EMBO/sd-character-level-ner huggingface dataset:
Figure 4(A) UMAP plot of Cluster 0 and Cluster 14 populations from the GW7 sample. (B) Heatmap of differentially expressed genes (DEGs) in Clusters 0 and 14 at GW7. On the right of the heatmap are the top-twelve DEGs. Selected top gene ontology (GO) terms related to the corresponding DEGs (right). (C) Single-cell resolution trajectory of EGFR+ transitional glial cells (Cluster 14) was constructed from sample (GW11T-NC, GW14T-NC, GW18T-NC, GW20T-NC, GW21T-NC) during the transitional period using Monocle. Linear arrangement of the transition trajectory with developmental stages represents the transition process of a regulatory cell type. The arrow indicates the developmental trend. (D) Violin plots produced by VlnPlot function of Seurat package representing changes in expression of EGFR, DLL1, MIAT, and FAM189A2 in cluster 14 with development based on snRNA counts data. (E) Overview of selected statistically significant interactions between neural cell types according to cell-cell communication analysis from statistical test of CellPhoneDB v.2.0. Size indicates P value and colour indicates the means of the average expression level of the receptor-ligand pairs. The EGFR+ glial cell type exhibited the most prominent interactions with Delta-Notch and EGFR signalling within the network.
Figure 7(A) SEC-profiles of the IFT-B1 and IFT-B2 complexes as well as the 15-subunit reconstituted IFT-B complex. IFT-B1 and IFT-B2 elute at similar volumes, but after mixing the resulting high-molecular weight complex is clearly shifted towards a smaller elution volume. The Coomassie-stained SDS-PAGE gel on the right shows the peak fractions of the individual runs. (B) Schematic representation of the IFT-B complex summarizing the results presented in this study. The six IFT-B proteins IFT172, IFT80, IFT57, IFT54, IFT38, and IFT20 form a hexameric complex (IFT-B2), where an IFT57/38 dimer (mediated by coiled-coil interactions) binds to IFT172 and IFT80 via its two CH-domains, and a dimer of IFT54/20 (again mediated by coiled-coil interactions) is held in the complex by binding to both IFT80 and IFT57/38. The IFT-B2 complex binds to the IFT-B1 complex via an interaction mediated by IFT88/52N (on IFT-B1) and IFT57/38 (on IFT-B2). In addition to the previously characterized tubulin/MT-binding module on IFT-B1 (IFT81CH/IFT74N), the N-terminal CH-domain of IFT54 is also able to bind tubulin and MTs, giving a total number of two binding sites per IFT-B complex. Note that several details, such as the exact binding sites for IFT80 and IFT57/38 on the IFT54/20 complex, as well as the directionalities of the coiled-coils in IFT57/38 and IFT54/20 remain to be determined.
Figure 4A. Genomic DNA sequence from FBXL4 knockout (KO), and wild-type (WT) RKO cells was compared to the NCBI reference FBXL4 coding sequence (CDS) NM_001278716.2 and showed a frameshift-causing deletion in the knockout cells. B. Steady-state protein levels in wild-type (WT) and FBXL4 knockout (KO) RKO cells. C. (Upper panel) Pulse-chase labeling of mitochondrial translation products in wild type (WT) and FBXL4 knockout (KO) cells. The cells were incubated for 1 hour with a 35S methionine-cysteine labeling mix in the presence of anisomycin to inhibit cytosolic translation (pulse). After removal of the medium, the cells were incubated in complete growth medium without inhibitors and labeled amino acids for 16 or 24 hours (chase). A representative experiment of 4 biological replicates is shown. A Coomassie-stained part of the gel is shown as loading control. (Lower panel) Quantification of the MTCO1-ND4 signal presented as mean ± SEM; n=4 biological replicates; Student's t-test; *, p<0.05. D. (Upper panel) Mitochondrial translation products were labeled in wild type (WT) and FBXL4 knock-out (KO) RKO cells for 60 minutes as in C, followed by 24 h chase in the presence of 5 μM epoxomicin (epoxo) or 20 mM ammonium chloride (NH4Cl). A representative experiment of n≥3 biological replicates is shown. A fragment of the Coomassie-stained gel is shown as loading control. (Lower panel) Quantification of the ND4-MTCO1 signal. n≥3 biological replicates, data are presented as mean ± SEM. Student's t-test; *, p<0.05. E-F. Steady-state levels of indicated proteins in wild type and FBXL4 knockout RKO cells treated with NH4Cl E-F. Steady-state levels of indicated proteins in wild type and FBXL4 knockout RKO cells treated with epoxomicin (Epoxo, F).
Immunohistochemistry indicates disturbed autophagic flow in neurons.Fig 4(A) Axonal spheroids stain diffusely positive for LC3B. IHCLC3B, scale bar 100 μm. (B,C) The granular cores of the spheroids are positive for (B) ubiquitin (arrow) and (C) p62. IHCubiquitin and p62, scale bars 20 and 100 μm, respectively. (D) Smooth axonal swellings in the cerebellar cortex contain ATG4D. Inset: control. IHCATG4D, scale bar 100 μm. (E) Affected neurons show increased perinuclear granular LC3B positivity. Inset: control. IHCLC3B, scale bar 100 μm. (F) Neuronalvacuoles are partially LAMP2 positive (arrow) and partially negative (arrow head). Inset: control. IHCLAMP2, scale bar 20 μm.
Figure 7A, B and C. Changes in mRNA expression of Ucp1 (A), Pgc1α (B) and Pparγ(C) from WT and TRPV2KO iBAT 4 h after intraperitoneal administration (i.p., arrows) of saline or a selective β3-adrenergic receptor agonist, BRL37344 (600 µg/kg body weight). Mean ± SEM, n = 5 - 7; * P < 0.05 vs. saline group; # P < 0.01 vs. WT BRL37344 administration group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction. D and E. Average traces of changes in iBAT temperature (D) or rectal body temperature (E) from WT and TRPV2KO mice after BRL37344 (600 µg/kg body weight) administration (i.p., arrows). Mean ± SEM, n = 5. * P < 0.05 vs. WT group. Unpaired Student's t-test. F and G. iBAT NE turnover rates (F) and rate constants k (G) in WT and TRPV2KO mice upon cold exposure (4oC) for 4 h. Data are presented as mean ± SEM, n = 8.
Figure 3(A) Immunostaining for Glial fibrillary acidic protein (GFAP), a marker of astrocytes, and Aβ plaques in coronal sections through the hippocampus of 6 month old wild type, 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A mice. Scale bar, 300μm. Right hand panels show high magnification of the indicated areas. Scale bar, 50μm. (B) Quantification of GFAP area in the hippocampus of wild type and 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows the percentage of hippocampal area occupied by GFAP immunostaining (mean ± SEM, N=5 mice per group). (C) Immunostaining for Ionized calcium binding adaptor molecule 1 (Iba1), a marker of microglial cells, and Aβ plaques in coronal sections through the hippocampus of 6 month old wild type, 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A. Scale bar, 300μm. Right hand panels show high magnification of the indicated areas. Scale bar, 10μm. (D) Quantification of Iba1 area in the hippocampus of wild type and 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows the percentage of hippocampal area occupied by Iba1 immunostaining (mean ± SEM, N=5 mice per group). (E) Immunostaining of reticulon 3 (RTN3), a marker of dystrophic neurites, and Aβ plaques in coronal sections through the hippocampus of 6 month old 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A mice. Scale bar, 40μm. (F) Quantification of RTN3-positive dystrophic neurite area in the hippocampus of 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows the percentage of Aβ plaque area that overlapped with RTN3 immunostaining (mean ± SEM, N=5 mice per group). (G) MitoSox staining, a mitochondrial superoxide indicator, and DAPI in coronal sections through the hippocampus of 6 month old wild type, 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A mice. Scale bar, 60μm. (H) Quantification of MitoSOX signal in the pyramidal cell layer of hippocampus of wild type and 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows MitoSox mean fluorescence intensity in arbitrary units (mean ± SEM, N=5 mice per group).
At an early stage of autophagy, UVRAG targets Beclin1 to facilitate autophagosome formation, whereas at the later stages, UVRAG interacts with C-Vps to promote autophagosome maturation.figf8At an early stage of autophagy, UVRAG targets Beclin1 to facilitate autophagosome formation, whereas at the later stages, UVRAG interacts with C-Vps to promote autophagosome maturation. UVRAG-C-Vps is also involved in the endosome-lysosome transition by activation of Rab7.
Figure 7  Figure 7. Contrasting models for histone chaperoning during nuclear import of H3.1 and H4. In the dimer import model, H3.1 and H4 form a heterodimer soon after synthesis in the cytosol. This heterodimer is bound by the chaperones NASP, ASF1, RbAp46 and HAT1, which further associate with IPO4 before translocation to the nucleus. In the monomer import model, H3 and H4 monomers are bound by Importin-β proteins before translocation to the nucleus, wherein they form a heterodimer and associate with the core histone chaperoning machinery. Additional cytosolic factors may also transiently interact with the monomeric histones. Expanded View Figure Legends
Figure 4Gating strategy of KLS (c-Kit+Lin-Sca-1+) cells in blood, using CD48 and CD150 to distinguish HSC (hematopoietic stem cells), MPP (multipotent progenitors) and HPC (hematopoietic progenitors). CoPP mobilizes higher numbers of HSC (KLS CD48-CD150+), MPP (KLS CD48-CD150-), HPC-1 (KLS CD48+CD150-) and HPC-2 (KLS CD48+CD150+) cells than G-CSF to the PB. Treatment with CoPP increases percentage of KLS cells in BM, what is related to increase in HPC populations, but not in LT-HSC fraction. In contrast, frequency of MPP tends to decrease (D) Scheme of common myeloid progenitor (CMP) differentiation towards granulocytes/monocytes, erythrocytes and platelets. The number of KLS- (c-Kit+Lin-Sca-1-) is higher in mice treated with CoPP than in mice treated with G-CSF. Numbers of lineage committed progenitors: GMP (granulocyte-macrophage progenitors, KLS- CD48+CD150-CD34+) and MEP (megakaryocyte-erythroid progenitors, KLS- CD48+CD150+CD34-) were higher after CoPP administration than after G-CSF. G-CSF and CoPP similarly increase EP numbers (erythrocyte progenitors, KLS- CD48-CD150-CD34-). Both G-CSF and CoPP treatments decrease the KLS- percentage in the BM, however the decrease after CoPP is smaller. G-CSF and CoPP decrease percentages of MEP and EP, but only G-CSF decreases the percentage of GMP. Results are shown as mean + individual values, one-way Anova with Bonferroni post-test, n = 7 mice per group.
Figure 2(A, B) Murine cardiac endothelial cells (MCEC) were treated with 2 mM D-β-hydroxybutyrate (β-OHB) or 2 mM acetoacetate (AcAc) for 24 hours. Quantification of concentrations of TCA cycle intermediates compared to control treatment. Data are presented as mean ± SD. n≥4; ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test. (C) Scheme of ketone body oxidation and the tricarboxylic acid (TCA) cycle. MCEC were incubated with 1 mM 13C4-β-hydroxybutyrate or 1 mM 13C4-acetoacetate for 24 hours or the same concentration of unlabeled ketone bodies ([12C] isotope). The [13C]-labeled fractions of the TCA cycle intermediates citrate, α-ketoglutarate and malate (marked with blue star) were quantified with an untargeted metabolomics approach indicating that 13C carbon from ketone bodies was incorporated into TCA cycle intermediates. (D) Quantifications of [13C]-labeled fractions of amino acids (asparagine, L-aspartate, L-glutamate, L-proline), putative lipid species (nervonic acid, 1-palmitoylglycerol, ceramide (d18:1/16:0)) and uridine diphosphate (UDP) species (uridine monophosphate, uridine 5'-diphosphate, UDP-N-acetylglucosamine) in MCEC treated with 1 mM 13C4-β-hydroxybutyrate or 1 mM 13C4-acetoacetate for 24 hours or the same concentration of unlabeled ketone bodies ([12C] isotope). Data are presented as mean ± SD. n≥4; ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.
Increased FAK activation in human pancreatic adenocarcinoma-associated fibroblasts.Figure 1(A-B) Representative Hematoxylin and Eosin (H&E) staining, and immunohistochemistry of phosphorylated FAK on tyrosine (Y) 397 (pY397 FAK), alpha-Smooth Muscle Actin (α-SMA), and Fibroblast Activated Protein alpha (FAP-α) in serial sections of human normal pancreas (Ct1), of human normal adjacent PDAC tissue (Ct2) and of human PDAC tissues (PDAC1-3); scale bar, 100 μm (A), and 300 µm (B). (C) Quantification of pY397 FAK level in cancer-associated fibroblasts from human PDAC microarray: 40 different PDAC patient samples and 10 normal human pancreas have been stained for pY397 FAK and double blinded quantified (based on intensity and positive cell number) on a scale between 0 (no staining) and 6 (Y397 FAK highly positive) in whole cell (left), cytoplasm (middle) and nucleus (right).
a) Immunoblot of autophagy-related proteins in U2OS cells expressing a control TALEN (U2OS-TAL2-C1) or two independent subcloned TALEN cell lines targeting ATG7 (U2OS-TAL2-A7, U2OS-TAL2-A8).f4(a) Immunoblot of autophagy-related proteins in U2OS cells expressing a control TALEN (U2OS-TAL2-C1) or two independent subcloned TALEN cell lines targeting ATG7 (U2OS-TAL2-A7, U2OS-TAL2-A8). p62 long and short refer to either long or short exposure times during film development. (b) U2OS cells expressing an ATG7-TALEN (TAL2-ATG7) or control TALEN (TAL2-CTL) were subjected to glutamine starvation for 6 days followed by 6 days recovery in glutamine-replete medium. Cells were then stained with SRB and absorbance was read at 540 nM. Graph shows average results from five clonal sub-lines for each condition. The statistical significance (P value) was determined by a two-tailed, paired Student's t-test. P0.05. (c) Representative images of SRB-stained clones in b d) IL-8 enzyme-linked immunosorbent assay (ELISA) of U2OS-TAL2-CTL-C1 (control) and U2OS-TAL2-ATG7-A8 (ATG7 knockout) following 24 h glutamine deprivation. (e) IL-8 U2OS of U2OS parental cells starved of glutamine for the indicated times with or without BafA1. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.
Figure 5A, Experimental design for the genome-wide DNA methylation analysis of wild-type and miiPSC as well as embryoid bodies (EBs) derived from them. Cells (two independent miiPSC clones and two iPSC technical replicates) were transiently treated with Dox for 5 days and then subjected to Dox withdrawal for 20 additional days before starting the EB formation protocol. Samples for DNA and RNA analysis were collected at the indicated time points before Dox (t=0), 5 days after Dox withdrawal (t=10), 20 days after Dox withdrawal (t=25) or 7 days after starting the EB generation protocol (t=32 days). B, Genome-wide DNA methylation data showing the number and total size of DNA methylation valleys (DMVs) and partially methylated domains (PMDs). C, DNA methylation distribution of the indicated samples, smoothed over 100 kb-blocks. D, Principal Component Analysis showing the distribution of methylation profiles in the indicated samples (color code as in panel B). E, Number of differentially methylated single CpG sites (DMPs) and differentially methylated regions (DMRs) in the indicated comparisons (color code as in panel B). F, Heat-scatterplots representing the mean methylation over all transcripts (transcription start sites, TSSs) in mm10, pairwise for "t=10 vs. t=0" (left panel) and "t=25 vs. t=0" (right panel). Note that especially for "t=25 vs. t=0" most of the points are below the diagonal, indicating broad hypomethylation across most of the genome. G, Heat-scatterplots representing the mean methylation over the TSS of the 282 genes included in the 2-cell signature (as in Fig. 1D). Analysis as in (F). H, The specific differentially methylated regions (DMR) at the Sirt6 locus were analyzed by PCR amplification and sequencing of bisulphite-modified DNA. Ten independent clones were sequenced per condition. The quantification of methylated (filled circles) vs. unmethylated (empty circles) CpGs is shown in the histogram in the indicated conditions. I, Heatmaps representing the methylation levels at the top Differentially Methylated Regions (DMRs; n=100; upper panel) or the Imprinting Control Regions (ICRs; n=103 different imprinting loci; lower panel) in the indicated samples (referred to panels A,B). Color code and scale of DNA methylation, from 0.0 (red) to 1.0 (green) is applicable to both heatmaps.
Fig.Figure 5RAW 247.6 macrophages loaded with a cytosolic chemical dye (Cyto dye) to detect global cytosolic Ca2+, and heterologously expressing (B) TPC1-G-GECO1.2 (TPC1-GG) or (C-E) TPC2-G-GECO1.2 (TPC2-GG) to detect peri-endo-lysosomal Ca2+ domains, which were not detected by (F) a TPC2 mutant (D276K)-G-GECO1.2 that blocks ion permeation or (A) unfused cytosolic G-GECO1.2 (Cyto GG). (A-F) Single-cell Ca2+ traces upon FcγR activation by opsonized 3-μm beads in -Ca2+o expressed as a percentage of the maximum fluorescence of the indicator, determined by adding 2 μM ionomycin and 10 mM CaCl2 at the end of each experiment (% Fmax) (D) TPC2-G-GECO1.2 activity was inhibited by 10 µM Ned-19, but was unaffected by (E) chelating global Ca2+ with EGTA/AM (100 μM for 2 min prior to bead addition). Summary data of the first spike from cells A-F (subsequent spikes show the same pattern; Appendix Fig. S4B), n = 87-274 cells; ***P<0.001 vs Cyto GG, ### P<0.001 vs TPC2-GG ctrl, ††† P<0.001 vs Cyto GG. Fusion of G-GECO1.2 to TPC2 did not alter the affinity of G-GECO1.2 for Ca2+ determined in permeabilized cells (n = 34-71 cells). (I) Single-cell Ca2+ traces upon FcγR activation by opsonized 3-μm beads in -Ca2+o expressed as a percentage of Fmax. (I, J) Pre-emptying the ER stores with CPA or thapsigargin (Tg) abolished the global cytosolic Ca2+ response evoked by beads, but a TPC2-G‑GECO1.2 component remained, n = 17 - 121 cells. No local Ca2+ signal was detected with TPC2-G-GECO1.2 when Ca2+ spiking was evoked by ER-Ca2+ release with 100 μM UTP (K, L) or 10 μM CPA (M); only a bystander response equivalent to unfused cytosolic G-GECO1.2 was detected (n = 121-182 cells; *P<0.05).
Luciferase transgene expression and vector biodistribution in tissue lysates.Figure 4Luciferase activity and vector copy numbers were determined in tissue lysates 14 days after vector administration (5x1010 genomic particles/mouse, age 8-12 weeks).A Vector-mediated luminescence. Transgene expression was measured in AAV-BR1 harboring the luciferase gene under control of the CAG promoter and control vectors (AAV-PPS and wild type rAAV2) in brain and off-target control organs (left panel). Comparison of luminescence in the brain mediated by AAV-BR1 and control vectors (right panel), ****p < 0.0001 (BR1: brain vs. all), ***p = 0.0002 (PPS: heart vs. brain/ muscle), ***p = 0.0001 (PPS: heart vs. lung/ liver/ spleen/ kidney), *p = 0.0101 (WT: heart vs. lung), *p = 0.0120 (WT: heart vs. spleen), *p = 0.0148 (WT: heart vs. kidney), *p = 0.0113 (WT: heart vs. muscle), **p = 0.0011 (Brain: BR1 vs. PPS), ***p = 0.0009 (Brain: BR1 vs. WT). Luciferase activity and vector copy numbers were determined in tissue lysates 14 days after vector administration (5x1010 genomic particles/mouse, age 8-12 weeks).B Biodistribution of AAV-BR1 and control vectors (AAV-PPS and wild type rAAV2) in brain and off-target control organs, excluding spleen (left panel). Genome copy numbers of AAV-BR1 and control vectors (AAV-PPS and wild type rAAV2) in the brain (right panel), ****p < 0.0001 (BR1: brain vs. liver/ kidney/ muscle), ***p = 0.0006 (BR1: brain vs. heart), ***p = 0.0003 (BR1: brain vs. lung), **p = 0.0025 (PPS: kidney vs. muscle), *p = 0.0378 (PPS: kidney vs. lung), *p = 0.0114 (PPS: kidney vs. liver), *****p < 0.0001 (WT: liver vs. all), **p = 0.0028 (Brain: BR1 vs. PPS/ WT).
Figure 1A. Patient 1 (older sibling) underwent 3 MRI studies, first and last are shown. Top row: 21 months; Bottom row: 4 years 7 months. The corpus callosum is thin on T1 weighted sagittal images (I, V, VI) with a saber-shape (long arrows). The shape remains consistent over time. The vermis is normal in configuration and size initially (I), but demonstrates volume loss on follow-up (V, VI) examination (short arrows). FLAIR axial image (II) is age appropriate, while (VII) demonstrates periatrial increased signal (arrow). There is delay in myelin maturation present on T1 (III, VIII) and T2 (IV, IX) weighted axial images (long arrows) on initial examination, with further slow myelin development over time. There is deficiency of periatrial white matter volume on both T1 (III, VIII) and T2 (IV, IX) weighted axial images (short arrows). Coronal T2 image (V) demonstrates abnormally increased signal of the dentate nuclei (arrow), unchanged on follow-up image (X). Superior vermian atrophy (short arrows) over time. B. Patient 2 (younger sibling). Top row: 4 years, 11 months; Bottom row: 9 years, 6 months. The corpus callosum is thin with a saber-shape (long arrows) configuration on T1 weighted sagittal (I) and T2 weighted sagittal (V, VI). The shape remains consistent over time, although the volume decreases slightly. The vermis demonstrates volume loss on both sagittal examinations (short arrows). There is iron deposition in the basal ganglia on FLAIR (VII) and T2 weighted coronal (VIII) and axial (IX) images at 9 years 8 months of age (arrow). This was not present on earlier imaging. Iron deposition was also present in the subthalamic nuclei (not shown). Myelin maturation is age appropriate on the initial T1 weighted axial image (III) and minimally delayed on T2 weighted image (IV) at 4 years 11 months of age (short arrow). Myelin is age appropriate (short arrow) on follow-up T2 weighted images (VIII, IX). The dentate nuclei (short arrows) are bright on T2 coronal images (V, VI, X) at both ages. There is progressive cerebellar hemisphere volume loss. C. Patient 3, 3 weeks old. Sagittal T1 weighted image (I) demonstrates a very thin severely hypoplastic remnant of corpus callosum (short arrow), a short cingulate sulcus (long arrow) and slender pons. FLAIR image (II) reveals faintly increased signal (short arrow) in the posterior limb of internal capsule (PLIC). There is lack of myelin maturation in the PLIC (short arrows) on T1-IR (III) and T2 (IV) weighted axial images. Focal widening of the interhemispheric CSF space (arrow) on T2 weighted image (IV) reflects absence of rostral fiber tracts). Cingulate gyrus is present (long arrow). No traversing callosal fibers are shown at this level (I).
Figure 4Biochemical characterization of recombinant OTULIN variants by means of differential scanning fluorimetry (DSF) A) DSF measurements with OTULINWT, OTULINM86I or OTULINW167S. Melting temperatures (Tm) are calculated from five independent experiments with standard deviations. *, P = 0.011; ****, P = 6.64x10-18, unpaired t-test. Biochemical characterization of recombinant OTULIN variants by means of surface plasmon resonance (SPR) measurements (B) SPR measurements and steady-state binding curves with calculated dissociation constants (Kd) after injection of a concentration series of OTULINC129A, OTULINC129A/M86I or OTULINC129A/W167S to CM5-immobilized di-ubiquitin chains. (C) Linear ubiquitin linkages isolated from A549 OTULIN KO cells by M1 TUBE assay were incubated with increasing concentrations of recombinant OTULINWT, OTULINM86I and OTULINW167S or catalytically inactive OTULINC129A as control for 1 hour. Afterwards, samples were subjected to analysis by western blot for the indicated proteins. Images are representative of three independent experiments.