EMBO/SourceData huggingface dataset:
f3(a, b) ITC titration profile of Beclin 1 CC domain with Atg14L (a) or UVRAG (b).c) Pull-down assay to assess the competition between Beclin 1-Atg14L and Beclin 1-UVRAG complexes. Increasing amount of His6-tagged Beclin 1 CC domain was added to a mixture of Atg14L and UVRAG proteins corresponding to their respective CC region. The Beclin 1-Atg14L/UVRAG complexes were pulled down by Ni2+- nitrilotriacetic acid (NTA) agarose beads and checked by SDS gel.(d) Co-IP experiments to assess the competition between Beclin 1-Atg14L and Beclin 1-UVRAG complexes in vivo. FLAG- or GFP-tagged Atg14L or UVRAG was transfected alone, or both were co-transfected into HEK 293T cells and their interaction with endogenous Beclin 1 was probed by co-IP using GFP antibody, followed by western blot with anti-Beclin 1 antibody.
f4(c) Self-association pattern of wild-type, MutM and MutStab constructs of Beclin 1 in vivo. For each construct, both GFP- and Myc-tagged full-length Beclin 1 are co-transfected into HEK 293T cells and their self-association is probed by co-IP using GFP antibody for IP, followed by western blot with anti-Myc antibody.(d). Temperature-dependent CD profile of Beclin 1 CC domain wild-type and MutStab constructs at 220 nm.
f5(a,b) Interaction of Beclin 1 wild-type and monomeric (MutM) mutants with Atg14L or UVRAG in cultured cells. HEK 293T cells were transfected with GFP-tagged Beclin 1 variant alone (a) or together with FLAG-UVRAG (b). The lysates were immunoprecipitated by GFP antibody, followed by western-blot analysis with anti-Atg14L antibody for detecting binding to endogenous Atg14L (a) or with anti-UVRAG antibody for detecting binding to the FLAG-tagged UVRAG protein (b).
f6(a, b) Interaction of MutStab mutants of Beclin 1 with Atg14L or UVRAG in vivo. HEK 293T cells were transfected with GFP-tagged Beclin 1 variant alone (b) or together with FLAG-UVRAG (b). The lysates were immunoprecipitated by GFP antibody, followed by western blot analysis with anti-Atg14L antibody for detecting binding to endogenous Atg14L (a) or with anti-UVRAG antibody for detecting binding to the FLAG-tagged UVRAG protein (b).
f7(a,b). Representative fluorescent images of HeLa cells co-tansfected with GFP-tagged Beclin 1 wild type, MutM L178A/L192A or MutStab E189L/A217L/E224L/A255L (green) and AsRed-Atg14L (red) (a) or FLAG-UVRAG (red) (b); Co-localization of AsRed-Atg14L with GFP-tagged Beclin 1 wild type or MutM is shown in discrete puncta.(c-e) Representative fluorescence images of HeLa cells co-transfected with Myc-Beclin 1 wild type, MutM L178A/L192A or MutStab E189L/A217L/E224L/A255L (blue), AsRed-Atg14L (red) and GFP-tagged Atg12 (c). The scale bar is 20 μM.(c-e) Representative fluorescence images of HeLa cells co-transfected with Myc-Beclin 1 wild type, MutM L178A/L192A or MutStab E189L/A217L/E224L/A255L (blue), AsRed-Atg14L (red) and GFP-tagged LC3 (d) and DFCP (e) (green). The scale bar is 20 μM.
Figure 1.(A) Confocal micrographs of adult brain of a young (2 d old) wild-type fly. Positive staining for Ref(2)P and ubiquitin is not evident. (B-D) Confocal micrographs of adult brain of an old (8 wk old) wild-type fly. Ref(2)P accumulates in protein aggregates that often colocalize with ubiquitin (arrows). The white rectangle in C indicates the area shown in D.(E and F) Western blot analysis of total cell lysates (E) and insoluble protein fraction (F) of wild-type young and old adult heads probed with anti-ref(2)P and anti-ubiquitin antibodies, demonstrating a significant increase in Ref(2)P and insoluble ubiquitinated protein levels in old flies.
Figure 2.(A-D) Electron micrographs of adult brain of old wild-type flies. Ref(2)P localizes in electron-dense structures ranging from 50 nm to 1 μm in diameter that are surrounded (arrows) or not (arrowhead) by a limiting membrane. (E and F) Electron micrographs of wild-type brain of old flies demonstrating that Ref(2)P forms shell-like structures that surround aggregated filamentous material (arrows). Bars: (A-C) 50 nm; (D) 100 nm; (E and F) 200 nm.
Figure 3.(A and B) atg8a mutants exhibiting a striking positive staining for Ref(2)P and ubiquitin staining in all the regions of the adult brain. As it is shown in high magnification of the optic lobe (medulla region; B), Ref(2)P- and ubiquitin-positive aggregates are observed (arrows). Genotypes: (A and B) atg8aKG07569/Y(C) Normal phenotype is observed in β2 and 6 dominant-negative temperature-sensitive mutants at 25°C. (D) Accumulation of Ref(2)P- and ubiquitin-positive aggregates are observed in β2 and 6 mutants at 29°C (arrows). (C and D) elavGal4/+; UAS-β6/+; UAS-β2/+(E and F) double mutants for atg8a and ref(2)P loss-of-function alleles lacking the PB1 (E) or UBA (F) domain do not form ubiquitinated protein aggregates. Ubiquitin exhibits a cytoplasmic staining pattern (arrows). Genotypes: (E) atg8aKG07569/Y; ref(2)Pod2/ref(2)Pod2, and (F) atg8aKG07569/Y; ref(2)Pod3/ref(2)Pod3.
Figure 4.(A and B) Protein aggregates in atg8aKG07569 mutants exhibit sequestosome morphology and are positively labeled for Ref(2)P (A) and ubiquitin (B). Protein aggregates are surrounded by small electron-dense vesicles (arrows).(C) Western blot analysis of cell lysates of wild type and atg8aKG07569 young adult heads demonstrating a significant increase of Ref(2)P protein levels in atg8aKG07569 flies.(D, left) Ref(2)P protein migration profile in ref(2)P loss-of-function alleles. Ref(2)P mutant protein for the PB1 domain has an apparent molecular mass of 85 kD (ref(2)Pod2/ref(2)Pod2) and the Ref(2)P mutant protein for the UBA domain has an apparent molecular mass of 69 kD (ref(2)Pod3/ref(2)Pod3). (D, right) Western blot analysis of insoluble protein fractions of adult heads of double mutants for atg8a and ref(2)P probed for ubiquitin. Genotypes: (1) atg8aKG07569/Y; ref(2)Pod2/Cyo, (2) atg8aKG07569/Y; ref(2)Pod2/ref(2)Pod2, (3) atg8aKG07569/Y; ref(2)Pod3/CyO, and (4) atg8aKG07569/Y; ref(2)Pod3/ref(2)Pod3. The insoluble ubiquitinated protein profile of the double mutants is diminished compared with the one of the respective control heterozygous for ref(2)P.(E and F) Neuronal cells mutant for atg1 (green) accumulate Ref(2)P- (E) and ubiquitin-positive (F) structures (arrows), whereas the normal surrounding cells do not exhibit positive staining. Genotypes: (E and F) yw/UAS-CD8-GFP, hs-flp; tubGal4/+; atg1Δ3D, FRT80/tubGal80, FRT80(G) Confocal micrographs of the optic lobe (medulla region) of young flies expressing a mutated form of human tau protein exhibiting Ref(2)P- and tau-positive aggregates (arrows). Genotypes: (G) elav/+;UAS-E14/+.
Figure 1B. Identification of highly variable genes (HVGs) for the time-point ZT2. The trend for the global relation (red line) between CV2 (variance/mean2) and mean expression is defined using all genes, minus small and lowly expressed genes (see material and methods for more detail) and used to identify HVG (blue). For each gene, a corrected CV2 is calculated: log2(CV2 /trend).C. Expression profiles (top) in the 14 seedlings over a 24 hour time course (with 12 time-points) for a non-variable gene (left), a highly variable gene (middle) and a gene with the level of variability changing across the 24 hour (right). Each dot is the mean normalised expression level for a single seedling. Variability profiles (bottom) of the log2(CV2 /trend) for the same genes are also shown. Blue stars indicate time-points for which the gene is identified as being highly variable.
Figure 2A. Number of genes identified as being highly variable for each time-point. These genes are separated between those that are also selected as highly variable in at least one other time-point (dark blue) and those highly variable in only one time-point (light blue). The top bar indicates time-points harvested during the day (orange), just before dusk (red), during the night (black) and just before dawn (blue).B. Distribution of the number of time-points at which genes are identified as highly variable (blue) or lowly variable (green). The distribution of average (black) and 95% confidence interval (dotted grey) for the thousand random sets is also represented.C. Heatmap of the percentage of HVGs shared between time-points. Red indicates a high percentage of HVGs in common between two time-points. The top and side bars indicates time-points harvested during the day (orange), just before dusk (red), during the night (black) and just before dawn (blue). D. Hierarchical clustering of HVGs based on the log2(CV2/trend) at each time point. The result is represented as a heatmap where yellow indicates a high log2(CV2/trend). The genes were separated into four clusters, indicated by the side colored bar. The top bar indicates time-points harvested during the day (orange), just before dusk (red), during the night (black) and just before dawn (blue). See Appendix Fig S3G for heatmaps of the log2(CV2/trend) with the same colour cutoffs for HVGs, LVGs and random genes. E. Heatmap of the mean normalised expression level for the genes in Fig 2D, keeping the same clustering organisation. The top bar indicates time-points harvested during the day (orange), just before dusk (red), during the night (black) and just before dawn (blue).
Figure 4C. Distribution of the number of TFs targeting a gene, based on the DAP-seq available dataset, for LVG (green) and HVG (blue). The distribution of average (black) and 95% confidence interval (dotted grey) for the thousand random sets is also represented.
Figure 5A. Proportion of genes marked with several chromatin marks among all genes passing size and expression level thresholds (black), HVGs (blue), LVGs (green) and the average for the thousand random sets (grey). Error bars indicate the 95% confidence interval for the thousand random sets. Blue and green stars indicate statistical differences in the number of marked genes compare to all genes, for variable and stable genes respectively (* Indicates a p-value < 0.05, *** Indicates a p-value < 0.001, Chi-square test).B. Average profile for H3K27me3, H2A.Z, H3K4me3 and H3K23ac at HVGs (blue), LVGs (green) and random genes (grey). C. Heatmap of the enrichment for H3K27me3, H2A.Z, H3K4me3 and H3K23ac for HVGs (top), random genes (middle) and LVG (bottom). Red means a high level and blue means a low level for the chromatin marks.
Figure 1(b) shows succinate quantification by NMR from tracheal aspirates collected in mechanically-ventilated patients with (pink bar, n=9) or without (orange bar, n=7) diagnostic of IAV pneumonia. *P < 0.05. (c) IL-6 and IL-8 measurements by ELISA from these same tracheal aspirates. Data information: Data are mean ± SEM and statistical analysis was performed using the Mann-Whitney U-test.
Figure 48-week-old female C57Bl/6 mice were infected intranasally with 150 pfu of influenza A/Scotland/20/74 (H3N2) virus (IAV) and treated or not simultaneously with 4 mg of succinate (Suc; by the intranasal route). Some mice were euthanized at 4 days post-infection and lungs were collected to determine: ; (b) the viral protein expression by Western blotting followed by a relative quantification (c), Data information: Statistical analysis was performed using the Mann-Whitney test c, (*P < 0.05).8-week-old female C57Bl/6 mice were infected intranasally with 150 pfu of influenza A/Scotland/20/74 (H3N2) virus (IAV) and treated or not simultaneously with 4 mg of succinate (Suc; by the intranasal route). Some mice were euthanized at 4 days post-infection and lungs were collected to determine: (h) tissue lesions in lung sections stained with hematoxylin-eosin and further assessed by microscopy. Scale bar: x6/20 µm. All data are represented as the mean or the mean ± SEM and are cumulative of 2 independent experiments with 5 animals each. Data information: Statistical analysis was performed using the Mann-Whitney test , h, , (*P < 0.05).
Figure 5Human bronchial epithelial BEAS-2B cells were infected with A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1. After 4 h, cells were treated or not with succinate (Suc; 4 mg/ml) up to 20 h. protein expression (c) were assessed by Western blotting to detect viral proteins (β-actin was used as a loading control), respectively.
Figure 6Human bronchial epithelial BEAS-2B cells were infected with influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h, then washed and treated or not with succinate (Suc; 4 mg/mL) for 20 h. (a Expression of IAV proteins (i.e. NP, NS1) was analyzed by confocal microscopy using specific antibodies. Viral proteins are stained in green, DNA in blue and actin in red. For a given antibody, pictures were taken on the exact same day with the same laser power settings, Pictures are representative of five independent experiments. Scale bar 10 µM. (b) Relative nuclear intensity of NS1 or NP was determined by using the Intensity Ratio Nuclei Cytoplasm Tool. Seven (NS1) or nine (NP) random images were collected per treatment with at least 5 cells per field. Data are represented as the mean ± SEM. Statistical analysis was performed using the Mann-Whitney test, (*P < 0.05).
Figure 8Human bronchial epithelial BEAS-2B cells were infected with the influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h, and subsequently treated or not with 4 mg/mL of succinate (Suc) for 20 h. (a, b) Cells were stained using a monoclonal antibody that specifically recognizes "NP-vRNA" complexes, but not RNA-free NP, and confocal microscopy (a) was performed (green staining). The value raw integrated density (RawIntDen, which is the sum of all pixel values in the ROI (region of interest)) was further measured (b). Data information: Data are represented as the mean ± SEM of 3 biological replicates or without (a 2 technical replicates each. 8 (b) random images were analysed per treatment with a minimum of 5 cells per field. Statistical analysis was performed using the Mann-Whitney test (b, (*P < 0.05).Human bronchial epithelial BEAS-2B cells were infected with the influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h, and subsequently treated or not with 4 mg/mL of succinate (Suc) for 20 h. (e) Human bronchial epithelial BEAS-2B cells were infected with a A/Scotland/20/74 (H3N2) virus (IAV) carrying a wild-type NP (NP WT) or with the corresponding mutated virus bearing a NP with a K87R substitution (NP K87R). Cells were infected by either virus at MOI=1 for 4 h, then washed and treated or not with 4 mg/mL of succinate for 20 h. Localization of NP proteins was analyzed by confocal immunofluorescence microscopy. Scale bar: 10 µM. Pictures are representative of three independent experiments. (f) Relative nuclear intensity of NP was determined by using the Intensity Ratio Nuclei Cytoplasm Tool. Data information: Data are represented as the mean ± SEM of 3 biological replicates without 2 technical replicates each. at least 10 (f) random images were analysed per treatment with a minimum of 5 cells per field. Statistical analysis was performed using the Mann-Whitney test (*P < 0.05).
Figure 2(B) The number of metabolites that change significantly when wild-type yeast and mutants in the predicted target are treated with the indicated drugs at a concentration of 10 µM are shown. Drugs targeting GPR1 are written in black text, with drugs targeting SNF3 and RGT2 written in purple and green respectively. Orange filled bars are predicted antagonists for GPR1.(C) The levels of trehalose compared to a DMSO treated control are shown for both wild-type yeast and gpr1 strains treated with predicted antagonists for GPR1. The bar height indicates mean values, and error bars indicate the standard deviation of the mean. Asterisks indicate a p-value of less than 0.05 from a two-sided student's T-test (n = 4 biological replicates) when comparing The WT and gpr1 reponses to the drug. Results are representative of 3 independent experiments.
Figure 3(C) The average quantified filamentous growth is shown for BY4741 and Σ1278b yeast spotted onto media supplemented with either a vehicle control (DMSO 0.1% (v/v) or the indicated compounds at a concentration of 20 µM across four biological replicates. Error bars represent the standard deviation of the mean between the averages of the replicates, and asterisks indicate p-value < 0.05 for a two-sided student's T-test comparing normalized values to the DMSO control (n = 4 biological replicates).(D) The average quantified filamentous growth is shown for BY4741 and Σ1278b yeast spotted onto media supplemented with either a vehicle control (DMSO 0.1% (v/v) or the indicated concentrations of ibuprofen. Error bars represent the standard deviation of the mean between the averages of the replicates, and asterisks indicate p-value < 0.05 for a two-sided student's T-test comparing normalized values to the DMSO control (n = 4 biological replicates).(E) The average relative amount of trehalose in ibuprofen-treated cells compared to a DMSO treatment is shown. The bar height indicates mean values, error bars indicate the standard deviation of the mean, and asterisks indicate p-value < 0.05 for a two-sided student's T-test comparing to the WT control (n = 8 biological replicates). Results are representative of 3 independent experiments.
Figure 1(A,B) PTPRN2 (A) and PLCB1 (B) expression levels were determined by qRT-PCR.(C) Bioluminescence imaging quantification of lung colonization by 40,000 LM2 breast cancer cells transduced with shRNAs targeting PTPRN2 or a control hairpin. For shCntrl, sh1PTPRN2, N = 5 mice/group. For sh2PTPRN2 N = 6 mice. Right, H&amp;amp;E staining of representative lung sections.(D) Bioluminescence imaging quantification of lung colonization by 40,000 LM2 cells transfected with siRNA targeting PLCβ1 or a control siRNA. For siCntrl, N = 5 mice. For si1PLCβ1, si2PLCβ1, N = 6 mice/group. Right, H&amp;amp;E staining of representative lung sections.(E,F) PTPRN2 (E) and PLCB1 (F) levels were analyzed in human breast cancers (stages I-IV) and normal breast tissue from TissueScan qPCR Array Breast Cancer Panels II and III (Origene, N = 97). Expression levels were normalized to levels in normal tissue for each gene.(G) Kaplan-Meier curve representing overall survival of a cohort of breast cancer patients (N = 528) as a function of their primary tumor's PTPRN2 and PLCB1 expression levels (data from the TCGA Research Network (Cancer Genome Atlas, 2012)). Patients whose primary tumors' PTPRN2 and PLCB1 expression levels were higher or lower than the median of the population were classified as low (blue) or high (red) expression.(H) Kaplan-Meier curve representing distal metastasis-free survival of a cohort of breast cancer patients (N = 1609) as a function of their primary tumor's PTPRN2 and PLCB1 expression levels (Data from KMPlot (Gyorffy et al., 2010)). Patients' primary tumors' PTPRN2 and PLCB1 expression levels were classified as low (blue) or high (red) expression.
Figure 2(A) Matrigel invasion by 50,000 LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA. Data normalized to control values. N = 6 inserts/group.(B) Migration assay by 100,000 LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA. Data normalized to control values. N = 6 inserts/group. Right, representative images of the migration assay. Scale bar, 100 μm.(C) Quantification of area covered by cells 24 h after a scratch was made through confluent cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA. N = 5 wells/group. Right, representative images of the scratch assay.(D,E) MDA-MB-231 cells transduced with PTPRN2, PTPRN2C945A, or control vector were subjected to the Matrigel invasion (D) and migration assays (E). N = 5 inserts/group.(F) Bioluminescence imaging quantification of lung colonization by 40,000 MDA-MB-231 cells overexpressing PTPRN2, PTPRN2C945A, or control vector. N = 5 mice/group. Right, H&amp;amp;E staining of representative lung sections.(G,H) MDA-MB-231 cells transduced with PLCβ1, PLCβ1H331Q, or control vector were subjected to the Matrigel invasion (G) and migration assays (H). N = 5 inserts/group.(I) Bioluminescence imaging quantification of lung colonization by 40,000 MDA-MB-231 cells overexpressing PLCβ1, PLCβ1H331Q, or control vector. For Cntrl and PLCβ1H331Q OE, N = 6 mice/group. For PLCβ1 OE, N = 5 mice. Right, H&amp;amp;E staining of representative lung sections.
Figure 3(A) Mean fluorescence intensity of membrane PI(4,5)P2 was analyzed in MDA-MB-231 and LM2 cells immunostained with PI(4,5)P2 antibody using fluorescence microscopy. N = 50 cells/group. Scale bar, 10 μm.(B) Bioluminescence imaging quantification of lung colonization by 40,000 LM2 cells treated with carrier incubated with PI(4,5)P2 or carrier alone for one hour and then immediately injected. Right, H&amp;amp;E staining of representative lung sections. N=6 mice/group.(C,D) MDA-MB-231 cells overexpressing PTPRN2, PLCβ1, or a control vector (C) or LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or a control siRNA (D) were immunostained for PI(4,5)P2 levels and analyzed by fluorescence microscopy. Mean fluorescence intensity of plasma membrane levels of the lipid were quantified. N = 50 cells/group. Left, representative immunofluorescence images of cells stained with anti-PI(4,5)P2 antibody (red) and 4',6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 10 μm.
Figure 4(A,B) LM2 cells transfected with siRNA targeting PTPRN2 (A), PLCβ1 (B) or a control siRNA were transfected with Lyn11-FRB and INPP5E-FKBP, treated with either DMSO or 100 nm rapamycin and subjected to the migration assay. N = 5 inserts/group.(C,D) MDA-MB-231 cells overexpressing PTPRN2 (C), PLCβ1 (D) or control vector were treated with carrier alone or carrier incubated with PI(4,5)P2 for one hour and then immediately subjected to the migration assay. N = 5 inserts/group.(C,D) MDA-MB-231 cells overexpressing PTPRN2 (C), PLCβ1 (D) or control vector were treated with carrier alone or carrier incubated with PI(4,5)P2 for one hour and then immediately subjected to the migration assay. N = 5 inserts/group.(E) Kaplan-Meier curve representing distal metastasis-free survival of a cohort of breast cancer patients (N = 1609) as a function of their primary tumor's mean PIP5K1A, PIP5KB, and PIP5KC expression levels (Data from KMPlot (Gyorffy et al., 2010)). Patients' primary tumors' combined PIP5K expression levels were classified as low (blue) or high (red) expression.(F,G) Migration (F) and Matrigel invasion (G) of LM2 cells transduced with a retroviral vector overexpressing PIP5K1A or control vector. Data normalized to control values. N = 5 inserts/group.