Figure 6RPE cells arrested in mitosis by nocodazole ( NZ ) were treated with DMSO , PLK1 inhibitor BI2536 ( 100 nM ) , Aurora - A inhibitor MLN8054 ( 0 . 25 μM ) , CDK1 inhibitor RO3306 ( 20 nM ) or CK1 inhibitor PF - 670462 ( 1 μM ) for 60 min , collected and analyzed by immunoblotting with indicated antibodies . Asynchronously growing RPE cells are shown for comparison ( left line ) . Staining for H3 - pS10 served as a marker of mitosis ( n = 3 ) . U2OS cells were trapped in mitosis by overnight treatment with nocodazole or PLK1 inhibitor BI2536 ( 100 nM ) and collected by mitotic shake off . Part of the nocodazole arrested cells was further treated with CK1 inhibitor PF - 670462 ( 1 μM ) for 60 min . Whole cell lysates were probed with indicated antibodies . Staining for pS10 - H3 served as a marker of mitosis .

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 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

Figure 6RPE cells arrested in mitosis by nocodazole (NZ) were treated with DMSO, PLK1 inhibitor BI2536 (100 nM), Aurora-A inhibitor MLN8054 (0.25 μM), CDK1 inhibitor RO3306 (20 nM) or CK1 inhibitor PF-670462 (1 μM) for 60 min, collected and analyzed by immunoblotting with indicated antibodies. Asynchronously growing RPE cells are shown for comparison (left line). Staining for H3-pS10 served as a marker of mitosis (n=3).U2OS cells were trapped in mitosis by overnight treatment with nocodazole or PLK1 inhibitor BI2536 (100 nM) and collected by mitotic shake off. Part of the nocodazole arrested cells was further treated with CK1 inhibitor PF-670462 (1 μM) for 60 min. Whole cell lysates were probed with indicated antibodies. Staining for pS10-H3 served as a marker of mitosis.


Figure 7RPE cells or RPE cells stably expressing indicated EGFP - FAM110A variants were transfected with control ( siNC ) , FAM110A or CSNK1D siRNA and grown for 48 h . Alternatively , cells were incubated with PF670462 for the last 12 h . Cells were treated with MG132 for 30 min prior fixation . Impaired chromosomal alignment was scored in > 30 metaphase cells per condition . Error bars indicate median ± SD . Statistical significance was determined by ANOVA ( n = 3 ) ( * * * * p < 0 . 0001 , * * * p < 0 . 001 ) . Representative immunoblot analysis of samples from ( D ) . Full arrow indicates migration of the EGFP - FAM110A - WT , empty arrow of endogenous FAM110A .

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 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 7RPE cells or RPE cells stably expressing indicated EGFP-FAM110A variants were transfected with control (siNC), FAM110A or CSNK1D siRNA and grown for 48 h. Alternatively, cells were incubated with PF670462 for the last 12 h. Cells were treated with MG132 for 30 min prior fixation. Impaired chromosomal alignment was scored in >30 metaphase cells per condition. Error bars indicate median ±SD. Statistical significance was determined by ANOVA (n=3) (**** p<0.0001, *** p<0.001).Representative immunoblot analysis of samples from (D). Full arrow indicates migration of the EGFP-FAM110A-WT, empty arrow of endogenous FAM110A.


Figure 8RPE1 were transfected with control siRNA or CSNK1D siRNA or were treated with PF670462 prior fixation . Cells were probed with rabbit polyclonal to FAM110A and mouse monoclonal to γ - Tubulin . Representative images are shown , scale bars indicate 10 μm . Quantification of ( A ) . Plotted is median FAM110A intensity at spindle poles positive for γ - tubulin ± SD . Each dot represents a single spindle pole . Statistical significance was determined by ANOVA ( n = 3 ) ( * * * * p < 0 . 0001 ) . Cells expressing the wild - type or mutant EGFP - FAM110A were fixed and stained for γ - tubulin and α - tubulin and analyzed by confocal microscopy ( z - stack 0 . 16 µm ) . Representative images of maximal projections of the metaphase cells are shown , scale bars indicate 10 μm . Merged images show signal of DAPI , γ - tubulin and EGFP . Insets show a single Z plane at the spindle pole .

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

Figure 8RPE1 were transfected with control siRNA or CSNK1D siRNA or were treated with PF670462 prior fixation. Cells were probed with rabbit polyclonal to FAM110A and mouse monoclonal to γ-Tubulin. Representative images are shown, scale bars indicate 10 μm.   Quantification of (A). Plotted is median FAM110A intensity at spindle poles positive for γ-tubulin ± SD. Each dot represents a single spindle pole. Statistical significance was determined by ANOVA (n=3) (**** p<0.0001).   Cells expressing the wild-type or mutant EGFP-FAM110A were fixed and stained for γ-tubulin and α-tubulin and analyzed by confocal microscopy (z-stack 0.16 µm). Representative images of maximal projections of the metaphase cells are shown, scale bars indicate 10 μm. Merged images show signal of DAPI, γ-tubulin and EGFP. Insets show a single Z plane at the spindle pole.


figf1 ( a ) Different VPS34 complexes were immunoprecipitated ( IP ) from MEFs in the presence ( N ) or absence ( − A ) of amino acids using the indicated antibodies and assayed for kinase activity ( left , top panel ) . Inputs were immunoblotted using the antibodies indicated ( left , lower panels ) . Quantification of VPS34 activity is from 3 biological repeats ( right panel ; data shown are mean + s . d . ) . AR , autoradiography . ( b ) Immunoprecipitation of three VPS34 complexes , normalized for VPS34 , was performed using the indicated antibodies under nutrient - rich ( N ) or starvation ( - A ) conditions . VPS34 - binding partners were analysed by western blotting . ( c ) ATG14L - containing VPS34 complexes were immunopurified from WT , ULK1 − / − 2KD ( ULK def . ) and FIP200 − / − MEFs , and measured for lipid kinase activity as in a . ( d ) VPS34 was immunopurified from WT , ULK - deficient and FIP200 − / − MEFs . A kinase assay was performed as in a . ( e ) Immunoprecipitation of Beclin - 1 - containing VPS34 complexes from WT , ULK - deficient and FIP200 − / − MEFs . A kinase assay was performed as in a . ( f ) LC3B puncta and PtdIns ( 3 ) P levels were analysed with anti - LC3B and the biotin - 2XFYVE domain probe . Representative immunofluorescence images of LC3B and 2XFYVE domain binding are shown ( scale bars , 10 μm ) . ( g ) Quantification of LC3B puncta from f . Details of quantification are provided in the Methods ; Data shown in g - i are mean + s . d . from a minimum of 6 unique fields of view from a representative experiment ( see statistical source data in Supplementary Table S1 ) . ( h ) Total PtdIns ( 3 ) P was quantified from the experiment in f . Error bars were calculated as in g . ( i ) Quantification of PtdIns ( 3 ) P that co - localizes with LC3B on amino - acid withdrawal from the experiment described in f . ( j ) VPS34 activity with or without ULK1 overexpression was assayed . A representative experiment of four repeats is shown .

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figf1(a) Different VPS34 complexes were immunoprecipitated (IP) from MEFs in the presence (N) or absence (−A) of amino acids using the indicated antibodies and assayed for kinase activity (left, top panel). Inputs were immunoblotted using the antibodies indicated (left, lower panels). Quantification of VPS34 activity is from 3 biological repeats (right panel; data shown are mean+s.d.). AR, autoradiography.(b) Immunoprecipitation of three VPS34 complexes, normalized for VPS34, was performed using the indicated antibodies under nutrient-rich (N) or starvation (-A) conditions. VPS34-binding partners were analysed by western blotting.(c) ATG14L-containing VPS34 complexes were immunopurified from WT, ULK1−/− 2KD (ULK def.) and FIP200−/− MEFs, and measured for lipid kinase activity as in a.(d) VPS34 was immunopurified from WT, ULK-deficient and FIP200−/− MEFs. A kinase assay was performed as in a.(e) Immunoprecipitation of Beclin-1-containing VPS34 complexes from WT, ULK-deficient and FIP200−/− MEFs. A kinase assay was performed as in a.(f) LC3B puncta and PtdIns(3)P levels were analysed with anti-LC3B and the biotin-2XFYVE domain probe. Representative immunofluorescence images of LC3B and 2XFYVE domain binding are shown (scale bars, 10 μm). (g) Quantification of LC3B puncta from f. Details of quantification are provided in the Methods; Data shown in g-i are mean+s.d. from a minimum of 6 unique fields of view from a representative experiment (see statistical source data in Supplementary Table S1). (h) Total PtdIns(3)P was quantified from the experiment in f. Error bars were calculated as in g. (i) Quantification of PtdIns(3)P that co-localizes with LC3B on amino-acid withdrawal from the experiment described in f.(j) VPS34 activity with or without ULK1 overexpression was assayed. A representative experiment of four repeats is shown.


figf2 ( a ) HEK293 cells were transfected with ATG14L , VPS34 and Beclin - 1 . ATG14L - containing VPS34 complexes were immunopurified and subjected to an in vitro ULK1 kinase assay in the presence of γ - 32P [ ATP ] . Bound ATG14L complexes and soluble ULK1 were separated and phosphorylation was detected by autoradiography ( AR , left panels ) . Western blotting was performed ( right panels ) . Results are representative of two unique experiments . ( b ) Full - length murine GST - Beclin - 1 and various truncations ( as labelled ) were subjected to an in vitro HA - ULK1 kinase assay . GST - Beclin - 1 6 ( S / T ) A has serine / threonine residues 4 , 7 , 10 , 14 , 29 and 42 mutated to alanine . ULK1 inputs were determined by western blotting , Beclin - 1 inputs by Coomassie ( Coom ) and target phosphorylation by autoradiography . ( c ) GST - Beclin - 1 ( 1 - 85 ) was subjected to an in vitro ULK1 kinase reaction and analysed by mass spectrometry . Ser 14 , Ser 15 in humans ( outlined ) , is the main phosphorylation site identified ( for mass spectrometry data , see Supplementary Fig . S2b , c ) . Mass spectrometry was performed on a single experiment . ( d ) Beclin - 1 Ser 14 is the main in vitro ULK1 phosphorylation site . Beclin - 1 WT , and S4A and S14A mutants were subjected to an in vitro ULK1 kinase assay . The reaction was developed by autoradiography and stained for Beclin - 1 input levels by Coomassie blue . Results are representative of two unique experiments . ( e ) HEK293 cells were transfected with the indicated plasmids under nutrient - rich conditions . Beclin - 1 was immunoprecipitated and immunoblotted with pBeclin - 1 ( Ser 14 ) , or anti - Beclin - 1 as a loading control . ULK1 inputs are included below immunoprecipitated samples . ( f ) Purified GST - Beclin - 1 ( 1 - 85 ) was subjected to in vitro phosphorylation by GST - ULK1 ( top panel ) and GST - ULK2 ( bottom panel ) . Reactions were immunoblotted with the indicated antibodies . ( g ) HEK293 cells were transfected with ATG14L , VPS34 and Beclin - 1 and grown under nutrient - rich conditions . ATG14L - containing VPS34 complexes were immunoprecipitated and lipid kinase activity was assayed as described in Fig . 1j . Inputs were immunoblotted with the indicated antibodies . Representative of four unique experiments . ( h ) Stable lines containing Beclin - 1 ( WT or S14A ) were used for Beclin - 1 immunoprecipitation . Binding partners were determined by SDS - PAGE analysis and western blot using the indicated antibodies . Uncropped images of blots are shown in Supplementary Fig . S4 .

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

figf2(a) HEK293 cells were transfected with ATG14L, VPS34 and Beclin-1. ATG14L-containing VPS34 complexes were immunopurified and subjected to an in vitro ULK1 kinase assay in the presence of γ-32P[ATP]. Bound ATG14L complexes and soluble ULK1 were separated and phosphorylation was detected by autoradiography (AR, left panels). Western blotting was performed (right panels). Results are representative of two unique experiments.(b) Full-length murine GST-Beclin-1 and various truncations (as labelled) were subjected to an in vitro HA-ULK1 kinase assay. GST-Beclin-1 6(S/T)A has serine/threonine residues 4, 7, 10, 14, 29 and 42 mutated to alanine. ULK1 inputs were determined by western blotting, Beclin-1 inputs by Coomassie (Coom) and target phosphorylation by autoradiography.(c) GST-Beclin-1 (1-85) was subjected to an in vitro ULK1 kinase reaction and analysed by mass spectrometry. Ser 14, Ser 15 in humans (outlined), is the main phosphorylation site identified (for mass spectrometry data, see Supplementary Fig. S2b,c). Mass spectrometry was performed on a single experiment.(d) Beclin-1 Ser 14 is the main in vitro ULK1 phosphorylation site. Beclin-1 WT, and S4A and S14A mutants were subjected to an in vitro ULK1 kinase assay. The reaction was developed by autoradiography and stained for Beclin-1 input levels by Coomassie blue. Results are representative of two unique experiments.(e) HEK293 cells were transfected with the indicated plasmids under nutrient-rich conditions. Beclin-1 was immunoprecipitated and immunoblotted with pBeclin-1(Ser 14), or anti-Beclin-1 as a loading control. ULK1 inputs are included below immunoprecipitated samples.(f) Purified GST-Beclin-1 (1-85) was subjected to in vitro phosphorylation by GST-ULK1 (top panel) and GST-ULK2 (bottom panel). Reactions were immunoblotted with the indicated antibodies.(g) HEK293 cells were transfected with ATG14L, VPS34 and Beclin-1 and grown under nutrient-rich conditions. ATG14L-containing VPS34 complexes were immunoprecipitated and lipid kinase activity was assayed as described in Fig. 1j. Inputs were immunoblotted with the indicated antibodies. Representative of four unique experiments.(h) Stable lines containing Beclin-1(WT or S14A) were used for Beclin-1 immunoprecipitation. Binding partners were determined by SDS-PAGE analysis and western blot using the indicated antibodies. Uncropped images of blots are shown in Supplementary Fig. S4.


figf3 ( a ) WT MEFs were cultured with or without amino acids . ATG14L - associated Beclin - 1 was immunoprecipitated and treated with lambda phosphatase treatment ( PPase ) as indicated . Western blotting ( immunoblotting , IB ) was performed with the indicated antibodies . Beclin - 1 Ser 14 phosphorylation was quantified ( shown under top panel ) and normalized to total Beclin - 1 . ( b ) WT MEFs were starved for the indicated times . Beclin - 1 was immunopurified by ATG14L immunoprecipitation and immunoblotted as indicated ( top panels ) . Whole - cell lysates were immunoblotted with pULK1 S757 ( mTORC1 - target site ) , pS6K and ULK1 antibodies ( bottom panels ) . Two unique experiments were performed . ( c ) WT or FIP200 − / − MEFs were incubated under nutrient - rich , amino - acid - deprived or Torin - 1 ( + T , an mTOR inhibitor ) conditions . Beclin - 1 was purified and immunoblotted as in Fig . 3b . ( d ) WT or ULK - deficient MEFs were incubated with or without amino acids . Beclin - 1 was purified and immunoblotted as in a . Two unique experiments were performed . Uncropped images of blots are shown in Supplementary Fig . S4 .

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figf3(a) WT MEFs were cultured with or without amino acids. ATG14L-associated Beclin-1 was immunoprecipitated and treated with lambda phosphatase treatment (PPase) as indicated. Western blotting (immunoblotting, IB) was performed with the indicated antibodies. Beclin-1 Ser 14 phosphorylation was quantified (shown under top panel) and normalized to total Beclin-1.(b) WT MEFs were starved for the indicated times. Beclin-1 was immunopurified by ATG14L immunoprecipitation and immunoblotted as indicated (top panels). Whole-cell lysates were immunoblotted with pULK1 S757 (mTORC1-target site), pS6K and ULK1 antibodies (bottom panels). Two unique experiments were performed.(c) WT or FIP200−/− MEFs were incubated under nutrient-rich, amino-acid-deprived or Torin-1 (+T, an mTOR inhibitor) conditions. Beclin-1 was purified and immunoblotted as in Fig. 3b.(d) WT or ULK-deficient MEFs were incubated with or without amino acids. Beclin-1 was purified and immunoblotted as in a. Two unique experiments were performed. Uncropped images of blots are shown in Supplementary Fig. S4.


figf4 ( a ) Beclin - 1 alone ( lanes 1 - 4 ) or Beclin - 1 and ATG14L ( lanes 5 - 8 ) were overexpressed in HEK293 cells . Beclin - 1 was purified either by direct immunoprecipitation ( lanes 1 - 4 ) or by ATG14L immunoprecipitation ( lanes 5 - 8 ) . Immunoprecipitated samples were subjected to an in vitro ULK1 kinase assay with increasing amounts of ULK1 . Reactions were immunoblotted with the indicated antibodies . Black line denotes discontinuous lanes from the same gel . Two unique experiments were performed . ( b ) Beclin - 1 alone or bound to ATG14L was purified as described in a . Equal amounts of ULK1 were added to each complex and reactions were quenched at the indicated time points . Western blotting was performed with the indicated antibodies . ( c ) An ATG14L - FLAG - 6His - inducible U2OS cell line was induced for 16 h in the presence of amino acids . Endogenous Beclin - 1 was immunoprecipitated and immunoblotted as in Fig . 3a . ATG14L input levels were detected by immunoblotting . Two unique experiments were performed . ( d ) HEK293 cells transfected with either ATG14L or Beclin - 1 , or both , in conjunction with ULK1 , were immunoprecipitated as indicated and blotted with the indicated antibodies . ( e ) HEK293 cells were transfected with Beclin - 1 and ULK1 in the presence of ATG14L WT or ATG14LΔCCD , which is defective in Beclin - 1 binding , under nutrient - rich conditions . Lysates were resolved by SDS - PAGE and blotted with the indicated antibodies . ( f ) HEK293 cells were transfected with ULK1 and Beclin - 1 in conjunction with either ATG14L WT , or one of two mutants ( ΔBATS , ΔN ) that are defective in phagophore localization . Samples were handled as in e . Uncropped images of blots are shown in Supplementary Fig . S4 . Lysates were resolved by SDS - PAGE and blotted with the indicated antibodies .

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figf4(a) Beclin-1 alone (lanes 1-4) or Beclin-1 and ATG14L (lanes 5-8) were overexpressed in HEK293 cells. Beclin-1 was purified either by direct immunoprecipitation (lanes 1-4) or by ATG14L immunoprecipitation (lanes 5-8). Immunoprecipitated samples were subjected to an in vitro ULK1 kinase assay with increasing amounts of ULK1. Reactions were immunoblotted with the indicated antibodies. Black line denotes discontinuous lanes from the same gel. Two unique experiments were performed.(b) Beclin-1 alone or bound to ATG14L was purified as described in a. Equal amounts of ULK1 were added to each complex and reactions were quenched at the indicated time points. Western blotting was performed with the indicated antibodies.(c) An ATG14L-FLAG-6His-inducible U2OS cell line was induced for 16 h in the presence of amino acids. Endogenous Beclin-1 was immunoprecipitated and immunoblotted as in Fig. 3a. ATG14L input levels were detected by immunoblotting. Two unique experiments were performed.(d) HEK293 cells transfected with either ATG14L or Beclin-1, or both, in conjunction with ULK1, were immunoprecipitated as indicated and blotted with the indicated antibodies.(e) HEK293 cells were transfected with Beclin-1 and ULK1 in the presence of ATG14L WT or ATG14LΔCCD, which is defective in Beclin-1 binding, under nutrient-rich conditions. Lysates were resolved by SDS-PAGE and blotted with the indicated antibodies.(f) HEK293 cells were transfected with ULK1 and Beclin-1 in conjunction with either ATG14L WT, or one of two mutants (ΔBATS,ΔN) that are defective in phagophore localization. Samples were handled as in e. Uncropped images of blots are shown in Supplementary Fig. S4. Lysates were resolved by SDS-PAGE and blotted with the indicated antibodies.


figf5 ( a ) HEK293 cells were transfected with Beclin - 1 , with or without UVRAG , in conjunction with ULK1 as indicated in the presence of amino acids . Lysates were immunoblotted with the indicated antibodies . A representative experiment of three repeats is shown . ( b ) UVRAG bridges the interaction between Beclin - 1 and ULK1 . HEK293 cells were transfected with Beclin - 1 , with or without UVRAG , in conjunction with ULK1 as indicated . Lysates were immunoprecipitated with anti - HA ( Beclin - 1 ) antibody and blotted with the indicated antibodies . A representative experiment of three repeats is shown . Uncropped images of blots are shown in Supplementary Fig . S4 .

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figf5(a) HEK293 cells were transfected with Beclin-1, with or without UVRAG, in conjunction with ULK1 as indicated in the presence of amino acids. Lysates were immunoblotted with the indicated antibodies. A representative experiment of three repeats is shown.(b) UVRAG bridges the interaction between Beclin-1 and ULK1. HEK293 cells were transfected with Beclin-1, with or without UVRAG, in conjunction with ULK1 as indicated. Lysates were immunoprecipitated with anti-HA(Beclin-1) antibody and blotted with the indicated antibodies. A representative experiment of three repeats is shown. Uncropped images of blots are shown in Supplementary Fig. S4.


figf6 ( a ) HEK293 cells were transfected with the indicated plasmids . Cells were grown in the presence of amino acids and treated with NH4Cl to block autophagic turnover where indicated . Lysates were immunoblotted with the indicated antibodies . ( b ) HEK293 cells transfected with Beclin - 1 ATG14L were grown in the presence or absence of amino acids . Lysates were immunoblotted with the indicated antibodies ( left panel ) and quantified by densitometry ( right panel ) . Data represent mean + s . d . of three unique experiments . ( c ) Beclin - 1 - shRNA reconstituted lines ( WT or mutant ) and controls ( scramble shRNA or Beclin - 1 shRNA ) were grown with or without amino acids and assessed for autophagy ( left panel ) and Beclin - 1 levels ( right panel ) . ( d ) Autophagosome ( denoted by arrowheads ) generation on amino - acid withdrawal was analysed by electron microscopy . Cell lines and conditions from c were used and representative images from the indicated amino - acid - starved lines are shown ; scale bars , 0 . 4 μm . ( d ) Autophagosome ( denoted by arrowheads ) generation on amino - acid withdrawal was analysed by electron microscopy . Cell lines and conditions from c were used and representative images from the indicated amino - acid - starved lines are shown ; scale bars , 0 . 4 μm . ( e ) Quantification of d . Fold induction was determined by arbitrarily making the nutrient - rich condition 1 ( solid bars ) for each line . Error bars represent the s . d . of the mean value over an average of 20 fields of view within a representative experiment . NS , not significant . ( f ) HA - Beclin - 1 WT or S14D was transiently expressed in FIP200 − / − MEFs grown under nutrient - rich conditions . Indirect immunofluorescence was performed using antibodies against endogenous LC3B and HA - Beclin - 1 . Scale bars , 20 μm . ( g ) Quantification of LC3B puncta from confocal in f . In the HA - Beclin - 1 - or HA - Beclin - 1 - S14D - transfected samples , only the HA - positive cells were counted for LC3B puncta . Error bars were processed as in e . Mean value shown ; P values determined by Student ' s t - test using 10 unique fields of view from f . Uncropped images of blots are shown in Supplementary Fig . S4 .

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

figf6(a) HEK293 cells were transfected with the indicated plasmids. Cells were grown in the presence of amino acids and treated with NH4Cl to block autophagic turnover where indicated. Lysates were immunoblotted with the indicated antibodies.(b) HEK293 cells transfected with Beclin-1 ATG14L were grown in the presence or absence of amino acids. Lysates were immunoblotted with the indicated antibodies (left panel) and quantified by densitometry (right panel). Data represent mean+s.d. of three unique experiments.(c) Beclin-1-shRNA reconstituted lines (WT or mutant) and controls (scramble shRNA or Beclin-1 shRNA) were grown with or without amino acids and assessed for autophagy (left panel) and Beclin-1 levels (right panel).(d) Autophagosome (denoted by arrowheads) generation on amino-acid withdrawal was analysed by electron microscopy. Cell lines and conditions from c were used and representative images from the indicated amino-acid-starved lines are shown; scale bars, 0.4 μm.(d) Autophagosome (denoted by arrowheads) generation on amino-acid withdrawal was analysed by electron microscopy. Cell lines and conditions from c were used and representative images from the indicated amino-acid-starved lines are shown; scale bars, 0.4 μm. (e) Quantification of d. Fold induction was determined by arbitrarily making the nutrient-rich condition 1 (solid bars) for each line. Error bars represent the s.d. of the mean value over an average of 20 fields of view within a representative experiment. NS, not significant.(f) HA-Beclin-1 WT or S14D was transiently expressed in FIP200−/− MEFs grown under nutrient-rich conditions. Indirect immunofluorescence was performed using antibodies against endogenous LC3B and HA-Beclin-1. Scale bars, 20 μm. (g) Quantification of LC3B puncta from confocal in f. In the HA-Beclin-1- or HA-Beclin-1-S14D-transfected samples, only the HA-positive cells were counted for LC3B puncta. Error bars were processed as in e. Mean value shown; P values determined by Student's t-test using 10 unique fields of view from f. Uncropped images of blots are shown in Supplementary Fig. S4.


figf7 ( a ) Bec - 1C . elegans were reconstituted with either WT or mutant GFP - BEC - 1 . Stable worm lines with Bec - 1 rescue were obtained and embryos were stained with anti - PGL - 1 antibody . Arrowheads indicate normal PGL - 1 staining in germline cells . ( b ) Quantification of PGL - 1 puncta outside germline cells ( left panel ) . Error bars represents s . d . between 3 unique embryos in a representative experiment . Reconstituted Bec - 1 ( WT and mutant ) levels in Bec − / − stable worms were compared by western blotting . Mean value presented . ( c ) Spectrum of defects in PGL granule degradation in bec - 1 mutant rescue embryos . Mutant embryos exhibited either high levels of diffuse PGL - 1 staining ( middle - left panel ; one - third of the embryos ) , or large punctuate PGL - 1 structures in somatic cells ( bottom - left panel ; two - thirds of the embryos ) . Both diffuse or punctuate PGL - 1 staining in somatic cells have been described in autophagy - deficient embryos . ( d ) Embryos from the lines described in a were labelled with anti - LGG - 1 , along with WT and unc - 51 worms . Representative embryos at ∼ 100 cell stage are shown . ( e ) Quantification of LGG - 1 per embryo from labelling in d . Error bars generated as in b . Mean value presented . Uncropped images of blots are shown in Supplementary Fig . S4 . Scale bar , 10 μm .

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

figf7(a) Bec-1C. elegans were reconstituted with either WT or mutant GFP-BEC-1. Stable worm lines with Bec-1 rescue were obtained and embryos were stained with anti- PGL-1 antibody. Arrowheads indicate normal PGL-1 staining in germline cells. (b) Quantification of PGL-1 puncta outside germline cells (left panel). Error bars represents s.d. between 3 unique embryos in a representative experiment. Reconstituted Bec-1 (WT and mutant) levels in Bec−/− stable worms were compared by western blotting. Mean value presented.(c) Spectrum of defects in PGL granule degradation in bec-1 mutant rescue embryos. Mutant embryos exhibited either high levels of diffuse PGL-1 staining (middle-left panel; one-third of the embryos), or large punctuate PGL-1 structures in somatic cells (bottom-left panel; two-thirds of the embryos). Both diffuse or punctuate PGL-1 staining in somatic cells have been described in autophagy-deficient embryos.(d) Embryos from the lines described in a were labelled with anti-LGG-1, along with WT and unc-51 worms. Representative embryos at ∼ 100 cell stage are shown. (e) Quantification of LGG-1 per embryo from labelling in d. Error bars generated as in b. Mean value presented. Uncropped images of blots are shown in Supplementary Fig. S4. Scale bar, 10 μm.


Figure 1 ( A ) 293T cells were cultured in complete medium made of dialyzed serum for 24 h which was then replaced with medium in which one amino acid was eliminated and cultured for 8 h . Then , WB was used to measure p - AMPK , p - ACC , p - Raptor and total AMPK , ACC , Raptor protein expression . " Nor " = normal medium . Actin served as the loading control . The red text " - Cys " indicates that cystine deficiency most significantly activates AMPK and its substrates . ( F ) WB analysis of p - AMPK , p - ACC , p - Raptor and total AMPK , ACC , Raptor protein in 293T cells transfected with shRNAs targeting LKB1 , CaMKK2 , TAK1 or a non - targeting control ( NTC ) that were treated with cystine - deficient medium for 8 h . Actin served as the loading control . ( I ) 293T cells cultured with cystine - deficient medium or complete medium for 8 h were harvested and subjected to immunoprecipitation with anti - AMPKα , followed by WB analysis with anti - AMPKα and anti - CaMKK2 .

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

Figure 1 (A) 293T cells were cultured in complete medium made of dialyzed serum for 24 h which was then replaced with medium in which one amino acid was eliminated and cultured for 8 h. Then, WB was used to measure p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor protein expression. "Nor" = normal medium. Actin served as the loading control. The red text "-Cys" indicates that cystine deficiency most significantly activates AMPK and its substrates. (F) WB analysis of p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor protein in 293T cells transfected with shRNAs targeting LKB1, CaMKK2, TAK1 or a non-targeting control (NTC) that were treated with cystine-deficient medium for 8 h. Actin served as the loading control. (I) 293T cells cultured with cystine-deficient medium or complete medium for 8 h were harvested and subjected to immunoprecipitation with anti-AMPKα, followed by WB analysis with anti-AMPKα and anti-CaMKK2.


Figure 2 ( E ) WB analysis of p - AMPK , p - ACC , p - Raptor and total AMPK ACC , Raptor protein expression in 293T cells transfected with shRNAs targeting CARS that were further treated with cystine - deficient medium for 8 h . Actin served as the loading control . ( J ) WB analysis of p - AMPK and total AMPK protein expression in CARS - overexpressing 293T cells and RCC4 cells that were treated with 1 µg / ml STO - 609 or DMSO for 8 h . Actin served as the loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (E) WB analysis of p-AMPK, p-ACC, p-Raptor and total AMPK ACC, Raptor protein expression in 293T cells transfected with shRNAs targeting CARS that were further treated with cystine-deficient medium for 8 h. Actin served as the loading control.  (J) WB analysis of p-AMPK and total AMPK protein expression in CARS-overexpressing 293T cells and RCC4 cells that were treated with 1 µg/ml STO-609 or DMSO for 8 h. Actin served as the loading control.


Figure 3 ( D , E ) WB analysis of p - AMPK , p - ACC , p - Raptor and total AMPK , ACC , Raptor , AMPKγ2 protein expression in 293T cells transfected with shRNAs targeting AMPKγ2 that were further treated with cystine - deficient medium for 8 h ( D ) or transfected with Flag - CARS for 48 h ( E ) . Actin served as the loading control . ( F ) 293T cells were transfected with HA - AMPKγ2 alone or with Flag - CARS for 48 h and then cultured with cystine - deficient medium or complete medium for 8 h . Cell lysates were immunoprecipitated with anti - Flag , and a WB analysis was performed .

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

Figure 3 (D, E) WB analysis of p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor, AMPKγ2 protein expression in 293T cells transfected with shRNAs targeting AMPKγ2 that were further treated with cystine-deficient medium for 8 h (D) or transfected with Flag-CARS for 48 h (E). Actin served as the loading control. (F) 293T cells were transfected with HA-AMPKγ2 alone or with Flag-CARS for 48 h and then cultured with cystine-deficient medium or complete medium for 8 h. Cell lysates were immunoprecipitated with anti-Flag, and a WB analysis was performed.


Figure 4 ( B ) The cell death level was determined in 293T cells with the same treatment conditions as in Figure 4A . Data are presented as the mean ( ± SD ) of three independent experiments . NS , not significant ; * P < 0 . 05 [ two - tailed Student ' s t - test ] , compared with the indicated groups . ( C , D ) The crystal violet assay ( C ) and apoptosis rate assay ( D ) were performed in 293T cells transfected with shRNAs targeting NTC , CARS , CaMKK2 or AMPKγ2 and further treated with cystine - deficient medium for 24 h with or without 1 mM AICAR . Data are presented as the mean ( ± SD ) of three independent experiments . NS , not significant ; * P < 0 . 05 [ two - tailed Student ' s t - test ] , compared with the indicated groups . ( F WB analysis of p - AMPK , total AMPK , CARS , AMPKγ2 , and CaMKK2 protein expression with 786 - O cells transfected with Flag - EV , Flag - CARS , Flag - CaMKK2 or Flag - AMPKγ2 and further treated with cystine - deficient medium for 8 h . Actin served as the loading control . Data are presented as the mean ( ± SD ) of three independent experiments . NS , not significant ; * P < 0 . 05 [ two - tailed Student ' s t - test ] , compared with the indicated groups .

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 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

Figure 4(B) The cell death level was determined in 293T cells with the same treatment conditions as in Figure 4A. Data are presented as the mean (± SD) of three independent experiments. NS, not significant; *P < 0.05 [two-tailed Student's t-test], compared with the indicated groups.(C, D) The crystal violet assay (C) and apoptosis rate assay (D) were performed in 293T cells transfected with shRNAs targeting NTC, CARS, CaMKK2 or AMPKγ2 and further treated with cystine-deficient medium for 24 h with or without 1 mM AICAR. Data are presented as the mean (± SD) of three independent experiments. NS, not significant; *P < 0.05 [two-tailed Student's t-test], compared with the indicated groups. (F WB analysis of p-AMPK, total AMPK, CARS, AMPKγ2, and CaMKK2 protein expression with 786-O cells transfected with Flag-EV, Flag-CARS, Flag-CaMKK2 or Flag-AMPKγ2 and further treated with cystine-deficient medium for 8 h. Actin served as the loading control. Data are presented as the mean (± SD) of three independent experiments. NS, not significant; *P < 0.05 [two-tailed Student's t-test], compared with the indicated groups.


Figure 1Percentage of circulating highly differentiated CD4 T cells within CD4 cells ( upper graph ) in age - matched healthy donors or NSCLC patients before undergoing immunotherapies . G1 and G2 , groups of patients classified according to high THD cells ( G1 , > 40 % CD4 THD cells ) and low THD cells ( G2 , < 40 % CD4 THD cells ) . Relevant statistical comparisons are shown by the test of Mann - Whitney . In green , objective responders ( OR ) . In red , no OR . Below the graph , correlation of objective responses to G1 and G2 groups by the Fisher ´ s exact test . Waterfall plot of change in lesion size in patients with measurable disease classified as having a G1 ( blue ) or G2 ( red ) profile . Dotted lines represents the limit to define significant progression ( upper line ) or significant regression ( lower line ) . Kaplan - Meier plot for PFS in patients treated with immunotherapies stratified only by G1 ( green ) and G2 ( red ) CD4 T cell profiles . Patients starting therapy with a G2 profile had an overall response rate ( ORR ) of 0 % and 82 % of them experienced progression or death by week 9 . ORR was 44 . 8 % for G1 patients , and the 12 - week PFS was 50 . 2 % .

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 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 1Percentage of circulating highly differentiated CD4 T cells within CD4 cells (upper graph) in age-matched healthy donors or NSCLC patients before undergoing immunotherapies. G1 and G2, groups of patients classified according to high THD cells (G1, >40% CD4 THD cells) and low THD cells (G2, <40% CD4 THD cells). Relevant statistical comparisons are shown by the test of Mann-Whitney. In green, objective responders (OR). In red, no OR. Below the graph, correlation of objective responses to G1 and G2 groups by the Fisher´s exact test.Waterfall plot of change in lesion size in patients with measurable disease classified as having a G1 (blue) or G2 (red) profile. Dotted lines represents the limit to define significant progression (upper line) or significant regression (lower line).Kaplan-Meier plot for PFS in patients treated with immunotherapies stratified only by G1 (green) and G2 (red) CD4 T cell profiles. Patients starting therapy with a G2 profile had an overall response rate (ORR) of 0% and 82% of them experienced progression or death by week 9. ORR was 44.8% for G1 patients, and the 12-week PFS was 50.2%.


Figure 2The scatter plot shows PD - 1 expression after co - culture of CD4 T cells from healthy donors or NSCLC patients , as indicated , with A459 - SC3 lung cancer cells . Relevant statistical comparisons with the test of Mann - Whitney are indicated . Upper graphs , flow cytometry density plots of PD - 1 and LAG - 3 co - expression in CD4 T cells from healthy donors , a G1 responder ( G1 R ) , a G1 non - responder ( G1 NR ) and a G2 non - responder as indicated , following stimulation with A549 - SC3 cells . Percentage of expressing cells are indicated within each quadrant . Below , same as in the upper graphs but as a scatter plot of the percentage of CD4 T cells that simultaneously co - express PD - 1 and LAG - 3 . Relevant statistical comparisons are shown with the test of Mann - Whitney . Upper flow cytometry histograms of Ki67 expression in CD4 T cells from the representative subjects as indicated on the right , after stimulation with A549 - SC3 cells . Vertical dotted line indicates the cut - off value of positive vs negative Ki67 expression . The percentage of Ki67 - expressing CD4 T cells is shown within the histograms . Below , same data represented as a scatter plot from a sample of G1 and G2 donors as indicated , with relevant statistical comparisons with the test Mann - Whitney . Proliferation of CD4 T cells stimulated by A549 - SC3 cells from the indicated patient groups . CD28 expression is shown together with the proliferation marker ki67 . Percentages of cells within each quadrant are shown . Same as in ( D ) but in the presence of an isotype control antibody or an anti - PD - 1 antibody with the equivalent sequence to pembrolizumab . The effects on CD4 T cells from a G1 and a G2 patient are shown , divided in CD28 high or low / negative subsets as indicated . Relevant statistical comparisons are shown with paired Student ´ s t test . Top , flow cytometry density plots of Ki67 expression in CD4 T cells from representative G1 or G2 patients after three cycles of therapy , activated by incubation with A549 - SC3 cells . Below , same as above but as a dot - plot graph . A comparison between proliferating CD4 T cells before and after therapy is shown in unpaired patient samples .

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

Figure 2The scatter plot shows PD-1 expression after co-culture of CD4 T cells from healthy donors or NSCLC patients, as indicated, with A459-SC3 lung cancer cells. Relevant statistical comparisons with the test of Mann-Whitney are indicated.Upper graphs, flow cytometry density plots of PD-1 and LAG-3 co-expression in CD4 T cells from healthy donors, a G1 responder (G1 R), a G1 non-responder (G1 NR) and a G2 non-responder as indicated, following stimulation with A549-SC3 cells. Percentage of expressing cells are indicated within each quadrant. Below, same as in the upper graphs but as a scatter plot of the percentage of CD4 T cells that simultaneously co-express PD-1 and LAG-3. Relevant statistical comparisons are shown with the test of Mann-Whitney.Upper flow cytometry histograms of Ki67 expression in CD4 T cells from the representative subjects as indicated on the right, after stimulation with A549-SC3 cells. Vertical dotted line indicates the cut-off value of positive vs negative Ki67 expression. The percentage of Ki67-expressing CD4 T cells is shown within the histograms. Below, same data represented as a scatter plot from a sample of G1 and G2 donors as indicated, with relevant statistical comparisons with the test Mann-Whitney.Proliferation of CD4 T cells stimulated by A549-SC3 cells from the indicated patient groups. CD28 expression is shown together with the proliferation marker ki67. Percentages of cells within each quadrant are shown.Same as in (D) but in the presence of an isotype control antibody or an anti-PD-1 antibody with the equivalent sequence to pembrolizumab. The effects on CD4 T cells from a G1 and a G2 patient are shown, divided in CD28 high or low/negative subsets as indicated. Relevant statistical comparisons are shown with paired Student´s t test.Top, flow cytometry density plots of Ki67 expression in CD4 T cells from representative G1 or G2 patients after three cycles of therapy, activated by incubation with A549-SC3 cells. Below, same as above but as a dot-plot graph. A comparison between proliferating CD4 T cells before and after therapy is shown in unpaired patient samples.


Figure 3Scatter plot graph with the percentage of lung cancer - specific systemic CD4 T cells quantified by an autologous DC - based antigen presentation assay ( See Materials and Methods ) , in a sample of G1 and G2 patients as indicated . Objective responses ( OR ) are shown in green . In red , patients with no OR . The scatter plot graph on the left represents the percentage of CD4 THD cells within lung - cancer specific CD4 T cells in a sample of patients from the indicated G1 / G2 groups . On the right , same as left but representing the percentage of CD28 + CD4 T cells within lung - cancer specific CD4 T cells . Objective responders ( OR ) are shown in green .

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

Figure 3Scatter plot graph with the percentage of lung cancer-specific systemic CD4 T cells quantified by an autologous DC-based antigen presentation assay (See Materials and Methods), in a sample of G1 and G2 patients as indicated. Objective responses (OR) are shown in green. In red, patients with no OR.The scatter plot graph on the left represents the percentage of CD4 THD cells within lung-cancer specific CD4 T cells in a sample of patients from the indicated G1/G2 groups. On the right, same as left but representing the percentage of CD28+ CD4 T cells within lung-cancer specific CD4 T cells. Objective responders (OR) are shown in green.


Figure 4Column graphs representing the percentage of CD4 T cells from NSCLC patients or age - matched healthy donors as represented in the graph , expressing the indicated cytokines after T cell stimulation with anti - CD3 / anti - CD28 antibodies . Data on total CD4 ( left graph ) , CD28 + subsets ( center graph ) and CD28negative subsets ( right graph ) are shown . Error bars correspond to standard deviations and bars represent means from 5 independent biological replicates ( patients ) . representing CD4 T cells expressing only one cytokinerepresenting CD4 T cells expressing only cytokine , twoCD4 T cells expressing only three cytokines

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Figure 4Column graphs representing the percentage of CD4 T cells from NSCLC patients or age-matched healthy donors as represented in the graph, expressing the indicated cytokines after T cell stimulation with anti-CD3/anti-CD28 antibodies. Data on total CD4 (left graph), CD28+ subsets (center graph) and CD28negative subsets (right graph) are shown. Error bars correspond to standard deviations and bars represent means from 5 independent biological replicates (patients).representing CD4 T cells expressing only one cytokinerepresenting CD4 T cells expressing only cytokine, twoCD4 T cells expressing only three cytokines simultaneously


Figure 5Expression of the proliferation marker Ki67 in CD8 T cells stimulated ex vivo by A549 - SC3 cells from the indicated patient groups , before the start of immunotherapy . Relevant statistical comparisons are shown with the test of Mann - Whitney . Dot plots of the percentage of Ki67 + proliferating CD8 T cells after ex vivo activation by A549 - SC3 cells . CD8 T cells were obtained from samples of G1 or G2 patients before immunotherapy and after three cycles of anti - PD - 1 therapy . Relevant statistical comparisons are shown with the test of Mann - Whitney . Green , objective responders ( OR ) and red , no ORs . Expression of the proliferation marker Ki67 in CD8 T cells stimulated ex vivo by A549 - SC3 cells from the indicated patient groups , before the start of immunotherapy . Relevant statistical comparisons are shown with the test of Mann - Whitney . but in the presence of an isotype control antibody or an anti - PD - 1 antibody molecularly equivalent to pembrolizumab . Relevant statistical comparisons are shown with comparisons carried out with paired Student ´ s t test . Change in CD8 CD28 + T cells from baseline to post - therapy in G1 patients ( left ) or in G2 patients ( right ) . Statistical comparisons were carried out with paired Student ´ s t test .

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Figure 5Expression of the proliferation marker Ki67 in CD8 T cells stimulated ex vivo by A549-SC3 cells from the indicated patient groups, before the start of immunotherapy. Relevant statistical comparisons are shown with the test of Mann-Whitney.Dot plots of the percentage of Ki67+ proliferating CD8 T cells after ex vivo activation by A549-SC3 cells. CD8 T cells were obtained from samples of G1 or G2 patients before immunotherapy and after three cycles of anti-PD-1 therapy. Relevant statistical comparisons are shown with the test of Mann-Whitney. Green, objective responders (OR) and red, no ORs.Expression of the proliferation marker Ki67 in CD8 T cells stimulated ex vivo by A549-SC3 cells from the indicated patient groups, before the start of immunotherapy. Relevant statistical comparisons are shown with the test of Mann-Whitney. but in the presence of an isotype control antibody or an anti-PD-1 antibody molecularly equivalent to pembrolizumab. Relevant statistical comparisons are shown with comparisons carried out with paired Student´s t test.Change in CD8 CD28+ T cells from baseline to post-therapy in G1 patients (left) or in G2 patients (right). Statistical comparisons were carried out with paired Student´s t test.


Figure 6Scatter plots of PD - 1 / LAG - 3 - expressing CD8 T cells after activation by A459 - SC3 cells in a sample of G1 and G2 patients within CD28 + and CD28negative populations as indicated in the figure . Dot - plot representing the percentage of proliferating CD4 T cells from a sample of G2 patients before starting immunotherapy , activated ex vivo by A549 - SC3 cells in the presence of the indicated antibodies . " Dual " represents the addition of both anti - PD - 1 and anti - LAG - 3 antibodies . Appropriate statistical comparisons are shown within the graph with two - way paired ANOVA . Data from CD28 + and CD28negative subsets are represented as indicated . Dot - plot representing the percentage of proliferating cells from a sample of G2 patients before starting immunotherapy , activated ex vivo by A549 - SC3 cells in the presence of the indicated antibodies . " Dual " represents the addition of both anti - PD - 1 and anti - LAG - 3 antibodies . Appropriate statistical comparisons are shown within the graph with two - way paired ANOVA . Data from CD28 + and CD28negative subsets are represented as indicated . for CD8 T cells .

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Figure 6Scatter plots of PD-1/LAG-3-expressing CD8 T cells after activation by A459-SC3 cells in a sample of G1 and G2 patients within CD28+ and CD28negative populations as indicated in the figure.Dot-plot representing the percentage of proliferating CD4 T cells from a sample of G2 patients before starting immunotherapy, activated ex vivo by A549-SC3 cells in the presence of the indicated antibodies. "Dual" represents the addition of both anti-PD-1 and anti-LAG-3 antibodies. Appropriate statistical comparisons are shown within the graph with two-way paired ANOVA. Data from CD28+ and CD28negative subsets are represented as indicated.Dot-plot representing the percentage of proliferating cells from a sample of G2 patients before starting immunotherapy, activated ex vivo by A549-SC3 cells in the presence of the indicated antibodies. "Dual" represents the addition of both anti-PD-1 and anti-LAG-3 antibodies. Appropriate statistical comparisons are shown within the graph with two-way paired ANOVA. Data from CD28+ and CD28negative subsets are represented as indicated. for CD8 T cells.


Figure 7Kaplan - Meier plot for PFS in patients undergoing immune checkpoint inhibitor therapies stratified by G1 / PD - L1 + tumors ( blue ) and remaining patients for whom their PD - L1 tumor status is known ( red ) . Same as ( A ) but including all patients in the study cohort . Remaining patients ( red ) also included G1 patients with PD - L1 low / negative tumors , G1 patients with unknown PD - L1 tumor status and G2 patients with either PD - L1 + or PD - L1 negative tumors .

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Figure 7Kaplan-Meier plot for PFS in patients undergoing immune checkpoint inhibitor therapies stratified by G1 / PD-L1+ tumors (blue) and remaining patients for whom their PD-L1 tumor status is known (red).Same as (A) but including all patients in the study cohort. Remaining patients (red) also included G1 patients with PD-L1 low/negative tumors, G1 patients with unknown PD-L1 tumor status and G2 patients with either PD-L1+ or PD-L1 negative tumors.


Figure 1B - H Live confocal images of ddaC neurons expressing mCD8 - GFP by ppk - Gal4 at WP stage or 16 h APF . Neurons expressing efa6 RNAi # 1 - # 3 ( C - E ) , efa612 mutant MARCM clones ( F ) , and efa612 / GX6w - transheterozygous mutant neurons ( G ) showed consistent dendrite pruning defects at 16 h APF , as compared to the wild - type neurons ( B ) . The dendrite pruning defects were fully rescued when overexpressing full - length Efa6 in the efa612 mutant clones ( H ) . Red arrowheads point to the ddaC somas .

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Figure 1B-H Live confocal images of ddaC neurons expressing mCD8-GFP by ppk-Gal4 at WP stage or 16 h APF. Neurons expressing efa6 RNAi #1-#3 (C-E), efa612 mutant MARCM clones (F), and efa612/GX6w- transheterozygous mutant neurons (G) showed consistent dendrite pruning defects at 16 h APF, as compared to the wild-type neurons (B). The dendrite pruning defects were fully rescued when overexpressing full-length Efa6 in the efa612 mutant clones (H). Red arrowheads point to the ddaC somas.


Figure 2Live confocal images of efa612 / GX6w - mutant ddaC neurons at WP stage and 16 h APF . Expression of Efa6FL ( C ) rescued the pruning defects in efa612 / GX6w - mutant ddaC neurons ( B ) . Red arrowheads point to the ddaC somas . Live confocal images of efa612 / GX6w - mutant ddaC neurons at WP stage and 16 h APF . Expression of Efa6Nterm ( D ) , Efa6ΔPH ( F ) and Efa6Nterm + CAAX ( H ) , but not Efa6Cterm ( E ) or Efa6ΔMTED ( G ) , rescued the pruning defects in efa612 / GX6w - mutant ddaC neurons Red arrowheads point to the ddaC somas . I - J Quantification of dendrite severing defects and unpruned dendrite lengths at 16 h APF .

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Figure 2Live confocal images of efa612/GX6w- mutant ddaC neurons at WP stage and 16 h APF. Expression of Efa6FL (C) rescued the pruning defects in efa612/GX6w- mutant ddaC neurons (B). Red arrowheads point to the ddaC somas.Live confocal images of efa612/GX6w- mutant ddaC neurons at WP stage and 16 h APF. Expression of Efa6Nterm (D), Efa6ΔPH (F) and Efa6Nterm+CAAX (H), but not Efa6Cterm (E) or Efa6ΔMTED (G), rescued the pruning defects in efa612/GX6w- mutant ddaC neurons Red arrowheads point to the ddaC somas. I-J Quantification of dendrite severing defects and unpruned dendrite lengths at 16 h APF.


Figure 3A - D Live confocal images of ddaC neurons at WP stage and 7 h APF . Efa6FL or Efa6Nterm - overexpressing neurons ( B , C ) showed precocious pruning phenotype at 7 h APF , compared to the control and Efa6ΔMTED - overexpressing neurons ( A , D ) in which most dendrites remained attached to the soma at the same time point . Red arrowheads point to the ddaC somas . Dorsal dendrites are marked by red arrows . E Quantification of precocious pruning defects as indicated by the percentage of dorsal dendrites attached to the soma at 7 h APF . F - I Confocal images of da sensory neurons expressing Efa6 - Ki - GFP ( F ) , or UAS - Efa6 - GFP driven by ppk - Gal4 ( H ) were co - stained with GFP ( Green ) and Nrg ( Magenta ) at wL3 stage . ( G , I ) Confocal images of the epidermal cells expressing Efa6 - Ki - GFP ( G ) or UAS - Efa6 - GFP driven by A58 - Gal4 ( I ) were co - stained with GFP ( Green ) and Dlg ( Magenta ) at wL3 stage . The ddaC soma is marked by dashed lines .

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Figure 3A-D Live confocal images of ddaC neurons at WP stage and 7 h APF. Efa6FL or Efa6Nterm-overexpressing neurons (B, C) showed precocious pruning phenotype at 7 h APF, compared to the control and Efa6ΔMTED-overexpressing neurons (A, D) in which most dendrites remained attached to the soma at the same time point. Red arrowheads point to the ddaC somas. Dorsal dendrites are marked by red arrows. E Quantification of precocious pruning defects as indicated by the percentage of dorsal dendrites attached to the soma at 7 h APF. F-I Confocal images of da sensory neurons expressing Efa6-Ki-GFP (F), or UAS-Efa6-GFP driven by ppk-Gal4 (H) were co-stained with GFP (Green) and Nrg (Magenta) at wL3 stage. (G, I) Confocal images of the epidermal cells expressing Efa6-Ki-GFP (G) or UAS-Efa6-GFP driven by A58-Gal4 (I) were co-stained with GFP (Green) and Dlg (Magenta) at wL3 stage. The ddaC soma is marked by dashed lines.


Figure 4A - F Representative kymographs of EB1 - GFP comets in the proximal dendrites of ddaC neurons at 96 h AEL . The horizontal arrow indicates the direction toward the soma , and the vertical arrow indicates the time . In contrast to the wild type ( A ) , the number of EB1 - GFP comets was significantly increased in efa612 / GX6w - mutant neurons ( B ) . The comet length and speed were significantly decreased in Efa6FL - and Efa6Nterm - overexpressing neurons , but not in Efa6ΔMTED - overexpression neurons ( C - F ) .

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Figure 4A-F Representative kymographs of EB1-GFP comets in the proximal dendrites of ddaC neurons at 96 h AEL. The horizontal arrow indicates the direction toward the soma, and the vertical arrow indicates the time. In contrast to the wild type (A), the number of EB1-GFP comets was significantly increased in efa612/GX6w- mutant neurons (B). The comet length and speed were significantly decreased in Efa6FL- and Efa6Nterm-overexpressing neurons, but not in Efa6ΔMTED-overexpression neurons (C-F).


Figure 5A - C Live confocal images of ddaC dendrites expressing tdEOS : : α - tubulin driven by ppk - Gal4 . The microtubule turnover rate was significantly reduced in efa6 RNAi neurons at wL3 ( A , right panels ) and WP stages ( B , right panels ) , and increased in Efa6 - overexpressing neurons at wL3 stage ( C , right panels ) , compared to the control neurons ( A - C , left panels ) .

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Figure 5A-C Live confocal images of ddaC dendrites expressing tdEOS::α-tubulin driven by ppk-Gal4. The microtubule turnover rate was significantly reduced in efa6 RNAi neurons at wL3 (A, right panels) and WP stages (B, right panels), and increased in Efa6-overexpressing neurons at wL3 stage (C, right panels), compared to the control neurons (A-C, left panels).


Figure 6A - H Live confocal images of ddaC neurons at WP stage and 16 h APF . Treatment with colchicine significantly suppressed the pruning defects of efa6 RNAi neurons ( A , B ) , while taxol treatment enhanced the pruning defects ( C , D ) . Removal of one copy of γTub23C ( F ) or expression of γTub23C RNAi ( H ) almost fully rescued the dendrite pruning defects in efa6 mutant ( E ) or RNAi neurons ( G ) , respectively . Red arrowheads point to the ddaC somas .

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Figure 6A-H Live confocal images of ddaC neurons at WP stage and 16 h APF. Treatment with colchicine significantly suppressed the pruning defects of efa6 RNAi neurons (A, B), while taxol treatment enhanced the pruning defects (C, D). Removal of one copy of γTub23C (F) or expression of γTub23C RNAi (H) almost fully rescued the dendrite pruning defects in efa6 mutant (E) or RNAi neurons (G), respectively. Red arrowheads point to the ddaC somas.


Figure 7A - F Live confocal images of ddaC neurons at WP stage and 16 h APF . stai RNAi neurons ( B ) , staiSK7 ( C ) , staiKO ( D ) mutant MARCM clones showed similar dendrite pruning defects , compared to the wild - type ddaC neurons ( A ) . Double RNAi of stai and efa6 significantly enhanced the dendrite pruning defects ( E ) , compared to their individual RNAi knockdown . Expression of stai RNAi in efa612 / GX6w - mutant background also significantly enhanced the dendrite pruning defects ( F ) , compared to the RNAi or mutant alone . Red arrowheads point to the ddaC somas . I - L Representative kymographs of EB1 - GFP comets driven by Gal44 - 77 in the proximal dendrites of ddaC neurons at 96 h AEL .

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Figure 7A-F Live confocal images of ddaC neurons at WP stage and 16 h APF. stai RNAi neurons (B), staiSK7 (C), staiKO (D) mutant MARCM clones showed similar dendrite pruning defects, compared to the wild-type ddaC neurons (A). Double RNAi of stai and efa6 significantly enhanced the dendrite pruning defects (E), compared to their individual RNAi knockdown. Expression of stai RNAi in efa612/GX6w- mutant background also significantly enhanced the dendrite pruning defects (F), compared to the RNAi or mutant alone. Red arrowheads point to the ddaC somas.I-L Representative kymographs of EB1-GFP comets driven by Gal44-77 in the proximal dendrites of ddaC neurons at 96 h AEL.


Figure 1A : HIF - 1α levels in HT29 primary tumors . Left : vehicle - treated controls Middle / left : Doublet LDM cyclophosphamide + capecitabine ( LDMCTX + LDMCPB ) Middle / right : LDM cyclophosphamide + MTD capecitabine ( LDMCTX + MTDCPB ) . Inset , high - magnification image of the region marked with a green asterisk ( * ) . Right : Quantification of the effect of monotherapies or doublet LDM / MTD regimens on HIF - 1α + area . F ( 5 , 12 ) = 8 . 791 and P = 0 . 001 for overall treatment by Brown - Forsythe ANOVA ; * , P = 0 . 046 vs vehicle ; # , P < 0 . 0001 vs LDMCTX + MTDCPB by Benjamini , Krieger and Yekuteli post hoc test . B : HIF - 2α levels in HT29 primary tumors . Left : Example of immunostaining in vehicle - treated controls . Inset , high - magnification image of the region marked with a green asterisk ( * ) . Right : Quantification of the effect of monotherapies or doublet LDM / MTD regimens on HIF - 2α + area . F ( 5 , 18 ) = 1 . 215 and P = 0 . 3424 ( not significant ) for overall treatment by Brown - Forsythe ANOVA . C : CA9 levels in HT29 primary tumors . Left : Example of immunostaining in vehicle - treated controls . Inset , high - magnification image of the region marked with a green asterisk ( * ) . Right : Quantification of the effect of monotherapies or doublet LDM / MTD regimens on CA9 + area . F ( 5 , 11 ) = 2 . 466 and P = 0 . 0961 ( not significant ) for overall treatment by Brown - Forsythe ANOVA . D : Correlation between HIF - 1α levels and proliferation indexes in HT29 primary tumors . Left and middle / left : HIF - 1α and Ki67 expression in vehicle - treated controls ; consecutive sections are shown . Middle / right : HIF - 1α + vs Ki67 + proliferation index scatterplot . Each point represents median values for each tumor . Regression line ( red ) and 95 % CI ( shaded blue area ) are indicated . F ( 1 , 27 ) = 34 . 45 and P = 10 - 4 ; slope ≠ 0 by F - test Right : Machine - learning quantification of the effect of monotherapies or doublet LDM / MTD regimens upon Ki67 + proliferative index . Median indexes per tumor are indicated . F ( 5 , 12 ) = 1 . 609 and P = 0 . 2312 ( not significant ) for overall treatment by Brown - Forsythe ANOVA .

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Figure 1A: HIF-1α levels in HT29 primary tumors. Left: vehicle-treated controls Middle/left: Doublet LDM cyclophosphamide + capecitabine (LDMCTX + LDMCPB) Middle/right: LDM cyclophosphamide + MTD capecitabine (LDMCTX + MTDCPB). Inset, high-magnification image of the region marked with a green asterisk (*). Right: Quantification of the effect of monotherapies or doublet LDM/MTD regimens on HIF-1α+ area. F(5,12) = 8.791 and P = 0.001 for overall treatment by Brown-Forsythe ANOVA; *, P= 0.046 vs vehicle; #, P< 0.0001 vs LDMCTX + MTDCPB by Benjamini, Krieger and Yekuteli post hoc test. B: HIF-2α levels in HT29 primary tumors. Left: Example of immunostaining in vehicle-treated controls. Inset, high-magnification image of the region marked with a green asterisk (*). Right: Quantification of the effect of monotherapies or doublet LDM/MTD regimens on HIF-2α+ area. F(5,18) = 1.215 and P = 0.3424 (not significant) for overall treatment by Brown-Forsythe ANOVA. C: CA9 levels in HT29 primary tumors. Left: Example of immunostaining in vehicle-treated controls. Inset, high-magnification image of the region marked with a green asterisk (*). Right: Quantification of the effect of monotherapies or doublet LDM/MTD regimens on CA9+ area. F(5,11) = 2.466 and P = 0.0961 (not significant) for overall treatment by Brown-Forsythe ANOVA. D: Correlation between HIF-1α levels and proliferation indexes in HT29 primary tumors. Left and middle/left: HIF-1α and Ki67 expression in vehicle-treated controls; consecutive sections are shown. Middle/right: HIF-1α+ vs Ki67+ proliferation index scatterplot. Each point represents median values for each tumor. Regression line (red) and 95% CI (shaded blue area) are indicated. F(1,27) = 34.45 and P = 10-4; slope ≠ 0 by F-test Right: Machine-learning quantification of the effect of monotherapies or doublet LDM/MTD regimens upon Ki67+ proliferative index. Median indexes per tumor are indicated. F(5,12) = 1.609 and P = 0.2312 (not significant) for overall treatment by Brown-Forsythe ANOVA.


Figure 2A : Effect of LDM and MTD chemotherapy on HT29 liver metastatic nodule size . Left : Histogram of pooled cross - sectional metastatic diameter ( Ø ) . Middle : Metastatic diameter ( Ø ) by chemotherapy regimen . A decreasing linear trend for median metastatic diameter was observed ( left to right , slope = - 143 . 2 , P = 0 . 043 by post - test for trend ) . Right : Dichotomized metastatic size at median diameter of vehicle - treated tumors . Liver nodule size was classified as small ( blue ) or large ( red ) . Individual nodule counts per group and size category ( small / large ) are plotted on the right ordinate . χ2 ( df = 5 ) = 6 . 80 ; P = 0 . 2361 ( not significant ) for overall effects on size ; χ2 ( df = 1 ) = 4 . 71 ; P = 0 . 0299 ( LDMCTX + LDMCPB vs vehicle ) . B : HIF - 1α levels in HT29 liver metastatic nodules . Left : vehicle - treated controls . Middle / left : Doublet LDM cyclophosphamide + capecitabine ( LDMCTX + LDMCPB ) . Middle / right : LDM cyclophosphamide + MTD capecitabine ( LDMCTX + MTDCPB ) . Right : Automatic quantification of the effect of monotherapies or doublet regimens on HIF - 1α + areas in individual metastatic nodules . F ( 2 , 13 ) = 4 . 796 and P = 0 . 028 for overall treatment by Brown - Forsythe ANOVA ; * , P = 0 . 038 LDMCTX + MTDCPB vs vehicle or P = 0 . 0123 LDMCTX + LDMCPB vs vehicle ; # , P = 0 . 0098 LDMCTX + LDMCPB vs LDMCTX + MTDCPB by Benjamini , Krieger and Yekuteli post hoc test . C : Correlation between metastatic and peri - metastatic HIF - 1α levels in the liver . Left : Nodule immunostaining and parenchymal HIF - 1α expression . Each point represents median values per nodule . Regression line ( blue ) and 95 % CI ( shaded blue area ) are indicated . F ( 1 , 57 ) = 150 . 7 and P < 0 . 0001 ; slope ≠ 0 by F - test . Right : Correlation between intra - metastatic and peri - metastatic parenchymatous ring in the liver . Each point represents median values per nodule . Regression line ( blue ) and 95 % CI ( shaded blue area ) are indicated . F ( 1 , 54 ) = 55 . 2 and P < 0 . 0001 ; slope ≠ 0 by F - test . . D : Correlation between metastatic and peri - metastatic HIF - 2α levels in the liver . Left : Nodule immunostaining and parenchymal HIF - 2α expression . Right : Correlation between intra - metastatic and peri - metastatic parenchymatous ring in the liver . Each point represents median values for each nodule . Regression line ( blue ) and 95 % CI ( shaded blue area ) are indicated . F ( 1 , 54 ) = 55 . 2 and P < 0 . 0001 ; slope ≠ 0 by F - test . n , number of nodules .

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 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 O O O O 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

Figure 2A: Effect of LDM and MTD chemotherapy on HT29 liver metastatic nodule size. Left: Histogram of pooled cross-sectional metastatic diameter (Ø). Middle: Metastatic diameter (Ø) by chemotherapy regimen. A decreasing linear trend for median metastatic diameter was observed (left to right, slope = -143.2, P = 0.043 by post-test for trend). Right: Dichotomized metastatic size at median diameter of vehicle-treated tumors. Liver nodule size was classified as small (blue) or large (red). Individual nodule counts per group and size category (small/large) are plotted on the right ordinate. χ2 (df=5) = 6.80; P= 0.2361 (not significant) for overall effects on size; χ2 (df=1) = 4.71; P= 0.0299 (LDMCTX + LDMCPB vs vehicle). B: HIF-1α levels in HT29 liver metastatic nodules. Left: vehicle- treated controls. Middle/left: Doublet LDM cyclophosphamide + capecitabine (LDMCTX + LDMCPB). Middle/right: LDM cyclophosphamide + MTD capecitabine (LDMCTX + MTDCPB). Right: Automatic quantification of the effect of monotherapies or doublet regimens on HIF-1α+ areas in individual metastatic nodules. F(2,13) = 4.796 and P = 0.028 for overall treatment by Brown-Forsythe ANOVA; *, P= 0.038 LDMCTX + MTDCPB vs vehicle or P= 0.0123 LDMCTX + LDMCPB vs vehicle; #, P= 0.0098 LDMCTX + LDMCPB vs LDMCTX + MTDCPB by Benjamini, Krieger and Yekuteli post hoc test. C: Correlation between metastatic and peri-metastatic HIF-1α levels in the liver. Left: Nodule immunostaining and parenchymal HIF-1α expression. Each point represents median values per nodule. Regression line (blue) and 95% CI (shaded blue area) are indicated. F(1,57) = 150.7 and P< 0.0001; slope ≠ 0 by F-test. Right: Correlation between intra-metastatic and peri-metastatic parenchymatous ring in the liver. Each point represents median values per nodule. Regression line (blue) and 95% CI (shaded blue area) are indicated. F(1,54) = 55.2 and P< 0.0001; slope ≠ 0 by F-test.. D: Correlation between metastatic and peri-metastatic HIF-2α levels in the liver. Left: Nodule immunostaining and parenchymal HIF-2α expression. Right: Correlation between intra-metastatic and peri-metastatic parenchymatous ring in the liver. Each point represents median values for each nodule. Regression line (blue) and 95% CI (shaded blue area) are indicated. F(1,54) = 55.2 and P< 0.0001; slope ≠ 0 by F-test. n, number of nodules.


Figure 3A : EMT6 - CDDP lung metastatic nodule size . Left : Histogram of cross - sectional metastatic diameter ( Ø ) . Middle : Metastatic diameter ( Ø ) by chemotherapy regimen . F ( 5 , 99 ) = 0 . 9310 and P = 0 . 4644 ( not significant ) for overall treatment by Brown - Forsythe ANOVA . Right : Dichotomized metastatic size at median diameter of vehicle - treated tumors . Lung nodule size was classified as small ( blue ) or large ( red ) . Individual nodule counts per group and size category ( small / large ) are plotted on the right ordinate . χ2 ( df = 5 ) = 2 . 631 ; P = 0 . 7567 ( not significant ) for overall effects on size . B : Hypoxic fraction in lung metastatic nodules . Pimonidazole adduct immunoreactivity was automatically quantified and expressed as fractional positive areas per nodule ( rightmost panel ) . F ( 5 , 110 ) = 7 . 544 and P < 0 . 0001 for overall treatment by Brown - Forsythe ANOVA ; * , P < 0 . 01 vs vehicle ; # , P = 0 . 0111 doublet LDM cyclophosphamide + capecitabine vs LDM cyclophosphamide + MTD capecitabine by Benjamini , Krieger and Yekuteli post hoc test . C : HIF - 1α levels in lung metastatic nodules . Automatic quantification of HIF - 1α levels measured as fractional positive areas per nodule ( rightmost panel ) . F ( 5 , 114 ) = 5 . 325 and P = 0 . 0004 for overall treatment by Brown - Forsythe ANOVA ; * , P < 0 . 01 vs vehicleby Benjamini , Krieger and Yekuteli post hoc test . D : Microvessel density in lung metastatic nodules . Automatic quantification of CD31 fractional areas per nodule ( rightmost panel ) . F ( 5 , 124 ) = 7 . 531 and P < 0 . 0001 for overall treatment by Brown - Forsythe ANOVA ; * , P < 0 . 01 ( LDM capecitabine , cyclophosphamide , or doublet capecitabine + cyclophosphamide vs vehicle ) , # , P = 0 . 0417 ( doublet LDM capecitabine + cyclophosphamide vs doublet LDM cyclophosphamide + MTD capecitabine ) by Benjamini , Krieger and Yekuteli post hoc test .

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

Figure 3A: EMT6-CDDP lung metastatic nodule size. Left: Histogram of cross-sectional metastatic diameter (Ø). Middle: Metastatic diameter (Ø) by chemotherapy regimen. F(5,99) = 0.9310 and P= 0.4644 (not significant) for overall treatment by Brown-Forsythe ANOVA. Right: Dichotomized metastatic size at median diameter of vehicle-treated tumors. Lung nodule size was classified as small (blue) or large (red). Individual nodule counts per group and size category (small/large) are plotted on the right ordinate. χ2 (df=5) = 2.631; P= 0.7567 (not significant) for overall effects on size. B: Hypoxic fraction in lung metastatic nodules. Pimonidazole adduct immunoreactivity was automatically quantified and expressed as fractional positive areas per nodule (rightmost panel). F(5,110) = 7.544 and P< 0.0001 for overall treatment by Brown-Forsythe ANOVA; *, P< 0.01 vs vehicle; #, P= 0.0111 doublet LDM cyclophosphamide + capecitabine vs LDM cyclophosphamide + MTD capecitabine by Benjamini, Krieger and Yekuteli post hoc test.C: HIF-1α levels in lung metastatic nodules. Automatic quantification of HIF-1α levels measured as fractional positive areas per nodule (rightmost panel). F(5,114) = 5.325 and P= 0.0004 for overall treatment by Brown-Forsythe ANOVA; *, P< 0.01 vs vehicleby Benjamini, Krieger and Yekuteli post hoc test.D: Microvessel density in lung metastatic nodules. Automatic quantification of CD31 fractional areas per nodule (rightmost panel). F(5,124) = 7.531 and P< 0.0001 for overall treatment by Brown-Forsythe ANOVA; *, P< 0.01 (LDM capecitabine, cyclophosphamide, or doublet capecitabine + cyclophosphamide vs vehicle), #, P= 0.0417 (doublet LDM capecitabine + cyclophosphamide vs doublet LDM cyclophosphamide + MTD capecitabine) by Benjamini, Krieger and Yekuteli post hoc test.


Figure 4B : HIFα - inducible colon cancer signature ( HIFi - CCS ) . Left and middle / left : List of HIFα targets and overall survival in TCGA ( left ) or aggregated microarray data ( middle / left ) from colon adenocarcinomas . Cases are classified as low ( blue ) or high ( red ) HIFi - CCS according to their relationship to the median . Kaplan - Meier analysis followed by log - rank test . Middle / right : HIFi - CCS and lymph node ( LN ) invasion status . Data are standardized as z - scores . t ( 194 ) = 4 . 399 and P < 0 . 0001 by Student t - test with Welch ' s correction . Right : HIFi - CCS is associated with accelerated distant metastasis . Cases are classified as low ( blue ) or high ( red ) HIFi - CCS according to their relationship to the median . Kaplan - Meier analysis followed by log - rank test . C : HIFα - inducible breast cancer signature ( HIFi - BCS ) . Left and middle / left : List of HIFα targets and overall survival in METABRIC ( left ) or recurrence - free survival in microarray ( middle / left ) from breast adenocarcinomas . Cases are classified as low ( blue ) or high ( red ) HIFi - BCS according to their relationship to the median . Kaplan - Meier analysis followed by log - rank test . Middle / right : HIFi - BCS and estrogen receptor ( ER ) status . Data are standardized as z - scores . t ( 112 ) = 3 . 789 and P = 0 . 0002 by Student t - test with Welch ' s correction . Right : HIFi - BCS is associated with worsened distant metastasis - free survival . Cases are classified as low ( blue ) or high ( red ) HIFi - BCS whenever they are below or above the distribution median . Kaplan - Meier analysis followed by log - rank test . D : Median RNAseq expression of individual HIFα targets as split by median HIFi - CCS ( left ) or HIFi - BCS ( right ) . Transcripts up / downregulated by ± 1 . 5 - fold are shown in blue or yellow , respectively . Error bars indicate 95 % CI of the median . Log2 scale ; n = 209 ( colon , TCGA ) or n = 609 ( breast , METABRIC ) .

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 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 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

Figure 4B: HIFα- inducible colon cancer signature (HIFi-CCS). Left and middle/left: List of HIFα targets and overall survival in TCGA (left) or aggregated microarray data (middle/left) from colon adenocarcinomas. Cases are classified as low (blue) or high (red) HIFi-CCS according to their relationship to the median. Kaplan-Meier analysis followed by log-rank test. Middle/right: HIFi-CCS and lymph node (LN) invasion status. Data are standardized as z-scores. t(194) = 4.399 and P< 0.0001 by Student t-test with Welch's correction. Right: HIFi-CCS is associated with accelerated distant metastasis. Cases are classified as low (blue) or high (red) HIFi-CCS according to their relationship to the median. Kaplan-Meier analysis followed by log-rank test. C: HIFα- inducible breast cancer signature (HIFi-BCS). Left and middle/left: List of HIFα targets and overall survival in METABRIC (left) or recurrence- free survival in microarray (middle/left) from breast adenocarcinomas. Cases are classified as low (blue) or high (red) HIFi-BCS according to their relationship to the median. Kaplan-Meier analysis followed by log-rank test. Middle/right: HIFi-BCS and estrogen receptor (ER) status. Data are standardized as z-scores. t(112) = 3.789 and P= 0.0002 by Student t-test with Welch's correction. Right: HIFi-BCS is associated with worsened distant metastasis-free survival. Cases are classified as low (blue) or high (red) HIFi-BCS whenever they are below or above the distribution median. Kaplan-Meier analysis followed by log-rank test. D: Median RNAseq expression of individual HIFα targets as split by median HIFi-CCS (left) or HIFi-BCS (right). Transcripts up/downregulated by ±1.5-fold are shown in blue or yellow, respectively. Error bars indicate 95% CI of the median. Log2 scale; n= 209 (colon, TCGA) or n= 609 (breast, METABRIC).


Figure 1B ) , C ) Volcano plots showing the fold change in average expression of all proteins and phosphopeptides respectively , in CKO cells compared to WT ( q = Benjamini - Hochberg corrected p - value , n = 24 time points over 3 days ) . Statistically significant changes ( q ≤ 0 . 05 ) are shown in red . Some proteins are labelled as space allows .

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

Figure 1B), C) Volcano plots showing the fold change in average expression of all proteins and phosphopeptides respectively, in CKO cells compared to WT (q = Benjamini-Hochberg corrected p-value, n= 24 time points over 3 days). Statistically significant changes (q ≤ 0.05) are shown in red. Some proteins are labelled as space allows.


Figure 2A ) Fold - change in relative amplitude ( RA ) was calculated for each of the proteins found to be rhythmic in both genotypes by RAIN analysis ( i . e . no RA cut - off ) . The mean fold change ( log ) in RA was an increase of 14 % in CKO cells compared to WT cells ( One sample t - test , * * * * p < 0 . 0001 ) . B ) For each genotype , all proteins were divided into 10 deciles of equal number , ranked by abundance . The mean abundance of each decile was plotted against the proportion of the decile that was rhythmic . Linear regression lines are shown for each genotype , and the slopes were significantly non - zero ( F test , WT * * p = 0 . 0016 , CKO * * * * p < 0 . 0001 ) . The slopes were also significantly different to each other ( F test , p = 0 . 0001 ) . C ) Fold - change in relative amplitude ( RA ) was calculated for each of the phosphopeptides found to be rhythmic in both genotypes by RAIN . On average , the RA was increased in CKO cells compared to WT cells by 6 % ( One sample t - test , * p < 0 . 05 ) . D ) The same analysis in B ) was carried out for phosphopeptides . The slopes were significantly non - zero in CKO but not WT ( F test , WT p = 0 . 3 ( ns . : non significant ) , CKO * p = 0 . 04 ) . The slopes were also significantly different to each other ( F test , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fold-change in relative amplitude (RA) was calculated for each of the proteins found to be rhythmic in both genotypes by RAIN analysis (i.e. no RA cut-off). The mean fold change (log) in RA was an increase of 14% in CKO cells compared to WT cells (One sample t-test, ****p<0.0001). B) For each genotype, all proteins were divided into 10 deciles of equal number, ranked by abundance. The mean abundance of each decile was plotted against the proportion of the decile that was rhythmic. Linear regression lines are shown for each genotype, and the slopes were significantly non-zero (F test, WT **p=0.0016, CKO ****p<0.0001). The slopes were also significantly different to each other (F test, p=0.0001). C) Fold-change in relative amplitude (RA) was calculated for each of the phosphopeptides found to be rhythmic in both genotypes by RAIN. On average, the RA was increased in CKO cells compared to WT cells by 6% (One sample t-test, *p<0.05). D) The same analysis in B) was carried out for phosphopeptides. The slopes were significantly non-zero in CKO but not WT (F test, WT p=0.3 (ns.: non significant), CKO *p=0.04). The slopes were also significantly different to each other (F test, p<0.0001).


Figure 3B ) Bar chart reports the number and % of detected proteins showing a significant difference in abundance for the listed comparisons by two - way ANOVA correcting for multiple comparisons , controlling for false discovery rate by the Benjamini , Krieger and Yekutieli method ( q = 0 . 05 ) . D - E ) Proteins with significant abundance change over time had no significant difference in mean absolute fold change between WT and CKO cells ( D , p = 0 . 47 ) , but significantly greater variance in absolute fold change between independent CKO biological replicates than between their WT counterparts ( E , * * p = 0 . 0013 ) ; Kolmogorov - Smirmov test ( n = 4 ) . Continuous lines = median , dotted lines = quartiles .

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 O O 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 3B) Bar chart reports the number and % of detected proteins showing a significant difference in abundance for the listed comparisons by two-way ANOVA correcting for multiple comparisons, controlling for false discovery rate by the Benjamini, Krieger and Yekutieli method (q=0.05).D-E) Proteins with significant abundance change over time had no significant difference in mean absolute fold change between WT and CKO cells (D, p=0.47), but significantly greater variance in absolute fold change between independent CKO biological replicates than between their WT counterparts (E, **p=0.0013); Kolmogorov-Smirmov test (n=4). Continuous lines = median, dotted lines = quartiles.


Figure 4A ) From the quantitative proteomics experiment , average abundance of catalytic proteasome subunits was calculated and normalised to WT means . Trypsin - like ( β2 ) , chymotrypsin - like ( β3 ) and caspase - like ( β1 ) catalytic subunits are shown . The average was calculated from all 24 time points of the proteomics experiment . Mean ± SD , 2 - way ANOVA with Holm - Sidak ' s multiple comparisons , * * * * p ≤ 0 . 0001 . B ) Representative Western blot using an antibody that recognises all 7 α subunits of the 20S proteasome , with anti - histone H3 as loading control . C ) Quantification of the blots in B ) , using all replicates ( n = 3 ) , normalised to WT mean . Mean ± SD , one - tailed Student ' s t test with Welch correction - prior prediction from experiment in A ) that CKO abundance would be lower , * p ≤ 0 . 05 .

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 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

Figure 4A) From the quantitative proteomics experiment, average abundance of catalytic proteasome subunits was calculated and normalised to WT means. Trypsin-like (β2), chymotrypsin-like (β3) and caspase-like (β1) catalytic subunits are shown. The average was calculated from all 24 time points of the proteomics experiment. Mean±SD, 2-way ANOVA with Holm-Sidak's multiple comparisons, ****p≤0.0001.B) Representative Western blot using an antibody that recognises all 7 α subunits of the 20S proteasome, with anti-histone H3 as loading control. C) Quantification of the blots in B), using all replicates (n=3), normalised to WT mean. Mean±SD, one-tailed Student's t test with Welch correction - prior prediction from experiment in A) that CKO abundance would be lower, *p≤0.05.