a total of 2 slides analyzed per ) . Data information : data are presented as mean ± . Each data point represents one .

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G) Histograms showing quantifications for n = 5 animals; 5 ROI covering the diencephalon on a total of 2 slides analyzed per animal). Data information: data are presented as mean ± SEM. Each data point represents one animal.


A - E of maximum projections ( A - D ) or single optical section ( E ) of forebrain sections stained as indicated for in astrocytes labelled in 8 mo ( A , B , E ) 3 mo ( C , D ) GLASTCreERT2 / Confetti mice at 21 dpt . Note that clusters of duplets ( D ) triplets ( C ) labelled in same color can be found only in DIE . Also note that astrocyte somata are often very close together , as also observed after live in vivo imaging of dividing astrocytes Somata of single are highlighted in circles . Data information : CTX : cerebral cortex ; DIE : diencephalon ; HC : hippocampus . Scale bars : 300 µm ( A ) , 100 µm ( B ) , 50 µm ( C , D ) , 20 µm ( E ) .

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A-E Micrographs of maximum intensity projections (A - D) or single optical section (E) of forebrain sections stained as indicated for fluorescent proteins in astrocytes labelled in 8 mo (A, B, E) and 3 mo (C, D) GLASTCreERT2/Confetti mice at 21 dpt. Note that clusters of duplets (D) and triplets (C) labelled in the same color can be found only in the DIE. Also note that astrocyte somata are often very close together, as also observed after live in vivo imaging of dividing astrocytes Somata of single cells are highlighted in circles. Data information: CTX: cerebral cortex; DIE: diencephalon; HC: hippocampus. Scale bars: 300 µm (A), 100 µm (B), 50 µm (C, D), 20 µm (E).


B of forebrain sections of 3 mo old GLASTCreERT2 / RPL22HA / eGFP 21 dpt mice stained for HA show maximum projections of HA + expressing with astroglial . CTX GM : cerebral cortex grey matter ; DIE : diencephalon . Scale bars : 100 µm . C of forebrain sections of 3 mo old GLASTCreERT2 / RPL22HA / eGFP mice 21dpt stained for HA S100β showing specificity of mouse with HA detected only in S100β + astrocytes . DIE : diencephalon . Scale bar : 70 μm . Data information : CTX GM : cerebral cortex grey matter ; DIE : diencephalon ; n = 3 biological replicates , tissue from 3 mice was

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B Micrographs of forebrain sections of 3 mo old GLASTCreERT2/RPL22HA/eGFP 21 dpt mice stained for HA show maximum intensity projections of HA+ expressing cells with astroglial morphology. CTX GM: cerebral cortex grey matter; DIE: diencephalon. Scale bars: 100 µm. C Micrographs of forebrain sections of 3 mo old GLASTCreERT2/RPL22HA/eGFP mice 21dpt stained for HA and S100β showing the specificity of the mouse line with HA detected only in S100β+ astrocytes. DIE: diencephalon. Scale bar: 70 μm. Data information: CTX GM: cerebral cortex grey matter; DIE: diencephalon; n=3 biological replicates, tissue from 3 mice was pooled


D Histogram showing expression of top 10 astrocyte specific markers genes in DIE ( blue bars ) CTX GM ( red bars ) samples . Data information : CTX GM : cerebral cortex grey matter ; DIE : diencephalon ; n = 3 biological replicates , tissue from 3 mice was

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D Histogram showing expression levels of the top 10 astrocyte specific markers genes in DIE (blue bars) and CTX GM (red bars) samples. Data information: CTX GM: cerebral cortex grey matter; DIE: diencephalon; n=3 biological replicates, tissue from 3 mice was pooled


C Example of a Smad4 / GFP double - positive astrocyte ( indicated by arrowhead ) in a single optical section of DIE from 3 mo old GLASTCreERT2 / eGFP mice 21 dpt immunostained as indicated on top of panel . Orthogonal projections of highlighted in left panel of C shown at higher magnification in right panel . Scale bar : 15 µm .

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C Example of a Smad4/GFP double-positive astrocyte (indicated by arrowhead) in a single optical section of DIE from 3 mo old GLASTCreERT2/eGFP mice 21 dpt immunostained as indicated on top of the panel. Orthogonal projections of the area highlighted in left panel of C shown at higher magnification in the right panel. Scale bar: 15 µm.


D Example of a proliferating ( PCNA + , arrowhead ) astrocyte in a single optical section of DIE from 3 mo old GLASTCreERT2 / eGFP mice 21 dpt immunostained as indicated on top with single channels shown in middle right panels . Scale bars : 15 µm .

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D Example of a proliferating (PCNA+, arrowhead) astrocyte in a single optical section of DIE from 3 mo old GLASTCreERT2/eGFP mice 21 dpt immunostained as indicated on top with single channels shown in middle and right panels. Scale bars: 15 µm.


E Histogram depicting of PCNA positive of GFP positive in DIE of genotype indicated on x - axis ( n = 6 per genotype ; 5 ROI covering diencephalon on a total of 3 slides were analyzed per ; PCNA negative positive comparing wt / wt vs . fl / fl , adjusted P - value = 0 . 002 ) . Data information : data are presented as mean ± . Each dot represents one n . * P < 0 . 05 , * * P < 0 . 01 nonparametric 2way ANOVA followed by Sidak ' s multiple comparisons

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E Histogram depicting percentage of PCNA positive cells of all GFP positive cells in the DIE of the genotype indicated on the x-axis (n = 6 animals per genotype; 5 ROI covering the diencephalon on a total of 3 slides were analyzed per animal; PCNA negative and positive cells comparing wt/wt vs. fl/fl animals, adjusted P-value = 0.002). Data information: data are presented as mean ± SEM. Each dot represents one n. * P < 0.05, ** P < 0.01 nonparametric 2way ANOVA followed by Sidak's multiple comparisons test


F , G Histograms depicting of neurosphere - generating obtained from DIE ( F ) or SEZ ( G ) of GLASTCreERT2 / Smad4fl / fl / eGFP or GLASTCreERT2 / Smad4WT / WT / eGFP , as indicated on x - axis ( n = 3 - 4 for SEZ , 4 to 8 technical replicates analyzed per ; 4 for DIE , 8 technical replicates analyzed per ) . Data information : data are presented as mean ± . F : GFP negative GFP positive comparing wt / wt vs fl / fl , adjusted P - value = 0 . 0408 ; G : GFP negative GFP positive comparing wt / wt vs fl / fl , ns ) . Each dot represents one n . * P < 0 . 05 , * * P < 0 . 01 nonparametric 2way ANOVA followed by Sidak ' s multiple comparisons

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F, G Histograms depicting percentage of the neurosphere-generating cells obtained from the DIE (F) or SEZ (G) of GLASTCreERT2/Smad4fl/fl/eGFP or GLASTCreERT2/Smad4WT/WT/eGFP, as indicated on the x-axis (n = 3-4 animals for SEZ, 4 to 8 technical replicates analyzed per animal; 4 animals for DIE, 8 technical replicates analyzed per animal). Data information: data are presented as mean ± SEM. F: GFP negative and GFP positive cells comparing wt/wt vs fl/fl animals, adjusted P-value = 0.0408; G: GFP negative and GFP positive cells comparing wt/wt vs fl/fl animals, ns). Each dot represents one n. * P < 0.05, ** P < 0.01 nonparametric 2way ANOVA followed by Sidak's multiple comparisons test


H Histogram depicting number of secondary neurospheres generated from 10000 Smad4f / f neurosphere at 7 days after infection with MLV - retrovirus containing either CAG - NLS - Cre or only CAG - GFP ( n = 11 cultures / , 3 technical replicates per condition analyzed ; control vs . Cre , P - value = 0 . 0149 ) . Note decrease in DIE astrocyte proliferation in vivo in neurosphere formation upon Smad4 deletion Data information : data are presented as mean ± . Each dot represents one n . * P < 0 . 05 , * * P < 0 . 01 nonparametric Mann - Whitney ) .

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H Histogram depicting the number of secondary neurospheres generated from 10000 Smad4f/f neurosphere cells at 7 days after infection with MLV-based retrovirus containing either CAG-NLS-Cre or only CAG-GFP (n = 11 cultures / animals, 3 technical replicates per condition analyzed; control vs. Cre, P-value = 0.0149). Note the decrease in DIE astrocyte proliferation in vivo and in neurosphere formation upon Smad4 deletion Data information: data are presented as mean ± SEM. Each dot represents one n. * P < 0.05, ** P < 0.01 nonparametric Mann-Whitney test).


Antibacterial competition . E . coli K12 recipient ( W3110 gfp + , kanR ) were mixed with indicated EAEC attacker ( 1 : 4 ) : WT , ∆ T6SS - 1 ∆ tle1 - tli1 carrying pBAD18 pBAD33 vectors , or producing indicated , spotted on SIM 0 . 02 % arabinose agar plates for 4 - h at 37 ° C . image of a representative spot is shown . number of recovered prey is indicated in upper graph ( in log of colony - forming units ( cfu ) ) . black , grey grey circles indicate values obtained from three different spots , average is indicated by bar . Western blot of production of Tle1V Tle1 ∆ 1 - 26 V is shown in inset . experiment was performed in triplicate a representative result is shown .

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Antibacterial competition assay. E. coli K12 recipient cells (W3110 gfp+, kanR) were mixed with the indicated EAEC attacker cells (1:4 ratio): WT, ∆T6SS-1 and ∆tle1-tli1 carrying pBAD18 and pBAD33 vectors, or producing the indicated proteins, and spotted on SIM 0.02 % arabinose agar plates for 4-h at 37°C. The image of a representative bacterial spot is shown. The number of recovered prey cells is indicated in the upper graph (in log of colony-forming units (cfu)). The black, dark grey and light grey circles indicate values obtained from three different spots, and the average is indicated by the bar. Western blot analysis of the production of Tle1V and Tle1∆1-26 V is shown in the inset. The experiment was performed in triplicate and a representative result is shown.


Co - immunoprecipitation . Lysates from E . coli K - 12 W3110 producing VSVG - tagged Tle1 ( Tle1V ) or VSVG - tagged Tle1 ∆ 1 - 26 truncated variant ( Tle1 ∆ 1 - 26 V ) were mixed with lysates from W3110 producing FLAG - tagged VgrG ( VgrGFL ) or not ( - , empty vector ) subjected to immunoprecipitation on anti - FLAG - coupled beads . mixed soluble lysates ( Input ) immunoprecipitated material ( IP ) were subjected to 12 . 5 % SDS - PAGE immunodetected with anti - FLAG ( upper panel ) anti - VSVG ( lower panel ) monoclonal antibodies . Molecular markers ( in kDa ) are indicated on left . This experiment was performed in triplicate a representative result is shown .

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Co-immunoprecipitation assay. Lysates from E. coli K-12 W3110 cells producing VSVG-tagged Tle1 (Tle1V) or VSVG-tagged Tle1∆1-26 truncated variant (Tle1∆1-26 V) were mixed with lysates from W3110 cells producing FLAG-tagged VgrG (VgrGFL) or not (-, empty vector) and subjected to immunoprecipitation on anti-FLAG-coupled beads. The mixed soluble lysates (Input) and the immunoprecipitated material (IP) were subjected to 12.5% SDS-PAGE and immunodetected with anti-FLAG (upper panel) and anti-VSVG (lower panel) monoclonal antibodies. Molecular weight markers (in kDa) are indicated on the left. This experiment was performed in triplicate and a representative result is shown.


Specific phospholipase A1 ( PLA1 ) activity measurements of Tle1H , SVgrG - Tle1H , isolated SVgrG . Thermomyces lanuginosus lipase ( TLL ) was used as positive standard for PLA1 activity . Mean values standard deviation from at least three independent assays are shown . Statistical relative to Tle1 activity is indicated . * * * P < 0 . 001 ( P = 0 , 0001998 ) , unpaired two - sample Fisher - Pitman permutation .

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

Specific phospholipase A1 (PLA1) activity measurements of Tle1H, SVgrG-Tle1H complex, and isolated SVgrG. Thermomyces lanuginosus lipase (TLL) was used as positive standard for PLA1 activity. Mean values and standard deviation from at least three independent assays are shown. Statistical analysis relative to the Tle1 activity is indicated. ***P<0.001 (P=0,0001998), unpaired two-sample Fisher-Pitman permutation test.


VgrG inhibition of Tle1H . Specific phospholipase A1 ( PLA1 ) activity measurements of purified Tle1H in presence of 1 : 10 molar of lysozyme from chicken egg ( Lyso ) increasing molar ( 0 , 1 , 5 , 10 ) of SVgrG or SVgrG1 - 490 . Inhibition experiments with SVgrG were repeated three times with independent preparations , each measured in triplicate . Inhibition experiments with SVgrG1 - 490 were repeated twice with two independent preparations , each measured in triplicate . Mean values standard deviation are shown . Statistical relative to Tle1 activity is indicated . ns , non - significant ( P > 0 . 05 ) , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , unpaired two - sample Fisher - Pitman permutation . ( P values : Lyso , 0 . 7006 ; VgrG 1 : 1 , 0 . 004946 ; VgrG 1 : 5 , 1 . 448e - 07 ; VgrG1 : 10 , 1 . 448e - 07 ; VgrG1 - 490 1 : 1 , 0 . 5627 ; VgrG1 - 490 1 : 5 , 0 . 2977 ; VgrG1 - 490 1 : 10 , 0 . 002631 ) .

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VgrG inhibition of Tle1H. Specific phospholipase A1 (PLA1) activity measurements of purified Tle1H in the presence of 1:10 molar ratio of lysozyme from chicken egg white (Lyso) and increasing molar ratio (0, 1, 5, and 10) of SVgrG or SVgrG1-490. Inhibition experiments with SVgrG were repeated three times with independent protein preparations, each measured in triplicate. Inhibition experiments with SVgrG1-490 were repeated twice with two independent protein preparations, each measured in triplicate. Mean values and standard deviation are shown. Statistical analysis relative to the Tle1 activity is indicated. ns, non-significant (P>0.05), *P<0.05, **P<0.01, ***P<0.001, unpaired two-sample Fisher-Pitman permutation test. (P values: Lyso, 0.7006; VgrG 1:1, 0.004946; VgrG 1:5, 1.448e-07; VgrG1:10, 1.448e-07; VgrG1-490 1:1, 0.5627; VgrG1-490 1:5, 0.2977; VgrG1-490 1:10, 0.002631).


Representative SDS - PAGE / Coomassie blue of Tle1H , SVgrG SVgrG1 - 490 purified used in B ( 5 μg / well ) .

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Representative SDS-PAGE / Coomassie blue staining of the Tle1H, SVgrG and SVgrG1-490 purified proteins used in B (5 μg/well).


( G ) . GFP - tagged ERMES retain their characteristic punctate structure in Δlam6 , suggesting that Lam6 is not an essential member . Scale bar represents 5 μm .

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(G). GFP-tagged ERMES complex proteins retain their characteristic punctate structure in Δlam6, suggesting that Lam6 is not an essential complex member. Scale bar represents 5 μm.


( A ) microscopy demonstrates that Lam6 - GFP co - localizes with ERMES ( Mdm34 - Cherry ) ( yellow arrows ) also localizes to non - ERMES locations in ( red arrows ) . These additional locations co - localized with NVJ ( Nvj1 - GFP ) as well as with vCLAMP ( GFP - Vps39 ) . Scale bar represents 5 μm .

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(A) Fluorescence microscopy demonstrates that Lam6-GFP co-localizes with ERMES (Mdm34-Cherry) (yellow arrows) and also localizes to non-ERMES locations in the cell (red arrows). These additional locations co-localized with the NVJ (Nvj1-GFP) as well as with the vCLAMP (GFP-Vps39). Scale bar represents 5 μm.


( B ) Overexpression tagging of Lam6 confirmed that it co - localizes with ERMES ( Mdm34 - GFP ) ( yellow arrows ) as well as to additional contact sites ( red arrows ) , NVJ ( marked by Nvj1 ) vCLAMP ( marked by Vps39 ) . Scale bar represents 5 μm .

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(B) Overexpression and tagging of Lam6 confirmed that it co-localizes with ERMES (Mdm34-GFP) (yellow arrows) as well as to additional contact sites (red arrows), NVJ (marked by Nvj1) and vCLAMP (marked by Vps39). Scale bar represents 5 μm.


( A ) microscopy demonstrates that overexpression of Cherry - Lam6 ( OE - Cherry - LAM6 ) results in an expansion of following three contact sites : ERMES ( Mdm34 - GFP ) , NVJ ( Nvj1 - GFP ) , vCLAMP ( GFP - Vps39 ) . This suggests that an increase in Lam6 in contact site is sufficient for its expansion . numbers represent average contact site ( 120 per sample ) . Scale bar represents 5 μm .

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(A) Fluorescence microscopy demonstrates that the overexpression of Cherry-Lam6 (OE-Cherry-LAM6) results in an expansion of the following three contact sites: ERMES (Mdm34-GFP), NVJ (Nvj1-GFP), and vCLAMP (GFP-Vps39). This suggests that an increase in Lam6 levels in the contact site is sufficient for its expansion. The numbers represent the average contact site size (120 cells per sample). Scale bar represents 5 μm.


( B ) Immuno - EM verified that GFP - Lam6 overexpression indeed causes an expansion of contact site . While vCLAMP was hardly visible in WT , it could be detected easily in OE ( albeit often had ER tubules invading it ) . NVJ underwent expansion as well as evoked PMN in OE strains , ER - mitochondria contact became large elongated instead of small distinct . N , nucleus ; M , mitochondria ; V , vacuole . Scale bar represents 200 nm ( see also Figure S3I ) .

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(B) Immuno-EM verified that GFP-Lam6 overexpression indeed causes an expansion of contact site size. While vCLAMP was hardly visible in WT cells, it could be detected easily in the OE strain (albeit often had ER tubules invading it). The NVJ underwent expansion as well as evoked PMN in OE strains, and the ER-mitochondria contact became large and elongated instead of small and distinct. N, nucleus; M, mitochondria; V, vacuole. Scale bar represents 200 nm (see also Figure S3I).


( A ) microscopy shows that ERMES contact ( as measured by number of Mdm34 - GFP per ) expanded in Δvps39 background relative to WT . However , expansion did not occur on background of Δvps39 Δlam6 , demonstrating that Lam6 is necessary for ERMES expansion under these conditions . Scale bar represents 5 μm . ( B ) Quantitation of ( A ) . Bars represent of containing specific number of / out of total counted for ( for WT , n = 234 ; for Δvps39 , n = 246 ; for Δvps39 Δlam6 , n = 271 ) .

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(A) Fluorescent microscopy shows that the ERMES contact (as measured by the number of Mdm34-GFP puncta per cell) expanded in the Δvps39 background relative to WT. However, the expansion did not occur on the background of Δvps39 Δlam6, demonstrating that Lam6 is necessary for ERMES expansion under these conditions. Scale bar represents 5 μm.(B) Quantitation of (A). Bars represent the percentage of cells containing the specific number of puncta/cell out of total cells counted for the strain (for WT, n = 234 cells; for Δvps39, n = 246; for Δvps39 Δlam6, n = 271).


( C ) microscopy demonstrates that downregulating ERMES contacts ( by growing GALp - MDM34 strains in glucose ) indeed caused expansion of vCLAMP ( GFP - Vps39 ) . However , this expansion was diminished in a Δlam6 background . Scale bar represents 5 μm ( see also Figure S4 ) .

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(C) Fluorescent microscopy demonstrates that downregulating ERMES contacts (by growing GALp-MDM34 strains in glucose) indeed caused expansion of vCLAMP (GFP-Vps39). However, this expansion was diminished in a Δlam6 background. Scale bar represents 5 μm (see also Figure S4).


C . of Cre activity by dual luciferase assays ( mean ± SD , n = 3 ) . Data information Npu : Nostoc punctiforme , Ssp : Synechocystis sp . , CDS : coding .

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C. Quantification of Cre activity by dual luciferase assays (mean±SD, n=3). Data information Npu: Nostoc punctiforme, Ssp: Synechocystis sp., CDS: coding sequence.


D . of tTA activity by dual luciferase assays ( mean ± SD , n = 3 ( N C ) or n = 7 ( N / C ) ) . Data information : Npu : Nostoc punctiforme CDS : coding .

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D. Quantification of tTA activity by dual luciferase assays (mean±SD, n=3 (N and C) or n=7 (N/C)). Data information: Npu: Nostoc punctiforme CDS: coding sequence.


A - B . Scheme of engineered Spc Ccsp loci after integration of split - Cre split - tTA effectors ( red mark = stop codon ) comparison of endogenous SPC or CCSP expression YFP or mCherry reporter expression in lung sections . Blue : DAPI . Scale bar : 100 µm .

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A-B. Scheme of engineered Spc and Ccsp loci after integration of split-Cre and split-tTA effectors (red mark = stop codon) and comparison of endogenous SPC or CCSP expression and YFP or mCherry reporter expression in lung sections. Blue: DAPI. Scale bar: 100 µm.


C - D . β - galactosidase H & E of lung sections from BASC tracer BASC viewer . Scale bars : 500 µm ( overview ) 20 µm ( boxed magnification ) .

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C-D. β-galactosidase and H&E staining of lung sections from BASC tracer and BASC viewer animals. Scale bars: 500 µm (overview) and 20 µm (boxed magnification).


E . Localization of β - gal + in adult BASC viewer . BADJ - associated " alveolar " β - gal + are expressed as of lineage - labeled ( means ± SD n = 3 ) .

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E. Localization of β-gal+ cells in adult BASC viewer animals. BADJ-associated and "alveolar" β-gal+ cells are expressed as percentage of all lineage-labeled cells (means±SD n=3).


F . Number of β - gal + in adult BASC tracer BASC viewer , normalized to number of terminal bronchioles ( TBs ) ( means ± SD n = 3 ) .

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F. Number of β-gal+ cells in adult BASC tracer and BASC viewer animals, normalized to the number of terminal bronchioles (TBs) (means±SD n=3).


A . Sequential microscopic imaging of epifluorescence β - galactosidase activity in lung sections of adult BASC viewer . Arrow highlights labeled BASC at BADJ ( magnified in box ) . Blue : DAPI . Scale bars : 100 µm ( overview ) 20 µm ( boxed magnification ) .

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A. Sequential microscopic imaging of epifluorescence and β-galactosidase activity in lung sections of adult BASC viewer animals. Arrow highlights labeled BASC at BADJ (magnified in box). Blue: DAPI. Scale bars: 100 µm (overview) and 20 µm (boxed magnification).


B . β - galactosidase of AT2 ( YFP + ) , Club ( mCherry + ) BASCs ( mCherry + / YFP + ) isolated from BASC viewer mice by - activated sorting ( FACS ) . Scale bar : 50 µm .

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B. β-galactosidase staining of AT2 cells (YFP+), Club cells (mCherry+) and BASCs (mCherry+/YFP+) isolated from BASC viewer mice by fluorescence-activated cell sorting (FACS). Scale bar: 50 µm.


C . Doxycycline - dependent reversible labeling of BASCs in BASC viewer mice . Scale bar : 200 µm .

C. Doxycycline-dependent reversible labeling of BASCs in BASC viewer mice. Scale bar: 200 µm.


A. Heatmaps for Spearman correlation.


C . MA Volcano plots , Top50 DEGs ( FDR < 0 . 05 , Log2FC > ± 0 . 585 , Means > 5 ) for visualization of pairwise differential gene expression of BASCs Club . D . MA Volcano plots , Top50 DEGs ( FDR < 0 . 05 , Log2FC > ± 0 . 585 , Means > 5 ) for visualization of pairwise differential gene expression of BASCs AT2 .

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C. MA and Volcano plots, and Top50 DEGs (FDR<0.05, Log2FC>±0.585, Means>5) for visualization of pairwise differential gene expression analysis of BASCs and Club cells. D. MA and Volcano plots, and Top50 DEGs (FDR<0.05, Log2FC>±0.585, Means>5) for visualization of pairwise differential gene expression analysis of BASCs and AT2 cells.


E . of - seq z - scores computed for Top50 DEGs in BASC , AT2 , Club ( FDR < 0 . 05 , Log2FC > ± 0 . 585 , Means > 5 ) .

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E. Heatmap of RNA-seq z-scores computed for the Top50 DEGs in BASC, AT2, and Club cells (FDR<0.05, Log2FC>±0.585, Means>5).


B . of β - gal + in FACS - purified AT2 Club fractions isolated from 3 months old BASC v - race mice ( mean ± SD , n = 3 ) example of β - galactosidase label distribution in lung sections . Scale bar : 100 µm .

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B. Quantification of β-gal+ cells in FACS-purified AT2 and Club cell fractions isolated from 3 months old BASC v-race mice (mean±SD, n=3) and example of β-galactosidase label distribution in lung sections. Scale bar: 100 µm.


C . BASCs emerge at early postnatal stages contribute to epithelial turnover throughout life . β - galactosidase of lung sections derived from BASC v - race collected at indicated time points . Arrow highlights β - gal + at P6 . Scale bar : 200 µm .

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C. BASCs emerge at early postnatal stages and contribute to epithelial turnover throughout life. β-galactosidase staining of lung sections derived from BASC v-race animals collected at indicated time points. Arrow highlights β-gal+ cell at P6. Scale bar: 200 µm.


A . β - galactosidase of cleared whole lung preparations isolated from control injured BASC v - race 21 days ( bleomycin , naphthalene ) or 35 days ( influenza ) following injury . Scale bar : 5 mm ( whole lungs ) 1 mm ( magnification ) .

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A. β-galactosidase staining of cleared whole lung preparations isolated from control and injured BASC v-race animals 21 days (bleomycin, naphthalene) or 35 days (influenza) following injury. Scale bar: 5 mm (whole lungs) and 1 mm (magnification).


B . Sequential microscopic imaging of epifluorescence β - galactosidase activity in lung sections of control injured BASC v - race mice . Clusters of BASC - derived AT2 ( YFP + ) Club ( mCherry + ) are highlighted . Blue : DAPI . Scale bar : 200 µm .

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B. Sequential microscopic imaging of epifluorescence and β-galactosidase activity in lung sections of control and injured BASC v-race mice. Clusters of BASC-derived AT2 (YFP+) and Club cells (mCherry+) are highlighted. Blue: DAPI. Scale bar: 200 µm.


C . Contribution of BASCs to different compartments . Bronchiolar alveolar accumulations of lineage - traced BASC derivatives are expressed as of clusters containing ≥ 10 β - gal + . Data depicted as mean ± SD , n = 4 ( bleomycin , naphthalene ) or n = 5 ( influenza ) .

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C. Contribution of BASCs to different cellular compartments. Bronchiolar and alveolar accumulations of lineage-traced BASC derivatives are expressed as percentage of all clusters containing ≥ 10 β-gal+ cells. Data depicted as mean±SD, n=4 (bleomycin, naphthalene) or n=5 (influenza).


A . β - galactosidase H & E of lung sections from control ( non - infected ) injured BASC viewer mice 7 days post infection ( 7 dpi ) with influenza . Scale bar : 200 µm . B . Abundance of β - gal + in control ( non - infected ) injured ( 7 dpi ) BASC viewer . Data depicted as mean ± SD , n = 4 , * p = 0 . 0286 ( Mann - Whitney ) , TB - terminal bronchiole .

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A. β-galactosidase and H&E staining of lung sections from control (non-infected) and injured BASC viewer mice 7 days post infection (7 dpi) with influenza virus. Scale bar: 200 µm. B. Abundance of β-gal+ cells in control (non-infected) and injured (7 dpi) BASC viewer animals. Data depicted as mean±SD, n = 4, *p = 0.0286 (Mann-Whitney test), TB - terminal bronchiole.


C . Club depletion / recovery after naphthalene treatment . Microscopic imaging of epifluorescence in lung sections from control injured BASC viewer isolated 3 , 7 21 days post naphthalene ( dpn ) . Blue : DAPI . Scale bar : 200 µm .

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C. Club cell depletion/recovery after naphthalene treatment. Microscopic imaging of epifluorescence in lung sections from control and injured BASC viewer animals isolated 3, 7 and 21 days post naphthalene (dpn). Blue: DAPI. Scale bar: 200 µm.


D . β - galactosidase of lung sections from control injured BASC viewer 3 , 7 21 days post naphthalene ( dpn ) . Scale bar : 200 µm .

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D. β-galactosidase staining of lung sections from control and injured BASC viewer animals 3, 7 and 21 days post naphthalene (dpn). Scale bar: 200 µm.


E . Abundance of β - gal + in control injured BASC viewer . of terminal bronchioles containing 0 , 1 , 2 , 3 , 4 , ≥ 5 β - gal + is shown . Data depicted as mean ± SD , n = 3 .

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E. Abundance of β-gal+ cells in control and injured BASC viewer animals. The percentage of terminal bronchioles containing 0, 1, 2, 3, 4, ≥ 5 β-gal+ cells is shown. Data depicted as mean±SD, n=3.


A . of lineage - traced Club in distal TBs β - galactosidase of cleared accessory lobe preparation ( scale bar : 250 µm ) isolated from injured BASC v - race mice at 21 dpn ( mean ± SD , n = 4 ) . Bottom panel : example of sequential microscopic imaging to quantify β - gal + mCherry + in distal TBs . Arrows denote last airway bifurcation before bronchioalveolar junction ( BADJ ) . Scale bar : 200 µm .

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A. Quantification of lineage-traced Club cells in distal TBs and β-galactosidase staining of cleared accessory lobe preparation (scale bar: 250 µm) isolated from injured BASC v-race mice at 21 dpn (mean±SD, n=4). Bottom panel: example of sequential microscopic imaging to quantify β-gal+ mCherry+ cells in distal TBs. Arrows denote last airway bifurcation before bronchioalveolar duct junction (BADJ). Scale bar: 200 µm.


B . β - galactosidase of FACS - purified Club from control injured BASC v - race . Scale bar : 50 µm . of lineage - traced Club is shown ( mean ± SD , n = 4 ( oil ctrl . ) or n = 5 ( 21 dpn ) , * p = 0 . 0159 ( Mann - Whitney ) .

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B. β-galactosidase staining of FACS-purified Club cells from control and injured BASC v-race animals. Scale bar: 50 µm. The percentage of lineage-traced Club cells is shown (mean±SD, n=4 (oil ctrl.) or n=5 (21 dpn), *p = 0.0159 (Mann-Whitney test).


C . Combined β - galactosidase ( blue ) acetylated tubulin ( cilia , brown color ) of lung sections from BASC v - race at 21 dpn . Red : DAPI ( pseudocolor ) . Examples of lineage - traced ciliated ( arrowheads ) Club ( asterisks ) are highlighted . Scale bar : 50 µm .

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C. Combined β-galactosidase (blue) and acetylated tubulin staining (cilia, brown color) of lung sections from BASC v-race animals at 21 dpn. Red: DAPI (pseudocolor). Examples of lineage-traced ciliated cells (arrowheads) and Club cells (asterisks) are highlighted. Scale bar: 50 µm.


D . β - galactosidase of cleared accessory lobe preparations ( scale bar : 5 mm ) of β - gal + bronchioles in lung sections from adult BASC v - race ( ctrl . ) BASC - ablated mice ( DTA ) . Data depicted as mean ± SD , n = 3 . E . β - galactosidase of cleared accessory lobe preparations ( scale bar : 5 mm ) microscopy of lung sections from uninjured naphthalene - exposed BASC v - race ( ctrl . ) BASC - ablated ( DTA ) mice 12 days post naphthalene ( dpn ) . Dotted lines depict bronchiolar boundaries . Scale bar : 100 µm . F . of Club recovery in injured BASC v - race ( ctrl . ) BASC - ablated ( DTA ) , expressed as of bronchiolar circumference re - populated by mCherry + at 12 dpn . Data depicted as mean ± SD , n = 5 , * * p = 0 . 0079 ( Mann - Whitney ) . DTA - diphtheria toxin fragment A .

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D. β-galactosidase staining of cleared accessory lobe preparations (scale bar: 5 mm) and quantification of β-gal+ bronchioles in lung sections from adult BASC v-race (ctrl.) and BASC-ablated mice (DTA). Data depicted as mean±SD, n=3. E. β-galactosidase staining of cleared accessory lobe preparations (scale bar: 5 mm) and fluorescence microscopy of lung sections from uninjured and naphthalene-exposed BASC v-race (ctrl.) and BASC-ablated (DTA) mice 12 days post naphthalene (dpn). Dotted lines depict bronchiolar boundaries. Scale bar: 100 µm. F. Quantification of Club cell recovery in injured BASC v-race (ctrl.) and BASC-ablated (DTA) animals, expressed as percentage of bronchiolar circumference re-populated by mCherry+ cells at 12 dpn. Data depicted as mean±SD, n=5, **p = 0.0079 (Mann-Whitney test). DTA - diphtheria toxin fragment A.


Assessment of mtDNA damage by quantitative PCR . HEK293 infected with control ( empty vector ) or shATG5 for 5 days . were then incubated with DMSO or 4 mM 3 - NPA for 2 h either immediately harvested or washed with fresh medium incubated for another 1 h . with or without washout were used for extraction of total . samples were used for amplification of 8 . 9 kb mtDNA fragment using quantitative PCR were normalized to amplification of a 221 bp mtDNA fragment . PCR products were quantitated by PicoGreen using Micro Plate Reader . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by two - student ' s t - , N . S . , not significant , * p < 0 . 05 , * * p < 0 . 01 . data in ( A ) were further calculated for frequency of mtDNA damage . equation was seen in " Materials Methods " . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by a two - student ' s t - , N . S . , not significant , * * p < 0 . 01 .

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Assessment of mtDNA damage by quantitative PCR. HEK293 cells infected with control (empty vector) or shATG5 for 5 days. Cells were then incubated with DMSO or 4 mM 3-NPA for 2 h and either immediately harvested or washed with fresh medium and incubated for another 1 h. Cells with or without washout were used for the extraction of total DNA. All DNA samples were used for amplification of 8.9 kb mtDNA fragment using quantitative PCR and were normalized to amplification of a 221 bp mtDNA fragment. PCR products were quantitated by PicoGreen staining using Micro Plate Reader. Data are presented as mean ± SD (n = 3 independent experiments), statistical significance was assessed by two-tailed student's t-test, N.S., not significant, *p < 0.05, **p < 0.01. The data in (A) were further calculated for the frequency of mtDNA damage. The equation was seen in "Materials and Methods". Data are presented as mean ± SD (n = 3 independent experiments), statistical significance was assessed by a two-tailed student's t-test, N.S., not significant, **p < 0.01.


Representative show 8 - oxo - dG . Control or shATG5 HeLa were treated with DMSO , 200 µM H2O2 , or 4 mM 3 - NPA for 2 h , then either fixed immediately or washed with fresh medium incubated for another 1 h . were immunostained with DAPI anti - 8 - oxo - dG antibody analyzed by confocal microscopy . Scale bar , 10 µm . of relative 8 - oxo - dG in ( C ) . Data are presented as mean ± SD ( n = 3 independent experiments , 20 per experiment ) , statistical significance was assessed by a two - student ' s t - , N . S . , not significant , * p < 0 . 05 , * * p < 0 . 01 .

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Representative images show 8-oxo-dG staining. Control or shATG5 HeLa cells were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h, and then either fixed immediately or washed with fresh medium and incubated for another 1 h. Cells were immunostained with DAPI and anti-8-oxo-dG antibody and analyzed by confocal microscopy. Scale bar, 10 µm. Quantification of the relative 8-oxo-dG fluorescence intensity in (C). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by a two-tailed student's t-test, N.S., not significant, *p < 0.05, **p < 0.01.


Control or shATG5 HEK293 stably expressing mito - Keima . Control were treated with DMSO , 200 µM H2O2 , or 4 mM 3 - NPA for 2 h , shATG5 were treated with 200 µM H2O2 as a negative control . were then imaged with 458 nm ( measuring mitochondria with a neutral pH ) 561 nm ( measuring mitochondria with an acidic pH ) excitation for mito - Keima by confocal microscopy . Right panels show pixel of red ( mitochondria within lysosomes ) green ( mitochondria in cytoplasm ) from a . Scale bar , 10 µm . of relative of red to green ( 561 nm / 458 nm ) of described in ( E ) . Data are presented as mean ± SD ( n = 3 independent experiments , 20 per experiment ) , statistical significance was assessed by a two - way ANOVA , N . S . , not significant , * * p < 0 . 01 .

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Control or shATG5 HEK293 cells stably expressing mito-Keima. Control cells were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h, and shATG5 cells were treated with 200 µM H2O2 as a negative control. Cells were then imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima by confocal microscopy. Right panels show the pixel intensity of red (mitochondria within lysosomes) and green (mitochondria in the cytoplasm) from a line. Scale bar, 10 µm. Quantification of the relative ratio of red to green fluorescence intensity (561 nm/458 nm) of the cells described in (E). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by a two-way ANOVA, N.S., not significant, **p < 0.01.


HeLa expressing GFP - LC3 were treated with DMSO , 200 µM H2O2 , or 4 mM 3 - NPA for 2 h . were stained with anti - Tom20 anti - antibodies , then analyzed by confocal microscopy . arrows were indicated LC3 punta colocalizing or contacting with mtDNA / Tom20 . Scale bar , 10 µm . of LC3 punta colocalizing or contacting with mtDNA / Tom20 in a . n = 30 from 3 coverslips , Data are presented as mean ± SD ( n = 30 ) , statistical significance was assessed by a one - way ANOVA with Tukey ' s multiple comparisons , * * * p < 0 . 001 .

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HeLa cells expressing GFP-LC3 were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h. Cells were stained with anti-Tom20 and anti-DNA antibodies, then analyzed by confocal microscopy. The white arrows were indicated the LC3 punta colocalizing or contacting with mtDNA/Tom20. Scale bar, 10 µm. Quantification of the LC3 punta colocalizing or contacting with mtDNA/Tom20 in a cell. n = 30 cells from 3 coverslips, Data are presented as mean ± SD (n = 30), statistical significance was assessed by a one-way ANOVA with Tukey's multiple comparisons test, ***p < 0.001.


Screening of mitophagy regulators for removing damaged mtDNA . HeLa were infected by lentiviral particles containing indicated knockdown vectors . Five days later , were treated with 200 µM H2O2 for 2 h . were then imaged with 458 nm ( measuring mitochondria with a neutral pH ) 561 nm ( measuring mitochondria with an acidic pH ) excitation for mito - Keima . ShATG5 was used as a negative control . Right panels for each image show FACS - mito - Keima dot plots . y - axis represents emission of mito - Keima at pH 4 . 0 ( lysosome ) , while x - axis indicates mito - Keima at pH 7 . 0 ( mitochondria ) . percentages of within different regions are indicated . Scale bar , 10 µm .

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Screening of mitophagy regulators for removing damaged mtDNA. HeLa cells were infected by lentiviral particles containing the indicated knockdown vectors. Five days later, cells were treated with 200 µM H2O2 for 2 h. Cells were then imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima. ShATG5 was used as a negative control. Right panels for each image show the FACS-based mito-Keima dot plots. The y-axis represents the fluorescence emission of mito-Keima at pH 4.0 (lysosome), while the x-axis indicates mito-Keima at pH 7.0 (mitochondria). The percentages of cells within the different regions are indicated. Scale bar, 10 µm.


Control or ATAD3B KO HeLa stably expressing mito - Keima were treated with 4 mM 3 - NPA for 2 h imaged with 458 nm ( measuring mitochondria with a neutral pH ) 561 nm ( measuring mitochondria with an acidic pH ) excitation for mito - Keima by confocal microscopy . Scale bar , 10 µm . of relative of red to green ( 561 nm / 458 nm ) of described in ( B ) . Data are presented as mean ± SD ( n = 3 independent experiments , 20 per experiment ) , statistical significance was assessed by two - student ' s t - , N . S . , not significant , * * p < 0 . 01 .

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Control or ATAD3B KO HeLa cells stably expressing mito-Keima were treated with 4 mM 3-NPA for 2 h and imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima by confocal microscopy. Scale bar, 10 µm. Quantification of the relative ratio of red to green fluorescence intensity (561 nm/458 nm) of the cells described in (B). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by two-tailed student's t-test, N.S., not significant, **p < 0.01.