EMBO/sd-character-level-ner huggingface dataset:
Inverse correlation of DNI and size of deletion and importance of positioning of deletion.Figure 6A. DNI efficiencies of ΔPrPs are inversely correlated to deletion size. Representative immunoblot showing PK-resistant PrPSc moiety of (3F4) MoPrP co-expressed with indicated ΔPrPs in 22L-ScN2a cells. Quantitative analysis is shown below as scheme. Relative PrPSc levels were calculated as proportion of PK-resistant (3F4) MoPrP co-transfected with each ΔPrP to that of co-transfected with empty vector control in the same experiment. Data from 4 (for Δ159-165, Δ159-167 and Δ159-167(169) only 3) independent experiments were statistically analyzed for mean and standard deviation (error bars). B. DNI of ΔPrP with a deletion in the C-terminal part of H1∼H2. Δ171-175 (see Fig. 7A) was transiently expressed in N2a cells (left panel) and DNI efficiency evaluated in 22L-ScN2a cells (right panel) (mAb 3F4). Despite the much shorter deletion size, Δ171-175 exerted only inefficient DNI, similar to Δ159-175 (right panel).
Model of 53BP1 phase separation upon DNA damage.Figure 7In accordance with current models on biomolecular condensation and liquid-liquid phase separation (Banani et al., 2016, Erdel & Rippe, 2018, Shin & Brangwynne, 2017), upon nucleation by upstream events triggered at sites of DNA damage (γH2AX formation, MDC1 recruitment, activation of RNF8 and RNF168), 53BP1 accumulates and phase separates, acting as scaffold for client molecules such as p53, which transiently interact with the 53BP1 compartment, where they find an environment permissive for their activation. As clients, with lower relative enrichment and higher mobility, they can dissociate again upon activation to carry out effector functions at distant sites in the nucleus. Phase separation of 53BP1 may also promote compartment fusions and clustering of break sites, with potential implications for mis-rejoining of DNA breaks and generation of chromosomal translocations.
Figure 6Structure-based flow chart for the virtual screening of small molecules interacting with the LARP6-RRM domain. Microscale thermophoresis (MST) analysis of the binding affinity of Bumetanide with MBP-LARP6 Microscale thermophoresis (MST) analysis of the binding affinity of Bumetanide with MBP control protein. Binding mode of the LARP6-RRM domain with Bumetanide. The protein is shown as cartoons and is green. Bumetanide and dominant residues are shown as sticks. Bumetanide is cyan and the dominant residues are green. , Effect of Bumetanide (2 μM) on direct interaction of CRTH2 N-terminal (CRTH2-2) with LARP6 RRM domain (LARP6-4) using GST and MBP pulldown assays. Effect of Bumetanide on LARP6 binding to Col1a1, Col1a2, and Col3a1 mRNA in primary mouse fibroblasts. *P < 0.05 vs DMSO, (unpaired two-tailed t test); n = 6 for all groups. Protocol for administration of Bumetanide to bleomycin-challenged mice. Bumetanide (2 mg/kg) was subcutaneously injected in mice daily for 14 days. Representative Masson's trichrome staining of lung sections from Bumetanide-treated control and F-CRTH2 KO mice. Scale bars, 100 µm. Quantification of collagen content in lung sections from Bumetanide-treated control (CRTH2flox/flox) and F-CRTH2 KO mice. *P < 0.05 vs control, #P < 0.05 vs Vehicle (two-way ANOVA); n = 7 for all groups. Effect of Bumetanide treatment on pulmonary hydroxyproline levels in control and F-CRTH2 KO mice. *P < 0.05 vs control; #P < 0.05 vs Vehicle (two-way ANOVA); n = 7 for all groups. Effect of Bumetanide treatment on Collagen (Col) I and III expression in lungs from control and F-CRTH2 KO mice.
Snai1 function is essential for in vivo intestinal regeneration after irradiation treatmentThe consequences of Snai1 knockout were analysed over a time course.Figure 5The consequences of Snai1 knockout were analysed over a time course. Immunohistochemistry for Snai1 revealed significant loss of expression at day 5 following induction compared to control. The epithelium is re‐populated by wild‐type cells as shown on day 60. Scale bars: 20 μm. To analyse the effect of Snai1 knockout after radiation treatment in the mouse small intestine, VillinCreERT2 Snai1+/+ (control) and VillinCreERT2 Snai1fl/fl (test) adult mice were irradiated on day 1 and injected with tamoxifen on 4 consecutive days to induce Cre‐mediated recombination. Tissue was harvested for analysis on day 5.
Figure 7(a) Speed of the EVL advancing front at different epiboly stages (φ angle) for two distinct simulated conditions: 1) a difference of elastic tension between the EVL and the yolk associated to a two-phase reduction of the contractile E-YSL width (from 20º to 5º) as epiboly advances (from 50º to 155º) (wild type - blue). The contractile E-YSL width decreases at a slower rate until 80º and shrinks afterwards at a rate 2.25 times faster, and 2) Same assumptions as in 1 but with no shrinkage of the E-YSL (constant 20º) (contractileless - red). (b) Experimental values for the EVL advancing front speeds at different epiboly stages (φ angle - top) and at different times (bottom) for wild type controls (blue) and Msn1yolk morphants with reduced E-YSL contractility (red). Experimental profiles closely approach to those inferred in the simulations (compare to (a)). (c) Timeline showing medial sections and power isolines for wild type (top) and Msn1yolk morphants (bottom) along epiboly. Mechanical parameters were inferred by HR from experimental velocity fields (Movie EV10). The RMSE of power is shown as a percentage at each time point. DCs in the Msn1yolk morphants undergo ingression on schedule at 50 % epiboly (yellow arrow). Importantly, as in wild type controls, the active work in morphants is detected at the E-YSL (red arrow). This power supply fades away as epiboly slows down and eventually fails. Scale bar 100 μm. (d) Tensional profiles normalized values in arbitrary units inferred by HR for wild type at 80 % epiboly and its temporal equivalent (9 hours after egg laying) for Msn1yolk morphants. The characteristic profiles of longitudinal and latitudinal stresses and the animal to vegetal stress gradient that are built up during epiboly are never properly developed in morphants.
Figure 4Incidence of spontaneous colitis observed in wild-type (WT) and CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice (n = 11 for each group; Representative images of HE staining of the colon dissected from a WT and Cd4cre A1fl/fl mouse. Scale bar, 200 μm. Histological scores for colitis were compared between WT (n = 4) and Cd4cre A1fl/fl (n = 7) mice Lymphocytes isolated from the colonic lamina propria of WT and Cd4cre A1fl/fl mice were stained with anti-IFNγ, anti-IL17 or anti-Foxp3 antibodies together with anti-CD4 antibodies and analyzed by flow cytometry. Representative images are shown (left panel). The absolute number of IFNγ-, IL17-, or Foxp3-expressing CD4+ T cells were compared between WT and Cd4cre A1fl/fl mice (right panel). Splenocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-IFNγ and anti-IL17 together with anti-CD4 antibodies and were gated on CD4+ cells. Representative flow cytometry images are shown (upper panel). The percentages of IFNγ- and IL17-secreting CD4+ T cells were compared between WT and Cd4cre A1fl/fl mice (lower panel). Splenocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-Foxp3 together with anti-CD4 antibodies and were gated on CD4+ cells. Representative flow cytometry images are shown (upper panel). The percentages of Foxp3-expressing CD4+ T cells were compared between WT and Cd4cre A1fl/fl mice (lower panel). Effector T (Teff) cells isolated from the spleen of a WT mouse were stimulated with soluble anti-CD3 (5 μg/ml) in the presence of mitomycin C-treated splenocytes and were cultured with regulatory T (Treg) cells isolated from WT and Cd4cre A1fl/fl mice at the indicated ratios. After 72 h, the proliferative activity was measured. Values are displayed as the mean ± SEM (n = 3 for each group; Mann-Whitney U-test, N.S.; not significant). The mean value for the suppression of proliferative activity of Teff cells in a condition without Treg cells was set as 0%.
Figure 3Representative immunofluorescence images of lung sections of KrasG12D mice (6 weeks post Ad-Cre inhalation) co-stained for ADAM17 and markers for club cells (CC10) (A), alveolar type II (surfactant protein-C, SP-C) (B) and type I (Podoplanin, Pdpn) (C) cells, total immune cells (CD45) (D) and endothelial cells (CD31) (E). DAPI nuclear staining is blue. Scale bars, 100μm. Arrowheads in merged images indicate dual-positive ADAM17-expressing cells. Quantification of immunofluorescence staining from A-E (n = 5 mice per stain). Representative images of H&E-stained lung sections from KrasG12D recipient mice reconstituted with KrasG12D (KrasKras) or KrasG12D:Adam17ex/ex bone marrow (KrasKras:ex/ex). Scale bar, 3mm. Quantification of lung parenchyma area occupied by tumor lesions in the indicated bone marrow chimeras (n = 6 mice per group).
Phosphomimetic VAMP8 reduces in vitro ensemble vesicle fusion.Figure 3A. Schematic of the in vitro ensemble content-mixing assay. v-SNARE vesicles (VAMP8) loaded with content-dye and unlabeled t-SNARE vesicles (SNAP23 and STX3). B. Ensemble content-mixing assay with v-SNAREvesicles (VAMP8) loaded with dye and unlabeled t-SNAREvesicles (SNAP23 and STX3). Reduced kinetics of content-mixing was observed with VAMP8Glu (T47E, T53E, S54E) compared to wild type VAMP8 (n = 3). As a control (black line), the content-mixing assay was performed by combining v-SNAREvesicles (VAMP8) loaded with dye with unlabeled t-SNAREvesicles prepared without SNAP23 and STX3. The slight steady increase of the control is due to a small amount of content-dye leakage and consequent dequenching of the dyes.
DNA with a 3' overhang increases SUMO conjugation to RPA.Figure 3Plot showing the initial rate of SUMO conjugation versus RPA-DNA concentration as obtained from single-turnover reactions to measure rates of SUMO conjugation to Rfa1 with different RPA-DNA complexes. Representative gels are shown in Appendix Fig S4. Histograms presenting Kd, k2 and specificity (k2/Kd) constant for SUMO conjugation to RPA-DNA.
Figure 6H&E staining (A) and histogram of the HF length (B) in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice injected with vehicles (Control), Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with CSA and vehicle (CSA); and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI treated with tamoxifen, CSA and intradermal Diphtheria Toxin (CSA+DT) starting from early Telogen (P49) and analyzed at the end of the treatments (P58). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Ki67 immunostaining (C) and histogram of the number of Ki67+ cells per HF (D) in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice injected with vehicles (Control), Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with CSA and vehicle; and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI treated with tamoxifen, CSA and intradermal DT, starting from early Telogen (P49) and analyzed at the end of the treatments (P58). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. LYVE1 immunostaining (E) and histogram of the LV caliber (F) analyzed in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice injected with vehicles (Control), Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with CSA and vehicle (CSA); and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with tamoxifen, CSA and intradermal DT, starting from early Telogen (P49) and analyzed at the end of the treatments (P58). Scale bar, 10 μm. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. H&E staining (G) and histogram of the HF length (H) in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice (Control), and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with tamoxifen and intradermal DT, starting from Anagen (P30) and analyzed at the end of the treatments (P37). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 200 μm. Data represent the mean value ± SEM. Cleaved caspase 3 immunostaining (I) and histogram of the number of cleaved caspase 3+ cells per HF (J) in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice (Control), and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with tamoxifen and intradermal DT, starting from Anagen (P30) and analyzed at the end of the treatments (P37). Scale bar, 50 μm. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.
Sorting neoblasts by DNA/RNA content reveals a relatively slow-cycling population.Figure 2A Dissociated cells from unirradiated, 1 dpi or 7 dpi animals (60 Gy of irradiation) were stained with Hoechst (DNA) and Pyronin Y (RNA) and sorted by FACS to reveal an RNAlow population. Percentages indicate the proportion of events within each gate. B Cell size and nucleus:cytoplasm ratio measured by CellMask (cytoplasm) and DAPI (nucleus) staining of FACS sorted cells at homeostasis (n ≥ 47 per sample). Representative images are shown, with dashed lines to indicate the area measured. Scale bars, 5 µm. C EdU pulse-chase time course of label-retention in sorted RNAlow and S/G2/M neoblasts (n ≥ 3 individual experiments per time point). Red and blue p values indicate the statistical significance of EdU dilution from 2 dpp to 14 dpp in S/G2/M or RNAlow neoblasts, respectively. Black p values indicate the statistical significance of EdU retention in RNAlow neoblasts compared to S/G2/M neoblasts. D Maximum projection confocal images showing examples of cells from the beginning and end of the EdU label retention timecourse in C. Red boxes denote examples of EdU- cells. Scale bars, 5 µm.
Figure 3(A-F) Pax2 (magenta, astrocytic progenitors) and Tuj1 (green, RGC axons) IF on E13.5, E15.5 and E18.5 OS/ONs cross-sections showing an increase of Pax2-expressing cells within the optic nerve (ON) of KO embryos at all stages. Histograms quantifying the average number of Pax2+ (G,H) cells in WT, HET and KO at different stages and axial length of the ON, as indicated. Astrocytic progenitors are significantly increased along the nerve of mutants. data are represented as means ± SEM; N=3-5. Statistical significance was obtained by Student t-test (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm Histograms quantifying the average number of NF1A+ (I) cells in WT, HET and KO at different stages and axial length of the ON, as indicated. Astrocytic progenitors are significantly increased along the nerve of mutants. data are represented as means ± SEM; N=3-5. Statistical significance was obtained by Student t-test (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm (J,K) NF1A (red, astrocyte precursors) IF on P7 WT and KO ON cross-sections illustrating a high astrocytic density in KO ON (delineated by thin dashed lines). (L) Histogram confirming the increased percentage of NF1A+ astrocytes in HET and KO ONs compared to WT. data are represented as means ± SEM; N=3-5. Statistical significance was obtained by Student t-test (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm (M-O") Tuj1 (green, RGC axons) and S100β (red, astrocytes) IF on P7 WT, HET and KO ONs. Arrowheads point to S100β+ astrocyte groups (N',O') and absence of Tuj1+ fibers (N'',O'') in HET and KO ONs. (P.Q) TEM images of WT and KO P8 ONs showing thick astrocytic processes filled with intermediate filaments (red dotted lines and arrowheads) in KO nerves. Scale bars (500nm) (R-S') S100β (green, astrocytes) and Sox2 (red, reactive/inflamed astrocytes) IF on P28 WT and HET ONs illustrating strong Sox2 expression in reactive astrocytes. (T) Graph quantifying the average number of Sox2+ astrocytes per P28 ON section in KO and WT animals. data are represented as means ± SEM; N=3-5. Statistical significance was obtained by Student t-test (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm (U,V) EM sections of P28 WT and HET ONs illustrating the expanded astrocytic processes (surrounded by red dotted lines) among myelinated fibers of P28 ONs. Scale bars: (U,V) (4µm).
Figure 2(A) Expression of diap-lacZ and ex-lacZ reporters upon depletion of B52 in the posterior part of the wing disc (hh-Gal4 driver). GFP expression labels the posterior domain. Arrows highlight the reduced expression of the reporters in the posterior domain. Scale bars: 100 µm. (B) Western blot showing overexpression level of V5-tagged Yki1 and V5-tagged Yki2 in total larvae, under the control of da-Gal4 driver, probed with V5 antibody. (C) Wing phenotype induced by depletion of B52 in the posterior domain of the wing disc combined with modulation of Hpo pathway. Dotted lines separate anterior (top) and posterior parts (bottom) of the wing. Flies were grown at 18°C. (D) Quantification of Posterior/Total wing area in male flies of the genotypes illustrated in panel C. UAS-GFP was used as a control. Each point represents a single wing, bars represent mean with standard deviation. *** p-value<0.001, **** p-value<0.0001 (unpaired two-tailed t-tests). Flies were grown at 18°C.
Pathway analysis of blood and muscle metabolites A - E Changed metabolic pathways in blood of IOSCA (A), MIRAS (B), PEO (C), MELAS/MIDD (D), IBM (E) and NMD (F) patients.Figure 5Changed metabolic pathways in blood of IOSCA (A) patients. Data information: Top 10 pathways with ≥ 10% of detected metabolites per pathway are shown. metab., metabolism. Changed metabolic pathways in blood of MIRAS (B) patients. Data information: Top 10 pathways with ≥ 10% of detected metabolites per pathway are shown. metab., metabolism. Changed metabolic pathways in blood of PEO (C) patients. Data information: Top 10 pathways with ≥ 10% of detected metabolites per pathway are shown. metab., metabolism. Changed metabolic pathways in blood of MELAS/MIDD (D) patients. Data information: Top 10 pathways with ≥ 10% of detected metabolites per pathway are shown. metab., metabolism. Changed metabolic pathways in blood of IBM (E) patients. Data information: Top 10 pathways with ≥ 10% of detected metabolites per pathway are shown. bio., biosynthesis Changed metabolic pathways in blood of NMD (F) patients. Data information: Top 10 pathways with ≥ 10% of detected metabolites per pathway are shown. metab., metabolism. Changed metabolic pathways in muscle of PEO (G) patients. Data information: Top 10 pathways with ≥ 10% of detected metabolites per pathway are shown. bio., biosynthesis; deg., degradation; metab., metabolism. Changed metabolic pathways in muscle of MIRAS (H) patients. Data information: Top 10 pathways with ≥ 10% of detected metabolites per pathway are shown. *5% metabolite coverage in the pathway. ; deg., degradation; metab., metabolism.
IP3 receptors are required for dendritic cell fast migrationRelative gene expression of IP3R1 (blue), IP3R2 (red), and IP3R3 (green) in IP3R‐silenced DCs.Figure 2A Relative gene expression of IP3R1 (blue), IP3R2 (red), and IP3R3 (green) in IP3R‐silenced DCs. DCs were infected with two lentiviruses encoding for different shRNAs for IP3R isoforms. ShScramble‐infected DCs were used as a control. Gene expression was determined by a quantitative PCR. It was calculated with respect to GAPDH expression and normalized to the levels observed in the shScramble. The median plus standard deviation of three independent experiments are shown. B Immunoblot for IP3R type 1 and IP3R type 3 in IP3R‐silenced DCs. Actin was used as a control. IP3R3 appears as two bands as previously described (22). C Immunoblot for IP3R type 1 (top) and IP3R type 3 (bottom) in DCs transduced with lentivirus encoding for different IP3R shRNAs. Actin was used as a control. D Relative IP3R2 expression in ShScramble‐infected DCs (gray), shIP3R1 (blue), and IP3R3 (green)‐silenced DCs. The experiment was performed as described in (A). E Table showing the nomenclature for shRNAs related to their isoform‐specific silencing. The shRNAs chosen to pursuit this study are highlighted in red. F Transmigration assay of shScramble‐, IP3R(1,3) A (blue)‐, IP3R(2,3) B (red)‐, and IP3R(1,3) C (green)‐expressing DCs. Cells were loaded in the upper chamber of a 5‐μm pore collagen‐coated transwell assay, recovered from the upper and lower chambers after 16 h, and counted. The median from three independent experiments is shown. G Quantification of the mean cell velocity of shIP3R(1,3) A (blue)‐, shIP3R(2,3) B (red)‐, and shIP3R(1,3) C (green)‐silenced immature DCs migrating in micro‐channels. shScramble‐infected DCs were used as a control (gray) (n > 100 cells from three independent experiments for shIP3R(1,3) A and shIP3R(1,3) C and two independent experiments for shIP3R(2,3) B). Boxes illustrate 10-90 percentiles of values, and whiskers represent the range of values. P‐values were calculated using a Kruskal-Wallis test. H Quantification of the mean cell velocity of shIP3R(1,3) C (green)‐silenced immature DCs migrating in micro‐channels in the presence of 2 mM BAPTA. shScramble‐infected DCs were used as a control (gray) (n > 70 cells from two independent experiments). P‐values were calculated using a Kruskal-Wallis test.
Figure 1(A) Schematic diagram of the domain organizations of Mre11 (top) and Rad50 (bottom). (B) The 25 bp duplex DNA with 2 nt 5' overhangs used in the crystallization. The two strands are labeled as "template" (orange, top) and "non-template" (dark yellow, bottom). In the text, bases are numbered from the 5' end of the "template" strand, and bases of the "non-template" strand are denoted by base pair numbers followed by the prime (') symbol. Disordered residues are shown in black. The protein-contacting residues are underlined. (C) The 3.1 Å 2Fo-Fc map (1.2 σ) around the DNA of the final refined ATPγS-MjMR-DNA complex structure. (D) A ribbon representation of the 3.1 Å ATPγS-MjMR-DNA structure showing the Mre11 nuclease, capping, and C-terminal three helix-bundle domain (light pink), Rad50 molecules (green and light blue), and the DNA (yellow and orange). ATPγS is shown in spheres. See Movie EV 1 for the movement of the complex.
Figure 3(A, B) HeLa cells harboring single copies of HR (pGC) or NHEJ (pEJ) repair substrates were transfected with either negative control (siCont) or siRNAs targeting CHD1, RAD51 or XRCC4. After 24 h of transfection, DSB was induced by transfecting cells with I-Sce-I-expressing vector (pCMV-I-SceI-3xNLS). After 48 h of transfection, GFP-positive cells were measured by flow cytometry. HR (A) or NHEJ (B) efficiency was calculated based on the fraction of GFP-positive cells and represented as mean values from three independent experiments as ± SD, 50,000 cells were counted for each condition. (C) CHD1-depleted cells show decreased RAD51 foci after DNA damage induction. The shCont or shCHD1 PC3 cells were irradiated and co-immunostained with γH2AX and RAD51 antibodies after the indicated time points. Scale bar 5 µm. (D) The number of RAD51 foci co-localized with γH2AX per cell (from C) were counted (n=50) and represented in a graph. The data is represented in mean value of three independent experiments as ± SD, more than 50 cells were counted for each condition. See also Figure EV3A-EV3F.
a) HeLa cells transfected for 24 h with GFP–Atg16L1 were fixed to reveal the Atg16L1 vesicles (green).figf4(a) HeLa cells transfected for 24 h with GFP-Atg16L1 were fixed to reveal the Atg16L1 vesicles (green). Scale bar, 10 μm. (b) HeLa cells transiently transfected for 20 h with GFP-Atg16L1 and mRFP-GPi were cultured in starvation medium for a further 2 h. TIRF image with GFP-Atg16L1 (green) and mRFP-GPi (red) is shown. Arrows indicate co-localization between Atg16L1 and GPi. The percentage co-localization of GFP-Atg16L1 with mRFP-GPi is shown in the graph. n = 2 cells. Scale bar, 5 μm. (c) HeLa cells transfected for 24 h with GFP-Atg16L1 were either left untreated or treated with 50 μM dynasore (Sigma) for 4 h and processed for immunogold electron microscopy with anti-GFP antibodies (15-nm gold particles) and anti-clathrin antibodies (10-nm gold particles). Co-localization can be seen in the boxed areas. Quantification of clathrin-coated structures (CCS) that were associated with Atg16L1 per 1,000 μm2 is shown in the graph. Three asterisks, P 0.0001. Scale bar, 100 nm. All error bars represent s.e.m.