EMBO/sd-character-level-ner huggingface dataset:
Figure 1Signal tracks showing cleavages generated by Med8-MNase, Med17-MNase, and PMED8-MNase in YPD at three highly expressed SAGA-dependent (A) and three highly expressed TFIID-dependent (B) loci. All time points for a given factor were scaled to the same data range, and PMED8-MNase tracks were scaled to the lower Mediator-MNase fusion range. Med14ChIP-seq and Med15ChIP-chip data are shown for comparison. TSSs are indicated by arrows.
Figure 8(A) Timeline of LV::S i.m.-i.n. immunization and challenge with SARS-CoV-2 Gamma in B6.K18-hACE2IP-THV mice (n = 5/group). The LVs used in this experiment were non-integrative. Olfactory bulbs, brains and lungs were collected at 3 dpi. (B) Brain or lung viral RNA contents, determined by conventional E-specific or sub-genomic Esg-specific qRT-PCR at 3 dpi. Two mice out of the 5 sham-vaccinated mice did not have detectable viral RNA in the lungs despite high viral RNA content in the brain and hACE2 mRNA expression levels comparable to that of the other mice in the same group. Data information: Statistical significance was evaluated by Mann-Whitney test (*= p < 0.05, **= p < 0.01, ****= p < 0.0001). (C) Neutralizing activity (EC50) of sera from individual LV::S-vaccinated mice against pseudo-viruses harboring SCoV-2 from the ancestral strain or D614G, Alpha, Beta or Gamma variants. Red asterisk indicates significance versus ancestral, blue asterisk indicates significance versus D614G variant, while orange asterisk indicates significance versus Alpha variant. Statistical comparisons were made at the respective boosting timepoint. In homologous settings, sera from mice immunized with LV::SBeta or LV::SGamma, fully inhibited pseudoviruses bearing S from Beta or Gamma, validating the assay for all pseudo-viruses used. Data information: Statistical significance was evaluated by Mann-Whitney test (*= p < 0.05, **= p < 0.01, ****= p < 0.0001). (D) Cytometric analysis of CD8+ T cells in pooled olfactory bulbs of LV::S i.m.-i.n. vaccinated and protected mice versus sham-vaccinated and unprotected mice. (E) Wild type or µMT KO mice (n = 5-9/group) were injected by LV::S or sham following the time line shown in (A), then pretreated with Ad5::hACE2 4 days before challenge with the ancestral SARS-CoV-2 strain. Lung viral RNA contents were determined at 3 dpi. Data information: Statistical significance was evaluated by Mann-Whitney test (*= p < 0.05, **= p < 0.01, ****= p < 0.0001). (F) T-splenocyte responses in LV::S-primed and -boosted C57BL/6 WT mice or sham controls (n = 3-5/group), evaluated by IFN-γ ELISPOT using 15-mer peptides encompassing SCoV-2 MHC-I-restricted epitopes. Data information: Statistical significance was evaluated by Mann-Whitney test (*= p < 0.05, **= p < 0.01, ****= p < 0.0001).
Figure 5(A) Hetero-assembly forms around 144DG11 and not around endogenous molecules as shown by the liquid crystals formed in experiments 1-3 (B) STRING network of targets at the interactome of 144DG11. Targets also show strong protein-protein interactions among themselves, as connectors show (see legend). SLC17A5, Solute Carrier Family 17 Member 5; LAMP-1, Lysosomal Associated Membrane Protein 1; LAMTOR4, Late endosomal/lysosomal adaptor, MAPK and MTOR activator 4; GBA, Glucosylceramidase; SCARB2 (aka LIMP2), Scavenger Receptor Class B Member 2; DPP4, Dipeptidyl peptidase-4; GAA, Lysosomal alpha-glucosidase; HEXA, Beta-Hexosaminidase Subunit Alpha. (C) Cellular thermal shift assay (CETSA) of different targets of the 144DG11hetero-assembly (B). Only LAMP1 was significantly protected by 144DG11 from heat-mediated denaturation (see right shift in its Tm in the lower panel) suggesting its specific interaction with 144DG11. n=3 biological replicates. Error bars represent S.D. (D) Surface plasmon resonance assays demonstrate dose and pH-responsive interaction between LAMP1 and 144DG11. Full interaction (but with apparent KD of 6.3mM) was demonstrated only at the lysosomal pH 4.5-5. (E) Upper panel, LAMP1 was deglycoslated as detailed in Materials & Methods. RNase B is a positive control for a glycosylated protein. Glycosylation status is shown after short (24 h) or long (72h) dialysis. The results demonstrate a full deglycosylation of both LAMP1 luminal part and RNase B, with unique bands appearing after a long dialysis. Lower panel, surface plasmon resonance, performed as in (D), showing that the luminal domain of deglycosylated LAMP1 specifically binds 144DG11 with apparent KD of 52.5 nM. (F) Three binding modes of 144DG11 (gray) according to LAMP1 grids predicted by SiteMap (green), fPocket (cyan) and FtSite (magenta). Two out of three binding modes (SiteMap and fPocket) are identical. In FtSite part of the molecule went through a rotation relative to the other two.
Figure 5(A). ChIP-qPCR analysis of KAP1 association with the indicated sites in wild type female mESCs (Rif1+/+, same as used in Fig. 3B but without OHT) and during early differentiation. ZFP629 is a well-characterised KAP1 associated region (positive control). Xite A and C indicate two regions within the Tsix enhancer Xite, Tsix region 1 indicates Tsix major promoter, Tsix region 2 indicates the Dxpas34 region, Tsix region 3 indicates a region slightly downstream of the Dxpas34 region. P1 and P2 indicate the two Xist promoters, 5' indicates a region 2 kb upstream of Xist TSS. Inter1 is an intergenic region. See Appendix Fig. S2C for the positions of the primers within Xist and Tsix. The data are presented as mean ±standard deviation from three (2d EB and 1d EB) and two (ESCs) independent experiments. Statistical significance was calculated by Student's two-tailed unpaired t test comparing RIF1 association to Xist P2 and P1 in 2d EB versus 1d EB (*p ≤ 0.05 and ns=not significant). In the inset, Tsix RNA levels were quantified by RT-qPCR during the differentiation of wild type female ESCs shown in Fig. 4F. The average of two experiments is shown. Tsix values are normalised to a geometric mean consisting of the expression of Rplp0, Ubiquitin and Sdha. Error bars indicate standard deviations. (B). Using allele-specific primers, ChIP-qPCR was used to measure the association of KAP1 with Xist P2 in the Fa2L cells (black) and a wild type female mESC line also harbouring one castaneus and one 129 X chromosome (grey). cast indicates association with the castaneus Xist P2 and 129 indicates association with the 129 Xist P2. Enrichments are presented relative to input DNA. Mean ±standard deviation from a minimum of three independent experiments. Statistical significance was determined by Student's two-tailed, paired t test. Below, schematic of the Xist/Tsix alleles in the Fa2L cells. (C). KAP1 association with Xist promoter in two independent Rif1+/+ (Rif1+/+ +OHT, black) and two Rif1-/- (Rif1F/F +OHT, grey) female mESC cell lines. Ezr is an additional region known to be associated with KAP1 in mESCs. Enrichments are presented relative to input DNA. Mean ±standard deviation from a minimum of three independent experiments per cell line are displayed. Statistical significance was determined using Student's two-tailed, unpaired t test comparing the KAP1 association with Xist P2 and P1 in Rif1+/+ versus Rif1-/- cells (**p ≤ 0.01). (D). Allele-specific KAP1 association with Xist P2 in Fa2L cells following knock down of Luciferase (Control, black) and Rif1 (Rif1 KD, grey). cast indicates association with the castaneus Xist P2 promoter and 129 indicates association with the 129 Xist P2 promoter. Enrichments are presented relative to input DNA. Average ±standard deviation of two independent experiments.
Figure 3Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. mRNA expression of MyHC I, MyHC IIa, PGC-1α, myoglobin, TnnT1 MyHC IIb, MyHC IIx and TnnT3 in the gastrocnemius muscle (n=5-6). Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. Immunoblots and quantification of MyHC-I, IIa and IIb protein expression in gastrocnemius (n=3-4). Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. Representative images and quantification of Laminin (green), MyHC-I and IIb immunofluorescent staining (red) in gastrocnemius (n=3). Scale bar in (C) represents 100 µm.
N-terminal proteoforms may display different stabilities.Figure 6A. Degradation profiles of AIMP2, MARE2 and AN32E dbTIS and aTIS proteoforms as representative examples of N-terminal proteoforms that differ in stability. B. MS spectra of the N-termini of two proteoforms originating from the aminoacyl tRNA synthase complex-interacting multifunctional protein 2 (AIMP2): a dbTIS indicative N-terminus with processed iMet and starting at the second amino acid Pro, an aTIS indicative peptide starting at position 3 and retaining its iMet. As evident from the MS spectra shown, these N-terminal proteoforms displayed largely different turnover rates. C. Differences in turnover times were calculated for 100 dbTIS and aTIS proteoform pairs and presented in ascending order. The extent of N-terminal truncation in each pair was colour-coded to visualize the absence of correlation between the number of lacking amino acids and the direction of turnover shift. D. An increase in disorder content between aTIS and dbTIS proteoforms can be observed for a multitude of dbTIS/aTIS pairs.
Figure 8A Immunoblot analysis of HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 in fed or starved conditions and treated as indicated with Baf A1 or PI3KC3 inhibitor SAR405. B Immunoblot analysis of HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 mLIR+∆623-691 for 24 hours or not, and then treated as indicated. C, Representative confocal images of HeLa CALCOCO1 KO cells transiently co-transfected with mCherry-CALCOCO1, Myc-VAPA and EGFP-LAMP1, and then treated as indicated before immunostaining with anti-Myc anti-body. Arrows indicate co-localization. Scale bars in C, 5 μm for large merged images and 1 μm for zoomed images. D, data are presented as mean ± sd. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as ***p ˂ 0.001; ns is not significant, n=3. E Model of ER-phagy mediated by CALCOCO1 dimers illustrating the dual LIR-LDS and UIR-UDS interaction with lipidated GABARAP subfamily proteins on the phagophore, and the FFAT-like motif mediated interaction with MSP domains of VAP proteins on tubular ER.
Figure 5Physical activity of male mice shown hourly over 24-hrs and as day and night time. Data shown are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice at 17-18wks of age (WT n=12, TG n=11, TG n=10) and shown as means + SEM energy expenditure of male mice shown hourly over 24-hrs and as day and night time. Data shown are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice at 17-18wks of age (WT n=12, TG n=11, TG n=10) and shown as means + SEM , total assimilated energy of male mice shown hourly over 24-hrs and as day and night time. Table showing mean values of assimilated energy (kJ) at day/night time and per 24-hrs. Energy balance calculated as delta of assimilated energy (AE) and energy expenditure (EE) and energy balance (J) per 24-hrs. Data shown are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice at 17-18wks of age (WT n=12, TG n=11, TG n=10) and shown as means + SEM Table showing mean values of energy balance (∆AE-EE, kJ) at day/night time and per 24-hrs. Data shown are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice at 17-18wks of age (WT n=12, TG n=11, TG n=10) and shown as means + SEM Respiratory quotient (RQ) shown hourly over 24-hrs (J), RQ amplitude (K) and metabolic flexibility via percentage relative cumulative frequency (PRCF) (L). Data shown are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice at 17-18wks of age (WT n=12, TG n=11, TG n=10) and shown as means + SEM
Figure 7A. Live imaging of MCCs shows the ability of wild type Ccdc108 and mutant proteins lacking the seven amino acid IFT interaction domain to localize to migrating centrioles. Embryos expressing GFP tagged Ccdc108 proteins (green) and Cetn4-RFP (purple) were imaged at stage 18. B. Live imaging of MCCs shows the ability of IFT-B complex proteins to localize to migrating centrioles. Embryos expressing GFP tagged IFT-B proteins (green) and Cetn4-RFP (purple) were imaged at stage 18. C-E. Ccdc108 depletion affects centriolar distribution of IFT-B proteins during centriole migration in MCCs. Embryos were co-injected with GFP-IFTs (C: Ift74; D: Ift80; E: Ift88; green), Cetn4-RFP (purple) and each morpholino, and imaged at stage 18. HA tagged Ccdc108 proteins were co-expressed to test for the rescue of phenotypic defects. Xenopus embryos were subjected to immunoblotting to evaluate the protein expression levels. α-tubulin (α-Tub) served as loading control. Quantitative plots show that Ccdc108-depleted MCCs showed a significant decrease of centriolar distribution of IFT-B proteins (Ift74, Ift80 and Ift88), and Ccdc108-IFT interaction deficient mutants are unable to rescue the decreased centriolar accumulation of IFT-B proteins. >40 MCCs from 6 embryos for each condition. Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001). Mean ± s.d. values are also presented.
LEMD2-regulated genes in cortical neurons contribute to cognitive function and risk of neurodevelopmental disorders.Figure 6MAGMA gene-set analysis of the differentially expressed genes upon LEMD2 depletion in cortical neurons using the summary statistics from multiple GWAS datasets. Phenotypes are listed on the y-axis, EA (educational attainment), IQ (cognitive ability/human intelligence), SZ (schizophrenia), ASD (autism spectrum disorder), CAD (coronary artery disease), ADHD, (attention deficit hyperactivity disorder), CD (Crohn's disease), MDD (major depressive disorder), Stroke, T2D (type 2 diabetes), AD (Alzheimer's disease), OSD (obsessive-compulsive disorder). P-values are shown above each data point, which represent beta values (x-axis). Horizontal bars indicate error bars (SE). P-values in red survived Bonferroni multiple test correction of p < 0.0125 for the 4 tests (IQ, EA, ASD, and, SZ). Analysis of LEMD2-regulated genes using data on de novo mutations. LEMD2 gene-set was enriched for LoF de novo mutations reported in schizophrenia (SZ) and Mis de novo, mutations reported in intellectual disability (ID); p-values in red survived Bonferroni multiple test correction for the 9 tests.
Figure 4(a) Upper panel: Single-cell UMAP of sequenced PBMCs from 2 NAFLD patients and 4 healthy subjects; n = 10,786 cells. We identified 15 distinct cell types by DICE annotation. Lower panel: Expression patterns of CXCR3, CXCR4, ACKR3, CX3CR1, CCR6 across different cell types in NAFLD and healthy PBMC samples. (b) Chemokine ligand-receptor mapping to identify potential immune cell interactors based on NAFLD endothelial chemokine signatures. (c) Graphic shows the percentage composition of identified cell types in NAFLD and healthy PBMCs single-cell analysis. (d) Upper panel: Representative flow cytometry dot plots, characterizing the isolated CD8+ T cells and CD8- immune cell fraction (mainly CD4+ T cells, NK cells and monocytes) from PBMCs of healthy and NAFLD subjects. Lower panel: Flow cytometry quantification of % of positively expressing CXCR4+ and CX3CR1+ cells within each immune subset. Sample sizes are n = 14 NAFLD; n = 6 healthy. (e) Heatmap of gene expressions of endothelial antigens relevant to interaction with T lymphocytes in NAFLD (n = 3) and healthy (n = 3) BOECs, with 3 biological replicates per donor. (f) Upper panel: Representative flow cytometry histograms of HLA-A/B/C, HLA-DM and IFN-γR1 expressions in NAFLD (red) and healthy (blue) BOECs. Grey, isotype control. Lower panel: Box-whisker plots reflect mean fluorescence intensity (a.u., arbituary unit), normalized to isotype control, as a readout for epitope density. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, non-significant (t-test). n = 4 donors; *p<0.05; ns, not significant (Mann-Whitney test for HLA-A/B/C and IFN-γR1; t-test for HLA-DM).
Inhibition of autophagy by cytoplasmic HMGB1.Figure 3.Inhibition of autophagy by cytoplasmic HMGB1. Hmgb1−/− and Hmgb1+/+ MEFs were enucleated by centrifugation after cytochalasin B treatment as described in Materials and methods and then were treated with HBSS for 3 h, and LC3 punctae formation was detected by a confocal microscope. (A) Representative images of LC3 punctae (white arrows) and HMGB1 (red arrows) in cytoplasts of Hmgb1−/− and Hmgb1+/+ MEFs are depicted. (B) The percentage of cells showing accumulation of LC3 punctae was reported (*, P < 0.05; n = 3). Data are means ± SEM.
Prediction of Nepali unknown samples using the 2-class and qPCR validation.Figure 5(a) PCA of sEF samples based on the 5-gene signature (based on gene array data) coloured by predicted class membership (EF: purple; green: rest). (b) Dot plot of prediction probability of being class EF versus the expression score calculated on the bases of the 5-gene signature (based on gene array data). (c) qPCR gene expression scores of the 5-gene signature (∆∆CT over PPIA) for CTRLs, 03NP-sEF, 03NP-SPT and 03NP-ST samples from Nepal. Yellow diamonds in the 03NP-sEF category represent the 9 patients classified as EF based on the random forest algorithm. (d) qPCR expression values (∆∆Ct over PPIA) of the 5-gene signature in control samples (Oxford and Nepal), S. Paratyphi A (03NP-SPT) or S. Typhi (03NP-ST) in Nepal, samples at day 7 after challenge of participants who stayed well following challenge with S. Typhi (nD7), or typhoid diagnosis after challenge (TD) in the Vi-TCV study (e) Combined qPCR expression score of the 5-gene signature. Black arrows indicate outlier samples. (f) Temperature and CRP for samples of which data was available (CRP was only measured in the Oxford CHIM). D0 = pre-challenge baseline Vi-TCV study; nD7 = day 7 samples of participants who stayed well following challenge (Vi-TCV study); SPT = S. Paratyphi A (03NP); ST = S. Typhi (03NP); TD = Typhoid diagnosis (Vi-TCV study). (g) Spearman's rank correlation of the 5-gene combined expression score and (left) temperature (only nD7 and TD samples from the Oxford CHIM - Vi-TCV and SPT and ST cases from Nepal at presentation to hospital were included) and (right) CRP (CRP was only available for Oxford CHIM - Vi-TCV samples and we excluded D0 baseline measures).
Figure 2Pyruvate kinase (PK) activity in FLAG immunoprecipitates from human endothelial cells expressing FLAG-tagged wild-type (WT), Y657F (YF) or Y657D (YD) eNOS; n=6 independent cell batches (1-way ANOVA and Tukey). PK activity in eNOS immunoprecipitates from HEK293 cells expressing WT, YF, YD or S633D (SD) eNOS; n=5-9 independent experiments (1-way ANOVA and Bonferroni). S-nitrosation of PKM2 in human endothelial cells expressing FLAG-tagged WT or YF eNOS and treated with H2O2 (30 µmol/L) for 15 min; n=8 independent cell batches (Unpaired Student's t test). Dithiothreitol (DTT) was included to demonstrate specificity of the S-NO signal. PKM2 S-nitrosation in HEK293 cells expressing YF or YD eNOS and either FLAG-tagged WT PKM2 or a C358S (CS) PKM2 mutant. SNO-FLAG-PKM2 was detected with an anti-FLAG antibody after biotin switch technique; n=4 independent experiments (2-way ANOVA & Bonferroni). DTT treatment was included to demonstrate specificity of the SNO signal. PK activity in eNOS immunoprecipitates from HEK293 cells co-expressing YF or YD eNOS and either WT PKM2 or the CS PKM2 mutant; n=6 independent experiments (2-way ANOVA & Bonferroni). PKM2 enzyme kinetics measured with increasing concentrations of phosphoenolpyruvate (PEP) in eNOS immunoprecipitates from HEK293 cells expressing YF or YD eNOS. The data were fit with the Michaelis-Menten equation to determine Vmax and Km; n=4 independent experiments (2-way ANOVA & Bonferroni). Quantification of pentose phosphate pathway (PPP) intermediates; gluconate-6-P (G6P), ribulose-5-P (Rl5P), ribose-5-P (R5P), sedoheptulose-7-P (S7P), fructose-6-P (F6P) and erythrose-4-P (E4P) in human endothelial cells expressing YF or YD eNOS; n=6-8 independent cell batches (Unpaired Student's t test). Link between the pentose phosphate pathway, generation of NADPH and reduction of GSSG to GSH. NADPH/NADP+ and GSH/GSSG ratios in human endothelial cells expressing YF or YD eNOS; n=6-8 independent cell batches (Unpaired Student's t test). NADPH/NADP+ and GSH/GSSG ratios in human endothelial cells treated with solvent (Sol) or the PKM2 inhibitor shikonin (SKN, 1 µmol/L) for 45 minutes; n=4 independent cell batches (Unpaired Student's t test).
a) Overall structure of GAL8 with bound Galbi peptide (cyan) and NAD (purple) in the asymmetric unit.f2(a) Overall structure of GAL8 with bound Galbi peptide (cyan) and NAD (purple) in the asymmetric unit. The other pair of crystallographic dimer is coloured grey. The first β-strand forming unique interaction is labelled as β1. (b) A topology diagram of GAL8. The β-strands in each N-CRD and C-CRD are numbered sequentially with prime (′) in the C-CRD. The β-strands with a solid line indicate a carbohydrate-binding side in each CRD and those of with a dashed line indicate an opposite side. The elongated purple and cyan ovals indicate NAD- and NDP52-binding sites, respectively. (c) Overall view of Galbi peptide (cyan) bound to the C-CRD of GAL8 (β-strands in yellow and residues in gray).
Figure 6A. Protein samples (20 μg) from Jurkat cells treated as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. WB are representative of at least three independent experiments.B-C. Quantitative analysis of the ratio between the protein levels of mitochondrial markers and Actin during AICD is shown. Data represent mean ± SE of three independent experiments. D. Jurkat cells were co-transfected with GFP-LC3 and Parkin-Cherry, 32h after AICD induction cells were fixed and immunostained with an anti-TOM20 antibody. Representative images for each condition are presented. Scale bar, 5μm.E. LC3-Parkin-mitochondria co-localization was calculated from 30 randomly selected images. Data represent mean ± SE of three independent experiments. Data referring to Ctrl and AICD-stimulated cells without rapamycin correspond to the ones presented in Fig.3C.
Figure 3 A-C Exponentially growing cell cultures (exp) were arrested in G1 with α-factor (αf) and released into YEPD with or without CPT (50 μM). Cell samples were harvested at the indicated time points to evaluate DNA contents by flow cytometr  A-C Exponentially growing cell cultures (exp) were arrested in G1 with α-factor (αf) and released into YEPD with or without CPT (50 μM). Cell samples were harvested at the indicated time points to evaluat nuclear division by fluorescence microscopy (B  A-C Exponentially growing cell cultures (exp) were arrested in G1 with α-factor (αf) and released into YEPD with or without CPT (50 μM). Cell samples were harvested at the indicated time points to evaluat Rad53 phosphorylation by western blot with anti-Rad53 antibodies (C)  D Exponentially growing cell cultures (exp) were treated with CPT (50 μM) for 1 hour, before being released in fresh medium without CPT. Western blot analysis with anti-Rad53 antibodies of protein extracts prepared at the indicated time points  E, F G1-arrested cell cultures (αf) were released in YEPD supplemented with CPT (50 μM) (E) or in YEPD supplemented with CPT (10 μM) with (+noc) or without (-noc) nocodazole (F). Western blot analysis with anti-Rad53 antibodies
We propose that growth factor stimulation results in the docking of the ERK cascade components, Raf, MEK and ERK onto the cytoplasmic face of autophagic structures.f8We propose that growth factor stimulation results in the docking of the ERK cascade components, Raf, MEK and ERK onto the cytoplasmic face of autophagic structures. ATG5-ATG12-positive preautophagosomes and LC3-II-positive membranes serve as scaffolds or cellular signalling platforms that facilitate efficient spatial coordination of the Raf-MEK-ERK cascade and thus facilitate growth factor-induced ERK phosphorylation.