Percentage 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 .

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

Percentage 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 ) .

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

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

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%.


The 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 .

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

The 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 .

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

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 .

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

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 .

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

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 .

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

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

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.


Scatter 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 .

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

Scatter 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


Column 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 ) .

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

Column 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 cytokine


representing CD4 T cells expressing only cytokine ,

representing CD4 T cells expressing only cytokine, two


CD4 T cells expressing only three cytokines simultaneously


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 .

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

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.


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 .

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

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 .

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

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 .

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

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.


Scatter 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 .

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

Scatter 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 .

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

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 .

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

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.


Kaplan - 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 ) .

Kaplan-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 .

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

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.


B - 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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.


Live 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 .

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

Live 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 .

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.


A - 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 .

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

A-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 .

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

F-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.


A - 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 ) .

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

A-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).


A - 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 ) .

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

A-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).


A - 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 .

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

A-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.


A - 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 .

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

A-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 .

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

I-L Representative kymographs of EB1-GFP comets driven by Gal44-77 in the proximal dendrites of ddaC neurons at 96 h AEL.


A : 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 .

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

A: 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: 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 .

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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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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.


A : 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 ) .

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A: 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 .

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

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 . .

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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 .

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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.


A : 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 .

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

A: 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 .

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

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 .

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

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.


B : 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 .

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B: 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 .

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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 ) .

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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).


B ) , 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 .

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B), 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.


A ) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A) 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).


B ) 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 ) .

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B) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D-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.


A ) 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 .

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

A) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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.


D - E ) From one time - course experiment , ions , cytosolic proteins and total protein were extracted in parallel samples . The presented experiment is representative of 3 separate time - course experiments that were carried out ( N = 3 ) . Representative experiment shown ( n = 4 technical replicates for [ K + ] , n = 3 technical replicates for the other traces ) . Blue lines highlight the antiphasic relationship between oscillations in cytosolic protein and potassium concentration . Mean ± SEM , p - values from RAIN , red lines are fits by a damped cosine compared with a straight line ( null hypothesis ) . Parallel PER2 : : LUC recordings were also performed and plotted as a phase marker .

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

D-E) From one time-course experiment, ions, cytosolic proteins and total protein were extracted in parallel samples. The presented experiment is representative of 3 separate time-course experiments that were carried out (N=3). Representative experiment shown (n=4 technical replicates for [K+], n=3 technical replicates for the other traces). Blue lines highlight the antiphasic relationship between oscillations in cytosolic protein and potassium concentration. Mean±SEM, p-values from RAIN, red lines are fits by a damped cosine compared with a straight line (null hypothesis). Parallel PER2::LUC recordings were also performed and plotted as a phase marker.


F ) Examples of key ion transporters are shown , as detected in the proteomics experiment . P values show the results of an F test comparing fits of damped cosine against straight line . All proteins except WT NKCC1 had RAIN p - values < 0 . 05 .

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

F) Examples of key ion transporters are shown, as detected in the proteomics experiment. P values show the results of an F test comparing fits of damped cosine against straight line. All proteins except WT NKCC1 had RAIN p-values <0.05.


A ) WT and CKO cells were treated with 500 nM tunicamycin ( TUN ) and lysed in RIPA buffer at time points between 0 - 24 hours afterwards . Western blots were carried out , probing for phosphorylated eIF2α , total eIF2α and actin . Representative blots shown , n = 4 . B ) Quantification of all replicates represented in ( A ) , where phosphorylated eIF2α is normalised to total eIF2α . Mean ± SD . 2 - way ANOVA ( TWA ) with Holm - Sidak ' s multiple comparisons .

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

A) WT and CKO cells were treated with 500 nM tunicamycin (TUN) and lysed in RIPA buffer at time points between 0-24 hours afterwards. Western blots were carried out, probing for phosphorylated eIF2α, total eIF2α and actin. Representative blots shown, n=4. B) Quantification of all replicates represented in (A), where phosphorylated eIF2α is normalised to total eIF2α. Mean±SD. 2-way ANOVA (TWA) with Holm-Sidak's multiple comparisons.


C ) Mice were housed in 12h : 12h light - dark cycles , with unrestricted feeding . Mouse lungs were collected 7 hours after transition to the light phase , 5 hours after intraperitoneal injection of mice with bortezomib ( BTZ , 2 . 5 mg / kg ) or vehicle ( Veh , 1 % DMSO in sterile PBS ) . Tissues were lysed in RIPA buffer and western blots were performed , probing for phosphorylated STAT3 , total STAT3 and actin . Male ( left ) and female ( right ) replicates shown , N = 2 mice for each condition . D ) Quantification of the blots shown in ( C ) , where phosphorylated STAT3 is normalised to total STAT3 . Mean ± SD . Multiple t - tests , corrected for multiple comparisons with the Holm - Sidak method , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 .

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

C) Mice were housed in 12h:12h light-dark cycles, with unrestricted feeding. Mouse lungs were collected 7 hours after transition to the light phase, 5 hours after intraperitoneal injection of mice with bortezomib (BTZ, 2.5 mg/kg) or vehicle (Veh, 1% DMSO in sterile PBS). Tissues were lysed in RIPA buffer and western blots were performed, probing for phosphorylated STAT3, total STAT3 and actin. Male (left) and female (right) replicates shown, N=2 mice for each condition. D) Quantification of the blots shown in (C), where phosphorylated STAT3 is normalised to total STAT3. Mean±SD. Multiple t-tests, corrected for multiple comparisons with the Holm-Sidak method, *p≤0.05, ***p≤0.001.


A ) WT PER2 : : LUC cells were treated with epoxomicin or vehicle control . Blue arrows show the time points where drug was washed off . Mean ( solid lines ) ± SD ( dotted lines ) . B ) Quantification of damping rate of the PER2 : : LUC recordings shown in A ) , for the duration of drug treatment . Mean ± SD . One - way ANOVA with Holm - Sidak ' s multiple comparisons , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 . C ) WT PER2 : : LUC cells were treated with tunicamycin or vehicle control . Mean ( solid lines ) ± SD ( dotted lines ) . D ) Quantification of damping rate of the PER2 : : LUC recordings shown in C ) , for the duration of drug treatment . Mean ± SD . One - way ANOVA with Holm - Sidak ' s multiple comparisons , * * * * p ≤ 0 . 0001 .

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

A) WT PER2::LUC cells were treated with epoxomicin or vehicle control. Blue arrows show the time points where drug was washed off. Mean (solid lines) ± SD (dotted lines). B) Quantification of damping rate of the PER2::LUC recordings shown in A), for the duration of drug treatment. Mean±SD. One-way ANOVA with Holm-Sidak's multiple comparisons, **p≤0.01, ****p≤0.0001. C) WT PER2::LUC cells were treated with tunicamycin or vehicle control. Mean (solid lines) ± SD (dotted lines). D) Quantification of damping rate of the PER2::LUC recordings shown in C), for the duration of drug treatment. Mean±SD. One-way ANOVA with Holm-Sidak's multiple comparisons, ****p≤0.0001.


E ) WT PER2 : : LUC cells were treated with radicicol or vehicle control . Blue arrows show the time points where drug was washed off . Mean ( solid lines ) ± SD ( dotted lines ) . F ) Quantification of damping rate of the PER2 : : LUC recordings shown in E ) , for the duration of drug treatment . Mean ± SD . One - way ANOVA with Holm - Sidak ' s multiple comparisons , * * * * p ≤ 0 . 0001 .

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

E) WT PER2::LUC cells were treated with radicicol or vehicle control. Blue arrows show the time points where drug was washed off. Mean (solid lines) ± SD (dotted lines). F) Quantification of damping rate of the PER2::LUC recordings shown in E), for the duration of drug treatment. Mean±SD. One-way ANOVA with Holm-Sidak's multiple comparisons, ****p≤0.0001.


I - J ) Representative double - plotted actograms of WT mice receiving drinking water with DMSO vehicle control ( N = 7 mice ) or 35 µg / mL ixazomib ( N = 8 mice ) . Two representative actograms are shown per condition . For the first 7 days the mice were in 12 : 12h light - dark cycles ( LD ) , and had drinking water supplemented with blackcurrant squash . Ixazomib / DMSO was added to this , and the lighting was changed to constant darkness ( DD ) . After 7 days the drug / vehicle was removed , and the mice remained in DD until the end of the experiment .

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

I-J) Representative double-plotted actograms of WT mice receiving drinking water with DMSO vehicle control (N=7 mice) or 35 µg/mL ixazomib (N=8 mice). Two representative actograms are shown per condition. For the first 7 days the mice were in 12:12h light-dark cycles (LD), and had drinking water supplemented with blackcurrant squash. Ixazomib/DMSO was added to this, and the lighting was changed to constant darkness (DD). After 7 days the drug/vehicle was removed, and the mice remained in DD until the end of the experiment.


CRH - Cre : : Ai9 reporter male mice fed on chow or 4 weeks on HFD were used to examine c - Fos expression in the PVH in 24hr fasting or refed after 24hr fasting . A ) Representative images showing CRH neurons ( red in the first colomn ) , c - Fos expression ( green in the second colomn ) , merged of red and green ( the third colomn ) and the amplied pictures for the boxed areas in the merged images ( the forth colomn ) . The experimental conditions for each group ( fasting verus refed , chow versus HFD ) are labelled on the left . Scale bar = 200µm .

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

CRH-Cre::Ai9 reporter male mice fed on chow or 4 weeks on HFD were used to examine c-Fos expression in the PVH in 24hr fasting or refed after 24hr fasting. A) Representative images showing CRH neurons (red in the first colomn), c-Fos expression (green in the second colomn), merged of red and green (the third colomn) and the amplied pictures for the boxed areas in the merged images (the forth colomn). The experimental conditions for each group (fasting verus refed, chow versus HFD) are labelled on the left. Scale bar=200µm.


B - C ) Comparison of average neuron numbers from cell counting on total number of c - Fos positive neurons in the PVH ( B , * * p = 0 . 0032 : chow fasting versus chow refed and * p = 0 . 023 , HFD fasting versus HFD refed ) and total number of c - Fos positive CRH neurons ( C , * * p < 0 . 0001 : chow fasting versus chow refed and p = 0 . 7158 : HFD fasting versus HFD refed ) , n = 3 - 4 each , data presented as mean + / - SEM , two way ANOVA tests .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) Comparison of average neuron numbers from cell counting on total number of c-Fos positive neurons in the PVH (B, **p=0.0032: chow fasting versus chow refed and *p=0.023, HFD fasting versus HFD refed) and total number of c-Fos positive CRH neurons (C, **p<0.0001: chow fasting versus chow refed and p=0.7158: HFD fasting versus HFD refed), n=3-4 each, data presented as mean +/-SEM, two way ANOVA tests.


Effects of HFD feeding on diurnal rhythmicity in feeding ( D and E ) The measurement was collected with male mice fed chow or after 4 weeks HFD feeding , n = 6 each , data presented as mean + / - SEM , * * p = 0 . 0066 in E and * * p = 0 . 0 . 0007 in G , unpaired Student ' s t tests .

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

Effects of HFD feeding on diurnal rhythmicity in feeding (D and E) The measurement was collected with male mice fed chow or after 4 weeks HFD feeding, n=6 each, data presented as mean +/- SEM, **p=0.0066 in E and **p=0.0.0007 in G, unpaired Student's t tests.


Effects of HFD feeding on diurnal rhythmicity in energy expenditure ( F and G ) . The measurement was collected with male mice fed chow or after 4 weeks HFD feeding , n = 6 each , data presented as mean + / - SEM , * * p = 0 . 0066 in E and * * p = 0 . 0 . 0007 in G , unpaired Student ' s t tests .

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

Effects of HFD feeding on diurnal rhythmicity in energy expenditure (F and G). The measurement was collected with male mice fed chow or after 4 weeks HFD feeding, n=6 each, data presented as mean +/- SEM, **p=0.0066 in E and **p=0.0.0007 in G, unpaired Student's t tests.


CRH - Cre mice received delivery of AAV - Flex - GCaMP6m viral vectors to bilateral PVH and optic fiber implantation targeting PVH neurons , and were used for in vivo live recording . ( H - I ) Representative traces showing recordings of activity changes in CRH neuron in response to stressor ( water spray ) in chow - fed controls ( H ) , and 4 weeks after HFD ( I ) , ( J ) Comparison of average responses in the two conditions , n = 4 each , * * p = 0 . 0006 , paired Student ' s t test .

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

CRH-Cre mice received delivery of AAV-Flex-GCaMP6m viral vectors to bilateral PVH and optic fiber implantation targeting PVH neurons, and were used for in vivo live recording. (H-I) Representative traces showing recordings of activity changes in CRH neuron in response to stressor (water spray) in chow-fed controls (H), and 4 weeks after HFD (I), (J) Comparison of average responses in the two conditions, n=4 each, **p=0.0006, paired Student's t test.


K - L ) Representative traces showing recordings of activity changes in CRH neurons in response to Dexamethasone ( Dex ) injections in chow - fed controls ( K ) and after 4 weeks on HFD ( L ) . M ) Comparison of average responses in the two conditions , n = 4 each , data presented as mean + / - SEM , * * P = 0 . 0012 , paired Student ' s t tests .

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

K-L) Representative traces showing recordings of activity changes in CRH neurons in response to Dexamethasone (Dex) injections in chow-fed controls (K) and after 4 weeks on HFD (L). M) Comparison of average responses in the two conditions, n=4 each, data presented as mean +/- SEM, **P=0.0012, paired Student's t tests.


CRH - Cre male mice received stereotaxic delivery of AAV - EF1a - Flex - EGFP - P2A - mNachBac vectors at 7 - 8 weeks of age and recordings were made at least 4 weeks after viral delivery on neurons with NachBac ( one side ) or control neurons ( contralateral side ) of the same animal ( A ) ;

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

CRH-Cre male mice received stereotaxic delivery of AAV-EF1a-Flex-EGFP-P2A-mNachBac vectors at 7-8 weeks of age and recordings were made at least 4 weeks after viral delivery on neurons with NachBac (one side) or control neurons (contralateral side) of the same animal (A);


typical action potentials ( APs ) in controls ( B , top panel ) and typical NachBac - mediated APs showing lower threshold and long duration ( B , bottom , the inset highlighting the long duration ) in NachBac

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

typical action potentials (APs) in controls (B, top panel) and typical NachBac-mediated APs showing lower threshold and long duration (B, bottom, the inset highlighting the long duration) in NachBac mice


the distribution of the membrane potential and firing cells in the two groups ( C , ratios in the bracket denoting neuron number out of total number recorded with the indicated behavior ) .

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

the distribution of the membrane potential and firing cells in the two groups (C, ratios in the bracket denoting neuron number out of total number recorded with the indicated behavior).


CRH - Cre male mice were injected with either AAV - Flex - GFP or AAV - EF1a - Flex - EGFP - P2A - mNachBac to examine c - Fos expression in the PVH in 24hr fasting or refed after fasting . ( D ) Representative images showing CRH neurons expressing GFP or NachBac ( GFP in the top 2 rows and NachBac in the bottom 2 rows of the first colomn ) , c - Fos expression ( red in the second colomn ) , merged of red and green ( the third colomn ) and the amplied pictures for the boxed areas in the merged images ( the forth colomn ) . The experimental conditions for each group ( fasting verus refed ) are labelled on the left .

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

CRH-Cre male mice were injected with either AAV-Flex-GFP or AAV-EF1a-Flex-EGFP-P2A-mNachBac to examine c-Fos expression in the PVH in 24hr fasting or refed after fasting. (D) Representative images showing CRH neurons expressing GFP or NachBac (GFP in the top 2 rows and NachBac in the bottom 2 rows of the first colomn), c-Fos expression (red in the second colomn), merged of red and green (the third colomn) and the amplied pictures for the boxed areas in the merged images (the forth colomn). The experimental conditions for each group (fasting verus refed) are labelled on the left.


( E - F ) Comparison of average neuron numbers from cell counting on total number of c - Fos positive neurons in the PVH ( E , * * p < 0 . 0001 : fasting versus refed ; * * p = 0 . 0026 : fasting versus fasting / NachBac ; * * p < 0 . 0001 : fasting / NachBac versus refed / NachBac ) and total number of c - Fos positive CRH neurons ( F , * * p < 0 . 0001 : fasting versus refed ; * * p < 0 . 0001 : fasting versus fasting / NachBac ; p = 0 . 3423 : fasting / NachBac versus refed / NachBac ) , n = 3 - 4 each , data presented as mean + / - SEM , two way ANOVA tests .

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

(E-F) Comparison of average neuron numbers from cell counting on total number of c-Fos positive neurons in the PVH (E, **p<0.0001: fasting versus refed; **p=0.0026: fasting versus fasting/NachBac; **p<0.0001: fasting/NachBac versus refed/NachBac) and total number of c-Fos positive CRH neurons (F, **p<0.0001: fasting versus refed; **p<0.0001: fasting versus fasting/NachBac; p=0.3423: fasting/NachBac versus refed/NachBac), n=3-4 each, data presented as mean +/- SEM, two way ANOVA tests.


CRH - Cre male mice received stereotaxic delivery of a mixture of AAV - Flex - GCaMP6m and AAV - EF1a - Flex - EGFP - P2A - mNachBac vectors at 7 - 8 weeks of age with optic fiber implantation targeting PVH neurons . ( G - H ) Representative traces showing typical responses of PVH CRH neurons to water spay ( arrows ) in NachBac mice ( G ) , and comparison of responsiveness to water spray between NachBac and control mice ( H ) .

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

CRH-Cre male mice received stereotaxic delivery of a mixture of AAV-Flex-GCaMP6m and AAV-EF1a-Flex-EGFP-P2A-mNachBac vectors at 7-8 weeks of age with optic fiber implantation targeting PVH neurons. (G-H) Representative traces showing typical responses of PVH CRH neurons to water spay (arrows) in NachBac mice (G), and comparison of responsiveness to water spray between NachBac and control mice (H).


( I - J ) Representative traces showing typical responses of PVH CRH neurons to water spay ( arrows ) in NachBac mice ( I ) , and comparison of responsiveness to water spray between NachBac and control mice ( J ) . Gray traces showed Ca2 + independent signal for system stability and red ones showed Ca2 + dependent signals for neuron activity , N = 5 - 7 each , males , data presented as mean + / - SEM , * * p < 0 . 0001 in both ( H and J ) , unpaired Student ' s t tests .

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

(I-J) Representative traces showing typical responses of PVH CRH neurons to water spay (arrows) in NachBac mice (I), and comparison of responsiveness to water spray between NachBac and control mice (J).Gray traces showed Ca2+ independent signal for system stability and red ones showed Ca2+ dependent signals for neuron activity, N=5-7 each, males, data presented as mean +/-SEM, **p<0.0001 in both (H and J), unpaired Student's t tests.


CRH - Cre male mice received stereotaxic delivery of AAV - EF1a - Flex - EGFP - P2A - mNachBac or control GFP vectors at 7 - 8 weeks of age and were maintained on chow diet for 7 weeks before switching to HFD . Weekly body weight gain after viral delivery ( A ) at 11 weeks after viral delivery .

CRH-Cre male mice received stereotaxic delivery of AAV-EF1a-Flex-EGFP-P2A-mNachBac or control GFP vectors at 7-8 weeks of age and were maintained on chow diet for 7 weeks before switching to HFD. Weekly body weight gain after viral delivery (A) at 11 weeks after viral delivery.


CRH - Cre male mice received stereotaxic delivery of AAV - EF1a - Flex - EGFP - P2A - mNachBac or control GFP vectors at 7 - 8 weeks of age and were maintained on chow diet for 7 weeks before switching to HFD . measurements of fat mass ( B ) and lean mass ( C ) at 11 weeks after viral delivery .

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

CRH-Cre male mice received stereotaxic delivery of AAV-EF1a-Flex-EGFP-P2A-mNachBac or control GFP vectors at 7-8 weeks of age and were maintained on chow diet for 7 weeks before switching to HFD. measurements of fat mass (B) and lean mass (C) at 11 weeks after viral delivery.


Measurements of energy expenditure during the 7th week after viral delivery . Comparison of real time O2 consumption traces during the 4 - day measurement period ( D ) , of average O2 consumption between the 2 groups ( E ) , and of the difference between day and night periods between the 2 groups ( F ) .

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Measurements of energy expenditure during the 7th week after viral delivery. Comparison of real time O2 consumption traces during the 4-day measurement period (D), of average O2 consumption between the 2 groups (E), and of the difference between day and night periods between the 2 groups (F).


( G - I ) Measurements of feeding during the 7th week after viral delivery . Comparison of real time feeding c traces during the 4 - day measurement period ( G ) , of average food intake between the 2 groups ( H ) , and of the difference in food intake between day and night periods between the 2 groups ( I ) .

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(G-I) Measurements of feeding during the 7th week after viral delivery. Comparison of real time feeding c traces during the 4-day measurement period (G), of average food intake between the 2 groups (H), and of the difference in food intake between day and night periods between the 2 groups (I).


CRH - Cre male mice received stereotaxic delivery of AAV - EF1a - DIO - Kir2 . 1 - P2A - dTomato vectors and AAV - Flex - GFP at 7 - 8 weeks of age and recording was made at least 4 weeks after viral delivery on neurons with Kir2 . 1 ( one side ) or control GFP neurons ( contralateral side ) of the same animal ( A )

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CRH-Cre male mice received stereotaxic delivery of AAV-EF1a-DIO-Kir2.1-P2A-dTomato vectors and AAV-Flex-GFP at 7-8 weeks of age and recording was made at least 4 weeks after viral delivery on neurons with Kir2.1 (one side) or control GFP neurons (contralateral side) of the same animal (A)


typical spontaneous neuron activity recorded in GFP controls ( B , top panel ) and Kir2 . 1 neurons ( B , bottom ) ; and comparison in resting membrane potentials between the two groups ( C , * * p < 0 . 0001 , unpaired Student ' s t test ) .

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typical spontaneous neuron activity recorded in GFP controls (B, top panel) and Kir2.1 neurons (B, bottom); and comparison in resting membrane potentials between the two groups (C, **p<0.0001, unpaired Student's t test).


CRH - Cre male mice were injected with either AAV - Flex - mCherry or AAV - EF1a - DIO - Kir2 . 1 - P2A - dTomato to examine c - Fos expression in the PVH in 24hr fasting or refed after fasting . ( D ) Representative images showing CRH neurons expressing mCherry or Kir2 . 1 ( mCherry in the top 2 rows and Kir2 . 1 in the bottom 2 rows of the second colomn ) , c - Fos expression ( green in the first colomn ) , merged of red and green ( the third colomn ) and the amplied pictures for the boxed areas in the merged images ( the forth colomn ) . The experimental conditions for each group ( fasting verus refed ) are labelled on the left .

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CRH-Cre male mice were injected with either AAV-Flex-mCherry or AAV-EF1a-DIO-Kir2.1-P2A-dTomato to examine c-Fos expression in the PVH in 24hr fasting or refed after fasting. (D) Representative images showing CRH neurons expressing mCherry or Kir2.1 (mCherry in the top 2 rows and Kir2.1 in the bottom 2 rows of the second colomn), c-Fos expression (green in the first colomn), merged of red and green (the third colomn) and the amplied pictures for the boxed areas in the merged images (the forth colomn). The experimental conditions for each group (fasting verus refed) are labelled on the left.


CRH - Cre male mice were injected with either AAV - Flex - mCherry or AAV - EF1a - DIO - Kir2 . 1 - P2A - dTomato to examine c - Fos expression in the PVH in 24hr fasting or refed after fasting . ( E - F ) Comparison of average neuron numbers from cell counting on total number of c - Fos positive neurons in the PVH ( E , * * p = 0 . 0001 : fasting versus refed ; * p = 0 . 0497 : refed versus refed / Kir2 . 1 ; * * p = 0 . 0025 : fasting / Kir2 . 1 versus refed Kir2 . 1 ) and total number of c - Fos positive CRH neurons ( F , * * p < 0 . 0001 : fasting versus refed ; p = 0 . 9586 : fasting / Kir2 . 1 versus refed / Kir2 . 1 ) , n = 3 - 4 each , two way ANOVA tests . All data presented as mean + / - SEM .

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CRH-Cre male mice were injected with either AAV-Flex-mCherry or AAV-EF1a-DIO-Kir2.1-P2A-dTomato to examine c-Fos expression in the PVH in 24hr fasting or refed after fasting. (E-F) Comparison of average neuron numbers from cell counting on total number of c-Fos positive neurons in the PVH (E, **p=0.0001: fasting versus refed; *p=0.0497: refed versus refed/Kir2.1; **p=0.0025: fasting/Kir2.1 versus refed Kir2.1) and total number of c-Fos positive CRH neurons (F, **p<0.0001: fasting versus refed; p=0.9586: fasting/Kir2.1 versus refed/Kir2.1), n=3-4 each, two way ANOVA tests. All data presented as mean +/- SEM.


CRH - Cre male mice received stereotaxic delivery of a mixture of AAV - Flex - GCaMP6m and AAV - EF1a - DIO - Kir2 . 1 - P2A - dTomato vectors at 7 - 8 weeks of age with optic fiber implantation targeting PVH neurons . ( G - H ) Representative traces showing typical responses of PVH CRH neurons to water spay ( arrows ) in Kir2 . 1 mice ( G ) , and comparison of responsiveness to water spray between Kir2 . 1 and control mice ( H ) .

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CRH-Cre male mice received stereotaxic delivery of a mixture of AAV-Flex-GCaMP6m and AAV-EF1a-DIO-Kir2.1-P2A-dTomato vectors at 7-8 weeks of age with optic fiber implantation targeting PVH neurons. (G-H) Representative traces showing typical responses of PVH CRH neurons to water spay (arrows) in Kir2.1 mice (G), and comparison of responsiveness to water spray between Kir2.1 and control mice (H).


CRH - Cre male mice received stereotaxic delivery of a mixture of AAV - Flex - GCaMP6m and AAV - EF1a - DIO - Kir2 . 1 - P2A - dTomato vectors at 7 - 8 weeks of age with optic fiber implantation targeting PVH neurons .

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CRH-Cre male mice received stereotaxic delivery of a mixture of AAV-Flex-GCaMP6m and AAV-EF1a-DIO-Kir2.1-P2A-dTomato vectors at 7-8 weeks of age with optic fiber implantation targeting PVH neurons.


CRH - Cre male mice received stereotaxic delivery of AAV - EF1a - DIO - Kir2 . 1 - P2A - dTomato or control AAV - Flex - mCherry vectors at 7 - 8 weeks of age and were maintained on chow diet for 7 weeks before switching to HFD . Weekly body weight gain after viral delivery ( A , * p < 0 . 0001 , unpaired Student ' s t test ) , at 15 weeks after viral delivery .

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CRH-Cre male mice received stereotaxic delivery of AAV-EF1a-DIO-Kir2.1-P2A-dTomato or control AAV-Flex-mCherry vectors at 7-8 weeks of age and were maintained on chow diet for 7 weeks before switching to HFD. Weekly body weight gain after viral delivery (A, *p<0.0001, unpaired Student's t test), at 15 weeks after viral delivery.


CRH - Cre male mice received stereotaxic delivery of AAV - EF1a - DIO - Kir2 . 1 - P2A - dTomato or control AAV - Flex - mCherry vectors at 7 - 8 weeks of age and were maintained on chow diet for 7 weeks before switching to HFD . measurements of fat mass ( B , * * p = 0 . 0006 , unpaired Student ' s t test ) and lean mass ( C , p = 0 . 3728 , unpaired Student ' s t test ) at 15 weeks after viral delivery .

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CRH-Cre male mice received stereotaxic delivery of AAV-EF1a-DIO-Kir2.1-P2A-dTomato or control AAV-Flex-mCherry vectors at 7-8 weeks of age and were maintained on chow diet for 7 weeks before switching to HFD. measurements of fat mass (B, **p=0.0006, unpaired Student's t test) and lean mass (C, p=0.3728, unpaired Student's t test) at 15 weeks after viral delivery.


( D - F ) Measurements of energy expenditure during the 7th week after viral delivery . Comparison of real time O2 consumption traces during the 4 - day measurement period ( D ) , of average O2 consumption between the 2 groups ( E , * p = 0 . 0007 , unpaired Student ' s t test ) , and of the difference between day and night periods between the 2 groups ( F , p = 0 . 0791 , unpaired Student ' s t test ) .

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(D-F) Measurements of energy expenditure during the 7th week after viral delivery. Comparison of real time O2 consumption traces during the 4-day measurement period (D), of average O2 consumption between the 2 groups (E, *p=0.0007, unpaired Student's t test), and of the difference between day and night periods between the 2 groups (F, p=0.0791, unpaired Student's t test).


( G - I ) Measurements of feeding during the 7th week after viral delivery . Comparison of real time feeding c traces during the 4 - day measurement period ( G ) , of average food intake between the 2 groups ( H , * * p = 0 . 0019 , unpaired Student ' s t test ) , and of the difference in food intake between day and night periods between the 2 groups ( I , * p = 0 . 0363 , unpaired Student ' s t test ) . n = 6 each . All data presented as mean + / - SEM .

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(G-I) Measurements of feeding during the 7th week after viral delivery. Comparison of real time feeding c traces during the 4-day measurement period (G), of average food intake between the 2 groups (H, **p=0.0019, unpaired Student's t test), and of the difference in food intake between day and night periods between the 2 groups (I, *p=0.0363, unpaired Student's t test). n=6 each. All data presented as mean +/- SEM.


( B ) BAX / BAK - deficient HeLa cells reconstituted with BAX and where indicated HA - Parkin were treated with antimycin A and oligomycin ( AO ) for 2 h and then 1 µM of each of the BH3 mimetics ABT - 737 and S63485 for 6 h . Cell death was assessed at the indicated times by LDH assay . Data is mean + / - SD of four independent experiments .

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(B) BAX/BAK-deficient HeLa cells reconstituted with BAX and where indicated HA-Parkin were treated with antimycin A and oligomycin (AO) for 2 h and then 1 µM of each of the BH3 mimetics ABT-737 and S63485 for 6 h. Cell death was assessed at the indicated times by LDH assay. Data is mean +/- SD of four independent experiments.


( C ) BAX / BAK - deficient HeLa cells ectopically expressing BAX and Parkin were treated with antimycin A and oligomycin ( AO , 3 h ) , MG132 ( 20 µM , 3 . 5 h ) and BH3 mimetics ( 1 µM of each of ABT - 737 and S63485 , 1 h ) . Cytosol and membrane fractions were immunoblotted as indicated . Graph shows densitometric analysis of non - ubiquitinated membrane proteins ( top , relative to untreated control ) or cytochrome c release ( bottom ; cytosol / cytosol + membrane , relative to untreated controls ) from three independent experiments . Error bars represent mean + / - SD .

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(C) BAX/BAK-deficient HeLa cells ectopically expressing BAX and Parkin were treated with antimycin A and oligomycin (AO, 3 h), MG132 (20 µM, 3.5 h) and BH3 mimetics (1 µM of each of ABT-737 and S63485, 1 h). Cytosol and membrane fractions were immunoblotted as indicated. Graph shows densitometric analysis of non-ubiquitinated membrane proteins (top, relative to untreated control) or cytochrome c release (bottom; cytosol/cytosol+membrane, relative to untreated controls) from three independent experiments. Error bars represent mean +/- SD.


( D ) BAX / BAK - deficient HeLa cells ectopically expressing BAX were treated and

(D) BAX/BAK-deficient HeLa cells ectopically expressing BAX were treated and analysed


( E ) UBA pull - down of ubiquitinated proteins following antimycin A and oligomycin ( AO ) treatment of BAX / BAK - deficient HeLa cells ectopically expressing HA - Parkin and BAX for 3 h . Representative immunoblot from two independent experiments .

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(E) UBA pull-down of ubiquitinated proteins following antimycin A and oligomycin (AO) treatment of BAX/BAK-deficient HeLa cells ectopically expressing HA-Parkin and BAX for 3 h. Representative immunoblot from two independent experiments.


( A ) Subcellular fractionation of cytosol and membrane fractions of HeLa cells expressing HA - Parkin following 2 h of antimycin A and oligomycin ( AO ) treatment .

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(A) Subcellular fractionation of cytosol and membrane fractions of HeLa cells expressing HA-Parkin following 2 h of antimycin A and oligomycin (AO) treatment.


( B ) Immunoblotting of whole cell lysates immunoblotted following treatment with antimycin A and oligomycin ( AO ) for 3 h with MG132 ( 20 µM ) . Graph shows densitometric analysis of non - ubiquitinated proteins relative to untreated control from three independent experiments . Error bars represent mean + / - SD .

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(B) Immunoblotting of whole cell lysates immunoblotted following treatment with antimycin A and oligomycin (AO) for 3 h with MG132 (20 µM). Graph shows densitometric analysis of non-ubiquitinated proteins relative to untreated control from three independent experiments. Error bars represent mean +/- SD.


( C ) UBA pull - down of ubiquitinated proteins following antimycin A and oligomycin ( AO ) treatment for 3 h and treatment with the non - specific DUB USP2 for 30 min at 37 ° C , representative immunoblot from two independent experiments .

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(C) UBA pull-down of ubiquitinated proteins following antimycin A and oligomycin (AO) treatment for 3 h and treatment with the non-specific DUB USP2 for 30 min at 37°C, representative immunoblot from two independent experiments.


( E ) Whole cell lysates of HA - Parkin and BAK variant - expressing HeLa cells following 3 h antimycin A and oligomycin ( AO ) treatment with 30 min pre - treatment with MG132 ( 20 µM ) , representative immunoblots from three independent experiments .

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(E) Whole cell lysates of HA-Parkin and BAK variant-expressing HeLa cells following 3 h antimycin A and oligomycin (AO) treatment with 30 min pre-treatment with MG132 (20 µM), representative immunoblots from three independent experiments.


( A ) HeLaParkin cells were treated with CCCP ( 10 μM ) , antimycin A and oligomycin ( AO ) for 2h or GTPP ( 10 μM ) for 6h . Immunoblots are representative of three independent experiments . Graph shows densitometric analysis of non - ubiquitinated proteins relative to untreated control from three independent experiments . Error bars represent mean + / -

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(A) HeLaParkin cells were treated with CCCP (10 μM), antimycin A and oligomycin (AO) for 2h or GTPP (10 μM) for 6h. Immunoblots are representative of three independent experiments. Graph shows densitometric analysis of non-ubiquitinated proteins relative to untreated control from three independent experiments. Error bars represent mean +/-


( B ) SH - SY5Y neuroblastoma cells expressing Cas9 together with a sgRNA targeting PRKN ( sgPRKN ) or a non - targeting control ( sgCon ) were assessed for Parkin expression by immunoblotting of cytosolic fractions .

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(B) SH-SY5Y neuroblastoma cells expressing Cas9 together with a sgRNA targeting PRKN (sgPRKN) or a non-targeting control (sgCon) were assessed for Parkin expression by immunoblotting of cytosolic fractions.


SH - SY5Y cells generated in ( B ) were treated with antimycin A and oligomycin ( AO ) for 3 h prior to cell lysis and immunoblotting . Mono and di - ubiquitinated BAK in Parkin - expressing cells following AO are indicated ( asterisk ) . Data is representative of three independent experiments .

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SH-SY5Y cells generated in (B) were treated with antimycin A and oligomycin (AO) for 3 h prior to cell lysis and immunoblotting. Mono and di-ubiquitinated BAK in Parkin-expressing cells following AO are indicated (asterisk). Data is representative of three independent experiments.


( D ) Domain architecture of human Parkin with positions of selected PD - associated mutations . Ubl , ubiquitin - like ; IBR , in - between - RING HeLa cells expressing wild - type Parkin or the indicated PD - associated Parkin mutants were treated with antimycin A and oligomycin ( AO ) for 2h . Immunoblots are representative of three independent experiments . Graph shows densitometric analysis of non - ubiquitinated proteins in AO - treated samples relative to untreated control from three independent experiments . Error bars represent mean + / - SD . All Parkin constructs were N - terminally FLAG - tagged except R104W .

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(D) Domain architecture of human Parkin with positions of selected PD-associated mutations. Ubl, ubiquitin-like; IBR, in-between-RING HeLa cells expressing wild-type Parkin or the indicated PD-associated Parkin mutants were treated with antimycin A and oligomycin (AO) for 2h. Immunoblots are representative of three independent experiments. Graph shows densitometric analysis of non-ubiquitinated proteins in AO-treated samples relative to untreated control from three independent experiments. Error bars represent mean +/- SD. All Parkin constructs were N-terminally FLAG-tagged except R104W.