EMBO/SourceData huggingface dataset:
Figure 4(C( In fasting, Akt sediments to glycerol gradient fractions containing USP1/UAF1, PHLPP1, and TSC1/TSC2. Muscle homogenates from fed or fasted mice were separated on 10%-40% glycerol gradient, and alternate fractions were analyzed by SDS-PAGE and immunoblot. Red rectangle marks fractions containing USP1/UAF1, PHLPP1, TSC1 and TSC2 in fed conditions.(D) During fasting, Akt is recruited to USP1-PHLPP1-TSC1 complex. TSC1 was immunoprecipitated from the soluble fraction of muscles expressing shLacz or USP1(C90S) from fed or fasted mice. Mouse IgG was used as a control for non-specific binding. Precipitates were analyzed by immunoblotting. Black lines indicate the removal of intervening lanes for presentation purposes.(E) During fasting, inhibition of USP1 reduces rates of protein synthesis. Mice were injected with puromycin, and soluble fractions of electroporated muscles were analyzed by immunoblotting using puromycin antibody. Right: Densitometric measurement of the presented blot. n = 3. *, P < 0.05 vs. shLacz by one-tailed t-test. Data are represented as mean ±SEM.(F) Inhibition of USP1 results in TSC1 accumulation. Soluble fraction of muscles expressing shLacz or USP1(C90S) from fasted mice were analyzed by SDS-PAGE and immunoblotting. Right: Densitometric measurement of the presented blot. n = 3. *, P < 0.05 vs. shLacz. Data is presented as protein content (AU) in USP1(C90S) vs. shLacz by one-tailed t-test. Data are represented as mean ±SEM.(G) mTOR inhibition with rapamycin results in reduced USP1 protein content and enhanced Akt phosphorylation at T308. However, a simultaneous downregulation of USP1 is required to promote TSC1 accumulation. Soluble fraction of muscles expressing shLacz or gUSP1 (in vivo CRISPR) from fed and fasted mice injected i.p. with rapamycin (6mg/kg body weight) or saline were analyzed by SDS-PAGE and immunoblot.
Figure 5(A) During fasting, inhibition of Dab2-Akt association reduces the interaction of Akt with USP1-PHLPP1-TSC1 complex. Left: TSC1 was immunoprecipitated from the soluble fraction of muscles expressing shLacz or Dab2-DN from fed or fasted mice. Mouse IgG was used as a control for non-specific binding. Precipitates were analyzed by immunoblotting. Right: densitometric measurement of PHLPP1 and TSC1 blots, and data is presented as PHLPP1/TSC1 ratio.(B) Dab2 promotes Akt deubiquitination in vivo. Left: Akt was immunoprecipitated from the soluble fraction of muscles expressing shLacz or Dab2-DN from fed and fasted mice using a specific antibody. Mouse IgG was used as a control for non-specific binding. Protein precipitates were analyzed by Western blot analysis using Akt antibody. Right: Densitometric measurements of presented blots. Data is presented as the ratio between ubiquitinated Akt to total Akt in each lane (n=2).(C( Dab2 promotes cleavage of K63-linked polyubiquitin chains on Akt in vivo. Akt was immunoprecipitated from the soluble fraction of muscles expressing shLacz or Dab2-DN from fed and fasted mice using a specific antibody. Mouse IgG was used as a control for non-specific binding. Protein precipitates were subjected to immunoblotting with an antibody against K63-linked polyubiquitin conjugates.(D) Inhibition of Dab2-Akt association enhances Akt phosphorylation in fasting. Soluble fraction of TA muscles transfected with shLacz or Dab2-DN from fed and fasted mice were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. Right: densitometric measurement of presented blots. Data are mean ±SEM, n=3. * p<0.05. vs. shLacz in fed conditions, by one-tailed t-test.
Figure 2(b) Experimental validation of predicted synergistic SR-based combinational therapies in head and neck cancer: A table summarizing the experimentally observed synergism between primary drugs and their predicted rescuer-targeting treatments in 5 HNSC cell lines, based on drug treatment experiments. Synergism was estimated using standard Fa-CI analysis. The table displays the average combination index (CI; synergism CI < 1, additivity effect CI = 1, antagonism CI > 1, NAN indeterminate CI) at 50% growth inhibition (Fraction affected). Combinations that are synergistic are colored blue (black otherwise) for each cell lines tested. The inset shows an example of CI calculation for BYL719 and Dasatinib combination in HN12 cell lines based on the corresponding dose matrix (number indicates % cell viability at 48h, n = 3), and Fa-CI curve.
Figure 3(a) Experimental testing of the predicted SR (DU) rescuers of DNMT1 via drug combination experiments in 18 NSCLC cell lines insensitive to Decitabine. The matrix displays drug interactions between Decitabine, a DNMT1 inhibitor, and inhibitors of its predicted rescuer genes (X-axis) across 18 NSCLC cell lines (Y-axis) that are insensitive to Decitabine. Row labels present rescuer genes and their inhibitors. Colors in the matrix show whether the interactions found are significantly synergistic (red), antagonistic (green) or non-significant (in gray). Values in the matrix show average synergism (<1 synergism and >1 antagonism Thirteen predicted DU-SR rescuers (red lines), two predicted DD SR rescuers (green lines) of DNMT1 and one random control (JAK3i) were tested.
Figure 6The transcriptomic alterations of rescuer genes post PD1/PDL1 blockade in patient tumor biopsies: Their post (vs. pre) treatment expression changes of DU/DD rescuers after anti-PD1 (b) Each panel displays the expression fold change of each predicted rescuer gene (rows) for different tumor samples (columns) and the P-value of over-all paired Wilcoxon test of the expression changes observed in paired samples. Significantly altered up/down-regulated genes are marked by (*). Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti-PD1 blockade in mice models.The transcriptomic alterations of rescuer genes post CTLA4 blockade in patient tumor biopsies: Their post (vs. pre) treatment expression changes of DU/DD rescuers after anti-CTLA4 (c) Each panel displays the expression fold change of each predicted rescuer gene (rows) for different tumor samples (columns) and the P-value of over-all paired Wilcoxon test of the expression changes observed in paired samples. Significantly altered up/down-regulated genes are marked by (*). Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti-PD1 blockade in mice models.The transcriptomic alterations of rescuer genes post PD1/PDL1 and CTLA4 blockade in patient tumor biopsies: Their post (vs. pre) treatment expression changes of DU/DD rescuers after PD1 + CTLA4 combination therapies (d). Each panel displays the expression fold change of each predicted rescuer gene (rows) for different tumor samples (columns) and the P-value of over-all paired Wilcoxon test of the expression changes observed in paired samples. Significantly altered up/down-regulated genes are marked by (*). Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti-PD1 blockade in mice models.
figf1a, Phase partitioning of Apg8. Wild-type and Δapg4Δapg8 cells producing Apg8FG and Δapg7 cells were subjected to phase partitioning (see Methods).b, MALDI mass spectrum of His6-Apg8. Purified His6-Apg8 (predominantly Apg8FG-X and a small amount of Apg8FG) was extracted with 0.4% β-octylglycoside from a polyacrylamide gel and desalted with ZipTipC4(Millipore) before mass measurement. The principal MH+ ion signal at m/z 15,003 is shifted in mass by about +635 from the MH+ value corresponding to His6-Apg8FG.c, MS/MS spectra of the C-terminal peptides of Apg8FG-X (top) and Apg8FG (bottom). C-terminal fragments were generated from Apg8FG-X and Apg8FG (see Methods). Each fragment that was observed only as non-18O-labelled signals was subjected to MS/MS. The MS/MS spectra, deconvoluted with MaxEnt3, were interpreted by SeqMS software26. Arrows show the sequences from the N and C terminus based on y″m and bl ions, respectively (where m and l denote positions counted from the C and N terminus) that were produced by cleavage of peptide bonds during MS/MS. Amino acids shown in three-letter code denote immonium ions. The nomenclature of these ions is as in previous work28.
figf2d, Thioester formation between Apg8 and Apg7. Cell extracts of Δapg7Δapg8 cells expressing Apg7-Myc and/or HA-Apg8 from 2µ plasmids were immunoprecipitated with anti-HA antibody, and immunoblotted (IB) with anti-Myc and anti-HA antibody. DTT-, no DTT; DTT+, 100 mM DTT; asterisk, Apg8-Apg7 conjugate.b, Interaction between Apg8 and Apg7. Δapg7Δapg8 cells were transformed with APG7-Myc and/or APG8 on 2µ plasmids. Cell lysates were immunoprecipitated with anti-Myc antibody, and immunoblotted with anti-Myc or anti-Apg8 antibody. Apg8 co-immunoprecipitated with Apg7-Myc.c, Effect of Apg8 C-terminal processing on the Apg8-Apg7 interaction. Δapg4 cells co-expressing Apg7-Myc and HA-Apg8 (FGR, FG and F) from 2µ plasmids. FGR, wild-type Apg8; FG, Apg8 lacking C-terminal arginine; F, Apg8 lacking C-terminal glycine and arginine. Cell lysates were immunoprecipitated with anti-HA antibody, and immunoblotted with anti-HA and anti-Myc antibody.
figf3b, Interaction between Apg8 and Apg3. Lysates of Δapg3 cells expressing APG3 and/or Myc-APG8 from 2µ plasmids were immunoprecipitated with anti-Apg3 antibody, and immunoblotted with anti-Apg3 and anti-Myc antibody.c, Effect of mutated Cys 234 in Apg3 on the interaction between Apg8 and Apg3. Lysates of Δapg3 cells expressing Myc-APG8 and either wild-type (wt) APG3 or mutant APG3 (C234S and C234A) from 2µ plasmids were immunoprecipitated with anti-Myc antibody, and immunoblotted with anti-Myc and anti-Apg3 antibodies.d, Thioester formation between Apg8 and Apg3. Δapg3, Δapg7 and Δapg10 cells were transformed with both Myc-APG8 and Apg3, or only Apg3, on 2µ plasmids. Cell extracts were immunoprecipitated with anti-Myc antibody, and subjected to immunoblot (IB) using anti-Myc and anti-Apg3 antibody. DTT-, no DTT; DTT+, 100 mM DTT; asterisk, Apg8-Apg3 conjugate; diamond, possible Myc-Apg8 dimer.
figf4a, Apg7 and Apg3 are necessary for attachment of Apg8 to PE. Δapg cell lysates were subjected to SDS-PAGE containing 6 M urea, and immunoblotted with anti-Apg8 antibody.b, Autophagic activity in apg3 mutants. Wild-type, Δapg3 and apg3C243A cells were grown in rich medium (open bar) and subjected to nitrogen starvation for 4 h (filled bar). The autophagic activity of each cell was assessed by measuring the alkaline phosphatase activity. The apg3C243A mutant showed defective autophagy.
Figure 1(A, B) BMDCs were infected with Pru parasites (at a MOI of 4 parasites/BMDC) or treated with tunicamycin (TN) for 16h and the expression of UPR target genes were analyzed by RT-qPCR. The results are expressed as (Log2) Fold change (A) or relative mRNA level (B) compared to non-infected (NI) conditions. IRE1α/XBP1s pathway: Ern1 (IRE1α), Xbp1s, Dnajb9 (ERdj4), Sec24D, Bloc1s1 and Sec61a1; PERK/ATF4 pathway: Atf4, Ddit3 (CHOP) and Ppp1r15a (GADD34); ATF6 pathway: Atf6 and Herpud1; classical ER chaperons: Hspa5 (BIP) and Hsp90b1 (GRP94). Results are normalized to the housekeeping gene Gapdh. Unpaired Student T-test, ns: p>0.05; **: p<0.01; ***: p<0.001; mean ± S.E.M (n=2-5 independent experiments depending of the studied gene, 3 mice / independent experiment).(C, D) Immunoblot analysis of IRE1α, PERK, Phospho-PERK (detected by the presence of a second upper band after migration on a 6% acrylamide gel), Phospho-eIF2α, CHOP and XBP1s protein expression in non-infected BMDCs, BMDCs treated with TN and Pru infected BMDCs (C) and in non-infected and Pru infected Flt3/Notch-DCs (D) for the indicated times. GAPDH and tubulin were used as loading controls. The same blots are shown in Figure EV3B-C.
Figure 2(D) mRNA relative expression level of Xbp1s, Dnajb9 (ERdj4), Ddit3 (CHOP) and Ppp1r15a (GADD34) from WT and MyD88KO BMDCs infected with Pru parasites for 16h compared to non-infected (NI) conditions. Results are normalized to the housekeeping gene Gapdh. Two-way ANOVA Tukey's multiple comparisons, ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001, ****: p<0.0001; mean ± S.E.M (n=4-5 mice pooled from 2 independent experiments).
Figure 4(E) mRNA relative expression level of Il-6, Il-12 and Il-23 from XBP1fl/flIRE1fl/fl, XBP1ΔDC IRE1truncDC and XBP1ΔDC BMDCs infected with Pru parasites for 16 h compared to non-infected (NI) conditions. Results are normalized to the housekeeping gene Gapdh. Two-way ANOVA Tukey's multiple comparisons, ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; mean ± S.E.M (n=3-5 mice pooled from 2 independent experiments).(I) Graph depicts the expression (indicated as the Mean Fluorescence Intensity) of H-2Kb by Pru Tomato SAG1‐OVA infected XBP1fl/fl IRE1fl/fl treated or not with 4μ8C, XBP1ΔDC IRE1truncDC and XBP1ΔDC BMDCs. Bar graphs depict mean ± S.E.M (n=3 mice, one representative experiment from 2 independent experiments). Tukey's ANOVA one-way test, ns: p>0.05.
Figure 5(A, B) Expression of VenusFP by splenic cDC1 (CD26+ CD11c+ MHCII+ XCR1+ CD172a-, upper panels) and cDC2 (CD26+ CD11c+ MHCII+ XCR1- CD172a+, lower panel) from WT (A) and ERAI (B) mice non-infected (NI) or infected with Pru (6 days post-infection (dpi)) reporting on IRE1α pathway activation.(E, F) RT-qPCR analysis of the indicated UPR genes among total RNA prepared from splenic cDC1 (E) and cDC2 (F) sorted by flow cytometry 6 dpi from wild-type mice, non-infected (NI) or infected with Pru parasites. Results were normalized to the housekeeping gene Gapdh and are expressed as relative mRNA level compared to the NI condition. Bar graphs depict mean ± S.E.M. Unpaired Student T-test, ns: p>0.05; *: p<0.05; **: p<0.01; mean ± S.E.M (n=3-5 mice pooled from 2 independent experiments).
Figure 6(A) Survival curves of XBP1fl/flIRE1fl/fl and XBP1ΔDC IRE1truncDC mice infected intraperitoneally with 500 Pru parasites. Log-rank (Mantel-Cox) test, p=0,0004; n=10 mice per genotype.(D, E) Quantification of the number of total (left histogram) and activated (right histogram) (IFNγ-positive) CD4+ T cells (D) and CD8+ T cells (E) in the spleen of control mice (XBP1fl/flIRE1fl/fl) and XBP1ΔDC IRE1truncDC infected for 6 days. Two-way ANOVA Tukey's multiple compare, ns: p> 0.05; *: p <0.05; **: p <0.01; ***: p <0.001; ****: p <0.0001; mean ± S.E.M (n=2 independent experiments combined).
Figure 1b, c Phospho-sites identified after TiO2 enrichment b and phospho-tyrosine immunoprecipitation (pY-IP) c. Left panel: pie chart presenting the proportion of identified phosphorylated serines (pS), threonines (pT) and tyrosines (pY) in the two data sets. Right panel: cumulative number of unique identified phospho-sites across the biological replicates (3 and 4 independent experiments for the TiO2 and pY-IP, respectively). Phosphorylated serines, threonines and tyrosines are presented individually next to the total number of identified sites (pSTY). The phospho-sites that were not previously reported in any species in the PhosphoSitePlus database (www.phosphosite.org) are reported in isolation in the graph on the right.
Figure 2 a, b Output of the statistical analyses of the TiO2-fractionated a and the pY-IP-fractionated b data sets. Left panel: number of phospho-sites significantly regulated upon TCR stimulation presented in bar plots for each time point, next to the total number of regulated sites across the entire time course (total). The number of phosphorylated threonines (pT) and serines (pS) regulated in the TiO2 data set are indicated in yellow and green, respectively. Right panel: representative volcano plot presenting the statistical significance distribution against the log2-transformed fold change between 300 seconds and the unstimulated control. For each condition, phospho-sites were considered significantly up- (red) or down- (blue) regulated when displaying a corrected P-value ≤ 0.05 (Anova test) and an absolute fold change ≥ 1.75 (see Materials and Methods for more detailed information).
Figure 4 a Heatmap showing kinase activities based on enrichment of their specific substrates for each cluster. Black dots indicate clusters with significantly enriched kinase activity (hypergeometric test P-value ≤ 0.05, fold-change ≥ 2, number of annotated sites ≥ 2; Grey squares: fold-change < 1).  d Immunoblot analysis of equal amounts of proteins from total lysates of CD4+ T cells treated with DMSO, U0126 (10 μM) or AKTi VIII (10 μM) and left unstimulated (-) or stimulated for 15, 120, or 300 sec with anti-CD3 and anti-CD4 antibodies, probed with the indicated phospho-specific antibodies. Anti-ERK1/2 immunoblot served as a loading control.  e Equal amounts of proteins from total lysates of CD4+ T cells left unstimulated (-) or stimulated for 120, 300 or 600 sec with anti-CD3 and anti-CD4 antibodies were analyzed by immunoblot with antibodies probing the indicated phospho-sites/proteins.
Figure 5 b Cas9-EGFP OT-I CD8+ T cells were transfected with control sgRNA (sgEGFP) or with two different sgRNA targeting Itsn2 (sgITSN2-1 or sgITSN2-2). Transfected cells were stimulated for 48 h with N4 peptide MHC tetramers (0.1 nM) in presence or absence of soluble anti-CD28 antibody or with IL-7 as control. Cell surface expression of CD69 and CD5 were analyzed by flow cytometry (Z-score normalization of the Geometric Means of fluorescence MFI per experiment) in four independent experiments. Comparison between sgEGFP and sgITSN2 conditions were performed using a paired t-test. Boxplot elements: center line corresponds to median, box limits correspond to the first and third quartiles, whiskers indicate variability from Q1-1.5. IQR to Q3+1.5 IQR. Outliers are shown as black dots. c Proliferation of OT-I Cas9-EGFP CD8+ T cells transfected with EGFP or with ITSN2 sgRNA activated for 48 h with N4 peptide MHC tetramers (0.01-0.1 nM) in the presence or absence of soluble anti-CD28 antibody or with PMA and ionomycin (PMA/Iono) or with IL-7. Data are presented as mean ± standard deviation (SD) from four proliferative measures of two independent sgEGFP and sgITSN2 transfections. Data are representative of three independent experiments.  d IFN-γ production of cells treated as in c was assessed by intracellular staining after 24 h of stimulation. e Proliferation of similar cells as in c activated for 48 h in vitro with a range of increasing doses of N4 peptide MHC tetramers in the presence of soluble anti-CD28 antibody.
Figure 6 a Immunoblot analysis of equal amounts of proteins from total lysates of Cas9-EGFP OT-I CD8+ T cells transfected with sgEGFP, sgITSN2-1 or sgITSN2-2 and left unstimulated (-) or stimulated for 2 or 5 min with N4 tetramers (20 nM). Upper panel: membrane probed with antibody to phosphorylated tyrosine (Anti-p-Tyr) or anti-LAT (loading control). Lower panel: membrane probed with antibodies to anti-CBL-pY774, anti-ZAP70-pY319/352, anti-AKT-pT308, anti-ERK1/2-pY204/T202 or anti-ERK1/2 (loading control).  b Heat map showing normalized levels (z-score) of indicated surface markers and protein phosphorylations (left margin) from Cas9-EGFP OT-I CD8+ T cells transfected with sgEGFP or sgITSN2-1 left unstimulated (-) or stimulated for 1, 3 or 10 min with N4 tetramers (1 or 10 nM) and analyzed by mass cytometry. Z-scores were calculated from hyperbolic arcsine (arcsinh) transformed intensities.  c Similar cells as in a stimulated with 1 nM of N4 peptide for the indicated time points (left panel) or stimulated with increased doses of N4 peptides for 4 h (right panel), were analyzed by FACS for surface TCR expression. Data are presented as mean ± SEM and comparisons were performed using a paired t-test (*, P ≤ 0.05, **, P ≤ 0.01; ***, P ≤ 0.001). A representative analysis of three independent experiments is shown.  d Heatmap showing scaled expression levels (z-score) of indicated surface markers and protein phosphorylations (left margin) from Cas9-EGFP OT-I CD8+ T cells transfected with sgEGFP or sgITSN2-1 and stimulated for 6 h with increasing doses of N4 peptides or PMA/Ionomycin (PI) analyzed by single-cell mass cytometry (left). Z-scores were calculated from hyperbolic arcsine (arcsinh) transformed intensities. Signal difference (mean arcsinh difference) between sgEGFP and sgITSN2-1 transfected cells is depicted on the right panel.
Figure 1(A) Differential interference contrast micrograph of live T. agilis cell. The arrow points to the cell anterior, where the rostrum is located; arrowheads point to some of the flagella.(B) Transmission electron micrographs of T. agilis centriole embedded in resin - longitudinal view; the hub (arrowhead) is visible in the cartwheel-bearing region (pink line), but not in the distal region (white line).(C) Transmission electron micrographs of T. agilis centriole embedded in resin in transverse view from distal end (left) and corresponding image circularized and symmetrized with the CentrioleJ plugin (middle), with schematic indicating principal architectural elements (right).
Figure 4(A) Transverse 2D slices through STA comprising 64 % and 36 % of sub-volumes of T. mirabilis central cartwheel at indicated height from proximal (0 nm) to distal (8.0 nm). Note filamentous cartwheel inner densities (fCID) in the center of the hub.(D, E) 2D longitudinal view of T. mirabilis central region of the cartwheel STA 64% (D) and 36% (E) classes in the region delineated by dashed boxes in (A). Arrowheads denote position of line scans along vertical axis at the hub level, with corresponding normalized pixel intensities (in arbitrary units). Some maxima are highlighted by dashed lines. The average distance between hub units in the 64 % class is 8.4 ± 0.6 nm (N=8; D); in the 36 % class, the distance between hub densities alternates between 3.2 ± 0.3 nm and 5.2 ± 0.3 nm and (both N=9; E). Dashed white line in (E) indicates the offset between two superimposed hub units at the level of spoke densities, which occurs every other hub unit pair.(H) (Top) 2D slice through STA transverse view of microtubule triplet in T. mirabilis, with insets showing pinhead (dashed green box) and A-C linker (dashed red box). (Bottom) Longitudinal 2D slice of STA centered on the pinhead (left) or A-C linker
Figure 5(E) Purified CrSAS-6[NL] protein self-organized into stacks and analyzed by cryo-ET; one longitudinal and one transverse view are highlighted by a dashed rectangle and a dashed circle, respectively. (F, G) 2D views through STA of in vitro self-assembled CrSAS-6[NL] proteins. Transverse sections at the indicated heights through one assembly unit (F), as well as longitudinal view (G) of the area delineated by a dashed box in (F); white arrowhead denotes position of line scan along the vertical axis at the level of the hub, with corresponding pixel intensities in arbitrary units (AU). The average distance between two hub elements is 4.5 ± 0.3 nm (N=7; dashed lines). (H) Longitudinal view of in vitro self-assembled CrSAS-6[NL] proteins STA 3D map. Note densities bridging successive hubs vertically (arrows). (I) Two CrSAS-6[HR] single rings fitted into the 3D map of in vitro self-assembled CrSAS-6[NL] proteins. Dashed box indicates longitudinal section through hub element (left), double chevron indicates viewing point of longitudinal external view (right). Note that the 3D map can accommodate rings stacked 4.4 nm apart (measured on fitted ring models), and that vertical densities linking hubs are partially occupied.