Figure 7Flt3 / ITD knock - in mouse were genotyped ( A ) . The spleen weight ( B and C , 4 mice each ) were measured in WT siblings and Flt3 / ITD - knock - in mice ( 8 mice each ) . data were presented in box plot . The whiskers , boxes , and central lines represented the minimum - to - maximum values , 25th - to - 75th percentile , and the 50th percentile ( median ) , respectively . * * P < 0 . 01 ( Student ' s t - test ) . The serum Fst level ( D ) were measured in WT siblings and Flt3 / ITD - knock - in mice ( 8 mice each ) . data were presented in box plot . The whiskers , boxes , and central lines represented the minimum - to - maximum values , 25th - to - 75th percentile , and the 50th percentile ( median ) , respectively . * * P < 0 . 01 ( Student ' s t - test ) . The engraftment of MOLM - 13 in NSG mice was confirmed at week 1 post injection ( E - F ) . Serum FST level was measured in MOLM - 13 - engrafted NSG mice at pre - injection and week 2 post injection ( G , PBS group , 3 mice ; MOLM - 13 group , 4 mice ) . Data information : data were presented as scatter dot plot . n . s : not significant , * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) . InAfter FST knockdown , serum FST level was also measured in MOLM - 13 - engrafted NSG mice at week 2 post injection ( H , 3 mice for each group ) . Data information : data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) . Serum FST level was significantly increased in primary AML - derived xenografted mouse at week 6 post injection . Human leukemic engraftments were confirmed by flow cytometry of human CD45 and mouse CD45 . 1 cells in recipient mouse BM aspiration ( J ) . Serum FST level was significantly increased in primary AML - derived xenografted mouse at week 6 post injection . Serum FST from pre - injection and post - engraftment mouse were measured ( K , one mouse for each primary AML sample ) . Data information : data were presented as scatter dot plot . n . s : not significant , * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) . L Correlation between serum FST levels and leukemia blast percentage from FLT3 / ITD - mutated AML at diagnosis . Correlation analysis ( Pearson correlation coefficient ) was performed by Graphpad Prism 6 . Serum FST decreased in CR and increased after relapse in 4 AML patients receiving Quizartinib monotherapy . Data information : data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) . N Serum FST continued to rise during disease progression from a patient who did not respond to Quizartinib . Patients in N were recruited in the QUANTUM - R and patient accrual has been completed . data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) .

O B-PANEL_START O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Flt3/ITD knock-in mouse were genotyped (A).The spleen weight (B and C, 4 mice each) were measured in WT siblings and Flt3/ITD-knock-in mice (8 mice each). data were presented in box plot. The whiskers, boxes, and central lines represented the minimum-to-maximum values, 25th-to-75th percentile, and the 50th percentile (median), respectively. **P<0.01 (Student's t-test).The serum Fst level (D) were measured in WT siblings and Flt3/ITD-knock-in mice (8 mice each). data were presented in box plot. The whiskers, boxes, and central lines represented the minimum-to-maximum values, 25th-to-75th percentile, and the 50th percentile (median), respectively. **P<0.01 (Student's t-test).The engraftment of MOLM-13 in NSG mice was confirmed at week 1 post injection (E-F).Serum FST level was measured in MOLM-13-engrafted NSG mice at pre-injection and week 2 post injection (G, PBS group, 3 mice; MOLM-13 group, 4 mice). Data information: data were presented as scatter dot plot. n.s: not significant, *P<0.05, **P<0.01 (Student's t-test). InAfter FST knockdown, serum FST level was also measured in MOLM-13-engrafted NSG mice at week 2 post injection (H, 3 mice for each group). Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).Serum FST level was significantly increased in primary AML-derived xenografted mouse at week 6 post injection. Human leukemic engraftments were confirmed by flow cytometry of human CD45 and mouse CD45.1 cells in recipient mouse BM aspiration (J).Serum FST level was significantly increased in primary AML-derived xenografted mouse at week 6 post injection. Serum FST from pre-injection and post-engraftment mouse were measured (K, one mouse for each primary AML sample). Data information: data were presented as scatter dot plot. n.s: not significant, *P<0.05, **P<0.01 (Student's t-test).L Correlation between serum FST levels and leukemia blast percentage from FLT3/ITD-mutated AML at diagnosis. Correlation analysis (Pearson correlation coefficient) was performed by Graphpad Prism 6.Serum FST decreased in CR and increased after relapse in 4 AML patients receiving Quizartinib monotherapy. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).N Serum FST continued to rise during disease progression from a patient who did not respond to Quizartinib. Patients in N were recruited in the QUANTUM-R and patient accrual has been completed. data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).


f1 ( a ) The ternary NDP52 - GAL8 - LC3C complex ( green line ) , binary NDP52 - GAL8 complex ( blue line ) and NDP52 complex alone ( red line ) by SEC - MALS . The dotted line represents measured MW and the vertical bars ( 1 and 2 ) indicate the gap in MW . The SEC - MALS result of NDP52 alone ( red line ) shows that the oligomeric state is ambiguous between dimer and trimer . However , the NDP52 - GAL8 complex shows a MW difference of 70 kDa compared with NDP52 alone , which confirmed that two molecules of monomeric GAL8 ( MW : ~ 33 . 5 kDa ; Supplementary Fig . S1b ) bound to homodimeric NDP52 . Furthermore , the ternary NDP52 - GAL8 - LC3C complex showed a MW difference of ~ 30 kDa compared with the binary NDP52 - GAL8 complex , which also confirmed that two additional molecules of monomeric LC3C bound to the binary complex . ( b ) Size - exclusion chromatography data of NDP52 - GAL8 - LC3C complex . The first peak ( marked by an orange bar ) represents the ternary complex , as shown by the SDS - polyacrylamide gel electrophoresis ( SDS - PAGE ) results marked by an orange bar ( lane 1 , 2 and 3 ) . The second merged peak ( marked by green and yellow bar ) and corresponding SDS - PAGE results show the remaining free GAL8 ( lane 4 , 5 , 6 and 7 ) and LC3C ( lane 5 , 6 , 7 and 8 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f1(a) The ternary NDP52-GAL8-LC3C complex (green line), binary NDP52-GAL8 complex (blue line) and NDP52 complex alone (red line) by SEC-MALS. The dotted line represents measured MW and the vertical bars (1 and 2) indicate the gap in MW. The SEC-MALS result of NDP52 alone (red line) shows that the oligomeric state is ambiguous between dimer and trimer. However, the NDP52-GAL8 complex shows a MW difference of 70 kDa compared with NDP52 alone, which confirmed that two molecules of monomeric GAL8 (MW: ~33.5 kDa; Supplementary Fig. S1b) bound to homodimeric NDP52. Furthermore, the ternary NDP52-GAL8-LC3C complex showed a MW difference of ~30 kDa compared with the binary NDP52-GAL8 complex, which also confirmed that two additional molecules of monomeric LC3C bound to the binary complex.(b) Size-exclusion chromatography data of NDP52-GAL8-LC3C complex. The first peak (marked by an orange bar) represents the ternary complex, as shown by the SDS-polyacrylamide gel electrophoresis (SDS-PAGE) results marked by an orange bar (lane 1, 2 and 3). The second merged peak (marked by green and yellow bar) and corresponding SDS-PAGE results show the remaining free GAL8 (lane 4, 5, 6 and 7) and LC3C (lane 5, 6, 7 and 8).


f2 ( a ) Overall structure of GAL8 with bound Galbi peptide ( cyan ) and NAD ( purple ) in the asymmetric unit . The other pair of crystallographic dimer is coloured grey . The first β - strand forming unique interaction is labelled as β1 . ( b ) A topology diagram of GAL8 . The β - strands in each N - CRD and C - CRD are numbered sequentially with prime ( ′ ) in the C - CRD . The β - strands with a solid line indicate a carbohydrate - binding side in each CRD and those of with a dashed line indicate an opposite side . The elongated purple and cyan ovals indicate NAD - and NDP52 - binding sites , respectively . ( c ) Overall view of Galbi peptide ( cyan ) bound to the C - CRD of GAL8 ( β - strands in yellow and residues in gray ) .

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f2(a) Overall structure of GAL8 with bound Galbi peptide (cyan) and NAD (purple) in the asymmetric unit. The other pair of crystallographic dimer is coloured grey. The first β-strand forming unique interaction is labelled as β1. (b) A topology diagram of GAL8. The β-strands in each N-CRD and C-CRD are numbered sequentially with prime (′) in the C-CRD. The β-strands with a solid line indicate a carbohydrate-binding side in each CRD and those of with a dashed line indicate an opposite side. The elongated purple and cyan ovals indicate NAD- and NDP52-binding sites, respectively. (c) Overall view of Galbi peptide (cyan) bound to the C-CRD of GAL8 (β-strands in yellow and residues in gray).


f3 ( a ) Magnified view showing the detailed interaction around Leu374 ( p ) . ( b ) Magnified view showing the detailed interaction around Tyr380 ( p ) . ( c ) Magnified view showing the importance of main - chain hydrogen bonds of the Galbi peptide . ( a - c ) Red dashed lines designate hydrogen bonds . ( d ) An FP assay using FITC - labelled Galbi peptides , including mutants ( L374A , Y380F and Y380A ) against an increasing concentration of GAL8 . ( e ) An FP assay using a FITC - labelled Galbi peptide against an increasing concentration of GAL8 mutants ( E268A , I270A , Y272A and Trp317 ) . ( d , e ) The KD values were calculated from at least three independent experiments . ND , not determined .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f3(a) Magnified view showing the detailed interaction around Leu374(p). (b) Magnified view showing the detailed interaction around Tyr380(p). (c) Magnified view showing the importance of main-chain hydrogen bonds of the Galbi peptide. (a-c) Red dashed lines designate hydrogen bonds.(d) An FP assay using FITC-labelled Galbi peptides, including mutants (L374A, Y380F and Y380A) against an increasing concentration of GAL8. (e) An FP assay using a FITC-labelled Galbi peptide against an increasing concentration of GAL8 mutants (E268A, I270A, Y272A and Trp317). (d,e) The KD values were calculated from at least three independent experiments. ND, not determined.


f4 ( a ) Sequence alignment of N - CRD and C - CRD of GAL8 . The residues important for interaction with the Galbi peptide and NAD are marked by yellow and magenta circles , respectively . ( b ) Detailed view of the peptide - binding site of C - CRD superposed with N - CRD . The residues for the Galbi peptide ( cyan ) interaction are labelled and the side chains of the corresponding N - CRD residues ( magenta ) are shown . ( c ) A detailed view of the NAD - binding site of N - CRD superposed with C - CRD . The residues for NAD ( purple ) interaction are labelled and the side chains of corresponding C - CRD residues ( yellow ) are shown .

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f4(a) Sequence alignment of N-CRD and C-CRD of GAL8. The residues important for interaction with the Galbi peptide and NAD are marked by yellow and magenta circles, respectively. (b) Detailed view of the peptide-binding site of C-CRD superposed with N-CRD. The residues for the Galbi peptide (cyan) interaction are labelled and the side chains of the corresponding N-CRD residues (magenta) are shown. (c) A detailed view of the NAD-binding site of N-CRD superposed with C-CRD. The residues for NAD (purple) interaction are labelled and the side chains of corresponding C-CRD residues (yellow) are shown.


f5 ( a ) Three GAL8 structures , including both CRDs , are available from PDB ( 4FQZ - lactose bound in only N - CRD ; 3VKL - lactose bound in both N - CRD and N - C - CRD ; 3VKM - sialylated lactose in N - CRD and N - CRD in C - CRD ) . The C - CRDs of these Gal8 structures were superposed with that of our Gal8 . For clarity , only Cα - traces of each structure ( our GAL8 - yellow ; 4FQZ - red ; 3VKM - blue ; and 3VKL - cyan ) are shown . The large rotation of the N - CRD domain relative to GAL8 compared with the other NDP52 structures is observed in the GAL8 - NDP52 complex . A representative angle between CRD of our Gal8 and that of the 4FQZ structure is indicated in degrees . ( b ) A 90 ° rotation of panel ( a ) along the horizontal axis showing a top view . Arg59 , which is a unique residue of Gal8CRD that provides carbohydrate specificity , is used as a standard for measuring the distance of domain movement .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f5(a) Three GAL8 structures, including both CRDs, are available from PDB (4FQZ-lactose bound in only N-CRD; 3VKL-lactose bound in both N-CRD and N-C-CRD; 3VKM-sialylated lactose in N-CRD and N-CRD in C-CRD). The C-CRDs of these Gal8 structures were superposed with that of our Gal8. For clarity, only Cα-traces of each structure (our GAL8-yellow; 4FQZ-red; 3VKM-blue; and 3VKL-cyan) are shown. The large rotation of the N-CRD domain relative to GAL8 compared with the other NDP52 structures is observed in the GAL8-NDP52 complex. A representative angle between CRD of our Gal8 and that of the 4FQZ structure is indicated in degrees. (b) A 90° rotation of panel (a) along the horizontal axis showing a top view. Arg59, which is a unique residue of Gal8CRD that provides carbohydrate specificity, is used as a standard for measuring the distance of domain movement.


Figure 1A αSMA mRNA levels in TGFβ - treated HFL1 fibroblasts and the effect of the IRE1α inhibitor 4μ8C . * P = 0 , 001 . B Collagen1α2 mRNA levels in TGFβ - treated HFL1 fibroblasts ± the IRE1α inhibitor 4μ8C . * P = 0 , 01 . C mRNA levels of ER - stress related genes in TGFβ - treated HFL1 fibroblasts . * P CHOP = 0 , 045 , * P Bip = 0 , 023 , * P spliced XBP - 1 P = 0 , 001 , compared to corresponding control . D Western blot of CHOP protein levels in TGFβ - treated HFL1 cells ± the IRE1α inhibitor 4μ8C . E Agarose gel showing the presence of spliced and unspliced XBP - 1 mRNA in TGFβ - treated HFL1 fibroblasts ± the IRE1α inhibitor 4μ8C . F Immunohistochemistry staining of αSMA in TGFβ - treated HFL1fibroblasts ± the IRE1α inhibitor 4μ8C . Scale bars = 20μm . G Quantification of secreted soluble collagen proteins produced by TGFβ - treated HFL1 cells ± the IRE1α inhibitor 4μ8C . * P = 0 , 049 . H αSMA mRNA expression in TGFβ - treated HFL1 cells overexpressing two RNase dead mutants ( K599A and K907A ) of IRE1α . * P K599A = 0 . 013 , * P K907A = 0 . 0008 compared to WT . I αSMA mRNA expression in TGFβ - treated IRE1α - / - or WT MEF cells . * P = 0 . 025 .

O B-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A αSMA mRNA levels in TGFβ-treated HFL1 fibroblasts and the effect of the IRE1α inhibitor 4μ8C. *P = 0,001.B Collagen1α2 mRNA levels in TGFβ-treated HFL1 fibroblasts ± the IRE1α inhibitor 4μ8C. *P = 0,01.C mRNA levels of ER-stress related genes in TGFβ-treated HFL1 fibroblasts. *P CHOP = 0,045, *P Bip = 0,023, *P spliced XBP-1 P = 0,001, compared to corresponding control.D Western blot of CHOP protein levels in TGFβ-treated HFL1 cells ± the IRE1α inhibitor 4μ8C.E Agarose gel showing the presence of spliced and unspliced XBP-1 mRNA in TGFβ-treated HFL1 fibroblasts ± the IRE1α inhibitor 4μ8C.F Immunohistochemistry staining of αSMA in TGFβ-treated HFL1fibroblasts ± the IRE1α inhibitor 4μ8C. Scale bars = 20μm.G Quantification of secreted soluble collagen proteins produced by TGFβ-treated HFL1 cells ± the IRE1α inhibitor 4μ8C. *P = 0,049.H αSMA mRNA expression in TGFβ-treated HFL1 cells overexpressing two RNase dead mutants (K599A and K907A) of IRE1α. *P K599A = 0.013, *P K907A = 0.0008 compared to WT.I αSMA mRNA expression in TGFβ-treated IRE1α -/- or WT MEF cells. *P = 0.025.


Figure 2A miR - 150 levels in TGFβ - treated HFL1 fibroblasts and the effect of the IRE1α inhibitor 4μ8C . * P = 0 . 046 compared to control . B miR - 150 levels in HFL1 fibroblasts overexpressing two RNase dead mutants ( K599A and K907A ) of IRE1α . * P = 0 . 0043 ( K599A ) , P = 0 . 043 ( K907A ) , compared to control . C miR - 150 levels in TGFβ - treated WT ( IRE1α + / + ) and IRE1α - / - MEF cells . * P = 0 . 018 . Results are expressed as fold change compared to the Ctrl . D miR - 150 levels in IRE1α - / - MEF cells that were either untransfected ( UT ) or transfected with miRNA inhibitor control ( siCtrl ) or with a miR - 150 - 5p miRNA inhibitor . * P = 0 . 034 compared to UT . E αSMA mRNA expression in TGFβ - treated IRE1α - / - MEF cells that were either untransfected ( UT ) or transfected with miRNA inhibitor control ( siCtrl ) or with a miR150 - 5p miRNA inhibitor . * P = 0 . 043 compared to UT . F miR - 150 levels in HFL1 fibroblasts transfected with a control plasmid ( Ctrl ) or a miR - 150 expression plasmid ( miR150 ) . * P = 0 . 038 . G Western blot of c - Myb in TGFβ - treated HFL1 fibroblasts that were either untransfected ( UT ) , transfected with a control plasmid ( pCtrl ) or a miR150 expression plasmid ( miR150 ) . * P = 0 . 0206 in UT Ctrl vs . UT TGFβ and P = 0 . 0126 in pCtrl Ctrl vs . pCtrl TGFβ . H Western blot of αSMA in the same experimental setup as in G . * P = 0 . 0405 in UT Ctrl vs . UT TGFβ , and P = 0 . 0284 in pCtrl Ctrl vs . pCtrl TGFβ . I c - Myb mRNA levels in untransfected HFL1 cells ( UT ) and in cells transfected with a control siRNA ( siCtrl ) or an siRNA targeting c - Myb ( sicMyb ) . * P = 0 . 00001 compared to UT . J αSMA mRNA expression in untransfected HFL1 cells ( UT ) and in cells transfected with a control siRNA ( siCtrl ) or with a siRNA targeting c - Myb ( sicMyb ) . * P = 0 . 0042 in UT Ctrl vs . UT TGFβ . L In vitro cleavage assay with recombinant IRE1α and miR - 150 ( left ) or XBP - 1 ( right ) . M miR - 150 expression in HFL - 1 cells that were pre - treated with TGFβ in the absence of presence of 4μ8C and then treated with Actinomycin D ( ActD ) for 4 and 24 hours . * P = 0 . 027 compared to TGFβ .

O B-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A miR-150 levels in TGFβ-treated HFL1 fibroblasts and the effect of the IRE1α inhibitor 4μ8C. * P = 0.046 compared to control.B miR-150 levels in HFL1 fibroblasts overexpressing two RNase dead mutants (K599A and K907A) of IRE1α. * P = 0.0043 (K599A), P = 0.043 (K907A), compared to control.C miR-150 levels in TGFβ-treated WT (IRE1α +/+) and IRE1α -/- MEF cells. *P = 0.018. Results are expressed as fold change compared to the Ctrl.D miR-150 levels in IRE1α -/- MEF cells that were either untransfected (UT) or transfected with miRNA inhibitor control (siCtrl) or with a miR-150-5p miRNA inhibitor. * P = 0.034 compared to UT.E αSMA mRNA expression in TGFβ-treated IRE1α -/- MEF cells that were either untransfected (UT) or transfected with miRNA inhibitor control (siCtrl) or with a miR150-5p miRNA inhibitor. *P = 0.043 compared to UT.F miR-150 levels in HFL1 fibroblasts transfected with a control plasmid (Ctrl) or a miR-150 expression plasmid (miR150). * P = 0.038.G Western blot of c-Myb in TGFβ-treated HFL1 fibroblasts that were either untransfected (UT), transfected with a control plasmid (pCtrl) or a miR150 expression plasmid (miR150). *P = 0.0206 in UT Ctrl vs. UT TGFβ and P = 0.0126 in pCtrl Ctrl vs. pCtrl TGFβ .H Western blot of αSMA in the same experimental setup as in G. * P = 0.0405 in UT Ctrl vs. UT TGFβ, and P = 0.0284 in pCtrl Ctrl vs. pCtrl TGFβ.I c-Myb mRNA levels in untransfected HFL1 cells (UT) and in cells transfected with a control siRNA (siCtrl) or an siRNA targeting c-Myb (sicMyb). * P = 0.00001 compared to UT.J αSMA mRNA expression in untransfected HFL1 cells (UT) and in cells transfected with a control siRNA (siCtrl) or with a siRNA targeting c-Myb (sicMyb). * P = 0.0042 in UT Ctrl vs. UT TGFβ.L In vitro cleavage assay with recombinant IRE1α and miR-150 (left) or XBP-1 (right).M miR-150 expression in HFL-1 cells that were pre-treated with TGFβ in the absence of presence of 4μ8C and then treated with Actinomycin D (ActD) for 4 and 24 hours. * P = 0.027 compared to TGFβ.


Figure 3A Analysis of ER size in HFL1 fibroblasts treated with TGFβ in the absence ( Ctrl ) or presence of the IRE1α inhibitor 4μ8C , or in HFL1 cells transfected with a siRNA control siRNA ( siCtrl ) or with siRNA targeting XBP - 1 ( siXBP - 1 ) . The ER was visualized with ER tracker Red and nuclei stained with Hoechst ( left , scale bars = 20 μm ) or by electron microscopy ( right , scale bars = 200 nm ) where red lines mark the ER . B Quantification of ER size relative to the nucleus from the experimental setup in A . * P = 0 . 0001 in Ctrl , and * P = 0 . 0223 in siCtrl . C XBP - 1 mRNA expression in untransfected HFL1 cells ( UT ) and in cells transfected with control siRNA ( siCtrl ) or siRNA targeting XBP - 1 ( siXBP - 1 ) . * P = 0 , 0001 . D Collagen protein levels in TGFβ treated HFL1 fibroblasts transfected as described in C . * P = 0 , 005 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O

Figure 3A Analysis of ER size in HFL1 fibroblasts treated with TGFβ in the absence (Ctrl) or presence of the IRE1α inhibitor 4μ8C, or in HFL1 cells transfected with a siRNA control siRNA (siCtrl) or with siRNA targeting XBP-1 (siXBP-1). The ER was visualized with ER tracker Red and nuclei stained with Hoechst (left, scale bars = 20 μm) or by electron microscopy (right, scale bars = 200 nm) where red lines mark the ER.B Quantification of ER size relative to the nucleus from the experimental setup in A. * P = 0.0001 in Ctrl, and * P = 0.0223 in siCtrl.C XBP-1 mRNA expression in untransfected HFL1 cells (UT) and in cells transfected with control siRNA (siCtrl) or siRNA targeting XBP-1 (siXBP-1). * P = 0,0001.D Collagen protein levels in TGFβ treated HFL1 fibroblasts transfected as described in C. * P = 0,005.


Figure 4A The effect of the IRE1α inhibitor 4μ8C on CCl4 induced liver fibrosis in C57BL / 6mice as measured by the histological metavir score . Ctrl ( n = 8 ) ; Ctrl + 4μ8C ( n = 6 ) ; CCl4 ( n = 6 ) ; CCl4 + 4μ8C ( n = 8 ) . * P = 0 , 0077 . B Representative liver tissue sections stained with sirius red for collagen ( top ) and αSMA ( bottom ) . Scale bars = 50 μm . C Percentage of αSMA positive cells in pericentral areas in livers from mice in the experimental setup in A . * P = 0 , 036 . D Liver to body weight ratio from the experimental setup in A . * P = 0 , 0031 . E BiP mRNA expression in liver tissue from mice presented in A . * P = 0 , 020 . F Representative gel showing levels of spliced and unspliced XBP - 1 in liver tissue from control and CCl4 - induced cirrhotic mice and the effect of 4μ8C treatment . G miR150 expression in liver tissue from mice in A . * P = 0 , 032 compared to untreated controls . H αSMA mRNA levels in primary stellate cells isolated from mouse liver and then treated or not with TGFβ ± 4μ8C . * P = 0 , 031 , n = 3 . I BiP mRNA levels in primary stellate cells isolated from mouse liver and then treated with or without TGFβ ± 4μ8C . * P = 0 , 008 , n = 3 . J Microscopic images of primary mouse hepatic stellate cells stained for BiP ( top ) or αSMA ( bottom ) . Scale bars = 20 μm . K Western blot showing BiP protein expression in primary mouse hepatic stellate cells . L Quantification of mRNA expression levels of CTGF and collagen in primary human hepatic stellate cells stimulated with TGFβ ± 4μ8C .

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Figure 4A The effect of the IRE1α inhibitor 4μ8C on CCl4 induced liver fibrosis in C57BL/6mice as measured by the histological metavir score. Ctrl (n = 8) ; Ctrl + 4μ8C (n = 6) ; CCl4 (n = 6); CCl4 + 4μ8C (n = 8). * P = 0,0077.B Representative liver tissue sections stained with sirius red for collagen (top) and αSMA (bottom). Scale bars= 50 μm.C Percentage of αSMA positive cells in pericentral areas in livers from mice in the experimental setup in A. * P = 0,036.D Liver to body weight ratio from the experimental setup in A. * P = 0,0031.E BiP mRNA expression in liver tissue from mice presented in A. * P = 0,020.F Representative gel showing levels of spliced and unspliced XBP-1 in liver tissue from control and CCl4-induced cirrhotic mice and the effect of 4μ8C treatment.G miR150 expression in liver tissue from mice in A. * P = 0,032 compared to untreated controls.H αSMA mRNA levels in primary stellate cells isolated from mouse liver and then treated or not with TGFβ ± 4μ8C. * P = 0,031, n = 3.I BiP mRNA levels in primary stellate cells isolated from mouse liver and then treated with or without TGFβ ± 4μ8C. * P = 0,008, n = 3.J Microscopic images of primary mouse hepatic stellate cells stained for BiP (top) or αSMA (bottom). Scale bars = 20 μm.K Western blot showing BiP protein expression in primary mouse hepatic stellate cells.L Quantification of mRNA expression levels of CTGF and collagen in primary human hepatic stellate cells stimulated with TGFβ ±4μ8C.


Figure 5A The effect of the IRE1α inhibitor 4μ8C on TGFβ - induced skin fibrosis in C57BL / 6 mice as measured by the percentage of sirius red areas in the hypodermis . Ctrl ( n = 6 ) ; Ctrl + 4μ8C ( n = 5 ) ; TGFβ ( n = 8 ) ; TGFβ + 4μ8C ( n = 8 ) . * P = 0 , 046 . B Representative skin tissue sections stained with sirius red for collagen ( top ) and αSMA ( bottom ) . Scale bars = 50 μm . C Percentage αSMA - positive cells in the hypodermis of mice from the experimental setup in A . * P = 0 , 024 . D Representative image of the area selected for laser capture microdissection ( scale bars = 100 μm ) . E Expression levels of αSMA mRNA in samples of hypodermis obtained by laser capture microdissection . Ctrl ( n = 5 ) , Ctrl + 4μ8C ( n = 5 ) , TGFβ ( n = 5 ) , TGFβ + 4μ8C ( n = 5 ) . * P = 0 , 044 . F miR - 150 expression levels in the same samples as shown in E . G Immunohistochemistry staining of αSMA in primary skinfibroblasts isolated from healthy mice after treatment with TGFβ ± 4μ8C . Scale bars = 20 μm . H αSMA mRNA expression in primary mouse skin fibroblasts after treatment with TGFβ ± 4μ8C . * P = 0 , 049 , n = 4 . I Agarose gel showing levels of spliced and unspliced XBP - 1 in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C . J BiP mRNA expression in primary mouse skin fibroblasts after treatment with TGFβ in the absence and presence of 4μ8C . * P = 0 , 00007 for TGFβ vs . Ctrl and P = 0 , 00002 for TGFβ vs . TGFβ + 4μ8C . n = 4 . K Western blot showing CHOP protein expression in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C . L miR - 150 mRNA levels in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C . * P = 0 , 00031 compared to untreated control , n = 3 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A The effect of the IRE1α inhibitor 4μ8C on TGFβ-induced skin fibrosis in C57BL/6 mice as measured by the percentage of sirius red areas in the hypodermis. Ctrl (n = 6); Ctrl + 4μ8C (n = 5); TGFβ (n = 8); TGFβ + 4μ8C (n = 8). * P = 0,046.B Representative skin tissue sections stained with sirius red for collagen (top) and αSMA (bottom). Scale bars = 50 μm.C Percentage αSMA-positive cells in the hypodermis of mice from the experimental setup in A. * P = 0,024.D Representative image of the area selected for laser capture microdissection (scale bars = 100 μm).E Expression levels of αSMA mRNA in samples of hypodermis obtained by laser capture microdissection. Ctrl (n = 5), Ctrl + 4μ8C (n = 5), TGFβ (n = 5), TGFβ + 4μ8C (n = 5). * P = 0,044.F miR-150 expression levels in the same samples as shown in E.G Immunohistochemistry staining of αSMA in primary skinfibroblasts isolated from healthy mice after treatment with TGFβ ± 4μ8C. Scale bars = 20 μm.H αSMA mRNA expression in primary mouse skin fibroblasts after treatment with TGFβ ± 4μ8C. * P = 0,049, n = 4.I Agarose gel showing levels of spliced and unspliced XBP-1 in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C.J BiP mRNA expression in primary mouse skin fibroblasts after treatment with TGFβ in the absence and presence of 4μ8C. * P = 0,00007 for TGFβ vs. Ctrl and P = 0,00002 for TGFβ vs. TGFβ + 4μ8C. n=4.K Western blot showing CHOP protein expression in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C.L miR-150 mRNA levels in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C. * P = 0,00031 compared to untreated control, n = 3.


Figure 6A CTGF mRNA expression levels in skin ( dSSc ) and lung ( LSSc ) fibroblasts isolated from scleroderma patients and treated with the IRE1α inhibitor 4μ8C . * P = 0 , 018 for dSSc ( n = 6 ) and P = 0 , 003 for LSSc ( n = 3 ) . B Collagen1α2 mRNA expression levels in the same samples as in A . * P = 0 , 0030 for dSSC and P = 0 , 0036 for LSSc . C Quantification of collagen protein levels in the same samples as in A . * P = 0 , 010 . D Immunohistochemistry staining to detect BiP protein in fibroblasts isolated from scleroderma patients treated or not with 4μ8C . E mRNA levels of αSMA in primary lung fibroblasts isolated from patients with either cystic fibrosis ( CF ) , chronic obstructive pulmonary disease ( COPD ) or asthma . * P = 0 , 00001 for asthma vs . healthy controls , and P = 0 , 00003 for ctrl vs . 4μ8C in asthma patients . F Quantification of mRNA levels for BiP in same samples as in E . * P = 0 , 0023 between asthma and healthy controls and P = 0 , 0031 between 4μ8C - treated and control treatment in fibroblasts from asthma patients .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A CTGF mRNA expression levels in skin (dSSc) and lung (LSSc) fibroblasts isolated from scleroderma patients and treated with the IRE1α inhibitor 4μ8C. * P = 0,018 for dSSc (n = 6) and P = 0,003 for LSSc (n = 3).B Collagen1α2 mRNA expression levels in the same samples as in A. * P = 0,0030 for dSSC and P = 0,0036 for LSSc.C Quantification of collagen protein levels in the same samples as in A. * P = 0,010.D Immunohistochemistry staining to detect BiP protein in fibroblasts isolated from scleroderma patients treated or not with 4μ8C.E mRNA levels of αSMA in primary lung fibroblasts isolated from patients with either cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD) or asthma. * P = 0,00001 for asthma vs. healthy controls, and P = 0,00003 for ctrl vs. 4μ8C in asthma patients.F Quantification of mRNA levels for BiP in same samples as in E. * P = 0,0023 between asthma and healthy controls and P = 0,0031 between 4μ8C-treated and control treatment in fibroblasts from asthma patients.


Figure 1 ( A ) RT - qPCR analysis of NimB1 , NimB2 , NimB3 , NimB4 and NimB5 transcripts , normalized to RpL32 , from carcass , macrophages , fat body and gut of wild type ( w1118 ) L3 wandering larvae . Data are represented as mean ± SD from three independent experiments . ( B ) Representative confocal imaging of NimB4 - sGFP macrophages of embryo at stage 16 ; lateral ( left ) and ventral ( right ) views ; Tissues were stained with anti - GFP ( green , corresponding to NimB4 - sGFP ) and anti - SIMU antibodies ( red , corresponding to macrophages ) . The arrows show the presence of NimB4 - sGFP inside the embryonic macrophages . Scale bars = 20

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) RT-qPCR analysis of NimB1, NimB2, NimB3, NimB4 and NimB5 transcripts, normalized to RpL32, from carcass, macrophages, fat body and gut of wild type (w1118) L3 wandering larvae. Data are represented as mean ± SD from three independent experiments.(B) Representative confocal imaging of NimB4-sGFP macrophages of embryo at stage 16; lateral (left) and ventral (right) views; Tissues were stained with anti-GFP (green, corresponding to NimB4-sGFP) and anti-SIMU antibodies (red, corresponding to macrophages). The arrows show the presence of NimB4-sGFP inside the embryonic macrophages. Scale bars = 20 μm


Figure 2 ( A - C ) Representative confocal imaging of CFSE - stained apoptotic bodies ( green ) incubated with secreted NimB4 - RFP ( B , C ) or SPvkg - RFP ( A ) . Apoptotic bodies were pre - incubated in absence ( A , B ) or presence ( C ) of annexin V ( 25 μg / mL ) . Scale bar = 10 μm . ( D ) Quantification of the colocalization of NimB4 - RFP or SPvkg - RFP with the apoptotic bodies in presence or absence of Annexin V , as measured by Pearson ' s correlation coefficient . Values from at least four independent experiments are represented as mean ± SD ( * * P < 0 . 01 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) .

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Figure 2(A-C) Representative confocal imaging of CFSE-stained apoptotic bodies (green) incubated with secreted NimB4-RFP (B, C) or SPvkg-RFP (A). Apoptotic bodies were pre-incubated in absence (A, B) or presence (C) of annexin V (25 μg/mL). Scale bar = 10 μm. (D) Quantification of the colocalization of NimB4-RFP or SPvkg-RFP with the apoptotic bodies in presence or absence of Annexin V, as measured by Pearson's correlation coefficient. Values from at least four independent experiments are represented as mean ± SD (** P<0.01 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).


Figure 3 ( A ) Representative confocal imaging of wild type ( w1118 ) , NimB4 sk2 and apoptosis - null NimB4 sk2 ; Df ( 3L ) H99 embryonic macrophages at stage 16 of development ; ventral and lateral view ; Tissues were co - stained with Dcp - 1 ( red , corresponding to apoptotic corpses ) and anti - SIMU ( green , corresponding to macrophages ) antibodies . The arrows show the presence of apoptotic cells inside embryonic macrophages . Scale bars = 20 μm ( C ) Representative projections from confocal stacks of entire third instar wild type ( w1118 ) and NimB4 sk2 larval brains stained with anti - Draper ( green ) and anti - Dcp1 ( red ) . The arrows show the presence of apoptotic cells in the central area of NimB4 sk2 larval brain . Scale bars = 100

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Representative confocal imaging of wild type (w1118), NimB4 sk2 and apoptosis-null NimB4 sk2; Df(3L) H99 embryonic macrophages at stage 16 of development; ventral and lateral view; Tissues were co-stained with Dcp-1 (red, corresponding to apoptotic corpses) and anti-SIMU (green, corresponding to macrophages) antibodies. The arrows show the presence of apoptotic cells inside embryonic macrophages. Scale bars = 20 μm(C) Representative projections from confocal stacks of entire third instar wild type (w1118) and NimB4 sk2 larval brains stained with anti-Draper (green) and anti-Dcp1 (red). The arrows show the presence of apoptotic cells in the central area of NimB4 sk2 larval brain. Scale bars = 100 μm


Figure 4 ( A ) Representative confocal imaging of wild type ( w1118 ) , NimB4 sk2 and draper macrophages incubated with fluorescently - labeled apoptotic cells ( red , CellTraceTM Red ) on ice for 1h and stained with AlexaFluorTM 488 phalloidin ( green ) . Scale bar : 10 μm . ( B ) Quantification of the number of apoptotic cells ( red ) binding to the macrophages ( green ) . Values from three independent experiments are represented as mean ± SD ( * * * P < 0 . 001 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) . ( D ) Ex vivo phagocytosis assay using Alexa555 fluorescent apoptotic bodies . Wild type , NimB4sk2 , NimB4 genomic rescue ( NimB4sk2 , [ NimB4 ] ) , draper and NimC11 , eater1 macrophages from L3 wandering were incubated with Alexa555 fluorescent apoptotic bodies for 30 , 60 or 120 min at room temperature . Phagocytosis was quantified by flow cytometry . Data are represented as mean ± SD from three independent experiments ( * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests ns : not significant ) . ( E ) Ex vivo phagocytosis assay using AlexFluor488 S . aureus Bioparticles . Wild type , NimB4sk2 and NimC11 , eater1 macrophages from L3 wandering were incubated with bioparticles for 30 , 60 or 120 min at room temperature . Phagocytosis was quantified by flow cytometry . Data are represented as mean ± SD from three independent experiments ( * * * * P < 0 . 0001 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Representative confocal imaging of wild type (w1118), NimB4 sk2 and draper macrophages incubated with fluorescently-labeled apoptotic cells (red, CellTraceTM Red) on ice for 1h and stained with AlexaFluorTM 488 phalloidin (green). Scale bar: 10 μm. (B) Quantification of the number of apoptotic cells (red) binding to the macrophages (green). Values from three independent experiments are represented as mean ± SD (*** P<0.001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant). (D) Ex vivo phagocytosis assay using Alexa555 fluorescent apoptotic bodies. Wild type, NimB4sk2, NimB4 genomic rescue (NimB4sk2, [NimB4]), draper and NimC11, eater1 macrophages from L3 wandering were incubated with Alexa555 fluorescent apoptotic bodies for 30, 60 or 120 min at room temperature. Phagocytosis was quantified by flow cytometry. Data are represented as mean ± SD from three independent experiments (* P<0.05,** P<0.01, **** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests ns: not significant). (E) Ex vivo phagocytosis assay using AlexFluor488 S.aureus Bioparticles. Wild type, NimB4sk2 and NimC11, eater1 macrophages from L3 wandering were incubated with bioparticles for 30, 60 or 120 min at room temperature. Phagocytosis was quantified by flow cytometry. Data are represented as mean ± SD from three independent experiments (**** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).


Figure 5 ( A ) Representative fluorescence microscopy images of wild type ( w1118 ) , NimB4sk2 , draper , eater1 and croquemort △ third instar larvae macrophages stained with LysoTracker Red ( live imaging ) . Overlay of fluorescence and differential interference contrast microscopy ( DIC ) . Scale bar = 10 μm . ( B ) Mean fluorescence intensity after staining with LysoTracker Red ( live confocal imaging ) . Values from at least three independent experiments are represented as mean ± SD ( * * P < 0 . 01 , * * * * P < 0 . 0001 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests ) . ( C ) Representative transmission electron micrographs of macrophages from wild type ( top , w1118 ) and NimB4sk2 ( bottom ) L3 wandering larvae . Scale bar : 2 μm ( D ) Quantification of the number of vesicles per macrophages in the electron micrographs . Values from at least three independent experiments are represented as mean ± SD ( * * * * P < 0 . 0001 by Mann - Whitney test ) . ( E ) Representative confocal imaging of Rab7EYFP immunostaining in wild type ( w1118 ) and NimB4 sk2 macrophages . Tissues were stained with anti - GFP ( red , Rab7 ) , counterstained with phalloidin ( gray ) and DAPI ( blue ) . The arrows indicate the enlarged vesicles are decorated with Rab7EYFP in the NimB4sk2 macrophages . Scale bar = 10 μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Representative fluorescence microscopy images of wild type (w1118), NimB4sk2, draper, eater1 and croquemort△ third instar larvae macrophages stained with LysoTracker Red (live imaging). Overlay of fluorescence and differential interference contrast microscopy (DIC). Scale bar = 10 μm. (B) Mean fluorescence intensity after staining with LysoTracker Red (live confocal imaging). Values from at least three independent experiments are represented as mean ± SD ( ** P<0.01, **** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests). (C) Representative transmission electron micrographs of macrophages from wild type (top, w1118) and NimB4sk2 (bottom) L3 wandering larvae. Scale bar: 2 μm (D) Quantification of the number of vesicles per macrophages in the electron micrographs. Values from at least three independent experiments are represented as mean ± SD (**** P<0.0001 by Mann-Whitney test). (E) Representative confocal imaging of Rab7EYFP immunostaining in wild type (w1118) and NimB4 sk2 macrophages. Tissues were stained with anti-GFP (red, Rab7), counterstained with phalloidin (gray) and DAPI (blue). The arrows indicate the enlarged vesicles are decorated with Rab7EYFP in the NimB4sk2 macrophages. Scale bar = 10 μm.


Figure 6 ( A ) Representative confocal imaging of localization of Lamp1 - mcherry and LysoTracker Green in wild type ( w1118 ) , NimB4 sk2 and draper △ 5 macrophages ( live imaging ) . The arrows indicate the colocalization ( w1118 ) or the clustering ( NimB4sk2 and draper △ 5 ) of Lamp1 - mcherry and LysoTracker Green signals . Scale bar : 10 μm . ( B ) Quantification of the colocalization of Lamp1 - mcherry with LysoTracker Green , as measured by Pearson ' s correlation coefficient between the two signals . Values from at least five independent experiments are represented as mean ± SD ( * P < 0 . 05 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests ) . ( C ) Ex vivo phagocytosis assay using apoptotic cells labelled with pHrodo ™ Red . Wild type ( w1118 ) , NimB4sk2 and draper △ 5 macrophages from L3 wandering larvae were incubated with pHrodo ™ Red apoptotic cells for 60 min at room temperature . Phagocytosis was quantified by flow cytometry . Data are represented as mean ± SD from three independent experiments ( * P < 0 . 05 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) . ( D ) Ex vivo phagocytosis assay using pHrodoTM Red S . aureus BioparticlesTM conjugates . Wild type , NimB4sk2 and NimC11 , eater1 Hml ∆ - Gal4 > UAS - GFP macrophages from L3 wandering larvae were incubated with pHrodoTM Red S . aureus BioparticlesTM for 30 , 60 or 120 min at room temperature . Phagocytosis was quantified by flow cytometry . Data are represented as mean ± SD from three independent experiments ( * P < 0 . 05by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Representative confocal imaging of localization of Lamp1-mcherry and LysoTracker Green in wild type (w1118), NimB4 sk2 and draper△5 macrophages (live imaging). The arrows indicate the colocalization (w1118) or the clustering (NimB4sk2 and draper△5) of Lamp1-mcherry and LysoTracker Green signals. Scale bar: 10 μm. (B) Quantification of the colocalization of Lamp1-mcherry with LysoTracker Green, as measured by Pearson's correlation coefficient between the two signals. Values from at least five independent experiments are represented as mean ± SD (* P<0.05 by ANOVA test followed by post hoc Dunnett's multiple comparison tests). (C) Ex vivo phagocytosis assay using apoptotic cells labelled with pHrodo™ Red. Wild type (w1118), NimB4sk2 and draper△5 macrophages from L3 wandering larvae were incubated with pHrodo™ Red apoptotic cells for 60 min at room temperature. Phagocytosis was quantified by flow cytometry. Data are represented as mean ± SD from three independent experiments (* P<0.05 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant). (D) Ex vivo phagocytosis assay using pHrodoTM Red S.aureus BioparticlesTM conjugates. Wild type, NimB4sk2 and NimC11, eater1 Hml∆-Gal4>UAS-GFP macrophages from L3 wandering larvae were incubated with pHrodoTM Red S. aureus BioparticlesTM for 30, 60 or 120 min at room temperature. Phagocytosis was quantified by flow cytometry. Data are represented as mean ± SD from three independent experiments (* P<0.05by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).


Figure 7 ( A - B ) Representative fluorescence microscopy images of wild type ( A ) or NimB4sk2 ( B ) macrophages expressing Rab5 ( middle panel ) or Rab7 ( right panel ) driven by Hml ∆ - Gal4 and stained with the LysoTracker Red ( live imaging ) . Overlay of fluorescence and DIC . Scale bar = 10 μm . ( C ) Quantification of the mean fluorescence intensity of LysoTracker in macrophages from larvae ( confocal live imaging ) . Values from at least three independent experiments are represented as mean ± SD ( * * P < 0 . 01 , * * * * P < 0 . 0001 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A-B) Representative fluorescence microscopy images of wild type (A) or NimB4sk2 (B) macrophages expressing Rab5 (middle panel) or Rab7 (right panel) driven by Hml∆-Gal4 and stained with the LysoTracker Red (live imaging). Overlay of fluorescence and DIC. Scale bar = 10 μm. (C) Quantification of the mean fluorescence intensity of LysoTracker in macrophages from larvae (confocal live imaging). Values from at least three independent experiments are represented as mean ± SD (** P<0.01, **** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).


f1a , MEFs of RagA + / + , RagAGTP / + and RagAGTP / GTP genotypes were deprived of amino acids for 1 h and re - stimulated for 10 min . Whole - cell protein lysates were immunoblotted for indicated proteins . b , Total RNA was extracted from RagA + / + ( n = 3 ) , RagAGTP / + ( n = 3 ) and RagAGTP / GTP ( n = 2 ) MEFs and RagA mRNA expression determined by quantitative PCR ( mean ± s . e . m . ) . c , Weights of RagA + / + ( n = 24 ) , RagAGTP / + ( n = 52 ) and RagAGTP / GTP ( n = 22 ) mice at birth ( data are scatter dots , mean ) . d , Representative photographs of RagA + / + , RagAGTP / + and RagAGTP / GTP neonates . Scale bar , 1 cm . e , Early suppression of mTORC1 signalling after birth was determined by immunoblotting of protein extracts from liver and heart of RagA + / + ( + / + ) , RagAGTP / + ( G / + ) and RagAGTP / GTP ( G / G ) neonates immediately after Caesarean section ( 0 h ) or after 1 h of fasting ( 1 h fast ) . f , Liver and heart extracts from RagA + / + , RagAGTP / + and RagAGTP / GTP neonates fasted for 10 h were analysed by immunoblotting for the indicated proteins . g , Survival curve of fasted neonates . Neonates obtained by Caesarean section and resuscitated were fasted and their survival monitored ( + / + : n = 13 ; G / + : n = 26 ; G / G : n = 10 ) . h , Survival curve of fasted neonates treated with rapamycin . Neonates obtained by Caesarean section and resuscitated were fasted and their survival monitored ( + / + : n = 13 ; G / + : n = 13 ; G / G : n = 6 ) . Numbers indicate the median survival for each curve . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 005 .

B-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f1a, MEFs of RagA+/+, RagAGTP/+ and RagAGTP/GTP genotypes were deprived of amino acids for 1 h and re-stimulated for 10 min. Whole-cell protein lysates were immunoblotted for indicated proteins.b, Total RNA was extracted from RagA+/+ (n = 3), RagAGTP/+ (n = 3) and RagAGTP/GTP (n = 2) MEFs and RagA mRNA expression determined by quantitative PCR (mean ± s.e.m.).c, Weights of RagA+/+ (n = 24), RagAGTP/+ (n = 52) and RagAGTP/GTP (n = 22) mice at birth (data are scatter dots, mean).d, Representative photographs of RagA+/+, RagAGTP/+ and RagAGTP/GTP neonates. Scale bar, 1 cm.e, Early suppression of mTORC1 signalling after birth was determined by immunoblotting of protein extracts from liver and heart of RagA+/+ (+/+), RagAGTP/+ (G/+) and RagAGTP/GTP (G/G) neonates immediately after Caesarean section (0 h) or after 1 h of fasting (1 h fast).f, Liver and heart extracts from RagA+/+, RagAGTP/+ and RagAGTP/GTP neonates fasted for 10 h were analysed by immunoblotting for the indicated proteins.g, Survival curve of fasted neonates. Neonates obtained by Caesarean section and resuscitated were fasted and their survival monitored (+/+: n = 13; G/+: n = 26; G/G: n = 10). h, Survival curve of fasted neonates treated with rapamycin. Neonates obtained by Caesarean section and resuscitated were fasted and their survival monitored (+/+: n = 13; G/+: n = 13; G/G: n = 6). Numbers indicate the median survival for each curve. *P<0.05; **P<0.01; ***P<0.005.


f2a , Glycaemia drop in RagA + / + , RagAGTP / + and RagAGTP / GTP and recovery in fasted RagA + / + and RagAGTP / + but not in RagAGTP / GTP neonates ( + / + : n = 5 , 18 , 4 , 5 , 9 , 8 ; G / + : n = 10 , 26 , 10 , 13 , 26 , 21 ; G / G : n = 7 , 20 , 9 , 10 , 16 , 11 , for 0 , 1 , 2 , 3 , 6 and 10 h , respectively ; mean ± s . e . m . ) . b , Rapamycin significantly increases glycaemia in RagAGTP / GTP fasted for 6 and 10 h ( mean ± s . e . m . ) . NS , not significant . c , Extension of survival by glucose injections in fasted RagAGTP / GTP neonates ( untreated : n = 10 ; glucose : n = 5 ) . d , Normal expression of genes involved in glucose metabolism in neonatal liver ( + / + : n = 2 , G / + : n = 5 ; G / G : n = 4 ; mean ± s . e . m . ) . e , Left : representative electron microscopy images showing abundant glycogen content in RagA + / + and RagAGTP / GTP hepatocytes before fasting ( 0 h , upper panels ) and more pronounced glycogen depletion after 10 h of fasting ( lower panels ) in RagAGTP / GTP neonates . Right : quantification of hepatic glycogen content ( + / + : n = 5 , 3 , 4 , 4 ; G / + : n = 11 , 7 , 14 , 15 ; G / G : n = 6 , 4 , 4 , 6 ; for 0 , 3 , 6 and 10 h , respectively ; mean ± s . e . m . ; AU , arbitrary units ) . . f , Partial rescue of hepatic glycogen content in RagAGTP / GTP neonates fasted for 10 h and treated with rapamycin ( rapa ) ( mean ± s . e . m . ) . g , Quantification of neonatal plasma amounts of branched - chain ( BCAA ) and essential amino acids at birth ( left ) , after 10 h fasting ( middle ) and after 10 h fasting with rapamycin treatment ( right ) ( n for + / + , G / + and G / G , respectively : n = 4 , 5 and 4 for 0 h ; n = 4 , 4 and 3 for 10 h ; n = 2 , 5 and 3 for rapamycin ; mean ± s . d . ) . Values are expressed relative to RagA + / + amounts at each time point . h , Extension of survival by injection of a combination of gluconeogenic amino acids ( a . a . ) in fasted RagAGTP / GTP neonates ( untreated : n = 10 ; amino acids : n = 8 ) . * P 0 . 05 ; * * P 0 . 01 ; ns , P > 0 .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f2a, Glycaemia drop in RagA+/+, RagAGTP/+ and RagAGTP/GTP and recovery in fasted RagA+/+ and RagAGTP/+ but not in RagAGTP/GTP neonates (+/+: n = 5, 18, 4, 5, 9, 8; G/+: n = 10, 26, 10, 13, 26, 21; G/G: n = 7, 20, 9, 10, 16, 11, for 0, 1, 2, 3, 6 and 10 h, respectively; mean ± s.e.m.).b, Rapamycin significantly increases glycaemia in RagAGTP/GTP fasted for 6 and 10 h (mean ± s.e.m.). NS, not significant.c, Extension of survival by glucose injections in fasted RagAGTP/GTP neonates (untreated: n = 10; glucose: n = 5).d, Normal expression of genes involved in glucose metabolism in neonatal liver (+/+: n = 2, G/+: n = 5; G/G: n = 4; mean ± s.e.m.).e, Left: representative electron microscopy images showing abundant glycogen content in RagA+/+ and RagAGTP/GTP hepatocytes before fasting (0 h, upper panels) and more pronounced glycogen depletion after 10 h of fasting (lower panels) in RagAGTP/GTP neonates. Right: quantification of hepatic glycogen content (+/+: n = 5, 3, 4, 4; G/+: n = 11, 7, 14, 15; G/G: n = 6, 4, 4, 6; for 0, 3, 6 and 10 h, respectively; mean ± s.e.m.; AU, arbitrary units).. f, Partial rescue of hepatic glycogen content in RagAGTP/GTP neonates fasted for 10 h and treated with rapamycin (rapa) (mean ± s.e.m.).g, Quantification of neonatal plasma amounts of branched-chain (BCAA) and essential amino acids at birth (left), after 10 h fasting (middle) and after 10 h fasting with rapamycin treatment (right) (n for +/+, G/+ and G/G, respectively: n = 4, 5 and 4 for 0 h; n = 4, 4 and 3 for 10 h; n = 2, 5 and 3 for rapamycin; mean ± s.d.). Values are expressed relative to RagA+/+ amounts at each time point.h, Extension of survival by injection of a combination of gluconeogenic amino acids (a.a.) in fasted RagAGTP/GTP neonates (untreated: n = 10; amino acids: n = 8). *P  0.05; **P  0.01; ns, P > 0.0


f3a , Top : representative micrographs of autophagosomes and autophagolysosomes in hepatocytes from RagA + / + neonates fasted for 1 h . Typical autophagosome with a double limiting membrane ( arrows ) ; autophagosome and several autolysosomes ( arrowheads ) . Scale bars , 5 µm . Bottom : frequency of the two types of organelle ( early : autophagosomes ; late : autophagolysosomes ) detected in cell profiles of hepatocytes and skeletal myocytes from RagA + / + and RagAGTP / GTPneonates . b , Protein extracts from livers of neonates at Caesarean section ( 0 h ) and fasted for 10 h were immunoblotted for autophagy markers LC3B and p62 . c , Protein extracts from skeletal muscle and heart from neonates at Caesarean section ( 0 h ) , fasted for 1 and 2 h , were immunoblotted for indicated proteins . d , Triggering of autophagy by amino - acid withdrawal in MEFs . MEFs were deprived of amino acids for the indicated time points , whole - cell protein extracts were obtained and mTORC1 activity and autophagic activity determined by immunoblotting . e , Recombinant LC3B - GFP was expressed in RagA + / + and RagAGTP / GTPMEFs and LC3B localization , in the presence and absence of amino acids , monitored by fluorescence microscopy . LC3B - GFP ( green fluorescent protein ) clustering , indicative of autophagy , was observed in amino acid - starved RagA + / + but not RagAGTP / GTPMEFs . Scale bar , 10 µm . f , Localization of recombinant TFEB - GFP was determined in MEFs as in e . Nuclear ( active ) TFEB was observed in RagA + / + MEFs upon amino - acid withdrawal .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f3a, Top: representative micrographs of autophagosomes and autophagolysosomes in hepatocytes from RagA+/+ neonates fasted for 1 h. Typical autophagosome with a double limiting membrane (arrows); autophagosome and several autolysosomes (arrowheads). Scale bars, 5 µm. Bottom: frequency of the two types of organelle (early: autophagosomes; late: autophagolysosomes) detected in cell profiles of hepatocytes and skeletal myocytes from RagA+/+ and RagAGTP/GTPneonates.b, Protein extracts from livers of neonates at Caesarean section (0 h) and fasted for 10 h were immunoblotted for autophagy markers LC3B and p62.c, Protein extracts from skeletal muscle and heart from neonates at Caesarean section (0 h), fasted for 1 and 2 h, were immunoblotted for indicated proteins.d, Triggering of autophagy by amino-acid withdrawal in MEFs. MEFs were deprived of amino acids for the indicated time points, whole-cell protein extracts were obtained and mTORC1 activity and autophagic activity determined by immunoblotting.e, Recombinant LC3B-GFP was expressed in RagA+/+ and RagAGTP/GTPMEFs and LC3B localization, in the presence and absence of amino acids, monitored by fluorescence microscopy. LC3B-GFP (green fluorescent protein) clustering, indicative of autophagy, was observed in amino acid-starved RagA+/+ but not RagAGTP/GTPMEFs. Scale bar, 10 µm.f, Localization of recombinant TFEB-GFP was determined in MEFs as in e. Nuclear (active) TFEB was observed in RagA+/+ MEFs upon amino-acid withdrawal.


f4a ) AMPKa1 / a2 double knockout ( DKO ) and wild - type ( WT ) MEFs were deprived of glucose for 1 h and re - stimulated for 10 min . Wholecell extracts were immunoblotted for the indicated proteins . b , Immortalized MEFs of the indicated genotypes were deprived of growth factors , glucose , amino acids or glucose and amino acids for 1 h and re - stimulated with glucose and / or amino acids for 10 min . Whole - cell lysates were immunoblotted for the indicated proteins . c , RagA + / + and RagAGTP / GTP immortalized MEFs were deprived of glucose or amino acids and surviving cells quantified in triplicate after 48 h . Cell number is indicated relative to cell number at the start of the treatment ; mean ± s . d . ; * * * P 0 . 005d , mTOR localization as detected by immunofluorescence . In HEK - 293T cells , glucose deprivation causes mTOR to localize to diffuse small puncta throughout the cytoplasm . Re - addition of glucose leads to mTOR shuttling to the lysosomal surface , co - localizing with the lysosomal protein Lamp2 . HEK - 293T - RagBGTP cells show mTOR localized at the lysosomal surface , regardless of glucose concentrations . Scale bars , 10 μm . e , Glucose and amino acids affect the binding of the v - ATPase to the Ragulator complex . HEK - 293T expressing Flag - p14 was deprived of glucose or amino acids for 90 min and re - stimulated for 20 min . Protein extracts and immunoprecipitates were immunoblotted for the indicated proteins . f , RagA + / + and RagAGTP / GTP primary MEFs were cultured for 1 h in media with the glucose and amino - acid concentrations measured in neonates at birth ( 0 h ) or after fasting for 1 h ( 1 h ) and whole - cell protein extracts were analysed by immunoblotting .

B-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f4a) AMPKa1/a2 double knockout (DKO) and wild-type (WT) MEFs were deprived of glucose for 1 h and re-stimulated for 10 min. Wholecell extracts were immunoblotted for the indicated proteins.b, Immortalized MEFs of the indicated genotypes were deprived of growth factors, glucose, amino acids or glucose and amino acids for 1 h and re-stimulated with glucose and/or amino acids for 10 min. Whole-cell lysates were immunoblotted for the indicated proteins.c, RagA+/+ and RagAGTP/GTP immortalized MEFs were deprived of glucose or amino acids and surviving cells quantified in triplicate after 48 h. Cell number is indicated relative to cell number at the start of the treatment; mean ± s.d.; ***P  0.005d, mTOR localization as detected by immunofluorescence. In HEK-293T cells, glucose deprivation causes mTOR to localize to diffuse small puncta throughout the cytoplasm. Re-addition of glucose leads to mTOR shuttling to the lysosomal surface, co-localizing with the lysosomal protein Lamp2. HEK-293T-RagBGTP cells show mTOR localized at the lysosomal surface, regardless of glucose concentrations. Scale bars, 10 μm.e, Glucose and amino acids affect the binding of the v-ATPase to the Ragulator complex. HEK-293T expressing Flag-p14 was deprived of glucose or amino acids for 90 min and re-stimulated for 20 min. Protein extracts and immunoprecipitates were immunoblotted for the indicated proteins.f, RagA+/+ and RagAGTP/GTP primary MEFs were cultured for 1 h in media with the glucose and amino-acid concentrations measured in neonates at birth (0 h) or after fasting for 1 h (1 h) and whole-cell protein extracts were analysed by immunoblotting.


Figure 1 ( A ) Binding of wild type DUBm - Ubp8 to monoubiquitin . ( B ) Binding of DUBmUbp8C146A to monoubiquitin . ( C ) Binding of DUBm - Ubp8C146S to monoubiquitin . ( D ) Binding of DUBm - Ubp8C146R to monoubiquitin . ( E ) Binding of DUBm - Ubp8C146A to K48 diubiquitin . ( F ) Binding of DUBm - Ubp8C146S to K48 diubiquitin .

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

Figure 1 (A) Binding of wild type DUBm-Ubp8 to monoubiquitin. (B) Binding of DUBmUbp8C146A to monoubiquitin. (C) Binding of DUBm-Ubp8C146S to monoubiquitin. (D) Binding of DUBm-Ubp8C146R to monoubiquitin. (E) Binding of DUBm-Ubp8C146A to K48 diubiquitin. (F) Binding of DUBm-Ubp8C146S to K48 diubiquitin.


Figure 3Binding was measured by fluorescence polarization using N - terminally TAMRA - labeled monoubiquitin . The dissociation constants for ubiquitin binding to USP4 WT and C311A are 92 ± 21 nM ( Clerici et . al , 2014 ) and 0 . 60 ± 0 . 17 nM , respectively . Error bars are s . d . calculated on five measurements per point .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Binding was measured by fluorescence polarization using N-terminally TAMRA-labeled monoubiquitin. The dissociation constants for ubiquitin binding to USP4 WT and C311A are 92 ± 21 nM (Clerici et. al, 2014) and 0.60 ± 0.17 nM, respectively. Error bars are s.d. calculated on five measurements per point.


Figure 4 ( A ) Equilibrium binding of OTUD1 C320A and C320R to K63 - linked diubiquitin was measured by fluorescence polarization using FlAsH tagged K63 - linked diubiquitin in which the proximal ubiquitin was fluorescently - labeled . Error bars indicate s . d . and are based on three measurements per data point . One representative experiment of two is shown . ( B ) Whole cell lysates of HEK293 cells expressing HA - tagged OTUD1 WT , C320R , and C320A were immunoblotted with indicated antibodies . One representative experiment of three 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 O

Figure 4 (A) Equilibrium binding of OTUD1 C320A and C320R to K63-linked diubiquitin was measured by fluorescence polarization using FlAsH tagged K63-linked diubiquitin in which the proximal ubiquitin was fluorescently-labeled. Error bars indicate s.d. and are based on three measurements per data point. One representative experiment of two is shown.  (B) Whole cell lysates of HEK293 cells expressing HA-tagged OTUD1 WT, C320R, and C320A were immunoblotted with indicated antibodies. One representative experiment of three is shown.


Figure 5 Polyubiquitin chains were co - immunoprecipitated with FLAG - PSMD4 , an ubiquitin receptor for the 26S proteasome , from cells expressing either USP14 wild - type , C114A , or C114R . One representative experiment of two 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

Figure 5 Polyubiquitin chains were co-immunoprecipitated with FLAG-PSMD4, an ubiquitin receptor for the 26S proteasome, from cells expressing either USP14 wild-type, C114A, or C114R. One representative experiment of two is shown.


Figure 1E Representative images of Notch activity indicated by Tg ( Tp1 : mVenus ) ( green ) and matured MG stained with GS ( magenta ) at different stages . Scale Bars : 20 μm . F Quantitative plots showing the numbers of Tp1 + ( left ) , GS + ( right ) cells in the developing retinas at different stages . We collected retinas from 60 hpf ( n = 3 dpf ( n = 12 ) , 5 dpf ( n = 12 ) , and 7 dpf ( n = 12 , Mean ± SEM ; * p < 0 . 05 , * * * p < 0 . 001 , t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1E Representative images of Notch activity indicated by Tg(Tp1:mVenus)( green) and matured MG stained with GS (magenta) at different stages. Scale Bars: 20 μm. F Quantitative plots showing the numbers of Tp1+ (left), GS+ (right) cells in the developing retinas at different stages. We collected retinas from 60 hpf (n = 3 dpf (n = 12), 5 dpf (n = 12), and 7 dpf (n = 12, Mean ± SEM; * p<0.05, ***p<0.001, t-test).


Figure 2A Representative images of Notch activation labeled by Tg ( Tp1 : mVenus ) ( green ) and mature MGs stained with glutamate synthase ( GS , magenta ) at 4 dpf in Notch ligands ( dla - / - / dlb - / - / dlc - / - / dld - / - / jag1b - / - / jag2b - / - ) mutants . B , C Quantitative plots showing the numbers of Notch activation ( Tp1 + ) cells ( B ) and mature MG ( GS + ) cells ( C ) in A at 4 dpf . In total , we collected 11 fish for wild - type fish ; 8 fish for jag2b - / - mutants ; 15 fish for dla - / - mutants ; 19 fish for dld - / - mutants ; 19 fish for dlc - / - mutants , 11 fish for dlb - / - mutants , 11 fish for jag1b - / - mutants ( mean ± SEM ; * * * p < 0 . 001 , t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A Representative images of Notch activation labeled by Tg(Tp1:mVenus)(green) and mature MGs stained with glutamate synthase (GS, magenta) at 4 dpf in Notch ligands (dla-/-/dlb-/-/dlc-/-/dld-/-/jag1b-/-/jag2b-/-) mutants.B, C Quantitative plots showing the numbers of Notch activation (Tp1+) cells (B) and mature MG (GS+) cells (C) in A at 4 dpf. In total, we collected 11 fish for wild-type fish; 8 fish for jag2b-/- mutants; 15 fish for dla-/- mutants; 19 fish for dld-/- mutants; 19 fish for dlc-/- mutants, 11 fish for dlb-/- mutants, 11 fish for jag1b-/- mutants (mean ± SEM; ***p<0.001, t-test).


Figure 4A Upper : representative images showing that GS + MGs ( magenta ) were rarely observed in 5 - dpf jag2b - / - mutants expressing H2B - CFP ( green ) driven by the crx promoter . Bottom : representative images showing that GS + MGs ( magenta ) were frequently rescued underneath PRs when jag2b expression ( green ) was driven by the crx promoter in 5 - dpf jag2b - / - mutants . The area of retina is labeled with solid line , and each layer structure is labeled with dashed lines and marked with outer nuclear layer ( ONL ) , inner nuclear layer ( INL ) , inner plexiform layer ( IPL ) , and ganglion cell layer ( GCL ) . Scale bars : 20μm . The remaining MGs are indicated by white arrowheads , and the rescued MGs are indicated by yellow arrows . B Quantification of GS + MGs ( magenta in A ) with ( green ) or without ( gray ) CFP expression driven by the crx promoter . Ctrl indicates H2B - CFP expression driven by the crx promoter ( n = 16 larvae ) ; rescue indicates jag2b - H2B - CFP expression driven by the crx promoter ( n = 21 larvae , mean ± SEM ; * p < 0 . 05 , ns , no significant difference , pair - wised t - test ) . C The proportions of jag2b - misexpressing PRs ( Green ) with underneath MGs ( GS + , magenta in A ) in 5 - dpf jag2b - / - mutants and all MGs in retina . In total , we analyzed retinae of 16 control ( Ctrl , jag2b - / - , crx : : h2b - ecfp expression ) and 21 rescue ( jag2b - / - , crx : : jag2b expression ) larvae ( Mean ± SEM ; * * * p < 0 . 001 , t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A Upper: representative images showing that GS+ MGs (magenta) were rarely observed in 5-dpf jag2b-/- mutants expressing H2B-CFP (green) driven by the crx promoter. Bottom: representative images showing that GS+ MGs (magenta) were frequently rescued underneath PRs when jag2b expression (green) was driven by the crx promoter in 5-dpf jag2b-/- mutants. The area of retina is labeled with solid line, and each layer structure is labeled with dashed lines and marked with outer nuclear layer (ONL), inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GCL). Scale bars: 20μm. The remaining MGs are indicated by white arrowheads, and the rescued MGs are indicated by yellow arrows. B Quantification of GS+ MGs (magenta in A) with (green) or without (gray) CFP expression driven by the crx promoter. Ctrl indicates H2B-CFP expression driven by the crx promoter (n = 16 larvae); rescue indicates jag2b-H2B-CFP expression driven by the crx promoter (n = 21 larvae, mean ± SEM; * p<0.05, ns, no significant difference, pair-wised t-test). C The proportions of jag2b-misexpressing PRs (Green) with underneath MGs (GS+, magenta in A) in 5-dpf jag2b-/- mutants and all MGs in retina. In total, we analyzed retinae of 16 control (Ctrl, jag2b-/- , crx::h2b-ecfp expression) and 21 rescue (jag2b-/-, crx::jag2b expression) larvae (Mean ± SEM; ***p<0.001, t-test).


Figure 5D Representative image showing the GS + , but vsx1 - expression in the progeny of Tp1 + Müller - like differentiating MGs at wild - type ( jag2b + / + ) . Layer structure indicated in ( A ) . E The percentage of the progeny of Müller - like Tp1 + differentiating MG precursors ( red + ) expression GS ( gray ) , vsx1 ( green ) , or both negative ( magenta ) in wild - type ( jag2b + / + ) fish ( n = 14 , Mean ± SEM ; * * * p < 0 . 001 , t - test ) . F Representative image showing vsx1 + , but GS - expression in the progeny of Tp1 + Müller - like differentiating MGs in jag2b - / - mutants . Layer structure indicated in ( A ) . G The percentage of the progeny of Müller - like Tp1 + differentiating MG precursors ( red + ) expressing GS ( gray ) , vsx1 ( green ) , or both negative ( magenta ) in jag2b - / - mutants ( n = 29 , mean ± SEM ; * * * p < 0 . 001 , t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5D Representative image showing the GS+, but vsx1- expression in the progeny of Tp1+ Müller-like differentiating MGs at wild-type (jag2b+/+). Layer structure indicated in (A). E The percentage of the progeny of Müller-like Tp1+ differentiating MG precursors (red+) expression GS (gray), vsx1 (green), or both negative (magenta) in wild-type (jag2b+/+) fish (n = 14, Mean ± SEM; ***p<0.001, t-test). F Representative image showing vsx1+, but GS- expression in the progeny of Tp1+ Müller-like differentiating MGs in jag2b-/- mutants. Layer structure indicated in (A). G The percentage of the progeny of Müller-like Tp1+ differentiating MG precursors (red+) expressing GS (gray), vsx1 (green), or both negative (magenta) in jag2b-/- mutants (n = 29, mean ± SEM; ***p<0.001, t-test).


Figure 6A Representative images of Notch activation labeled by Tg ( Tp1 : mVenus ) ( green ) and mature MGs stained with glutamate synthase ( GS , magenta ) at 4 dpf in Notch receptors ( notch1a - / - / notch1b - / - / notch3 - / - ) mutants . Scale Bars : 20 μm . B Quantitative plots showing the numbers of cells with active Notch ( Tp1 + ) and mature MG ( GS + ) cells at 4 dpf . In total , we collected 4 fish for wild - type fish ; 7 fish for notch1a - / - mutants ; 5 fish for notch1b - / - mutants ; 18 fish for notch3 - / - mutants ( Mean ± SEM ; * * p < 0 . 01 ; * * * p < 0 . 001 , t - test ) . C Representative images showing that GS + MGs ( magenta ) were rarely observed when H2B - CFP expression ( green ) was driven by the gfap promoter in 5 - dpf jag2b - / - mutants . The area between the upper dashed and solid lines is the outer nuclear layer ( ONL ) . The area in the middle two dash lines is the inner plexiform layer ( IPL ) . INL , inner nuclear layer ; GCL , ganglion cell layer . D Representative images showing that GS + MGs ( magenta ) were frequently rescued underneath cells with notch3 expression ( green ) driven by the gfap promoter in 5 - dpf jag2b - / - mutants . Layer structure is similar as ( C ) . E The cell number of GS + MGs ( magenta ) with or without CFP expression driven by gfap promoter . Ctrl : H2B - CFP expression ( green ) driven by the gfap promoter ( n = 5 fish ) ; rescue : notch1b ( n = 13 fish ) and notch3 ( n = 17 fish ) NICD - H2B - CFP expression ( green ) driven by the gfap promoter ( Mean ± SEM ; * p < 0 . 05 , * * * p < 0 . 001 , ns , no significant difference , pair - wised t - test ) . F The proportions of notch1b and notch3 NICD - misexpressing cells ( green ) with underneath MGs ( GS + , magenta ) in 5 - dpf jag2b - / - mutants , indicating that gfap promoter - driven notch1b ( n = 13 fish ) or notch3 ( n = 17 fish ) NICD expression could rescue MG loss in jag2b - / - mutants ( Mean ± SEM ; * * * p < 0 . 001 , t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A Representative images of Notch activation labeled by Tg(Tp1:mVenus)( green) and mature MGs stained with glutamate synthase (GS, magenta) at 4 dpf in Notch receptors (notch1a-/-/notch1b-/-/notch3-/-) mutants. Scale Bars: 20 μm. B Quantitative plots showing the numbers of cells with active Notch (Tp1+) and mature MG (GS+) cells at 4 dpf. In total, we collected 4 fish for wild-type fish; 7 fish for notch1a-/- mutants; 5 fish for notch1b-/- mutants; 18 fish for notch3-/- mutants (Mean ± SEM; ** p<0.01; ***p<0.001, t-test).C Representative images showing that GS+ MGs (magenta) were rarely observed when H2B-CFP expression (green) was driven by the gfap promoter in 5-dpf jag2b-/- mutants. The area between the upper dashed and solid lines is the outer nuclear layer (ONL). The area in the middle two dash lines is the inner plexiform layer (IPL). INL, inner nuclear layer; GCL, ganglion cell layer. D Representative images showing that GS+ MGs (magenta) were frequently rescued underneath cells with notch3 expression (green) driven by the gfap promoter in 5-dpf jag2b-/- mutants. Layer structure is similar as (C). E The cell number of GS+ MGs (magenta) with or without CFP expression driven by gfap promoter. Ctrl: H2B-CFP expression (green) driven by the gfap promoter (n = 5 fish); rescue: notch1b (n = 13 fish) and notch3 (n = 17 fish) NICD-H2B-CFP expression (green) driven by the gfap promoter (Mean ± SEM; * p<0.05, ***p<0.001, ns, no significant difference, pair-wised t-test). F The proportions of notch1b and notch3 NICD-misexpressing cells (green) with underneath MGs (GS+, magenta) in 5-dpf jag2b-/- mutants, indicating that gfap promoter-driven notch1b (n = 13 fish) or notch3 (n = 17 fish) NICD expression could rescue MG loss in jag2b-/- mutants (Mean ± SEM; ***p<0.001, t-test).


Figure 1Gating strategy for sorting of EpCAMhighCD24lowSca ‑ 1 + cells and rMC from lung homogenate of adult mice . Time - course of epithelial stem / progenitor cell proliferation and differentiation within BALO at days 8 , 11 and 21 of co - culture with rMC . Clonal expansion of EpCAMhighCD24lowSca ‑ 1 + cells derived from either GFP - expressing mice or tdTomato - expressing ROSAmT / mG mice at day 21 of co ‑ culture . Percentages of SCGB1A1 + SFTPC + ( BALO ) , SCGB1A1 + SFTPC - ( bronchiolospheres ) and SCGB1A1 - SFTPC + ( alveolospheres ) organoids per well at day 21 of culture derived from EpCAMhighCD24lowSca - 1 + cells isolated from Scgb1a1mCherrySftpcYFP reporter mice ( n = 4 biological replicates ) . Representative confocal images of days 8 to 21 of culture showing endogenous SCGB1A1 and SFTPC expression during BALO formation derived from EpCAMhighCD24lowSca - 1 + SCGB1A1 + SFTPC + cells isolated from Scgb1a1mCherrySftpcYFP reporter mice . Heat map ( left ) and tSNE plot ( right ) ( F ) of digested day 21 BALO cultures depicting four distinct clusters ( C1 , airway , blue ; C2 , alveolar , purple ; C3 , MYO , orange , and C4 , LIF , green ) . Violin plots of selected genes representing airway associated - genes ( G ) ( Itgb4 , Trp63 , Krt7 , and Sox2 ) and alveoli associated ‑ genes ( H ) ( Cxcl15 , Lyz2 , Sftpc , and Hopx ) . Each violin plot shows the frequency distribution of the mean transcript level ( log2 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Gating strategy for sorting of EpCAMhighCD24lowSca‑1+ cells and rMC from lung homogenate of adult mice.Time-course of epithelial stem/progenitor cell proliferation and differentiation within BALO at days 8, 11 and 21 of co-culture with rMC.Clonal expansion of EpCAMhighCD24lowSca‑1+ cells derived from either GFP-expressing mice or tdTomato-expressing ROSAmT/mG mice at day 21 of co‑culture.Percentages of SCGB1A1+SFTPC+ (BALO), SCGB1A1+SFTPC- (bronchiolospheres) and SCGB1A1-SFTPC+ (alveolospheres) organoids per well at day 21 of culture derived from EpCAMhighCD24lowSca-1+ cells isolated from Scgb1a1mCherrySftpcYFP reporter mice (n=4 biological replicates).Representative confocal images of days 8 to 21 of culture showing endogenous SCGB1A1 and SFTPC expression during BALO formation derived from EpCAMhighCD24lowSca-1+SCGB1A1+SFTPC+ cells isolated from Scgb1a1mCherrySftpcYFP reporter mice.Heat map (left) and tSNE plot (right) (F) of digested day 21 BALO cultures depicting four distinct clusters (C1, airway, blue; C2, alveolar, purple; C3, MYO, orange, and C4, LIF, green).Violin plots of selected genes representing airway associated-genes (G) (Itgb4, Trp63, Krt7, and Sox2) and alveoli associated‑genes (H) (Cxcl15, Lyz2, Sftpc, and Hopx). Each violin plot shows the frequency distribution of the mean transcript level (log2).


Figure 2mRNA expression analysis of epithelial cell differentiation markers Sftpc ( AEC II ) , Hopx ( AEC I ) , Foxj1 ( ciliated cells ) , Muc5ac ( secretory cells ) and p63 ( basal cells ) in BALO at days 0 , 10 , and 21 of culture ( n = 3 biological replicates with pooled cells from 4 cultures per replicate ) Electron microscopy of BALO alveoli showing two cuboidal epithelial cells ( AEC II ) connected by tight junctions ( red square ) with numerous mitochondria ( M ) and lamellar bodies ( LB ) . A LIF with numerous lipid droplets ( * ) and a MYO with cisterns of rough ER ( red arrow ) are located at the basal side ( left ) . The alveolar lumen is filled with lamellar surfactant including tubular myelin ( white arrow ) ( lower right ) . Exocytosis of a lamellar body ( * ) from an AEC II ( upper right ) . Scale bars indicate 500 nm . Electron microscopy of bronchiolar - like airway depicting columnar ciliated cells ( red arrowhead ) with basal bodies ( black arrowhead ) at the left side of the longitudinally sectioned lumen . The boxed area indicates an apical junctional complex between two ciliated cells . 1 = tight junction , 2 = adherens junction . Scale bar indicates 500 nm ( in insert : 100 nm ) . Staining of lamellar bodies in BALO with RFP LysoTracker . Scale bars represent 100 μm . Western blot analysis of the surfactant proteins : pro - SPC , mature SPC , pro - SPB , mature SPB , and SPA in lung homogenate ( LH ) , AEC and day 21 BALO ( n = 3 biological replicates ) . F - G . Alveolar diameter ( F ) and number of alveoli ( G ) in BALO at days 15 , 21 , 30 and 40 of culture was measured from n = 5 BALO in n = 3 biological replicates with tdTomato + rMC . H - I . Representative scheme and images of day 40 BALO alveoli ( H ) and airway ( I ) . BALO alveolar - like structures ( H ) are shown ( h ' ) ( red arrowheads ) in semithin section ( 0 . 5 µm ) stained with Toluidine blue . Scale bar indicates 100 μm ( left ) . Electron microscopy showing AEC I ( h ' ' ) ultrastructure within BALO . Scale bars indicate 2500 nm ( center ) and 1000 nm ( right ) . An airway - like structure ( i ' ) is shown with secretory and ciliated cells ( red arrowheads ) in semithin sections ( 0 . 5 µm ) longitudinally cut and stained with Toluidine blue . Scale bar indicates 50 μm ( far left ) . Electron microscopy of a bronchiolar - like airway ( I ) depicting pseudostratified epithelium ( i ' ' ) with a basal - like cell ( BC ) , not reaching the lumen in which cilia ( C ) are seen , located between a secretory ( SC ) and a ciliated cell ( Ci ) . Scale bars indicate 1000 nm ( left and right ) . Mature cilia ( i ' ' ' ) in BALO at higher magnification depicting the 9x2 + 2 structure with central doubled microtubules ( insert , red arrowhead ) , a characteristic for motile cilia . Scale bar indicates 100 nm ( far right ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2mRNA expression analysis of epithelial cell differentiation markers Sftpc (AEC II), Hopx (AEC I), Foxj1 (ciliated cells), Muc5ac (secretory cells) and p63 (basal cells) in BALO at days 0, 10, and 21 of culture (n=3 biological replicates with pooled cells from 4 cultures per replicate)Electron microscopy of BALO alveoli showing two cuboidal epithelial cells (AEC II) connected by tight junctions (red square) with numerous mitochondria (M) and lamellar bodies (LB). A LIF with numerous lipid droplets (*) and a MYO with cisterns of rough ER (red arrow) are located at the basal side (left). The alveolar lumen is filled with lamellar surfactant including tubular myelin (white arrow) (lower right). Exocytosis of a lamellar body (*) from an AEC II (upper right). Scale bars indicate 500 nm.Electron microscopy of bronchiolar-like airway depicting columnar ciliated cells (red arrowhead) with basal bodies (black arrowhead) at the left side of the longitudinally sectioned lumen. The boxed area indicates an apical junctional complex between two ciliated cells. 1 = tight junction, 2 = adherens junction. Scale bar indicates 500 nm (in insert: 100 nm).Staining of lamellar bodies in BALO with RFP LysoTracker. Scale bars represent 100 μm.Western blot analysis of the surfactant proteins: pro-SPC, mature SPC, pro-SPB, mature SPB, and SPA in lung homogenate (LH), AEC and day 21 BALO (n=3 biological replicates).F-G. Alveolar diameter (F) and number of alveoli (G) in BALO at days 15, 21, 30 and 40 of culture was measured from n=5 BALO in n=3 biological replicates with tdTomato+ rMC.H-I. Representative scheme and images of day 40 BALO alveoli (H) and airway (I). BALO alveolar-like structures (H) are shown (h') (red arrowheads) in semithin section (0.5 µm) stained with Toluidine blue. Scale bar indicates 100 μm (left). Electron microscopy showing AEC I (h'') ultrastructure within BALO. Scale bars indicate 2500 nm (center) and 1000 nm (right). An airway-like structure (i') is shown with secretory and ciliated cells (red arrowheads) in semithin sections (0.5 µm) longitudinally cut and stained with Toluidine blue. Scale bar indicates 50 μm (far left). Electron microscopy of a bronchiolar-like airway (I) depicting pseudostratified epithelium (i'') with a basal-like cell (BC), not reaching the lumen in which cilia (C) are seen, located between a secretory (SC) and a ciliated cell (Ci). Scale bars indicate 1000 nm (left and right). Mature cilia (i''') in BALO at higher magnification depicting the 9x2+2 structure with central doubled microtubules (insert, red arrowhead), a characteristic for motile cilia. Scale bar indicates 100 nm (far right).