EMBO/sd-character-level-ner huggingface dataset:
Figure 7A) WT PER2::LUC cells were treated with epoxomicin or vehicle control. Blue arrows show the time points where drug was washed off. Mean (solid lines) ± SD (dotted lines). B) Quantification of damping rate of the PER2::LUC recordings shown in A), for the duration of drug treatment. Mean±SD. One-way ANOVA with Holm-Sidak's multiple comparisons, **p≤0.01, ****p≤0.0001. C) WT PER2::LUC cells were treated with tunicamycin or vehicle control. Mean (solid lines) ± SD (dotted lines). D) Quantification of damping rate of the PER2::LUC recordings shown in C), for the duration of drug treatment. Mean±SD. One-way ANOVA with Holm-Sidak's multiple comparisons, ****p≤0.0001. E) WT PER2::LUC cells were treated with radicicol or vehicle control. Blue arrows show the time points where drug was washed off. Mean (solid lines) ± SD (dotted lines). F) Quantification of damping rate of the PER2::LUC recordings shown in E), for the duration of drug treatment. Mean±SD. One-way ANOVA with Holm-Sidak's multiple comparisons, ****p≤0.0001. G-H) WT and CKO cells were treated with 0.3 µM cycloheximide (CHX) or DMSO vehicle control, and kept in constant conditions, n=4, Mean±SD. Panels above show baseline-subtracted raw data, and panels below show data detrended with a 24-hour moving average. I-J) Representative double-plotted actograms of WT mice receiving drinking water with DMSO vehicle control (N=7 mice) or 35 µg/mL ixazomib (N=8 mice). Two representative actograms are shown per condition. For the first 7 days the mice were in 12:12h light-dark cycles (LD), and had drinking water supplemented with blackcurrant squash. Ixazomib/DMSO was added to this, and the lighting was changed to constant darkness (DD). After 7 days the drug/vehicle was removed, and the mice remained in DD until the end of the experiment. K-L) Quantification of the amplitude of wheel-running activity and period length from the experiment shown in I), J), including pre-treatment, during treatment and post-drug treatment. Mean±SEM. 2-way ANOVA with Holm-Sidak's multiple comparisons, *p≤0.05, ***p≤0.001, ****p≤0.0001, TWAINT = two-way ANOVA interaction. M) Schematic diagram: our data suggests that CRY deficiency is associated with proteome imbalance, which in turn disrupts circadian rhythmicity of physiology.
Figure 6(A and B) XO8 GSCs and XO8 DFCs were treated with 50 nM MG132 for indicated hours. The expression of genes was assessed by real-time PCR using total RNA extracted from MG132-treated cells. Mean +S.D. values from the three independent experiments are shown after normalization to that of control cells treated with DMSO. (A and B) XO8 GSCs and XO8 DFCs were treated with 50 nM MG132 for indicated hours. The expression of genes was assessed by real-time PCR using total RNA extracted from MG132-treated cells. Mean +S.D. values from the three independent experiments are shown after normalization to that of control cells treated with DMSO. (C) XO8 GSCs and XO8 DFCs treated with 50 nM MG132 as described in (A) were subjected to immunoblotting. (D) XO8 GSCs were treated with MG132, salinosporamide A, epoxomicin, or bortezomib for 24 hrs. Cell lysates were subjected to immunoblot analysis with anti-PARP-1, anti-CHOP, anti-ATF3, anti-ATF3, and anti-BiP antibodies. (E) XO8 cells were treated with 50 nM MG132 in the absence or presence of 5μM actinomycin D (ActD) for 6 h. The level of ATF3 mRNA was measured using semi-quantitative real-time PCR in comparison with β-actin.
x expression.Figure 1A, Top: schema of analyzed TR regions (purple), located downstream of the annotated open reading frame (grey), between the annotated stop codon (red triangle) and the next in-frame stop codon (orange triangle); Bottom: histogram of average PhastCons conservation scores (x-axis) of putative TR regions of all RefSeq-annotated transcripts. The scores of the four human Argonaute protein family members are highlighted. B, Multiple sequence alignment of the AGO1 putative TR region across vertebrates. C, Multiple sequence alignment of the corresponding predicted amino acid sequence. The unique peptide targeted by the polyclonal antibody is indicated by the red line. The green and blue lines indicate peptide sequences obtained after tryptic digestion, in which cleavage is exclusively at Arginine (R) and Lysine (K) (further described below in panels E and F). Red asterisks indicate stop codons. D, Western blot showing AGO1x expression in three cell lines. For comparison, a parallel blot was probed with an antibody directed against canonical AGO1. Tubulin served as loading control. E,F, Annotated MS/MS spectrum of peptides specific for the endogenous AGO1x, "QNAVTSLDR", depicted in green (E) and "LSKPQELCHPNPEEAR", depicted in blue (F). The Mascot Ion Score (text color corresponds to peptides marked in Fig 1C for reference) as well as the annotated fragments (blue = y-ions; red = b-ions) together with the corresponding amino acids are indicated.
Figure 6A. Representative pictures of CD31 (red) immunostaining of Control and CriptoMy-LOF TA sections at day 5 (left panel) and 30 (right panel) after single injury (CTX-I). B-D. Quantification of CD31+ capillary number per area (mm2; B), average of capillary cross-sectional area (µm2; C) and percentage of small (<20 µm2) and large (>100 µm2) capillaries (D) in Control and CriptoMy-LOF injured muscles at indicated time points after single injury (CTX-I). Data are mean±SEM (n=5 biological replicates; *P<0.05; **P<0.01; ***P<0.001, Student's t-test). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x. E. Representative pictures of CD31 (red) immunostaining of Control and CriptoMy-LOF TA sections at day 5 (left panel) and 30 (right panel) after re-injury (CTX-II). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x. F. Representative pictures of mdx-Control and mdx-CriptoMy-LOF diaphragm sections immunostained with VEcad (red). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x. G-H. Quantification of CD31+ capillary number per area (mm2; G) and average of capillary cross-sectional area (µm2; H) in Control and CriptoMy-LOF injured muscles at the indicated time points after re-injury (CTX-II) Data are mean±SEM (n=6 biological replicates; *P<0.05; ***P<0.001, Student's t-test). I-J. Quantification of capillary number per area (mm2; I) and average of capillary cross-sectional area (µm2; J) in mdx-Control and mdx-CriptoMy-LOF diaphragm muscles. Data are mean±SEM (n=5 biological replicates; ***P<0.001, Student's t-test).
Intramuscular injection of AAV2.Figure 7A. Glycogen assay in TFEB-injected gastrocnemii and in the contralateral muscles. In TFEB-injected musclesglycogen levels were significantly decreased compared to those in untreated muscles. * indicates statistically significant differences (p = 1.0 × 10−4; n = 6; Student's t-test). B. PAS staining of TFEB-treated muscle shows a reduction of lysosomalglycogen stores (puncta) compared to those in untreated muscle. Original magnification: 20×. C. LAMP1 staining of TFEB-injected gastrocnemii and of the contralateral untreated muscles. In TFEB-treated muscles, the size of LAMP1-positive vesicles was reduced. Bar: 2 µm.
Figure 2A, MCF10A WT cells were pretreated with AZD6738(250 nM) and RO-3306 (9 μM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies. B, Same condition except that RO-3306 is replaced by PD-0332991 (1µM). C, MCF10A WT cells were pretreated with DMSO or AZD6738(250 nM) for 2 hours, and then irradiated with 20 Gy, 24 hours later, cell cycle distribution was measured by flow cytometry. EdU (10 μM) was added into medium 2 hours before harvesting for flow cytometry. D, MCF10A WT cells were pretreated with DMSO or AZD6738(250 nM) for 2 hours, and then irradiated with 20 Gy, and cells were harvested at indicated time points for immunoblotting. E, MCF10A control cells and CDC25C-WT/constitutively active mutant CDC25C-S216A over-expressed cells were pretreated with DMSO or AZD6738(250 nM) for 2 hours, and then irradiated with 20 Gy, 24 hours later, cells were harvested for immunoblotting with indicated antibodies. F, Same condition except that cells were harvested 72 hours later.
Figure 4(A) Western Blot analysis of the crypt-derived chromatin fractions incubated with soluble lysates from villi or crypts for the indicated times. (B) Table showing the proteases differentially expressed in the villus compartment as determined by RNA-seq analysis of intestinal crypt (purified EphB2 high cells) and villus (EphB2 negative/low) cells. Predicted cleavage sites for each protease in the histone H4 sequence was determined using the PeptideCutter-ExPASy and PROSPER software. (C) Western blot analysis of mCherry-H4 with the indicated mutations transfected in HCT-116 cells. Quantification of relative amounts of truncated H4 relative to the WT. (D, E) Western Blot analysis of mCherry-H4 transfected in HCT-116 cells treated for 16 hours with TLCK (D) or AdaAhx₃L₃VS (E) at the indicated concentrations. (F) Western Blot analysis of a cleavage experiment incubating modified H4 peptides (P1-P9) with soluble lysates from villi or crypts in the presence (+) or absence (-) of the commercial protease inhibitors cocktail. Lanes indicated as P correspond to the control peptide without lysate incubation. Notice the electrophoretic shift of peptides incubated with villus lysates (compared with control peptides) suggestive of post-translational modifications or binding to proteins absent from crypt lysates. (G) Quantification of the relative cleavage of specific H4 peptides incubated with villus-derived lysates from 3 independent experiments performed.
Fig.Figure 5A Experimental timelines for CSDS, ARL67156 treatment and immunostaining. B Immunostaining for BrdU (blue) and DCX (green) in the DG region of control, susceptible and ARL67156 treatment mice. Arrows indicate the BrdU+ DCX+ cells. Scale bar: 50 μm. C, D Quantification of BrdU+ cells (C) and BrdU+ DCX+ cells (D) in the DG of susceptible mice with ARL67156 compared with vehicle treatment (n = 4 mice/group. For BrdU+ Treatment F2, 9 = 10.07, P =0.0051; CSDS - ACSF vs Ctrl - ACSF, P = 0.0147; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0018; BrdU+ DCX+ Treatment F2, 9 = 9.005, P = 0.0071; CSDS - ACSF vs Ctrl - ACSF, P = 0.0211; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0024, one-way ANOVA with Fisher's LSD test). E Experimental timelines for neurogenesis examination with LV-siCD39. F Confocal images of BrdU (blue) and DCX (red) in mice treatment with LV-siCD39 in the DG. Arrows indicate the BrdU+ DCX+ cells. Scale bar: 50 μm. G, H Quantitative of BrdU+ (G) and BrdU+ DCX+ (H) cells in the DG of susceptible mice with LV-siCD39 compared with LV-GFP treatment (n = 3, 6, 6 mice. For BrdU+ Treatment F2, 12 = 15.79, P =0.0004; CSDS - LV-GFP vs Ctrl - LV-GFP, P = 0.0012; CSDS - LV-siCD39 vs CSDS - LV-GFP, P = 0.0003; BrdU+ DCX+ Treatment F2, 12 = 12.79, P = 0.0011; CSDS - LV-GFP vs Ctrl - LV-GFP, P = 0.05; CSDS - LV-siCD39 vs CSDS - LV-GFP, P = 0.0003, one-way ANOVA with Fisher's LSD test).
Figure 6(A) (top) Cartoon representation and surface plot of a superpositioning of CrIFT52N and MmIFT52N crystal structures. (bottom) Rotated 180 degrees compared to top panel. Conservation is mapped from 5 different species (see alignment in Appendix Fig. S5) and conserved residues are labeled (Cr numbering). The two residues (K130 and R204) found to disrupt interaction with IFT-B2 (panel (B)) are labeled in larger font. (B) Coomassie stained SDS PAGE gel of a pull-down of IFT-B2 using WT or mutant IFT88/521-335-GST. Whereas single point mutations slightly reduced the amounts of pulled down IFT-B2, the K130E/R204E double mutation reduced it to background levels (compare lanes 2 and 6).
Figure 3(A) Principal component analysis of DNA methylation data at 17,937,401 CpGs for CD4+ naïve T cells (gray), WT Treg cells (blue) and Tet2/3 DKO Treg cells (red). The percentage of variation explained by each component is shown in parentheses. (B) Scatter plots of DNA methylation data showing the comparisons between WT Treg vs CD4+ naïve T cells (left) and WT Treg vs Tet2/3 DKO Treg cells (right). The differentially methylated CpGs (DM-CpGs) are shown in color (blue: WT Treg; black: CD4+ naïve T cells; red: Tet2/3 DKO) and the numbers are shown on top of each graph. (C) K-means clustering of DNA methylation data for all the samples using CpGs differentially methylated between CD4+ naïve T cells vs WT Treg cells and WT Treg vs Tet2/3 DKO Treg cells (left); the number (N) of CpGs in each cluster is shown in parenthesis. Middle: CpG loci proximal to selected genes are annotated. Right: the average DNA hydroxymethylation levels at each CpGs as measured by CMS-IP. (D) The average DNA methylation levels at selected consensus transcription factor-binding motifs (gray: CD4+ naïve T cells; blue: WT Treg cells; red: Tet2/3 DKO Treg cells). Standard error is denoted in transparency of the corresponding colors. (E) Genome browser view of DNA methylation patterns of Hopx and Il10 loci for CD4+ naïve T cells, WT Treg cells and Tet2/3 DKO Treg cells. Differentially hydroxymethylated regions (DhmRs) and differentially methylated CpGs (DM-CpGs) are shown on top of the tracks. Conservation track (Cons) and denotation of all CpGs are shown at the bottom. WGBS data are from two biological replicate samples.
Figure 4(A) Patient n= 4 (F7/7F-healthy carrier, F7/2M-RIRCD, F7/5F-healthy carrier, F7/1M-RIRCD without a clear second mutation) and control fibroblasts (n=2) were grown for 12 days in media containing 0.02 mM cysteine and 5% dialyzed FBS. Proteins belonging to mitochondrial complexes (NDUFA9-Complex I, SDHA-Complex II, MTCO1-Complex IV and MTCO2-Complex IV) were analysed via Western Blotting. VCL was used as a loading control and the densitometry analysis was done based on the mitochondrial proteinTOM20. (B) Densitometry analysis of proteins from (A) showing no significant changes in OXPHOS proteins upon glutamine/ glutamic acid depletion. Control bar represents an average of the two control fibroblast lines employed (F008 and F154). (C) Patient (F7/7F-healthy carrier, F7/2M-RIRCD, F7/5F-healthy carrier, F7/1M-RIRCD without a clear second mutation) and control fibroblasts were grown for 12 days in media with no added glutamine or glutamic acid and 5% dialyzed FBS. Cells were lysed and the above mentioned mitochondrial proteins were checked via Immunoblotting. VCL was used as a loading control while the densitometry analysis (D) was done based on the mitochondrial proteinTOM20. Control bar represents an average of the two control fibroblast lines employed (F008 and F154). Although we could observe significant changes upon cysteine depletion in all conditions, the phenotype was exacerbated in the digenic fibroblasts compared to the controls.
signaling and promotes GSC self-renewal through competitively binding miR-200s A and B.Figure 4A and B. Correlation of expression between LINC00115 and ZEB1 from TCGA GBM RNA-Seq dataset (A) and REMBRANDT GBM array dataset (B). ***, p < 0.001, by two-tailed t-test. C. LINC00115 knockdown reduced ZEB1 mRNA expression in 1123 and 528 GSCs. Data information: , data are representative of three independent experiments. Error bars, ± SD. **, p < 0.01, by One-way ANOVA D. Effects of LINC00115 knockdown on ZEB1, vimentin, and E-cadherin protein expression. Data information: data are representative of three independent experiments. Error bars, ± SD. **, E. Effects of re-expression of shRNA-resistant LINC00115 WT (L115-WT), miR-200 binding mutant (L115-mut) with or without miR-200b on ZEB1 signaling activation. Data information data are representative of three independent experiments. Error bars, ± SD. F. Neuro-like sphere formation assay of effects of re-expression of shRNA-resistant LINC00115 WT and mutant with or without miR-200b. Data information: data are representative of three independent experiments. Error bars, ± SD. **, p < 0.01, by two-tailed t-test in F. G. Kaplan-Meier analysis of animal implantation with 1123 GSCs with indicated modifications. n = 10. Statistical analysis was performed by log-rank test. H and I. GSEA of EMT signature genes using ranked gene expression changes in TGF-β1-treated 1123/shLINC00115 GSCs versus the control (H) or in 1123 GSCs with ectopic expression of LINC00115 or an empty vector (EV) (I). NES, normalized enrichment score.
TBK1 promotes EGF-stimulated mTORC1 signaling and catalytic activity in a manner dependent on site-specific mTOR phosphorylation.Figure 4A. mTOR S2159 phosphorylation is required for EGF stimulated mTORC1 signaling: HEK293 cells were co-transfected with vector control, wild type, or rapamycin-resistant (RR) AU1-mTOR alleles (RR or RR/S2159A) together with HA-S6K1. Cells were serum starved (20 hr.), treated -/+ rapamycin (30 min) to ablate endogenous mTORC1 function, and stimulated -/+EGF [25 ng/mL] (30 min). HA-S6K1 was immunoprecipitated, and immunoprecipitates (IP) and whole cell lysate (WCL) were immunoblotted (IB) as indicated. B. TBK1 is required for EGF stimulated mTOR auto-phosphorylation: TBK1+/+ and TBK1-/- MEFS were serum starved, EGF stimulated, and analyzed as in A. C. EGF time course analysis of mTOR auto-phosphorylation: TBK1+/+ and TBK1-/- MEFS were serum starved, EGF stimulated for 0-60 min, and analyzed as in A. D. TBK1 is required for EGF stimulated mTOR auto-phosphorylation within mTORC1: TBK1+/+ and TBK1-/- MEFS were serum starved, pre-treated with Ku-0063794 [1 μM], and EGF stimulated as in A. Raptor was immunoprecipitated, and IPs and WCL were analyzed. E. mTOR S2159 phosphorylation is required for EGF stimulated mTOR auto-phosphorylation: HEK293 cells were transfected with Myc-mTOR wild type (WT), S2159A, and kinase dead (KD). Cells were then serum starved, pre-treated with Ku-0063794 (30 min), and stimulated -/+ EGF as in A. F. Model: EGF-receptor signaling increases mTORC1 signaling through at least three pathways in MEFs, the PI3K/Akt, MAPK, and TBK1 pathways.
TFEB mediates pTau reduction in a PTEN-dependent mannerWestern blot analysis of total Tau and PHF1-positive pTau levels as a function of wild-type or the C124S or G129E mutant PTEN expression.Figure 9A Western blot analysis of total Tau and PHF1‐positive pTau levels as a function of wild‐type or the C124S or G129E mutant PTEN expression. Empty vector (Ctrl)‐ or TFEB‐transfected cells were used as negative and positive controls, respectively. γ‐tubulin was used as a loading control. B Quantization of blots in A and normalized for loading to γ-tubulin. TFEB and PTEN transfection significantly reduced pTau as probed by PHF1 (P = 0.0095 and 0.0092, respectively), as well as total Tau (P = 0.0052 and 0.034, respectively), n = 3, Student's t-test. C Representative immunofluorescence imaging showing that cells with wild‐type PTEN (WT), but not the C124S or G129E mutants, were correlated with reduced PHF1‐positive Tau. Scale bar: 10 μm. D Western blot analysis of total Tau, PHF1‐positive pTau and LC3 levels in response to TFEB or TFEB and PTEN shRNA expression. Transfection of a scramble PTEN shRNA was used as negative controls. γ‐tubulin was used as a loading control. E Quantization of blots in D and normalized for loading to γ-tubulin. Comparing with cells transfected with scramble+pEGFP, scramble+TFEB transfection reduces PHF1 and total Tau intensity (P = 0.038 and 0.035, respectively) and increases PTEN and LC3II (P = 0.04 and 0.0076, respectively) (white slashed bars). shRNA significantly reduce PTEN when transfected with either pEGFP or TFEB (P = 0.0089 and 0.043, respectively), but TFEB's effect in reducing pTau drops from 60% reduction in the presence of scramble sequence to 30% reduction when combined with shRNA of PTEN. All statistics here are Student's t-test, n = 3. Each bar represents average ± s.e.m.
Gata6 is mutated or downregulated in human sebaceous carcinomas with a high mutational burden.Figure 7(A) Differentiation status (poor, moderate or well-differentiated), circumscription (not infiltrative, focally infiltrative or infiltrative), log10 total number of mutations and Gata6 mutations in 32 human sebaceous carcinomas (SebC) separated in 3 subgroups: pauci-mutational, MSI-related and UV-related For Gata6 mutations, the most deleterious mutation is displayed. n.a.: not assessed. (B) Schematic representation of Gata6 protein displaying the different protein domains: PEST, GATA-type transcription activator, Zinc-finger (ZnF), Nuclear Localization Sequence (NLS) and the different Gata6 missense mutations and deletions found in human SebC. Heat-map displaying the predicted effect (deleterious or neutral) of W8C, S33C, G61R and S511F point mutations as assessed by PredictSNP, Polyphen-1, Polyphen-2, SIFT, SNAP and SNAP2 algorithms (right panel). Percentages indicate the level of confidence of the predictions. (C) Gata6 expression (measured in Transcripts Per Million, TPM) in human pauci-mutational (pauci-mut) SebC (n=4), MSI-related SebC (n=4), UV-related SebC (n=5) samples. Data are means ± SD. (*) p-value ≤ 0.05, Student's t-test. (D-F) Graphs showing the number of mutations per megabase (Mutations/Mb) (D), the number of Somatic Single-Nucleotide Variants (SSNV) (E) and indel (F) of Gata6mut (n=3) or Gata6wt (n=6) MSI-related SebC (left panels) and of Gata6mut (n=3) or Gata6wt (n=7) UV-related SebC (right panels). Data are means ± SD. (G) Heat-map displaying the mean expression level of MMR genes (measured in TPM) in Gata6wt (n=10) and Gata6mut (n=3) SebC.
Figure 3A Localisation of HERK1, ANJ, LRE, FER and NTA in the synergid cell of wild-type (Col-0; WT), herk1 anj and lre-5 in unfertilised ovules, as shown by pFER::HERK1-GFP, pANJ::ANJ-GFP, pLRE::LRE-Citrine, pFER::FER-GFP and pMYB98::NTA-GFP. DIC and fluorescence images are shown, left to right, respectively. White and red dotted lines delineate the egg cell and synergid cells, respectively. Scale bars = 25 µm. B Localisation of NTA in the synergid cell of wild-type and herk1 anj plants before (upper panels) and after (lower panels) pollen tube arrival. In green, NTA localisation as shown by pMYB98::NTA-GFP fluorescence. In magenta, callose of the filiform apparatus and pollen tube stained with SR2200. From left to right, images shown are DIC, merged fluorescence images, and merged images of DIC and fluorescence. White and red dotted lines delineate the pollen tube and synergid cells, respectively. Scale bars = 25 µm. M, micropyle. Profile of relative fluorescence intensity of NTA-GFP along the synergid cells of wild-type and herk1 anj ovules (C) before (virgin) and after (pollinated) pollen arrival. Data shown are means ± SEM, n = 25. *** p<0.001 (Student's t-test). FA, filiform apparatus. D Profile of relative fluorescence intensity of NTA-GFP along the synergid cells of wild-type and lre-5 ovules (D) before (virgin) and after (pollinated) pollen arrival. Data shown are means ± SEM, n = 25. *** p<0.001 (Student's t-test). FA, filiform apparatus.
a, Correlative light and electron microscopy analysis of STX17 knockdown HeLa cells expressing GFP–LC3 (white arrows) infected with GAS (red arrows).f4a, Correlative light and electron microscopy analysis of STX17 knockdown HeLa cells expressing GFP-LC3 (white arrows) infected with GAS (red arrows). DAPI, 4′,6-diamidino-2-phenylindole. b, Electron micrographs of STX17 knockdown cells. White arrows denote autolysosomes, black arrows denote autophagosome, white arrowheads mark isolation membranes. c, Immunogold electron micrographs of STX17 knockdown HeLa cells stained for GFP-LC3. d, GFP-ATG5 dots (arrows) in STX17 knockdown HeLa cells. e, Quantification of staining in d. f, Immunoblot of p62 in STX17 knockdown cells. Tubulin was used as a loading control. g, Degradation of long-lived proteins in STX17 knockdown cells. St, starved; wor, wortmannin. All error bars denote s.d. Scale bars, 1 µm (a, b), 200 nm (c) and 5 µm (d).