figf1 ( a ) Flow cytometry of LPS - stimulated B cells stained with LysoTracker Red . ( b ) Autophagic flux of GFP - LC3 splenic B cells stimulated with LPS and then left untreated ( UT ) or treated for 1 h with bafimolycin A1 ( BafA1 ) , assessed by live - cell fluorescence microscopy ( left ) and automated quantification of spots ( right ) . Scale bar ( left ) , 5 μm . ( c ) Immunoblot analysis of the processing of endogenous unconjugated LC3 - I to lipid - conjugated LC3 - II in LPS - stimulated B cells with ( + NH4Cl ) or without treatment with NH4Cl ( left ) . Actin serves as a loading control throughout . Right , quantification of LC3 - II band intensity in the absence of NH4Cl , presented relative to that of actin . ( d ) Quantitative RT - PCR analysis of transcripts encoding autophagy - related molecules in ASCs 3 d after stimulation with LPS , presented relative to that in unstimulated B cells . ( e ) Immunoblot analysis of p62 in LPS - stimulated B cells ( bottom ) . Above , quantification of band intensity ( as in c ) . ( f ) Live - cell fluorescence microscopy of CD138 + PCs purified from the spleen of GFP - LC3 mice 5 d after the injection of NP - Ficoll ( PCs ) and of splenic B cells ( B cells ) and bone marrow CD138 + PCs ( BM PCs ) purified from unstimulated GFP - LC3 mice ( right ) and automated quantification of spots ( left ) . Scale bar ( right ) , 5 μm . * P 0 . 05 , * * P 0 . 01 and * * * P 0 . 001 ( Student ' s t - test ) . Data are representative of three or more independent experiments ( a , c - e ) or one experiment or more with > 40 cells ( b , f ; average and s . e . m . ) .

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 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 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 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

figf1(a) Flow cytometry of LPS-stimulated B cells stained with LysoTracker Red.(b) Autophagic flux of GFP-LC3 splenic B cells stimulated with LPS and then left untreated (UT) or treated for 1 h with bafimolycin A1 (BafA1), assessed by live-cell fluorescence microscopy (left) and automated quantification of spots (right). Scale bar (left), 5 μm.(c) Immunoblot analysis of the processing of endogenous unconjugated LC3-I to lipid-conjugated LC3-II in LPS-stimulated B cells with (+NH4Cl) or without treatment with NH4Cl (left). Actin serves as a loading control throughout. Right, quantification of LC3-II band intensity in the absence of NH4Cl, presented relative to that of actin.(d) Quantitative RT-PCR analysis of transcripts encoding autophagy-related molecules in ASCs 3 d after stimulation with LPS, presented relative to that in unstimulated B cells.(e) Immunoblot analysis of p62 in LPS-stimulated B cells (bottom). Above, quantification of band intensity (as in c).(f) Live-cell fluorescence microscopy of CD138+ PCs purified from the spleen of GFP-LC3 mice 5 d after the injection of NP-Ficoll (PCs) and of splenic B cells (B cells) and bone marrow CD138+ PCs (BM PCs) purified from unstimulated GFP-LC3 mice (right) and automated quantification of spots (left). Scale bar (right), 5 μm. *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test). Data are representative of three or more independent experiments (a,c-e) or one experiment or more with >40 cells (b,f; average and s.e.m.).


figf2 ( a ) Proliferation of B cells purified from Atg5f / fCD19 - Cre and Atg5f / f control mice and activated for 3 d with LPS , assessed by flow cytometry analysis of dilution of the membrane dye CFSE . ( b ) Size of B cells purified as in a and activated for various times ( horizontal axis ) with LPS , assessed by the intensity of forward scatter ( FSC ) by flow cytometry . ( c ) CD138 expression of B cells purified and activated as in a . APC , allophycocyanin ; PE , phycoerythrin . ( d ) Immunoblot analysis of ER proteins in lysates of Atg5f / f and Atg5f / fCD19 - Cre B cells 3 d after stimulation with LPS ( left ) , and quantification of band intensity ( right ; as in Fig . 1c ) . IgM - H , IgM heavy chain . NS , not significant ; * P 0 . 05 and * * P 0 . 01 ( Student ' s 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 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

figf2(a) Proliferation of B cells purified from Atg5f/fCD19-Cre and Atg5f/f control mice and activated for 3 d with LPS, assessed by flow cytometry analysis of dilution of the membrane dye CFSE. (b) Size of B cells purified as in a and activated for various times (horizontal axis) with LPS, assessed by the intensity of forward scatter (FSC) by flow cytometry. (c) CD138 expression of B cells purified and activated as in a. APC, allophycocyanin; PE, phycoerythrin.(d) Immunoblot analysis of ER proteins in lysates of Atg5f/f and Atg5f/fCD19-Cre B cells 3 d after stimulation with LPS (left), and quantification of band intensity (right; as in Fig. 1c). IgM-H, IgM heavy chain. NS, not significant; *P 0.05 and **P 0.01 (Student's t-test).


figf3 ( a ) Electron microscopy of Atg5f / f and Atg5f / fCD19 - Cre B cells 3 d after stimulation with LPS ( left and middle ) , and quantification of the ER by stereology ( right ) . Scale bars ( left and middle ) , 1 μm . ( b ) G6Pase electron - microscopy cytochemistry of differentiating PCs 3 d after stimulation with LPS ( left ) , and quantification of G6Pase + ( ER ) area , relative to total cell area ( right ) . Scale bars ( left ) , 1 μm . ( c ) Quantitative RT - PCR analysis of transcripts encoding spliced XBP - 1 ( sXBP - 1 ) , total XBP - 1 ( tXBP - 1 ) and BiP in differentiating PCs at 0 d and 3 d after stimulation with LPS , presented relative to that of unstimulated Atg5f / f B cells at day 3 . ( d ) Quantitative RT - PCR analysis of CHOP mRNA in Atg5f / f and Atg5f / fCD19 - Cre B cells stimulated for 3 d with LPS ( left and middle ) , and in Atg5f / f B cells stimulated for 3 d with LPS and treated for the final 24 h with tunicamycin ( Atg5f / f 24 h tun ; 0 . 5 μg / ml ; positive control ) , presented relative to that in Atg5f / f B cells stimulated for 3 d with LPS . ( e ) Immunoblot analysis ( left ) of ER proteins in Atg5f / f B cells left untreated ( UT ) or treated with lysosomal protease inhibitors ( 10 μM leupeptin and E - 64d ) for the final 8 h of the third day of LPS - induced differentiation ( Leu + E - 64d ( 8 h ) ) . Right , quantification of band intensity ( presented as in Fig . 1c ) . * P 0 . 05 , * * P 0 . 01 and * * * P 0 . 001 ( Student ' s 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 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 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 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

figf3(a) Electron microscopy of Atg5f/f and Atg5f/fCD19-Cre B cells 3 d after stimulation with LPS (left and middle), and quantification of the ER by stereology (right). Scale bars (left and middle), 1 μm.(b) G6Pase electron-microscopy cytochemistry of differentiating PCs 3 d after stimulation with LPS (left), and quantification of G6Pase+ (ER) area, relative to total cell area (right). Scale bars (left), 1 μm.(c) Quantitative RT-PCR analysis of transcripts encoding spliced XBP-1 (sXBP-1), total XBP-1 (tXBP-1) and BiP in differentiating PCs at 0 d and 3 d after stimulation with LPS, presented relative to that of unstimulated Atg5f/f B cells at day 3.(d) Quantitative RT-PCR analysis of CHOP mRNA in Atg5f/f and Atg5f/fCD19-Cre B cells stimulated for 3 d with LPS (left and middle), and in Atg5f/f B cells stimulated for 3 d with LPS and treated for the final 24 h with tunicamycin (Atg5f/f 24 h tun; 0.5 μg/ml; positive control), presented relative to that in Atg5f/f B cells stimulated for 3 d with LPS.(e) Immunoblot analysis (left) of ER proteins in Atg5f/f B cells left untreated (UT) or treated with lysosomal protease inhibitors (10 μM leupeptin and E-64d) for the final 8 h of the third day of LPS-induced differentiation (Leu + E-64d (8 h)). Right, quantification of band intensity (presented as in Fig. 1c). *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test).


figf4 ( a ) Enzyme - linked immunosorbent assay ( ELISA ) of IgM in supernatants of Atg5f / fCD19 - Cre and Atg5f / f B cells stimulated for 3 d with LPS and plated for 4 h ( as trypan blue - negative cells at a density of 1 × 106 cells per ml in dedicated media ) , presented relative to results obtained for Atg5f / f cells . ( b , c ) Immunoglobulin assembly ( b ) and total intracellular ( IC ) and secreted ( Sec ) IgM heavy chain ( IgM - H , c ) in Atg5f / f and Atg5f / fCD19 - CreB cells stimulated for 3 d with LPS , then pulse - labeled and chased ( time , above lanes ) , followed by immunoprecipitation of the μ - chain from lysates and SDS - PAGE ( left ) in nonreducing conditions ( b ) or reducing conditions ( c ) . Right , quantification of intracellular IgM polymers by densitometry ( b ) and of secretion efficiency , estimated as the amount of radioactive secreted μ - chain at the end of the chase relative to intracellular μ - chain at the end of the pulse ( c ) . AU , arbitrary units . ( b , c ) Immunoglobulin assembly ( b ) and total intracellular ( IC ) and secreted ( Sec ) IgM heavy chain ( IgM - H , c ) in Atg5f / f and Atg5f / fCD19 - CreB cells stimulated for 3 d with LPS , then pulse - labeled and chased ( time , above lanes ) , followed by immunoprecipitation of the μ - chain from lysates and SDS - PAGE ( left ) in nonreducing conditions ( b ) or reducing conditions ( c ) . Right , quantification of intracellular IgM polymers by densitometry ( b ) and of secretion efficiency , estimated as the amount of radioactive secreted μ - chain at the end of the chase relative to intracellular μ - chain at the end of the pulse ( c ) . AU , arbitrary units . ( d ) Quantitative RT - PCR analysis of mRNA encoding Blimp - 1 and the secreted μ - chain in Atg5f / f and Atg5f / fCD19 - Cre B cells stimulated for 3 d with LPS , presented relative to that of Atg5f / f cells . ( e ) Quantitative RT - PCR analysis of transcripts encoding spliced XBP - 1 , BiP , Blimp - 1 and secreted μ - chain in Atg5f / f B cells stimulated for 3 d with LPS and given no further treatment ( − tun ) or treated for the final 24 h with tunicamycin ( tun 24 h ; 0 . 5 μg / ml ) , presented relative to results obtained with LPS - stimulated cells given no further treatment . * P 0 . 05 , * * P 0 . 01 and * * * P 0 . 001 ( Student ' s 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf4(a) Enzyme-linked immunosorbent assay (ELISA) of IgM in supernatants of Atg5f/fCD19-Cre and Atg5f/f B cells stimulated for 3 d with LPS and plated for 4 h (as trypan blue-negative cells at a density of 1 × 106 cells per ml in dedicated media), presented relative to results obtained for Atg5f/f cells.(b,c) Immunoglobulin assembly (b) and total intracellular (IC) and secreted (Sec) IgM heavy chain (IgM-H, c) in Atg5f/f and Atg5f/fCD19-CreB cells stimulated for 3 d with LPS, then pulse-labeled and chased (time, above lanes), followed by immunoprecipitation of the μ-chain from lysates and SDS-PAGE (left) in nonreducing conditions (b) or reducing conditions (c). Right, quantification of intracellular IgM polymers by densitometry (b) and of secretion efficiency, estimated as the amount of radioactive secreted μ-chain at the end of the chase relative to intracellular μ-chain at the end of the pulse (c). AU, arbitrary units.(b,c) Immunoglobulin assembly (b) and total intracellular (IC) and secreted (Sec) IgM heavy chain (IgM-H, c) in Atg5f/f and Atg5f/fCD19-CreB cells stimulated for 3 d with LPS, then pulse-labeled and chased (time, above lanes), followed by immunoprecipitation of the μ-chain from lysates and SDS-PAGE (left) in nonreducing conditions (b) or reducing conditions (c). Right, quantification of intracellular IgM polymers by densitometry (b) and of secretion efficiency, estimated as the amount of radioactive secreted μ-chain at the end of the chase relative to intracellular μ-chain at the end of the pulse (c). AU, arbitrary units.(d) Quantitative RT-PCR analysis of mRNA encoding Blimp-1 and the secreted μ-chain in Atg5f/f and Atg5f/fCD19-Cre B cells stimulated for 3 d with LPS, presented relative to that of Atg5f/f cells.(e) Quantitative RT-PCR analysis of transcripts encoding spliced XBP-1, BiP, Blimp-1 and secreted μ-chain in Atg5f/f B cells stimulated for 3 d with LPS and given no further treatment (−tun) or treated for the final 24 h with tunicamycin (tun 24 h; 0.5 μg/ml), presented relative to results obtained with LPS-stimulated cells given no further treatment. *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test).


figf5 ( a ) Intracellular ATP in fresh lysates of Atg5f / f and Atg5f / fCD19 - Cre splenic B cells activated for 3 d with LPS ( 3 ) or left untreated ( 0 ) . ( b ) Death of Atg5f / f and Atg5f / fCD19 - Cre splenic B cells activated for various times ( horizontal axis ) with LPS , quantified by flow cytometry by propidium iodide ( PI ) staining . ( c ) Quantitative PCR analysis of genomic DNA in differentiating B cells from Atg5f / f and Atg5f / fCD19 - Cre mice , assessing nonrecombinant ( Undeleted ; left ) and recombinant ( Deleted ; right ) loxP - flanked Atg5 alleles ; results are presented relative to those of unstimulated Atg5f / fCD19 - Cre B cells . ( d ) Quantitative RT - PCR analysis of Atg5 mRNA in differentiating Atg5f / f and Atg5f / fCD19 - Cre B cells at 0 and 3 d after stimulation with LPS , presented relative to that in untreated Atg5f / fCD19 - Cre B cells . * P 0 . 05 , * * P 0 . 01 and * * * P 0 . 001 ( Student ' s 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 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 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

figf5(a) Intracellular ATP in fresh lysates of Atg5f/f and Atg5f/fCD19-Cre splenic B cells activated for 3 d with LPS (3) or left untreated (0).(b) Death of Atg5f/f and Atg5f/fCD19-Cre splenic B cells activated for various times (horizontal axis) with LPS, quantified by flow cytometry by propidium iodide (PI) staining.(c) Quantitative PCR analysis of genomic DNA in differentiating B cells from Atg5f/f and Atg5f/fCD19-Cre mice, assessing nonrecombinant (Undeleted; left) and recombinant (Deleted; right) loxP-flanked Atg5 alleles; results are presented relative to those of unstimulated Atg5f/fCD19-Cre B cells.(d) Quantitative RT-PCR analysis of Atg5 mRNA in differentiating Atg5f/f and Atg5f/fCD19-Cre B cells at 0 and 3 d after stimulation with LPS, presented relative to that in untreated Atg5f/fCD19-Cre B cells. *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test).


figf6 ( a ) ELISA of anti - pneumovax IgM titers in diluted serum 1 day before and 4 , 10 and 14 d after intraperitoneal immunization of Atg5f / f and Atg5f / fCD19 - Cre mice with 1 μg pneumovax . ( b ) ELISA of anti - pneumovax IgG3 titers in serum 14 d after injection as in a . Results are presented as absorbance ( A ) . * P 0 . 05 , * * P 0 . 01 and * * * P 0 . 001 ( Student ' s 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 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

figf6(a) ELISA of anti-pneumovax IgM titers in diluted serum 1 day before and 4, 10 and 14 d after intraperitoneal immunization of Atg5f/f and Atg5f/fCD19-Cre mice with 1 μg pneumovax.(b) ELISA of anti-pneumovax IgG3 titers in serum 14 d after injection as in a. Results are presented as absorbance (A). *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test).


figf7 ( a ) Frequency of CD138hiB220lo cells ( numbers above outlined areas , bottom ) among the IRF4hi population ( numbers above outlined areas , top ) , assessed by flow cytometry ( left ) . 488 , Alexa Fluor 488 . Right , quantification of bone marrow PCs ( each symbol represents an individual mouse ; small horizontal lines indicate mean ( and s . e . m . ) . ( b ) Quantitative RT - PCR analysis of mRNA encoding IRF4 and Blimp - 1 in CD138hiB220lo bone marrow PCs and B220hiCD138lo spleen B lymphocytes ( control ) sorted from Atg5f / f and Atg5f / fCD19 - Cre mice , to confirm PC phenotype ; results are presented relative to that of histone H3 mRNA ( loading control ) . ( c ) Quantitative PCR analysis of genomic DNA assessing undeleted and deleted loxP - flanked Atg5 alleles in sorted B cells and PCs ( as in b ; presented as in Fig . 5c ) . ( d ) Immunofluorescence of endogenous LC3 ( left , top row ) and immunoglobulin content ( left , bottom row ) in bone marrow PCs sorted from Atg5f / f and Atg5f / fCD19 - Cre mice , and quantitative RT - PCR analysis of Atg5 mRNA in bone marrow PCs and spleen B cell populations ( right ) sorted from Atg5f / f and Atg5f / f CD19 - Cre mice . Nuclei ( left ) are stained blue with Hoechst . Scale bar ( left ) , 5 μm . * P 0 . 05 , * * P 0 . 01 and * * * P 0 . 001 ( Student ' s 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 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 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

figf7(a) Frequency of CD138hiB220lo cells (numbers above outlined areas, bottom) among the IRF4hi population (numbers above outlined areas, top), assessed by flow cytometry (left). 488, Alexa Fluor 488. Right, quantification of bone marrow PCs (each symbol represents an individual mouse; small horizontal lines indicate mean (and s.e.m.).(b) Quantitative RT-PCR analysis of mRNA encoding IRF4 and Blimp-1 in CD138hiB220lo bone marrow PCs and B220hiCD138lo spleen B lymphocytes (control) sorted from Atg5f/f and Atg5f/fCD19-Cre mice, to confirm PC phenotype; results are presented relative to that of histone H3 mRNA (loading control). (c) Quantitative PCR analysis of genomic DNA assessing undeleted and deleted loxP-flanked Atg5 alleles in sorted B cells and PCs (as in b; presented as in Fig. 5c).(d) Immunofluorescence of endogenous LC3 (left, top row) and immunoglobulin content (left, bottom row) in bone marrow PCs sorted from Atg5f/f and Atg5f/fCD19-Cre mice, and quantitative RT-PCR analysis of Atg5 mRNA in bone marrow PCs and spleen B cell populations (right) sorted from Atg5f/f and Atg5f/f CD19-Cre mice. Nuclei (left) are stained blue with Hoechst. Scale bar (left), 5 μm. *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test).


Figure 1Representative confocal images of the FtsZ · SlmA · SBS condensates formed in 150 g / L dextran 500 or Ficoll 70 , and absence of condensates in dilute solution . Scale bars : 20 μm , except for images at higher magnification in dextran ( 5 μm , second row ) . Turbidity of FtsZ · SlmA · SBS in buffer ( n = 3 ) , in 150 g / L dextran 500 ( n = 5 ) or Ficoll 70 ( n = 3 ) and in 50 g / L PEG 8 ( n = 3 ) . Data correspond to the average ± S . D .

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

Figure 1Representative confocal images of the FtsZ·SlmA·SBS condensates formed in 150 g/L dextran 500 or Ficoll 70, and absence of condensates in dilute solution. Scale bars: 20 μm, except for images at higher magnification in dextran (5 μm, second row).Turbidity of FtsZ·SlmA·SBS in buffer (n = 3), in 150 g/L dextran 500 (n = 5) or Ficoll 70 (n = 3) and in 50 g/L PEG 8 (n = 3). Data correspond to the average ± S.D.


Figure 2Formation of condensates as a function of FtsZ and SlmA · SBS concentration , as measured by turbidity , in working buffer ( 300 mM KCl ) . SlmA concentration was fivefold that of SBS ( except at 0 . 5 μM SBS , where SlmA concentration was 3 μM ) . Data are the average of 2 independent measurements . Errors ( S . D . ) , symmetrical , are depicted as white discs . Representative confocal images of the FtsZ · SlmA · SBS condensates at the specified salt concentrations . The concentrations of FtsZ , SlmA and SBS were 12 , 5 and 1 µM , respectively . Scale bars : 5 μm . Data information : All measurements in 150 g / L dextran 500 .

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

Figure 2Formation of condensates as a function of FtsZ and SlmA·SBS concentration, as measured by turbidity, in working buffer (300 mM KCl). SlmA concentration was fivefold that of SBS (except at 0.5 μM SBS, where SlmA concentration was 3 μM). Data are the average of 2 independent measurements. Errors (S.D.), symmetrical, are depicted as white discs.Representative confocal images of the FtsZ·SlmA·SBS condensates at the specified salt concentrations. The concentrations of FtsZ, SlmA and SBS were 12, 5 and 1 µM, respectively. Scale bars: 5 μm. Data information: All measurements in 150 g/L dextran 500.


Figure 3Representative confocal images showing final state after addition of FtsZ - Alexa 488 to FtsZ · SlmA · SBS complexes ( FtsZ labeled with Alexa 647 ) in 150 g / L dextran . Below , images showing the stepwise diffusion of FtsZ - Alexa 488 into the condensates containing FtsZ - Alexa 647 at the indicated times in seconds ( time zero , beginning of visualization for that particular condensate ) and corresponding intensity profiles at selected times in the green channel . The profile in the red channel is shown as a reference and varies slightly among images . Scale bars : 5 μm ( top row ) and 4 μm ( bottom row ) . Stepwise diffusion of FtsZ - Alexa 488 into FtsZ · SlmA · SBS condensates ( FtsZ labeled with Alexa 647 ) at the indicated times in seconds ( time zero , beginning of visualization for those particular condensates ) in 100 g / L PEG . Scale bars : 4 μm . Assembly of FtsZ fibers upon GTP addition ( 0 . 5 mM ) to FtsZ · SlmA · SBS condensates and condensates formed after FtsZ fibers disassembly at the indicated times in minutes ( time zero , GTP addition ) in 150 g / L dextran . Scale bars : 5 μm . Scheme of the dynamic process on the right . The number of condensates decreases upon fibers formation , and they rearrange upon GTP depletion and fibers disassembly .

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 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 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

Figure 3Representative confocal images showing final state after addition of FtsZ-Alexa 488 to FtsZ·SlmA·SBS complexes (FtsZ labeled with Alexa 647) in 150 g/L dextran. Below, images showing the stepwise diffusion of FtsZ-Alexa 488 into the condensates containing FtsZ-Alexa 647 at the indicated times in seconds (time zero, beginning of visualization for that particular condensate) and corresponding intensity profiles at selected times in the green channel. The profile in the red channel is shown as a reference and varies slightly among images. Scale bars: 5 μm (top row) and 4 μm (bottom row).Stepwise diffusion of FtsZ-Alexa 488 into FtsZ·SlmA·SBS condensates (FtsZ labeled with Alexa 647) at the indicated times in seconds (time zero, beginning of visualization for those particular condensates) in 100 g/L PEG. Scale bars: 4 μm.Assembly of FtsZ fibers upon GTP addition (0.5 mM) to FtsZ·SlmA·SBS condensates and condensates formed after FtsZ fibers disassembly at the indicated times in minutes (time zero, GTP addition) in 150 g/L dextran. Scale bars: 5 μm. Scheme of the dynamic process on the right. The number of condensates decreases upon fibers formation, and they rearrange upon GTP depletion and fibers disassembly.


Figure 4B - D . Representative confocal images of the microdroplets without ( B ) and with GTP ( C and D ) . The concentrations of FtsZ , SlmA and SBS were 12 , 5 and 1 μM , respectively ( B and C ) or 6 , 3 and 0 . 5 μM respectively ( D ) . 1 mM ( C ) or 2 mM GTP ( D ) . Scale bars : 40 μm except in images on the far right ( 20 μm ) , which are either a magnification of the indicated region in the merged image ( B ) or independent images at higher magnification ( C , D ) .

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

Figure 4B-D. Representative confocal images of the microdroplets without (B) and with GTP (C and D). The concentrations of FtsZ, SlmA and SBS were 12, 5 and 1 μM, respectively (B and C) or 6, 3 and 0.5 μM respectively (D). 1 mM (C) or 2 mM GTP (D). Scale bars: 40 μm except in images on the far right (20 μm), which are either a magnification of the indicated region in the merged image (B) or independent images at higher magnification (C, D).


Figure 5B , C . Representative confocal images of the microdroplets stabilized by the E . coli lipid mixture containing the biphasic PEG / DNA mixture and the FtsZ · SlmA · SBS complex without and with 2 mM GTP , respectively . Last image in ( B ) focuses in the lipid interface to show the high density of condensates . Scale bars : 20 μm except in images on the far right ( 10 μm ) , which are an independent image at higher magnification ( B ) or a magnification of the indicated region in the merged image ( C ) .

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

Figure 5B,C. Representative confocal images of the microdroplets stabilized by the E. coli lipid mixture containing the biphasic PEG/DNA mixture and the FtsZ·SlmA·SBS complex without and with 2 mM GTP, respectively. Last image in (B) focuses in the lipid interface to show the high density of condensates. Scale bars: 20 μm except in images on the far right (10 μm), which are an independent image at higher magnification (B) or a magnification of the indicated region in the merged image (C).


Figure 1B ) Proliferation assay of H3122 parental and isogenic drug - resistant cells , treated with the indicated concentrations of crizotinib for 72h . P < 0 . 0001 was calculated for IC50 shift as indicated in the methods . C ) Gene - set enrichment analysis after RNA - seq of H3122 sensitive vs CrizR1 crizotinib resistant cells . D ) Epithelial to mesenchymal transition enrichment plot from the RNA - seq of C ) , using the HALLMARK gene collection . E ) H3122 parental and drug - resistant isogenic cell lines were RNA - extracted and gene expression was analysed by RT - qPCR . On the right hand side is a western blot analysis of the AXL and EML4 - ALK protein levels from the same cell lines . AXL : CrizR1 vs H3122 P < 0 . 001 , CrizR4 vs H3122 P < 0 . 001 , CrizR5 vs H3122 P = 0 . 03 , CeritR vs H3122 P = 0 . 2 ; LOX : CrizR1 vs H3122 P < 0 . 001 , CrizR4 vs H3122 P < 0 . 001 , CrizR5 vs H3122 P < 0 . 001 , CeritR vs H3122 P = 0 . 008 ; SNAI2 : CrizR1 vs H3122 P < 0 . 001 , CrizR4 vs H3122 P < 0 . 001 , CrizR5 vs H3122 P = 0 . 003 , CeritR vs H3122 P = 0 . 003 ; VIM : CrizR1 vs H3122 P < 0 . 001 , CrizR4 vs H3122 P < 0 . 001 , CrizR5 vs H3122 P < 0 . 001 , CeritR vs H3122 P < 0 . 001 . F ) Gene expression analysis by RT - qPCR of CrizR1 cells treated with the indicated concentrations of bemcentinib for 48h . LOX : P = 0 . 03 ; SNAI2 : P = 0 . 02 ; VIM : P = 0 . 03 . G ) Proliferation assay of CrizR1 and CrizR4 cells treated with the indicated concentrations of bemcentinib ± 1uM crizotinib for 72h . * * * = P < 0 . 001 for IC50 shift , as indicated in the methods ( n = 4 ) . H ) Annexin V + apoptotic assay in CrizR1 cells treated with 1uM crizotinib , 2 . 5uM bemcentinib , or combination . STE - 1 parental cells were used as crizotinib drug control . crizotinib : P = 0 . 2 ; bemcentinib : P = 0 . 4 ; combination : P = 0 . 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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

Figure 1B) Proliferation assay of H3122 parental and isogenic drug-resistant cells, treated with the indicated concentrations of crizotinib for 72h. P < 0.0001 was calculated for IC50 shift as indicated in the methods.C) Gene-set enrichment analysis after RNA-seq of H3122 sensitive vs CrizR1 crizotinib resistant cells. D) Epithelial to mesenchymal transition enrichment plot from the RNA-seq of C), using the HALLMARK gene collection. E) H3122 parental and drug-resistant isogenic cell lines were RNA-extracted and gene expression was analysed by RT-qPCR. On the right hand side is a western blot analysis of the AXL and EML4-ALK protein levels from the same cell lines.AXL: CrizR1 vs H3122 P <0.001, CrizR4 vs H3122 P <0.001, CrizR5 vs H3122 P=0.03, CeritR vs H3122 P=0.2; LOX: CrizR1 vs H3122 P <0.001, CrizR4 vs H3122 P <0.001, CrizR5 vs H3122 P <0.001, CeritR vs H3122 P=0.008; SNAI2: CrizR1 vs H3122 P <0.001, CrizR4 vs H3122 P <0.001, CrizR5 vs H3122 P =0.003, CeritR vs H3122 P =0.003; VIM: CrizR1 vs H3122 P <0.001, CrizR4 vs H3122 P <0.001, CrizR5 vs H3122 P <0.001, CeritR vs H3122 P <0.001.F) Gene expression analysis by RT-qPCR of CrizR1 cells treated with the indicated concentrations of bemcentinib for 48h. LOX: P=0.03; SNAI2: P =0.02; VIM: P =0.03.G) Proliferation assay of CrizR1 and CrizR4 cells treated with the indicated concentrations of bemcentinib ± 1uM crizotinib for 72h. *** = P < 0.001 for IC50 shift, as indicated in the methods (n=4).H) Annexin V+ apoptotic assay in CrizR1 cells treated with 1uM crizotinib, 2.5uM bemcentinib, or combination. STE-1 parental cells were used as crizotinib drug control. crizotinib: P =0.2; bemcentinib: P =0.4; combination: P=0.3.


Figure 2A ) GSEA enrichment analysis using the KEGG gene set identifiers . Shown are the significantly dysregulated pathways ( P < 0 . 05 ) . B ) Cell cycle enrichment plot from A ) . C ) Western blot analysis of H3122 parental and isogenic drug - resistant cell lines for the indicated proteins . D ) Western blot analysis of Ste - 1 parental and isogenic Alectinib resistant cell lines for the indicated proteins . E ) Proliferation assay of CrizR1 cells treated with the indicated concentrations of alvocidib ± 1uM crizotinib for 72h . P = 0 . 2 ( n = 4 ) . F ) As above , in a proliferation assay , H3122 and CrizR1 cells were treated in parallel with DMSO or 1uM crizotinib as drug control . H3122 vs DMSO P < 0 . 0001 , CrizR1 vs DMSO P = 0 . 1 ( n = 4 ) . Proliferation assay of CrizR1 , CrizR4 , CrizR5 , CeritR isogenic drug - resistant cell lines , treated with the indicated concentrations of alvocidib ( G ) for 72h . P > 0 . 05 was calculated for IC50 shift , as indicated in the methods . H ) Proliferation assay of AlecR isogenic drug - resistant cell lines , treated with the indicated concentrations of dinaciclib ( H ) for 72h . P > 0 . 05 was calculated for IC50 shift , as indicated in the methods .

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 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 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 O O O 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

Figure 2A) GSEA enrichment analysis using the KEGG gene set identifiers. Shown are the significantly dysregulated pathways (P < 0.05). B) Cell cycle enrichment plot from A). C) Western blot analysis of H3122 parental and isogenic drug-resistant cell lines for the indicated proteins.D) Western blot analysis of Ste-1 parental and isogenic Alectinib resistant cell lines for the indicated proteins.E) Proliferation assay of CrizR1 cells treated with the indicated concentrations of alvocidib ± 1uM crizotinib for 72h. P=0.2 (n=4).F) As above, in a proliferation assay, H3122 and CrizR1 cells were treated in parallel with DMSO or 1uM crizotinib as drug control. H3122 vs DMSO P <0.0001, CrizR1 vs DMSO P=0.1 (n=4).Proliferation assay of CrizR1, CrizR4, CrizR5, CeritR isogenic drug-resistant cell lines, treated with the indicated concentrations of alvocidib(G) for 72h. P >0.05 was calculated for IC50 shift, as indicated in the methods.H) Proliferation assay of AlecR isogenic drug-resistant cell lines, treated with the indicated concentrations of dinaciclib (H) for 72h. P >0.05 was calculated for IC50 shift, as indicated in the methods.


Figure 3A ) Proliferation assay of CrizR1 , CrizR4 , CrizR5 , CeritR , and AlecR isogenic drug - resistant cell lines , treated with the indicated concentrations of THZ1 for 72h . P < 0 . 05 was calculated for IC50 shift , as indicated in the methods . B ) Crystal violet staining of EML4 / ALK parental and drug - resistant cells . Cells were treated with the indicated drugs until the vehicle control reached confluence , then fixed and stained . C ) CrizR1 cells were treated with the indicated drugs for 72h , then cells were stained with Annexin V / PI . Flow cytometry was then used to quantify Annexin V + cells . Alvocidib : early apoptosis P = 0 . 002 , late apoptosis P = 0 . 006 , alive P = 0 . 0008 ; Dinaciclib : early apoptosis P = 0 . 005 , late apoptosis P = 0 . 02 , alive P = 0 . 002 .

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 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

Figure 3A) Proliferation assay of CrizR1, CrizR4, CrizR5, CeritR, and AlecR isogenic drug-resistant cell lines, treated with the indicated concentrations of THZ1 for 72h. P <0.05 was calculated for IC50 shift, as indicated in the methods.B) Crystal violet staining of EML4/ALK parental and drug-resistant cells. Cells were treated with the indicated drugs until the vehicle control reached confluence, then fixed and stained.C) CrizR1 cells were treated with the indicated drugs for 72h, then cells were stained with Annexin V/PI. Flow cytometry was then used to quantify Annexin V+ cells. Alvocidib: early apoptosis P=0.002, late apoptosis P=0.006, alive P=0.0008; Dinaciclib: early apoptosis P=0.005, late apoptosis P=0.02, alive P=0.002.


Figure 4A ) Indicated cell lines were treated with DMSO , 200nM alvocidib or 25nM dinaciclib for 48h , then cells were stained with Annexin V / PI . Flow cytometry was then used to quantify Annexin V + cells ( n = 2 ) . Alvocidib : CrizR1 P = 0 . 005 , AlecR P = 0 . 001 , PC9 P = 0 . 05 , A549 P = 0 . 002 , H1299 P = 0 . 03 , H460 P = 0 . 07 ; P dinaciclib : CrizR1 P = 0 . 02 , AlecR P = 0 . 005 , PC9 P = 0 . 06 , A549 P = 0 . 08 , H1299 P = 0 . 09 , H460 P = 0 . 01 . ( B ) CrizR1 cells were treated with DMSO or 200nM alvocidib for 24h and cell extracts were hybridized to a 43 - antibody array and analysed by immunoblotting . Graphs depict the significant changes from two independent experiments . C ) CrizR1 / CeritR / AlecR cells were treated with DMSO or 200nM alvocidib or 50nM dinaciclib for 6h and cell extracts were analysed by western blotting . D ) ( Top ) Western blot analysis of CrizR1 cells treated with siScrambled or siRNA for CDK7 or CDK9 for 72h . ( Bottom ) CrizR1 were treated as above , or with DMSO / alvocidib / THZ1 and cells were stained with Annexin V / PI and analysed by flow cytometry for Annexin V + cells 72h post - transfection ( n = 2 ) . Annexin siCDK7 P = 0 . 2 , siCDK9 P = 0 . 015 , alvocidib P = 0 . 0001 , THZ1 P = 0 . 004 . E ) H3122 parental and CrizR1 cells were treated with DMSO , 200nM alvocidib or 250ng / ml actinomycin D for 6 hours . RNA was extracted and the mRNA levels of the indicated genes were quantified by RT - qPCR . CDK1 : DMSO vs H3122 DMSO P = 0 . 002 , Actinomycin vs DMSO P = 0 . 0003 , Alvocidib vs DMSO P = 0 . 0005 ; CDK2 : DMSO vs H3122 DMSO P = 0 . 0001 , Actinomycin vs DMSO P < 0 . 0001 , Alvocidib vs DMSO P = 0 . 0005 ; CDK6 : DMSO vs H3122 DMSO P = 0 . 0005 , Actinomycin vs DMSO P = 0 . 0004 , Alvocidib vs DMSO P = 0 . 0004 ; CDK9 : DMSO vs H3122 DMSO P = 0 . 0003 , Actinomycin vs DMSO P = 0 . 0002 , Alvocidib vs DMSO P = 0 . 0003 ; CCNB1 : DMSO vs H3122 DMSO P = 0 . 02 , Actinomycin vs DMSO P = 0 . 0002 , Alvocidib vs DMSO P = 0 . 0003 ; CCNE1 : DMSO vs H3122 DMSO P = 0 . 0004 , Actinomycin vs DMSO P = 0 . 0004 , Alvocidib vs DMSOF ) RT - qPCR analysis of MCL - 1 and Survivin expression after treatment of CrizR1 cells with 100nM alvocidib , 25nM dinaciclib and 50 nM THZ1 . RNA was extracted after 24h of treatment . MCL - 1 alvocidib vs control P = 0 . 0001 , dinaciclib vs control P = 0 . 0001 , THZ1 vs control P = 0 . 0001 ; Survivin : alvocidib vs control P = 0 . 01 , dinaciclib vs control P = 0 . 01 , THZ1 vs control P = 0 . 0001 . G ) ( Top ) Western blot analysis of CrizR1 cells treated with siScrambled or with a pool of 4 different siRNAs targeting Survivin ( siBIRC5 ) . ( Bottom ) Cells were stained with Annexin V / PI and analysed by flow cytometry for Annexin V + cells 72h post - transfection . Annexin : early apoptosis P = 0 . 0003 , late apoptosis P = 0 . 5 , alive P < 0 . 0001 . H ) The CCLE RNA - seq dataset was used and RPKM values were plotted for the Cyclin D1 and Survivn genes , indicating high expression in H3122 cells compared with other LUAD cells .

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

Figure 4A) Indicated cell lines were treated with DMSO, 200nM alvocidib or 25nM dinaciclib for 48h, then cells were stained with Annexin V/PI. Flow cytometry was then used to quantify Annexin V+ cells (n=2). Alvocidib: CrizR1 P=0.005, AlecR P =0.001, PC9 P =0.05, A549 P =0.002, H1299 P =0.03, H460 P =0.07; P dinaciclib: CrizR1 P=0.02, AlecR P =0.005, PC9 P =0.06, A549 P =0.08, H1299 P =0.09, H460 P =0.01.(B) CrizR1 cells were treated with DMSO or 200nM alvocidib for 24h and cell extracts were hybridized to a 43-antibody array and analysed by immunoblotting. Graphs depict the significant changes from two independent experiments.C) CrizR1/CeritR/AlecR cells were treated with DMSO or 200nM alvocidib or 50nM dinaciclib for 6h and cell extracts were analysed by western blotting.D) (Top) Western blot analysis of CrizR1 cells treated with siScrambled or siRNA for CDK7 or CDK9 for 72h. (Bottom) CrizR1 were treated as above, or with DMSO/alvocidib/THZ1 and cells were stained with Annexin V/PI and analysed by flow cytometry for Annexin V+ cells 72h post-transfection (n=2). Annexin siCDK7 P=0.2, siCDK9 P=0.015, alvocidib P=0.0001, THZ1 P =0.004.E) H3122 parental and CrizR1 cells were treated with DMSO, 200nM alvocidib or 250ng/ml actinomycin D for 6 hours. RNA was extracted and the mRNA levels of the indicated genes were quantified by RT-qPCR. CDK1: DMSO vs H3122 DMSO P=0.002, Actinomycin vs DMSO P=0.0003, Alvocidib vs DMSO P=0.0005; CDK2: DMSO vs H3122 DMSO P =0.0001, Actinomycin vs DMSO P<0.0001, Alvocidib vs DMSO P=0.0005; CDK6: DMSO vs H3122 DMSO P =0.0005, Actinomycin vs DMSO P =0.0004, Alvocidib vs DMSO P =0.0004; CDK9: DMSO vs H3122 DMSO P=0.0003, Actinomycin vs DMSO P =0.0002, Alvocidib vs DMSO P =0.0003; CCNB1: DMSO vs H3122 DMSO P=0.02, Actinomycin vs DMSO P=0.0002, Alvocidib vs DMSO P=0.0003; CCNE1: DMSO vs H3122 DMSO P =0.0004, Actinomycin vs DMSO P =0.0004, Alvocidib vs DMSOF) RT-qPCR analysis of MCL-1 and Survivin expression after treatment of CrizR1 cells with 100nM alvocidib, 25nM dinaciclib and 50 nM THZ1. RNA was extracted after 24h of treatment. MCL-1 alvocidib vs control P=0.0001, dinaciclib vs control P=0.0001, THZ1 vs control P =0.0001; Survivin: alvocidib vs control P =0.01, dinaciclib vs control P=0.01, THZ1 vs control P =0.0001.G) (Top) Western blot analysis of CrizR1 cells treated with siScrambled or with a pool of 4 different siRNAs targeting Survivin (siBIRC5). (Bottom) Cells were stained with Annexin V/PI and analysed by flow cytometry for Annexin V+ cells 72h post-transfection. Annexin: early apoptosis P =0.0003, late apoptosis P =0.5, alive P <0.0001.H) The CCLE RNA-seq dataset was used and RPKM values were plotted for the Cyclin D1 and Survivn genes, indicating high expression in H3122 cells compared with other LUAD cells.


Figure 5A ) CrizR1 cells were treated with vehicle control , 200nM alvocidib or 100nM THZ1 for 6 hrs , then chromatin was precipitated with an anti - RNA polymerase II antibody and sequenced . Plotted is the average number of peaks per condition . GSEA analysis using the HALLMARK gene collection for the differentially enriched peaks around the TSS ( ± 1kb ) with ( B ) alvocidib Shown on the right is the plot of the " MYC targets " - enriched signature . GSEA analysis using the HALLMARK gene collection for the differentially enriched peaks around the TSS ( ± 1kb ) with ( C ) THZ1 treatment . Shown on the right is the plot of the " MYC targets " - enriched signature . D ) Gene tracks of RNA polymerase II occupancy at the MCL1 , BIRC5 ( Survivin ) , CCND1 and MYC genes . E ) qPCR of MYC and CCND1 upon treatment of CrizR1 cells with Alvocidib or THZ1 for 6h . The graph represents the mean fold change ± SD . MYC : alvocidib vs DMSO P = 0 . 03 , THZ1 vs DMSO P < 0 . 0001 ; CCND1 : alvocidib vs DMSO P = 0 . 001 , THZ1 vs DMSO P = 0 . 003 . F ) BIM is upregulated upon MYC silencing in CrizR1 cells . G ) Annexin V assay showing the percentage of apoptotic CrizR1 cells upon MYC downregulation using siRNA . Early apoptosis P = 0 . 002 , late apoptosis P = 0 . 04 , alive P = 0 . 0008 . H ) Annexin V assay showing the percentage of apoptotic CrizR1 cells after treatment with trametinib , rapamycin or both . Trametinib : early apoptosis P = 0 . 002 , late apoptosis P = 0 . 05 , alive P = 0 . 009 ; rapamycin : early apoptosis P = 0 . 04 , late apoptosis P = 0 . 8 , alive P = 0 . 003 ; tram + rapa : early apoptosis P = 0 . 0001 , late apoptosis P = 0 . 04 , alive P = 0 . 01 .

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 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 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 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 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

Figure 5A) CrizR1 cells were treated with vehicle control, 200nM alvocidib or 100nM THZ1 for 6 hrs, then chromatin was precipitated with an anti-RNA polymerase II antibody and sequenced. Plotted is the average number of peaks per condition.GSEA analysis using the HALLMARK gene collection for the differentially enriched peaks around the TSS (±1kb) with (B) alvocidib Shown on the right is the plot of the "MYC targets"-enriched signature.GSEA analysis using the HALLMARK gene collection for the differentially enriched peaks around the TSS (±1kb) with (C) THZ1 treatment. Shown on the right is the plot of the "MYC targets"-enriched signature.D) Gene tracks of RNA polymerase II occupancy at the MCL1, BIRC5 (Survivin), CCND1 and MYC genes.E) qPCR of MYC and CCND1 upon treatment of CrizR1 cells with Alvocidib or THZ1 for 6h. The graph represents the mean fold change ± SD. MYC: alvocidib vs DMSO P =0.03, THZ1 vs DMSO P <0.0001; CCND1: alvocidib vs DMSO P =0.001, THZ1 vs DMSO P =0.003.F) BIM is upregulated upon MYC silencing in CrizR1 cells.G) Annexin V assay showing the percentage of apoptotic CrizR1 cells upon MYC downregulation using siRNA. Early apoptosis P =0.002, late apoptosis P=0.04, alive P =0.0008.H) Annexin V assay showing the percentage of apoptotic CrizR1 cells after treatment with trametinib, rapamycin or both. Trametinib: early apoptosis P =0.002, late apoptosis P =0.05, alive P =0.009; rapamycin: early apoptosis P =0.04, late apoptosis P =0.8, alive P =0.003; tram+rapa: early apoptosis P =0.0001, late apoptosis P =0.04, alive P =0.01.


Figure 6Tumour growth of in vivo xenografts of H3122 CrizR1 cell lines in response to crizotinib and correspondent tumour weights ( P . O . control n = 5 ; I . P . control n = 4 ; crizotinib n = 8 Tumour weights , crizotinib P = 0 . 44Tumour growth of in vivo xenografts of H3122 CrizR1 cell lines in response to alvocidib and correspondent tumour weights ( P . O . control n = 5 ; I . P . control n = 4 and alvocidib n = 6 ) . Tumour weights , , alvocidib P = 0 . 03 . C ) Tumour growth of in vivo xenografts of Ste - 1 AlectR cell lines in response to alvocidib and correspondent tumor weights ( I . P . control n = 6 ; alvocidib n = 4 ) . Tumour weights alvocidib P = 0 . 004 . D ) Number of cleaved caspase - 3 positive nuclei in alvocidib treated tumours compared to controls . E ) Ki67 and p21 staining of CrizR1 xenograft tumours . Scale bar = 200μm . Quantitative analysis of IHC staining is presented on the right . Ki67 P = 0 . 0004 ; p21 P = 0 . 02 . F ) qPCR of Survivin ( P = 0 . 0009 ) , MCL - 1 ( P = 0 . 04 ) , MYC ( P = 0 . 007 ) and BIM ( P = 0 . 03 ) in Ste - 1 AlectR cells xenografts treated with alvocidib ( Control n = 6 ; alvocidib n = 4 ) . G ) Proliferation assay of H3122 parental and isogenic drug - resistant cell lines , treated with DMSO , 100nM lorlatinib , 200nM alvocidib or 50nM dinaciclib for 72h ( n = 4 ) . H ) Growth curve of CrizR1 xenograft tumors in response to lorlatinib ( Control n = 7 ; lorlatinib n = 7 ) .

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 O O O O O O O O O O O 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 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 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 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 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 6Tumour growth of in vivo xenografts of H3122 CrizR1 cell lines in response to crizotinib and correspondent tumour weights (P.O. control n=5; I.P. control n=4; crizotinib n=8 Tumour weights, crizotinib P= 0.44Tumour growth of in vivo xenografts of H3122 CrizR1 cell lines in response to alvocidib and correspondent tumour weights (P.O. control n=5; I.P. control n=4 and alvocidib n=6). Tumour weights, , alvocidib P=0.03.C) Tumour growth of in vivo xenografts of Ste-1 AlectR cell lines in response to alvocidib and correspondent tumor weights (I.P. control n=6; alvocidib n=4). Tumour weights alvocidib P=0.004.D) Number of cleaved caspase-3 positive nuclei in alvocidib treated tumours compared to controls.E) Ki67 and p21 staining of CrizR1 xenograft tumours. Scale bar = 200μm. Quantitative analysis of IHC staining is presented on the right. Ki67 P=0.0004; p21 P=0.02.F) qPCR of Survivin (P=0.0009), MCL-1 (P=0.04), MYC (P=0.007) and BIM (P=0.03) in Ste-1 AlectR cells xenografts treated with alvocidib (Control n=6; alvocidib n=4).G) Proliferation assay of H3122 parental and isogenic drug-resistant cell lines, treated with DMSO, 100nM lorlatinib, 200nM alvocidib or 50nM dinaciclib for 72h (n=4).H) Growth curve of CrizR1 xenograft tumors in response to lorlatinib (Control n=7; lorlatinib n=7).


Figure 1 ( A ) γδ T cell concentration in the peripheral blood from healthy donors and indicated metastatic cancer patients . ( B ) RT - PCR analysis of the expression of NKG2D in γδ T cells isolated from indicated samples . ( C ) RT - PCR analysis of the expression of IFN - γ and TNF - α in γδ T cells isolated from the peripheral blood of healthy controls and metastatic cancer patients . ( D ) RT - PCR analysis of the expression of miR - 125b - 5p and miR - 99a - 5p in γδ T cells isolated from indicated samples . ( E ) RT - PCR analysis of the expression of miR - 181a ( - 5p and - 2 - 3p ) in γδ T cells isolated from indicated samples .

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 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

Figure 1(A) γδ T cell concentration in the peripheral blood from healthy donors and indicated metastatic cancer patients.(B) RT-PCR analysis of the expression of NKG2D in γδ T cells isolated from indicated samples.(C) RT-PCR analysis of the expression of IFN-γ and TNF-α in γδ T cells isolated from the peripheral blood of healthy controls and metastatic cancer patients. (D) RT-PCR analysis of the expression of miR-125b-5p and miR-99a-5p in γδ T cells isolated from indicated samples. (E) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) in γδ T cells isolated from indicated samples.


Figure 2 ( A ) RT - PCR analysis of the expression of miR - 181a ( - 5p and - 2 - 3p ) in γδ T cells isolated from thymic biopsies , cultured for 11 days with IL - 7 or IL - 2 ( n = 12 - 15 independent biological samples ) . ( B ) RT - PCR analysis of the expression of miR - 181a ( - 5p and - 2 - 3p ) in γδ T cells freshly isolated from thymic biopsies ( Thymus ) and PBLs ( n = 6 - 14 independent biological samples ) . ( C - D ) RT - PCR analysis of the expression of miR - 181a ( - 5p and - 2 - 3p ) in γδ T cells isolated from thymus ( C ) or from PBLs ( D ) and cultured with the indicated cytokines , respectively for 11 days ( C ) or for 4 - 6 days ( D ) . When indicated , γδ PBLs were also co - cultured with an anti - TGF - β blocking antibody . Data are normalized to the value obtained in IL - 7 cultures ( n = 4 - 10 independent biological samples ) . ( E - F ) RT - PCR analysis of the expression of miR - 181a ( - 5p and - 2 - 3p ) versus FACS analysis of the expression of TNF - α ( left panel ) , IFN - γ ( middle panel ) and NKGD2 ( right panel ) in γδ T cells isolated from thymus ( E ) and PBLs ( F ) . Samples were either freshly isolated or cultured for 11 days ( γδ thymocytes ) or for 6 days ( γδ PBLs ) with the indicated cytokines ( n = 5 independent biological samples ) .

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 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 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

Figure 2(A) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) in γδ T cells isolated from thymic biopsies, cultured for 11 days with IL-7 or IL-2 (n=12-15 independent biological samples). (B) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) in γδ T cells freshly isolated from thymic biopsies (Thymus) and PBLs (n=6-14 independent biological samples).(C-D) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) in γδ T cells isolated from thymus (C) or from PBLs (D) and cultured with the indicated cytokines, respectively for 11 days (C) or for 4-6 days (D). When indicated, γδ PBLs were also co-cultured with an anti-TGF-β blocking antibody. Data are normalized to the value obtained in IL-7 cultures (n=4-10 independent biological samples).(E-F) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) versus FACS analysis of the expression of TNF-α (left panel), IFN-γ (middle panel) and NKGD2 (right panel) in γδ T cells isolated from thymus (E) and PBLs (F). Samples were either freshly isolated or cultured for 11 days (γδ thymocytes) or for 6 days (γδ PBLs) with the indicated cytokines (n=5 independent biological samples).


Figure 3 ( D ) FACS analysis of the expression of Annexin V in miR - 181a versus empty transduced ( GFP + ) and untransduced ( GFP - ) γδ thymocytes , cultured with IL - 7 plus IL - 2 for 11 days ( n = 5 independent biological samples ) . ( E ) RT - PCR analysis of the expression of Bcl2 and Bcl - xL ( normalized to the values obtained with the empty virus ) in transduced ( GFP + ) γδ thymocytes , cultured with IL - 7 plus IL - 2 for 11 days ( n = 5 independent biological samples ) . ( F ) FACS - derived expression of indicated surface and intracellular markers in miR - 181a versus empty transduced ( GFP + ) and untransduced ( GFP - ) γδ T thymocytes , cultured with IL - 7 plus IL - 2 for 11 days ( n = 7 - 11 independent biological samples ) .

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 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 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

Figure 3(D) FACS analysis of the expression of Annexin V in miR-181a versus empty transduced (GFP+) and untransduced (GFP-) γδ thymocytes, cultured with IL-7 plus IL-2 for 11 days (n=5 independent biological samples).(E) RT-PCR analysis of the expression of Bcl2 and Bcl-xL (normalized to the values obtained with the empty virus) in transduced (GFP+) γδ thymocytes, cultured with IL-7 plus IL-2 for 11 days (n=5 independent biological samples).(F) FACS-derived expression of indicated surface and intracellular markers in miR-181a versus empty transduced (GFP+) and untransduced (GFP-) γδ T thymocytes, cultured with IL-7 plus IL-2 for 11 days (n=7-11 independent biological samples).


Figure 4 ( B ) RT - PCR analysis of the expression of the indicated genes associated with Mapk , Notch , Irf4 or Stat1 signaling in sorted GFP + γδ thymocytes transduced with empty or miR - 181a viral vector . Data are normalized to the value obtained with the empty virus . Samples were cultured with IL - 7 plus IL - 2 for 11 days ( n = 3 - 5 independent biological samples ) . ( C ) Dual luciferase reporter assay performed to verify binding between miR - 181a and Map3k2 , Notch2 , Irf4 or Stat1 mRNA . HEK293 T cells were co - transfected with a pmirGLO Dual - Luciferase miRNA Target Expression Vector containing either the WT or mutated ( Mut ) 3 ′ UTR target sites plus miR - 181a . A negative construct ( without miR - 181a binding sites ) was included . Firefly / Renilla ratios were normalized to those obtained for the empty vector ( n = 4 independent biological samples ) . ( E - G ) RT - PCR analysis in circulating γδ T cells of Map3k2 or Notch2 expression ( E ) , Nkg2d or Tnfa expression ( F ) , and NKG2D or TNF - α protein expression ( G ) after siRNA - mediated knockdown of Map3k2 or Notch2 . In each experiment , a control condition was also used where a nontargeting negative control siRNA was transfected . γδ were cultured with IL - 7 for 4 days ( n = 6 independent biological samples ) .

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 O O O O O O O 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

Figure 4(B) RT-PCR analysis of the expression of the indicated genes associated with Mapk, Notch, Irf4 or Stat1 signaling in sorted GFP+ γδ thymocytes transduced with empty or miR-181a viral vector. Data are normalized to the value obtained with the empty virus. Samples were cultured with IL-7 plus IL-2 for 11 days (n=3-5 independent biological samples).(C) Dual luciferase reporter assay performed to verify binding between miR-181a and Map3k2, Notch2, Irf4 or Stat1 mRNA. HEK293 T cells were co-transfected with a pmirGLO Dual-Luciferase miRNA Target Expression Vector containing either the WT or mutated (Mut) 3′ UTR target sites plus miR-181a. A negative construct (without miR-181a binding sites) was included. Firefly/Renilla ratios were normalized to those obtained for the empty vector (n=4 independent biological samples).(E-G) RT-PCR analysis in circulating γδ T cells of Map3k2 or Notch2 expression (E), Nkg2d or Tnfa expression (F), and NKG2D or TNF-α protein expression (G) after siRNA-mediated knockdown of Map3k2 or Notch2. In each experiment, a control condition was also used where a nontargeting negative control siRNA was transfected. γδ were cultured with IL-7 for 4 days (n=6 independent biological samples).


Figure 1 ( A ) Western analysis for on - target effects for Everolimus ( EV , P - RPS6 ) and CX - 5461 ( p53 ) . Each lane represents equal amounts of protein from lymph node tissue isolated from a single mouse that received drug vehicles ( everolimus vehicle : 1 % methylcellulose ; CX - 5461 vehicle : 25 mM NaH2PO 4 ) ( V / V ; mouse # 1 - 6 ) , 5 mg / kg everolimus ( EV ; mouse # 7 - 12 ) , 35 mg / kg CX - 5461 ( mouse # 13 - 18 ) or both drugs ( CX - 5461 + EV ; mouse # 19 - 24 ) for 2 hours ( n = 6 per treatment group ) . Actin was used as a loading control . ( C ) Enrichment analysis by MetaCore ® GeneGO of genes in " translation up " ( red ) and " translation down " ( blue ) categories identified by anota2seq analysis comparing lymph node cells isolated from mice in CX - 5461 + EV treatment group with the V / V group ( n = 6 ) . Ratio " values were obtained by dividing the number of significant genes in our data assigned to a molecular process by the total number of genes in the process in MetaCore ® ' s database . ( D ) Genes implicated in " Translation : initiation " and " Translation : Elongation - Termination " processes based on Fig

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 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

Figure 1(A) Western analysis for on-target effects for Everolimus (EV, P-RPS6) and CX-5461 (p53). Each lane represents equal amounts of protein from lymph node tissue isolated from a single mouse that received drug vehicles (everolimus vehicle: 1% methylcellulose; CX-5461 vehicle: 25 mM NaH2PO4) (V/V; mouse #1-6), 5 mg/kg everolimus (EV; mouse #7-12), 35 mg/kg CX-5461 (mouse #13-18) or both drugs (CX-5461+EV; mouse #19-24) for 2 hours (n=6 per treatment group). Actin was used as a loading control.(C) Enrichment analysis by MetaCore® GeneGO of genes in "translation up" (red) and "translation down" (blue) categories identified by anota2seq analysis comparing lymph node cells isolated from mice in CX-5461+EV treatment group with the V/V group (n=6). Ratio" values were obtained by dividing the number of significant genes in our data assigned to a molecular process by the total number of genes in the process in MetaCore®'s database. (D) Genes implicated in "Translation: initiation" and "Translation: Elongation-Termination" processes based on Fig 1C


Figure 2 ( B ) Cell viability analysis of the indicated early - passage Eµ - Myc lymphoma cells treated with indicated compound ( s ) for 48 hours as determined by Beckman ® Coulter counter . ( E ) Intracellular ATP levels determined using the liquid chromatography - mass spectrometry in CTRL , EV , CX and CMB early - passage cell lines . ( F ) Basal extracellular acidification rate ( ECAR ) and oxygen consumption rate ( OCR ) in the early - passage cell lines determined using the Seahorse XF96 Extracellular Flux Analyzer ; graphs represent mean ± SEM of 6 - 8 technical replicates for each biological replicate ( n = 3 ) . ( G ) Propidium iodide ( PI ) exclusion analysis of early - passage CX - 5461 - everolimus combination therapy - resistant ( CMB ) , CX - 5461 - resistant ( CX ) and drug - naïve ( CTRL ) lymphoma cells treated with indicated concentrations of metformin for 48 hours . ( H ) CellTiterGLO ® - based assay measuring cellular ATP levels of the CX - 5461 - resistant ( CX ) and CX - 5461 - everolimus combination therapy - resistant ( CMB ) cells treated with metformin as indicated for 48 hours . ( I ) PI analysis of CTRL and CMB cells treated with CX - 5461 and everolimus in the presence and absence of metformin for 48 hours as indicated . ( J ) PI analysis of CTRL and CX cells treated with CX - 5461 in the presence and absence of metformin for 48 hours as indicated .

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 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 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 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

Figure 2(B) Cell viability analysis of the indicated early-passage Eµ-Myc lymphoma cells treated with indicated compound(s) for 48 hours as determined by Beckman® Coulter counter.(E) Intracellular ATP levels determined using the liquid chromatography-mass spectrometry in CTRL, EV, CX and CMB early-passage cell lines.(F) Basal extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) in the early-passage cell lines determined using the Seahorse XF96 Extracellular Flux Analyzer; graphs represent mean ± SEM of 6-8 technical replicates for each biological replicate (n=3).(G) Propidium iodide (PI) exclusion analysis of early-passage CX-5461-everolimus combination therapy-resistant (CMB), CX-5461-resistant (CX) and drug-naïve (CTRL) lymphoma cells treated with indicated concentrations of metformin for 48 hours.(H) CellTiterGLO®-based assay measuring cellular ATP levels of the CX-5461-resistant (CX) and CX-5461-everolimus combination therapy-resistant (CMB) cells treated with metformin as indicated for 48 hours.(I) PI analysis of CTRL and CMB cells treated with CX-5461 and everolimus in the presence and absence of metformin for 48 hours as indicated.(J) PI analysis of CTRL and CX cells treated with CX-5461 in the presence and absence of metformin for 48 hours as indicated.


Figure 3 ( B ) Abundance of intracellular 3 ' 5 ' - cyclic AMP as measured by a liquid chromatography ( LC ) - mass spectrometry ( MS ) analysis . Graphs represent mean ± SEM of n = 3 ( 5 - 6 technical replicates each ) . ( C ) Western analysis of EPAC1 and EPAC2 abundance , as well as active GTP - bound RAP1 levels in the indicated early - passage cells during their log - phase growth period ( n = 3 ) . Actin and total RAP1 were used as loading controls . ( D ) Propidium iodide ( PI ) exclusion assays of CMB cells treated with CX - 5461 and everolimus as indicated in the presence or absence of a selective PKA inhibitor H89 for 48 hours . ( E ) PI exclusion analysis of early - passage drug - naïve ( CTRL ) lymphoma cells treated with CX - 5461 and everolimus in the presence or absence of selective PKA activator 6 - Bnz - cAMP for 48 hours . ( F ) PI exclusion analysis of the CMB cells treated with CX - 5461 and everolimus as indicated in the presence or absence of EPAC1 inhibitor CE3F4 or EPAC2 inhibitor ESI - 05 for 48 hours . ( G ) PI exclusion analysis of early - passage drug - naïve ( CTRL ) cells treated with CX - 5461 and everolimus in the presence or absence of the selective EPAC activator 8 - pCPT - 2 - O - Me - cAMP for 48 hours .

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 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 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 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 3(B) Abundance of intracellular 3'5'-cyclic AMP as measured by a liquid chromatography (LC)-mass spectrometry (MS) analysis. Graphs represent mean ± SEM of n=3 (5-6 technical replicates each).(C) Western analysis of EPAC1 and EPAC2 abundance, as well as active GTP-bound RAP1 levels in the indicated early-passage cells during their log-phase growth period (n=3). Actin and total RAP1 were used as loading controls.(D) Propidium iodide (PI) exclusion assays of CMB cells treated with CX-5461 and everolimus as indicated in the presence or absence of a selective PKA inhibitor H89 for 48 hours.(E) PI exclusion analysis of early-passage drug-naïve (CTRL) lymphoma cells treated with CX-5461 and everolimus in the presence or absence of selective PKA activator 6-Bnz-cAMP for 48 hours.(F) PI exclusion analysis of the CMB cells treated with CX-5461 and everolimus as indicated in the presence or absence of EPAC1 inhibitor CE3F4 or EPAC2 inhibitor ESI-05 for 48 hours.(G) PI exclusion analysis of early-passage drug-naïve (CTRL) cells treated with CX-5461 and everolimus in the presence or absence of the selective EPAC activator 8-pCPT-2-O-Me-cAMP for 48 hours.


Figure 4 ( A ) A heatmap illustrating the abundances of polysome - associated mRNAs that are associated with the mRNA translation / protein synthesis based on the Gene Ontology Resource Database in drug - naïve ( CTRL ) or CX - 5461 - everolimus combination therapy - resistant ( CMB ) cell lines ( n = 3 ) . The colours illustrate the expression values of indicated mRNAs that were normalized using voom ( red : high expression ; blue : low expression ) . ( B ) Polysome profiles demonstrating increased ribosome abundance in CMB cells as compared to CTRL and CX - 5461 - resistant ( CX ) cells ( representatives of n = 3 ) . ( C ) Quantitation of polysome : monosome ratio in the indicated early - passage Eµ - Myc lymphoma cells as determined by measuring the area under the curve using ImageJ software . Graph represents mean ± SEM of n = 3 . ( F ) Polysomal abundance of mRNAs ( selected based on a nominal p - value < 0 . 01 cut - off in either the CMB vs . CTRL or CX vs . CTRL as analysed by limma ) encoding components of mitochondrial oxidative phosphorylation based on polysome profiling data . Red denotes upregulation , blue denotes downregulation ( n = 3 ; genes with an adjusted P value ( false discovery rate ; FDR ) ≤ 0 . 05 were considered to be significant , and are denoted with a black border ) .

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

Figure 4(A) A heatmap illustrating the abundances of polysome-associated mRNAs that are associated with the mRNA translation/protein synthesis based on the Gene Ontology Resource Database in drug-naïve (CTRL) or CX-5461-everolimus combination therapy-resistant (CMB) cell lines (n=3). The colours illustrate the expression values of indicated mRNAs that were normalized using voom (red: high expression; blue: low expression).(B) Polysome profiles demonstrating increased ribosome abundance in CMB cells as compared to CTRL and CX-5461-resistant (CX) cells (representatives of n=3). (C) Quantitation of polysome:monosome ratio in the indicated early-passage Eµ-Myc lymphoma cells as determined by measuring the area under the curve using ImageJ software. Graph represents mean ± SEM of n=3. (F) Polysomal abundance of mRNAs (selected based on a nominal p-value < 0.01 cut-off in either the CMB vs. CTRL or CX vs. CTRL as analysed by limma) encoding components of mitochondrial oxidative phosphorylation based on polysome profiling data. Red denotes upregulation, blue denotes downregulation (n=3; genes with an adjusted P value (false discovery rate; FDR) ≤ 0.05 were considered to be significant, and are denoted with a black border).


Figure 5 ( A ) Western analysis demonstrating the effects of metformin treatment for 48 hours on the levels of active GTP - bound RAP1 in CX and CMB cells ( n = 3 ) and its quantitation . ( B , C ) Proportion of green fluorescent protein ( GFP ) - positive CMB ( clone # 8 ) cells in ( B ) lymph node and ( C ) spleen of transplanted C57BL / 6 mice treated as indicated for 6 hours on day 12 post - transplant . ( D ) Kaplan - Meier curve of C57BL / 6 mice transplanted with CX - 5461 - everolimus - resistant ( CMB # 8 ) early - passage Eµ - Myc lymphoma cells treated with vehicles ( everolimus vehicle : 1 % methylcellulose ; CX - 5461 / metformin vehicle : 25 mM NaH2PO 4 ) ( n = 6 ) ; CX - 5461 ( 35 mg / kg every twice weekly ) and everolimus ( 5 mg / kg daily ) ( n = 8 ) , metformin ( 400 mg / kg twice daily ) ( n = 6 ) , or CX - 5461 , everolimus and metformin ( 35 mg / kg twice weekly , 5 mg / kg daily and 400 mg / kg twice daily , respectively ) ( n = 8 ) . Light grey : 5 - day metformin pre - treatment period , dark grey : treatment period . Data were analyzed by a log - rank ( Mantel - Cox ) test . Vehicle vs . CX - 5461 - everolimus : P = 0 . 0006 , Vehicle vs . CX - 5461 - everolimus - metformin : P = 0 . 0001 . CX - 5461 - everolimus vs . CX - 5461 - everolimus - metformin : P = 0 . 0003 .

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 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 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

Figure 5(A) Western analysis demonstrating the effects of metformin treatment for 48 hours on the levels of active GTP-bound RAP1 in CX and CMB cells (n=3) and its quantitation.(B, C) Proportion of green fluorescent protein (GFP)-positive CMB (clone #8) cells in (B) lymph node and (C) spleen of transplanted C57BL/6 mice treated as indicated for 6 hours on day 12 post-transplant.(D) Kaplan-Meier curve of C57BL/6 mice transplanted with CX-5461-everolimus-resistant (CMB #8) early-passage Eµ-Myc lymphoma cells treated with vehicles (everolimus vehicle: 1% methylcellulose; CX-5461/metformin vehicle: 25 mM NaH2PO4) (n=6); CX-5461 (35 mg/kg every twice weekly) and everolimus (5 mg/kg daily) (n=8), metformin (400 mg/kg twice daily) (n=6), or CX-5461, everolimus and metformin (35 mg/kg twice weekly, 5 mg/kg daily and 400 mg/kg twice daily, respectively) (n=8). Light grey: 5-day metformin pre-treatment period, dark grey: treatment period. Data were analyzed by a log-rank (Mantel-Cox) test. Vehicle vs. CX-5461-everolimus: P = 0.0006, Vehicle vs. CX-5461-everolimus-metformin: P = 0.0001. CX-5461-everolimus vs. CX-5461-everolimus-metformin: P = 0.0003.


Figure 1Kaplan - Meier analysis showing cases from the Australian cohort ( n = 73 ) delineated on the basis of expression of a set of hazardous genes , termed PC - 1 . Patients with the highest expression had a significantly poorer prognosis ( red line , median survival : 11 . 6 months ) compared with those patients with the lowest expression ( blue line , median survival : 34 . 4 months , P = 0 . 01 ) . The black line shows those with medium expression . Kaplan - Meier analysis showing that cases in the Australian cohort that fell in the highest quartile of a hypoxia signature ( red line , median survival : 11 . 4 months ) have significantly decreased survival compared with those in the lowest quartile ( blue line , median survival : 25 . 2 months ; P = 0 . 03 ) . Kaplan - Meier analysis showing that cases in the APGI cohort ( n = 266 ) with high LOX expression above the 3rd quantile ( red line ) have significantly decreased survival compared to those with low expression below the 1st quantile ( blue line ) . Kaplan - Meier analysis showing that cases in the Glasgow cohort with high LOX expression ( red line , n = 24 ) have significantly decreased survival compared with those with low expression ( blue line , n = 23 ; P = 0 . 005 ) . Kaplan - Meier analysis showing that fibrillar collagen is significantly associated with reduced survival in human PDAC ( 20 months vs . 28 . 3 months , P = 0 . 033 ) . Mean decay distance of the second harmonic generation ( SHG ) signal emitted by human PDAC - associated collagen . Mean decay distance is represented by boxplots showing the second and third quartile of the data with the whiskers indicating the maximum and minimum data points . Outliers are indicated by individual markers . Stage T3 tumors have a higher collagen mean decay distance score compared with Stage T2 tumors ( P = 0 . 02 ) . Lymph node - positive tumors have a higher collagen mean decay distance score vs . lymph node - negative tumors ( P = 0 . 047 ) . Tumors showing vascular invasion have a higher collagen mean decay distance score than non - vascular invasive tumors ( P = 0 . 037 ) .

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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1Kaplan-Meier analysis showing cases from the Australian cohort (n = 73) delineated on the basis of expression of a set of hazardous genes, termed PC-1. Patients with the highest expression had a significantly poorer prognosis (red line, median survival: 11.6 months) compared with those patients with the lowest expression (blue line, median survival: 34.4 months, P = 0.01). The black line shows those with medium expression.Kaplan-Meier analysis showing that cases in the Australian cohort that fell in the highest quartile of a hypoxia signature (red line, median survival: 11.4 months) have significantly decreased survival compared with those in the lowest quartile (blue line, median survival: 25.2 months; P = 0.03).Kaplan-Meier analysis showing that cases in the APGI cohort (n = 266) with high LOX expression above the 3rd quantile (red line) have significantly decreased survival compared to those with low expression below the 1st quantile (blue line).Kaplan-Meier analysis showing that cases in the Glasgow cohort with high LOX expression (red line, n = 24) have significantly decreased survival compared with those with low expression (blue line, n = 23; P = 0.005).Kaplan-Meier analysis showing that fibrillar collagen is significantly associated with reduced survival in human PDAC (20 months vs. 28.3 months, P = 0.033).Mean decay distance of the second harmonic generation (SHG) signal emitted by human PDAC-associated collagen. Mean decay distance is represented by boxplots showing the second and third quartile of the data with the whiskers indicating the maximum and minimum data points. Outliers are indicated by individual markers. Stage T3 tumors have a higher collagen mean decay distance score compared with Stage T2 tumors (P = 0.02). Lymph node-positive tumors have a higher collagen mean decay distance score vs. lymph node-negative tumors (P = 0.047). Tumors showing vascular invasion have a higher collagen mean decay distance score than non-vascular invasive tumors (P = 0.037).


Figure 2PC - 1 signature is predictive of metastatic disease in mouse models of pancreatic cancer . Log - transformed expressions of signature transcripts from mouse tumor microarrays were mean - centered across samples and scaled to unit variance . These values were then multiplied by the matching loading values from the PC - 1 signature and summarized for each sample across all transcripts to yield the risk score for that sample . Inverted invasion assays were performed with PDAC tumor cell lines from KPC and KPflC mice . Tumor cell lines bearing mutant p53R172H ( KPC ) invade significantly further than tumor cells with deletion of 1 copy of p53 ( KPflC ) ( P ≤ 0 . 01 ) . Data are shown as the average of four wells + SEM . Introduction of shRNA targeting Lox into KPC tumor cells significantly inhibits invasion ( P ≤ 0 . 01 by unpaired Student ' s t - test ) . Data are shown as the average of four wells + SEM . Introduction of exogenous LOX into KPflC tumor cells significantly promotes invasion ( left panel , P ≤ 0 . 01 by unpaired Student ' s t - test ) . LOX expression was assessed by immunoblotting ( right panel ) . Columns indicate the mean of four well and error bars indicate SEM .

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 O O 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 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

Figure 2PC-1 signature is predictive of metastatic disease in mouse models of pancreatic cancer. Log-transformed expressions of signature transcripts from mouse tumor microarrays were mean-centered across samples and scaled to unit variance. These values were then multiplied by the matching loading values from the PC-1 signature and summarized for each sample across all transcripts to yield the risk score for that sample.Inverted invasion assays were performed with PDAC tumor cell lines from KPC and KPflC mice. Tumor cell lines bearing mutant p53R172H (KPC) invade significantly further than tumor cells with deletion of 1 copy of p53 (KPflC) (P ≤ 0.01). Data are shown as the average of four wells + SEM.Introduction of shRNA targeting Lox into KPC tumor cells significantly inhibits invasion (P ≤ 0.01 by unpaired Student's t-test). Data are shown as the average of four wells + SEM.Introduction of exogenous LOX into KPflC tumor cells significantly promotes invasion (left panel, P ≤ 0.01 by unpaired Student's t-test). LOX expression was assessed by immunoblotting (right panel). Columns indicate the mean of four well and error bars indicate SEM.


Figure 3A Representative fluorescence images showing the SHG signal from cross - linked collagen in untreated , LOX antibody - treated or isotype control - treated organotypic cultures of primary fibroblasts and rat tail fibrillar collagen I . B Chart showing the percentage of cross - linked collagen by depth , based on SHG signal , in organotypic matrices treated as indicated . Error bars indicate SEM . C Barchart showing total cross - linked collagen in organotypic matrices treated as indicated . The experiment was performed in triplicate with three different zones imaged for 80 μm , at 2 . 5 - μm sections each . Data are shown as total pixels ± SEM . D Kaplan - Meier ( non - parametric ) survival curve showing significantly extended tumor - free survival of KPC mice treated with LOX antibody ( 1 mg / kg i . p . , twice weekly ) + gemcitabine ( 100 mg / kg i . p . , twice weekly ) ( green line , n = 17 ) , compared with gemcitabine ± isotype control antibody - treated mice ( blue line , n = 22 , P = 0 . 014 ) or LOX antibody alone ( red line , n = 9 ) . Treatment was initiated when mice were 70 days old ( randomization was not used when recruiting the mice ) and the mice were treated twice weekly . Censored mice did not develop PDAC . E H & amp ; amp ; E - stained section of PDAC harvested from LOX + gemcitabine - treated KPC mouse . Note necrosis . F Boxplot showing quantification of necrosis in H & amp ; amp ; E sections of PDACs harvested from these mice , as indicated . At least 30 fields of view from at least five mice per cohort were scored . G - H H & amp ; amp ; E - stained sections of PDAC harvested from LOX + gemcitabine - treated KPC mice . Note stromal collapse ( G ) and marked dilation of blood vessels ( H ) . I Representative H & amp ; amp ; E image showing a section of PDAC harvested from a control - treated KPC mouse .

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

Figure 3A Representative fluorescence images showing the SHG signal from cross-linked collagen in untreated, LOX antibody-treated or isotype control-treated organotypic cultures of primary fibroblasts and rat tail fibrillar collagen I.B Chart showing the percentage of cross-linked collagen by depth, based on SHG signal, in organotypic matrices treated as indicated. Error bars indicate SEM.C Barchart showing total cross-linked collagen in organotypic matrices treated as indicated. The experiment was performed in triplicate with three different zones imaged for 80 μm, at 2.5-μm sections each. Data are shown as total pixels ± SEM.D Kaplan-Meier (non-parametric) survival curve showing significantly extended tumor-free survival of KPC mice treated with LOX antibody (1 mg/kg i.p., twice weekly) + gemcitabine (100 mg/kg i.p., twice weekly) (green line, n = 17), compared with gemcitabine ± isotype control antibody-treated mice (blue line, n = 22, P = 0.014) or LOX antibody alone (red line, n = 9). Treatment was initiated when mice were 70 days old (randomization was not used when recruiting the mice) and the mice were treated twice weekly. Censored mice did not develop PDAC.E H&amp;E-stained section of PDAC harvested from LOX + gemcitabine-treated KPC mouse. Note necrosis.F Boxplot showing quantification of necrosis in H&amp;E sections of PDACs harvested from these mice, as indicated. At least 30 fields of view from at least five mice per cohort were scored.G-H H&amp;E-stained sections of PDAC harvested from LOX + gemcitabine-treated KPC mice. Note stromal collapse (G) and marked dilation of blood vessels (H).I Representative H&amp;E image showing a section of PDAC harvested from a control-treated KPC mouse.


Figure 4A Outer left panels : Immunohistochemistry analysis of macrophage infiltration ( by F4 / 80 staining ) in tumors from isotype control + gemcitabine - treated KPC mice vs . LOX antibody + gemcitabine - treated KPC mice . Inner left panels : Immunohistochemistry analysis of neutrophil infiltration ( by MPO staining ) in tumors from isotype control + gemcitabine - treated KPC mice vs . LOX antibody + gemcitabine - treated KPC mice . Inner right panels : Immunohistochemistry analysis of tumorvasculature ( by CD31 staining ) in tumors from isotype control + gemcitabine - treated KPC mice vs . LOX antibody + gemcitabine - treated KPC mice . Outer right panels : Immunohistochemistry analysis of tenascin C expression in tumors from isotype control + gemcitabine - treated KPC mice vs . LOX antibody + gemcitabine - treated KPC mice . B - D Plots showing quantification of macrophage infiltration ( B ) , neutrophil infiltration ( C ) and tumorvasculature ( D ) in tumors from KPC mice treated as indicated . At least 30 fields of view from at least four mice per cohort were scored , and scoring was conducted blind . P - values were calculated using Mann - Whitney U - test and median is indicated by horizontal lines . E Correlation of LOX protein with tenascin C expression in 47 cases of PDAC ( Spearman ' s rho correlation coefficient = 0 . 61 ; P < 0 . 0001 ) .

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

Figure 4A Outer left panels: Immunohistochemistry analysis of macrophage infiltration (by F4/80 staining) in tumors from isotype control + gemcitabine-treated KPC mice vs. LOX antibody + gemcitabine-treated KPC mice. Inner left panels: Immunohistochemistry analysis of neutrophil infiltration (by MPO staining) in tumors from isotype control + gemcitabine-treated KPC mice vs. LOX antibody + gemcitabine-treated KPC mice. Inner right panels: Immunohistochemistry analysis of tumorvasculature (by CD31 staining) in tumors from isotype control + gemcitabine-treated KPC mice vs. LOX antibody + gemcitabine-treated KPC mice. Outer right panels: Immunohistochemistry analysis of tenascin C expression in tumors from isotype control + gemcitabine-treated KPC mice vs. LOX antibody + gemcitabine-treated KPC mice.B-D Plots showing quantification of macrophage infiltration (B), neutrophil infiltration (C) and tumorvasculature (D) in tumors from KPC mice treated as indicated. At least 30 fields of view from at least four mice per cohort were scored, and scoring was conducted blind. P-values were calculated using Mann-Whitney U-test and median is indicated by horizontal lines.E Correlation of LOX protein with tenascin C expression in 47 cases of PDAC (Spearman's rho correlation coefficient = 0.61; P < 0.0001).


Figure 1mRNA expression of ANG in colonic tissues from normal controls ( n = 25 ) and UC ( n = 27 ) or CD ( n = 46 ) patients . Representative western blotting of ANG in colonic tissues from normal controls and UC or CD patients . Representative images showing ANG immunohistochemical ( IHC ) staining in normal control and UC or CD sample . Corresponding statistical analysis of ANG expression score in normal controls ( n = 10 ) and UC ( n = 10 ) or CD ( n = 15 ) patients . Representative images showing hematoxylin - eosin ( HE ) staining and ANG IHC staining in mild and severe IBD samples . Statistical analyses of ANG expression scores in mild ( n = 18 ) and severe ( n = 19 ) UC ( F ) or mild ( n = 20 ) and severe ( n = 20 ) CD ( G ) 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 B-PANEL_START 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 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 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 1mRNA expression of ANG in colonic tissues from normal controls (n = 25) and UC (n = 27) or CD (n = 46) patients.Representative western blotting of ANG in colonic tissues from normal controls and UC or CD patients.Representative images showing ANG immunohistochemical (IHC) staining in normal control and UC or CD sample.Corresponding statistical analysis of ANG expression score in normal controls (n = 10) and UC (n = 10) or CD (n = 15) patients.Representative images showing hematoxylin-eosin (HE) staining and ANG IHC staining in mild and severe IBD samples.Statistical analyses of ANG expression scores in mild (n = 18) and severe (n = 19) UC (F) or mild (n = 20) and severe (n = 20) CD (G) patients.