EMBO/SourceData huggingface dataset:
figf1a, Etoposide-treated Atg5 -/- MEFs were assessed by EM (left) and by Lamp2immunofluorescence (IF; middle). Right: reverse Lamp2 immunofluorescence image merged with the electron microscopy image. Arrows indicate Lamp2-positive autolysosomes. Magnified photos are provided in Supplementary Fig. 5b, The size of each autophagic vacuole (AV) (n = 30 cells) in MEFs treated with etoposide. The total number of AVs in 30 cells was 483 in WT cells and 494 in Atg5-/- cells.c, Percentage autophagic area (n ≥ 20 cells) in MEFs treated with etoposide.d, e, Representative macroautophagy in etoposide-treated Atg5-/- MEFs. Conventional (d) and quick freezing and freeze-substitution (e) techniques were used. The autolysosomes (arrows) contain multilamellar bodies (arrowheads).f, The number of autophagosomes (white columns) and autolysosomes (black columns) in MEFs (n = 10 cells) treated with etoposide.g, Reduction of percentage autophagic area by 3-methyladenine (3-MA; n = 22 cells).. h, Time-course analysis of long-lived protein degradation (n = 4).i, Inhibition of long-lived protein degradation by bafilomycin A1, protease inhibitor (PI) cocktail and 3-MA (*P 0.05; n = 4).j, k, Similar experiments to those in c and i were performed by starvation. Red and blue lines in b, c, g and j indicate means and s.e.m., respectively. The dashed line in c, g and j indicates the autophagic cell border (6%). Black error bars indicate s.d.
figf2a, The production of LC3-II. Gapdh was used as a loading control.b, c, Punctate GFP-LC3 fluorescence in WT MEFs but not in Atg5-/- MEFs. b, Representative photographs. c, Graphical presentation of the percentages of WT MEFs and Atg5-/- MEFs with punctate GFP-LC3 fluorescence.b, c, Punctate GFP-LC3 fluorescence in WT MEFs but not in Atg5-/- MEFs. b, Representative photographs. c, Graphical presentation of the percentages of WT MEFs and Atg5-/- MEFs with punctate GFP-LC3 fluorescence.d, The same GFP-LC3-transfected Atg5-/- MEFs were assessed by IF and by EM. The magnified photo is the area indicated by the squares. Arrows indicate AVs.e, Atg5-/- MEFs were incubated without (NT) or with etoposide and immunostained with an anti-Lamp2 antibody (green). Nuclei were counterstained (red). The percentages of cells with punctate Lamp2 immunostaining are shown (means ± s.d., n = 4).f, Microarray analysis of the Atg5-/- MEFs during treatment with etoposide.g, h, Induction of Ulk1 in the etoposide-treated Atg5-/- MEFs was assessed by qPCR (g) and by immunoblotting (h).i-l, Atg5-/- MEFs were transfected with the siRNAs related to the Ulk1 complex (i, j) for 24 h and treated with etoposide for 18 h. Induction of macroautophagy was assessed by Lamp2 IF (i, k)i-l, Atg5-/- MEFs were transfected with the siRNAs related to the Ulk1 complex (i, j) for 24 h and treated with etoposide for 18 h. Induction of macroautophagy was assessed by EM (j, l).i-l, Atg5-/- MEFs were transfected with the siRNAs related to the PI(3)K complex (k, l) and a beclin 1 plasmid (k) for 24 h and treated with etoposide for 18 h. Induction of macroautophagy was assessed by Lamp2 IF (i, k).i-l, Atg5-/- MEFs were transfected with the siRNAs related to the PI(3)K complex (k, l)for 24 h and treated with etoposide for 18 h. Induction of macroautophagy was assessed by EM (j, l).
figf3a-d, Electron micrographs of etoposide-treated Atg5-/- MEFs. a, AVs were observed near the Golgi apparatus (G). Inset, the isolation membrane (I) was extended from the Golgi stack. b, Developing autophagosome. rER, rough endoplasmic reticulum. c, d, Isolation membrane fusing with vesicles containing thick membrane (d) and a complete autophagosome (c) is generated.e, f, Co-localization of Lamp2 with GFP-M6PR, GFP-Stx7 or GFP-Rab9 in etoposide-treated Atg5-/- MEFs. Arrows indicate co-localized dots. Original magnification, × 200.g-i, Atg5-/- MEFs were transfected with the indicated siRNAs for 24 h and treated with etoposide. Induction of macroautophagy was assessed by Lamp2 IF (g) and by EM (h), and the number of isolation membranes was calculated (i). In g, error bars indicate s.d. (n = 4). In h and i, red and blue lines indicate means and s.e.m., respectively; n = 35 cells each.j, No effect of silencing of Rab9 (siRab9) on the production of LC3-II in etoposide-treated WT MEFs.
figf4a-e, Typical autophagic structures in the tissues of an Atg5-/- embryo (E14.5): a, midbrain; b-d, liver; c, d, developing autophagosomes; e, heart. Arrows indicate the autophagic vacuoles.f, g, k, l, Electron micrographs of wild-type (WT) and Atg5-/- (KO) reticulocytes (f, g) and erythrocytes (k, l). An isolation membrane (asterisk), autophagosomes (arrows) and autolysosomes (arrowheads) are shown. Inset, engulfed mitochondria.h-j, m-o, The numbers of autophagic vacuoles (h, m) and autophagic vacuoles containing mitochondria (i, n) and persisting mitochondria (j, o) in reticulocytes (h-j) and erythrocytes (m-o). Error bars indicate s.d. (*P  0.05; n = 55 cells).f, g, k, l, Electron micrographs of wild-type (WT) and Atg5-/- (KO) reticulocytes (f, g) and erythrocytes (k, l). An isolation membrane (asterisk), autophagosomes (arrows) and autolysosomes (arrowheads) are shown. Inset, engulfed mitochondria.
Figure 1(A) GAPC enzymatic activity was measured in wild-type crude extracts treated with H2O (mock), and 100 µM of Tyr-Asp, Tyr, Asp, and Ile-Glu. (B-D) The enzymatic activities of GAPC (B), GAPA/B (C), and GAPN (D) were measured in the wild type and double k.o. mutant gapc1gapc2 treated with H2O (mock), and 100 µM of Tyr-Asp. Data information: Data are mean ± SEM of n=4 (A-D; four technical replicates) , significance was assessed using unpaired two-tailed Student t-test.
Figure 2(A) Fresh weight quantification of Arabidopsis and tobacco seedlings. Seedlings were pretreated with mock (water), Tyr-Asp (100 μM), Tyr and Asp (100 μM), Ser-Leu (100 μM), or Gly-Pro (100 μM) for 1 h prior an oxidative stress (catechin or H2O2). Each treatment was compared to its respective control, corresponding to plants grown in one 24-well plate (represented by the adjoined bars). Data information: Data are mean ± SEM of n=10-12 (seedlings). Unpaired two-tailed Student t-test was performed to compare treatments with control. Scale bar: 10 cm.(B) Catechin- (left upper panel) and (catechin-) Tyr-Asp-treated tobacco plants (right upper panel) grown for four days in liquid media. H2O2- (left upper panel) and (H2O2-) Tyr-Asp-treated tobacco plants (right upper panel) grown for two days in liquid media. All plants were 2-weeks-old before starting the stress treatment. Data information: Data are mean ± SEM of n=10-12 (seedlings).
Figure 6(C) PEPCK activity was measured in a protein lysate prepared from 3-week-old Arabidopsis leaves in absence (control, C) or presence of 500 µM of the different dipeptides. Measurements were performed in the carboxylation direction, as described in the Methods section. Data are mean ± SE of 4 independent lysate preparations. Unpaired two-tailed Student t-tests were performed to compare dipeptide treatments with the control.
Figure 1(A) Ribbon diagram of the HsAtg4B-LC3(1-120) complex. HsAtg4B is coloured salmon red, and LC3 is coloured green.(B) Structural comparison between free and HsAtg4B‐bound LC3. Crystal structure of free LC3(1-120) (PDB code 1UGM) is superimposed on the HsAtg4B‐bound LC3. HsAtg4B‐bound LC3 is coloured green, and free LC3 is coloured grey.(C) Structural comparison between free and LC3‐bound HsAtg4B. Crystal structure of free HsAtg4B (PDB code 2CY7) is superimposed on the LC3‐bound HsAtg4B. LC3‐bound HsAtg4B is coloured salmon red, and free HsAtg4B is coloured grey. The regions with large conformational differences between free and LC3‐bound forms are coloured black (free) and red (LC3 bound). All the figures representing molecular structures were generated with PyMOL ( DeLano, 2002).
Figure 2(A) Interaction between HsAtg4B and the ubiquitin core of LC3. HsAtg4B is coloured salmon red, and LC3 is coloured green. The side chains of the residues involved in the HsAtg4B-LC3 interaction are shown as stick models. Possible hydrophilic interactions are shown as broken lines.(B) Interaction between HsAtg4B and the C‐terminal tail of LC3. Trp142 and the regulatory loop of HsAtg4B are coloured blue, whereas the remainder is coloured as in (A). As the side chain of Cys74 has two conformations, both conformations are shown. Possible hydrophilic interactions are shown as broken lines.(C) In vitro proteolysis assay. Left, LC3 mutants fused to GST were incubated with HsAtg4B. Right, LC3-GST was incubated with HsAtg4B mutants. Procedures are described in detail in Materials and methods.
Figure 3(A) Annealed Fo−Fc electron density map for the C‐terminal region (residues 119-122) of LC3(1-124). Left, LC3(1-124) bound to the HsAtg4B H280A mutant; right, LC3(1-124) bound to the HsAtg4B C74S mutant. Structure of the C‐terminal region of LC3 is also shown as a stick model, in which carbon, nitrogen and oxygen atoms are coloured green, blue and red, respectively.(B) Structure of the C‐terminal tail of LC3(1-124) and the catalytic site of HsAtg4B. Left, LC3(1-124) bound to HsAtg4B H280A mutant, right, LC3(1-124) bound to the HsAtg4B C74S mutant. HsAtg4B is shown as a ribbon model, whereas LC3 is shown as a stick model. The side chains of the residues comprising the catalytic triad and Tyr54 of HsAtg4B are also shown as a stick model. Carbon, nitrogen, oxygen and sulphur atoms are coloured green, blue, red and yellow, respectively. Mutated catalytic residues are coloured cyan.(C) Structural comparison between the HsAtg4B-LC3 precursor and SENP2-SUMO‐3 precursor complexes. Left, structure of the HsAtg4B-LC3 precursor complex; right, structure of the SENP2-SUMO‐3 precursor complex. HsAtg4B and SENP2 are shown as a ribbon model, whereas LC3 and SUMO‐3 precursors are shown as a stick model. The side chains of the important residues at the catalytic site are also shown as a stick model. Atom colouring is the same as in (A, B).
Figure 4(A) Surface model of free (left) and LC3(1-124)‐bound (right) HsAtg4B. W142 and the regulatory loop are coloured blue, and the N‐terminal tail is coloured cyan. The catalytic Cys74 is coloured red. LC3(1-124) is shown as a ribbon model.(B) In vitro proteolysis assay using N‐terminal tail‐deleted HsAtg4B. LC3-GST was incubated with either wild‐type or N‐terminally truncated HsAtg4B and was subjected to SDS–PAGE analysis.(C) Structural comparison between free and LC3(1-124)‐bound HsAtg4B and SENP2-SUMO‐3 precursor complexes. Left, free HsAtg4B; middle, the HsAtg4B-LC3 precursor complex; right, the SENP2-SUMO‐3 precursor complex. The three structures are shown in the same orientation. Model colouring is the same as in Figure 3.(D) urface model of LC3(1-120) bound to HsAtg4B. Atom colouring is the same as in (A).
Figure 5(A) Overall structure of Hs bound to both substrate and non‐substrate LC3. Substrate LC3 is designated as LC3(S) and coloured green, whereas non‐substrate LC3 is designated as LC3(N) and coloured blue.(B) Stereo‐view of the N‐terminal tail of HsAtg4B bound to the WXXL‐binding site of LC3. The side chains of the residues involved in the interaction between HsAtg4B and LC3 are labelled and shown with a stick model. Model colouring is the same as in (A).(C) Structural comparison of the N‐terminal tail-LC3 interaction with the p62-LC3 interaction. The structure of LC3 complexed with a p62 peptide is superimposed on that complexed with the N‐terminal tail peptide. As the structure of LC3 is almost identical to each other, only that bound to the N‐terminal tail peptide of Hs is shown.(D) Chemical shift perturbations of the LC3 backbone amide groups upon complex formation with the N‐terminal‐tail peptide. The combined 1H and 15N chemical shift differences, calculated using the equation Δp.p.m.=[(ΔδHN)2+(ΔδN/5)2]1/2 were plotted against residue numbers.(E) Mapping of chemical shift perturbation results on the crystal structure of LC3 bound to HsAtg4B. The residues with Δp.p.m. >0.4 are shown in blue, 0.4>Δp.p.m.>0.3 in cyan and 0.3>Δp.p.m. >0.2 in green.
Figure 2B. Immunoblot shows GST-GGA-GAT domain bound Arf1 (active; top) and total Arf1 (input lysates; bottom) from equal aliquots of lysates of HeLa cells that were stimulated with EGF for the indicated time points prior to lysis.C. Graphs display the model fitting for Arf1 activation dynamics. The experimentally determined Arf1 activation (in B) dynamics are displayed as black dots with error bars, representing mean ± S.E.M (n=3 biological replicates), and numerical simulation is shown by the blue continuous line.E. HeLa cells expressing Arf1-HA were serum starved overnight (E, top) and subsequently stimulated with EGF for 5 min (E, bottom) prior to fixation with PFA. Fixed cells were stained for Arf1 (HA; green) and GIV (red) and nuclei (DAPI; blue). Panels on the left show overlay of all 3 stains and representative RGB plots of sections through the Arf1-stained pixels. Panels on the right display the magnified 3D surface plots of the boxed regions in the left panels. Scale bar = 10 µm.
Figure 3C. Equal aliquots (~45 µg) of whole cell lysates of control (shControl; top) and GIV-GEM depleted (shGIV; bottom) HeLa cells were analyzed for GIV and tubulin (loading control) by immunoblotting (IB).D. Control (sh Control; top) and GIV-GEM depleted (shGIV; bottom) HeLa cells were co-transfected with Gαi1-YFP, Gβ1-CFP and Gγ2 (untagged) and live cells were analyzed by FRET imaging at steady-state, after being serum starved in 0.2% FBS overnight and then after stimulation with 50 nM EGF. Representative freeze-frame FRET images are shown. FRET image panels display intensities of acceptor emission due to efficient energy transfer in each pixel. FRET scale is shown in the inset. Golgi and PM regions of interest are indicated with arrows. Scale bar = 10 µm. See also Figure EV2A-B for free-frame images for additional time points in control HeLa cells.F. HeLa cells starved with 0.2% FBS overnight or stimulated subsequently with 50 nM EGF were fixed and stained for active Gαi (green; anti-Gαi:GTP mAb) and Man II (red) and analyzed by confocal microscopy. Activation of Gαi was detected exclusively after EGF stimulation. When detected, active Gαi colocalizes with Man II (yellow pixels in merge panel). See also Figure EV2C-D for additional time points and stimulus. Scale bar = 7.5 µm.G. Model fit for the fold change of active tGTPase (denoted as tG*). Experiment data is the fold change of ∆FRET/CFP in D and is shown as mean ± S.E.M (n=8; 3-5 cells from 2 independent biological experiments). Continuous lines display the model simulation results after parameter fittingI. Immunoblot shows bound Arf1 (active; top) and total Arf1 (input lysates; bottom) from equal aliquots of lysates of control (sh Control) and GIV-depleted (shGIV) HeLa cells. Cells were stimulated with EGF for the indicated time points prior to lysis. Bar graphs in Figure EV2E display the fold change in Arf1 activity normalized to t0 min. 'Low' and 'high' indicate exposures.
Figure 5D-E. The secretion (D) or the cell number (E) as a function of stimulus in coupled and uncoupled switches. The stimulus =0, 0.00046, 0.0046, 0.046, 0.115, 0.23 and 0.46 correspond to varying doses of EGF in simulations, ranging from 0, 0.1 nM, 1 nM, 10 nM, 25 nM, 50 nM, and 100 nM, respectively. The error bar denotes the S.D based on 1000 repeated independent simulations of ODEs when noise is in the stimulus and connections.F. The bar plot depicts cell numbers achieved by cells with either coupled or uncoupled switches, at different levels of stimulus. For the first two bars, the height and error bars are the mean and S.D. of cell number when stimulus =0.046 in E, respectively. For the last two bars, the height and error bars are the mean and S.D. of cell number when stimulus =0 in E, respectively.
Figure 6B. Immunoblots showing intracellular (left) and secreted (in the media; right) GFP-MMP9 at 24 h after stimulation with varying doses of EGF. Tubulin, used as a loading control, confirms the presence of a similar number of plated cells in the assay.C. Left: Graph displays experimentally determined secretion of GFP-MMP9 in response to varying doses of EGF in control (shControl) and GIV-depleted (shGIV) HeLa cells (as in B), and quantified by band densitometry. Results are expressed as mean ± S.E.M; n = 3 biological replicates. p values were determined by two-sided unpaired t-test. Right: Schematic diagram of dose responses (mG* and secretion) for the single switch and coupled switches. Coupled switches stretch the range of proportionate responses. Single mG switch results in misaligned responses. DoRA, dose response alignment.G-H. Control (parental) and GIV-depleted (GIV KO) HeLa cells grown in different concentrations of serum (FBS%) were treated or not with varying concentrations of BFA (µM) as indicated. Line graphs in 3D (G) depict the formazan absorbance expressed as a measure of cell viability from the HeLa cells in various conditions tested. Bar graphs (H) depict the cell number in serum-free growth conditions that are supported exclusively by autocrine secrete-and-sense loop (without BFA; BFA = 0.0 µM) or when such loop is interrupted (BFA = 0.1 µM). Results are expressed as mean ± S.E.M; n = 3 biological replicates. Statistical significance was determined by one way ANOVA.I-K. Control (parental) and GIV-depleted (GIV KO) MDA MB-231 cells grown in different concentrations of serum (FBS%) were treated or not with varying concentrations of BFA (µM) as in G-H. Line graphs in 3D (I) depict the formazan absorbance expressed as a measure of cell viability from the MDA MB-231 cells in various conditions tested. Bar graphs (J) depict the viability of the MDA MB-231 cells in serum-free growth conditions that are supported exclusively by autocrine secrete-and-sense loop (without BFA; BFA = 0.0 µM) or when such loop is interrupted (BFA = 0.1 µM). Results are expressed as mean ± S.E.M; n = 3 biological replicates. Statistical significance was determined by one way ANOVA. Immunoblots (K) of equal aliquots of whole cell lysates confirm the depletion of GIV compared to tubulin (loading control). See also Appendix Figure S5D-H for dot plots and early and late apoptotic fractions. Results are expressed as mean ± S.E.M; n = 3 biological replicates.
Figure 1(A) SA-β-gal staining of U2-OS cells, harvested at indicated times post irradiation (IR); h, hours; D, days; UT, untreated. n = 3. Scale bar: 50 μm. Bar Graph: Significance was determined by Brown Forsythe ANOVA with Dunnett's 3T test, using n = 3 biological replicates. SD shown. *** p < 0.001, n.s. = not significant.(B) Nuclear fractions from U2-OS cells were harvested at indicated time points post IR (20 Gy) as in (A). Top panel: NF-κB DNA binding was analyzed by EMSA. Lower panels: Western blot of indicated NF-κB subunits and PARP1 as loading control. The two phases of nuclear translocation(C) Gene expression analysis of two NF-κB phases following damage. RNA-Seq analysis (n = 5 biological replicates per group) of U2-OS cells, either untreated or analyzed 1.5 hours or 7 days after irradiation (20 Gy), as indicated. The heatmap shows significantly regulated genes (log2 value > 0.5 and p value < 0.05). Black arrow points to NFKBIA. Significance determined by ANOVA with Bonferroni correction for multiple testing. See also Dataset EV1.(D) Female mice 12-16 weeks old (n = 6 mice per condition (with three technical repeats per mouse), were irradiated (5 Gy) and sacrificed after 1 hour (first phase), 7 days (second phase) or left untreated. Skin or renal tissue RNA was analyzed by RT-qPCR, shown as a mean +/- SD. Significance was confirmed by ANOVA with Tukey multiple comparisons test. SD shown: * = p < 0.05, ** = p < 0.01.(E) In situ hybridization using an anti-sense IκBα/Nfkbia riboprobe on longitudinal skin sections from male and female mice (5-8 weeks of age) treated as in (D) for the time indicated. Representative sections shown from n = 6 mice per condition. Bu, bulge region (dashed lines), HS, hair shaft, IFE, interfollicular epidermis. Scale bar: 50 μm. (F) Quantitation of (E) from n = 6 mice per condition and 3-4 sections per mouse. Bulge regions positive for Nfkbia were counted and presented as percentage of total. One way ANOVA analysis with Tukey multiple comparisons test was performed. SD shown. ** = p < 0.01. (G) Skin sections as in (E) analyzed by immunofluorescence with IκBα (red) or IL-6 antibody (green) and nuclear DAPI staining (blue). Dotted lines delineate hair follicles, Bu, bulge region, HS, hair shaft, IFE, interfollicular epidermis, SG, sebaceous gland. Scale bar: 50 μm. (H) Quantitation of (G) from n = 6 mice per condition and 3-4 sections per mouse. Bulge regions positive for IκBα or IL-6 were counted and presented as percentage of total. One way ANOVA analysis with Tukey multiple comparisons test was performed. SD shown. * = p < 0.05, *** = p < 0.001.
Figure 2(A) Left panel: IκBα western blot of U2-OS cells. Lane 1, untreated (ut); Lanes 2-4, single IR (20 Gy) at indicated time points prior to harvest; Lane 5, IR at 5 days prior to harvest, plus 1.5 hours prior to harvest. Right panel: Samples treated as in the left panel, analyzed by RT-qPCR. Significance from n = 3 replicates per condition determined by ANOVA analysis with Tukey multiple comparisons test. SD shown. *** = p < 0.001.(B) NFKBIA knockdown by dox-inducible shRNA in U2-OS cells. Cells were pre-treated with Dox to induce knockdown. For knockdown efficiency see Fig. S2F. Cells were irradiated (20 Gy) or not, as indicated and described above. NF-κB activity was analyzed by EMSA. ns, non-specific band. A representative gel from n = 3 biological replicates is shown.(E) GSEA was performed comparing the IκBα IEC‐KO gene list (n = 4 villin-Cre x floxed IκBα mice and n = 4 littermate controls (GSE 139251) Mikuda et al, 2020(), with a gene signature based on a list of SASP factors Coppe et al, 2010(). NES: normalized enrichment score. FDR: false discovery rate.(F) RT-qPCR analysis of indicated genes was performed using RNA extracted from the duodenum of 5 and 8 weeks old control villin-Cre (n = 6) or villin-Cre x floxed IκBα mice (n = 6) both male and female. Expression is shown as fold change between littermates, paired t-test. SD shown. * p < 0.05 ** = P < 0.01.
Figure 3(A) Nuclear fractions from either untreated or irradiated (20 Gy) U2-OS cells, 1.5 hours or 7 days prior to harvest, were analyzed by EMSA with supershift analysis. For supershifting, lysates were incubated with antibodies or water, as indicated. Arrows point to shift location of a given subunit, due to antibody binding. ns, non-specific band. All antibodies were previously tested for supershift compatibility. Representative gel shown from n = 3 replicates.(B) Expression of SASP targets of NF-κB obtained from RNA-seq analysis (Dataset EV1B), was quantitated using RT-qPCR from the shRNA (RELA) stably expressing U2-OS cells. For knockdown efficiency see Appendix Fig S4A. Cells were treated with doxycycline (Dox) to induce knockdown of p65. Heatmap represents targets normalized to the untreated Scrambled control. Expression is shown as log2 change with p values < 0.05. Statistical analysis performed using ANOVA with Bonferroni correction for multiple testing. First-phase and second-phase samples were irradiated (20 Gy) 1.5h and 7 Days prior to harvest, respectively. Analysis based on n = 3 biological replicates.(C) Cell duplication was measured at time points indicated in U2-OS cells bearing either scrambled control or Dox-inducible shRNA against RELA (n = 3 biological replicates). Treatment with Dox was initiated at two days prior to IR (20 Gy) or after IR. Statistical significance in total duplication number between groups at day 6 was determined by ANOVA with Tukey multiple comparisons test. SD shown. * = P < 0.05, *** = p < 0.001.