EMBO/sd-character-level-ner huggingface dataset:
Figure 8(A, B) Confocal z-projections of adult DIP-δT2A-Gal4/1-119 trans-heterozygous mutant brains, in which γ-KCs are labeled by membrane bound tandem tomato (mtdT-HA; green) driven by R71G10-QF2 (γ-QF2), and DIP-δ-Gal4 (expressed in DIP-δ+ neurons) either drives expression or UAS-DIP-α (B) or not (A). Grey boxes below the images describe the relevant components within PAM-DANs and γ-KCs. (C, D) Confocal z-projections of adult DIP-δT2A-Gal4/1-119 trans-heterozygous mutant (D) or DIP-δ1-119/+ heterozygous (C) brains, which express MiMIC mediated Dpr12GFSTF (Dpr12-GFP; green), in which DIP-δ-Gal4 either drives expression or UAS-DIP-α (D) or not (C). Grayscale panels represent single channels, as indicated. The γ-lobe is outlined in white. (E, F) Confocal z-projections of adult dpr12Δ50-81 homozygous mutant brains, in which DIP-δ-Gal4 drives expression of either UAS-mtdT (E) or UAS-DIP-α-T2A-tdT (F; expected to induce expression of DIP-α as well as tdT encoded by a polycistronic message). In orange are longitudinal sections across the γ-lobe at the indicated location. The γ-lobe is outlined in white, as determined by FasII staining (grey in E'-F'). Cyan is tdT within DIP-δ+ cells. Grey boxes below the images describe the relevant components within PAM-DANs and γ-KCs. (G) Ranking of regrowth for A, B, E, F and Figure EV8A-B. Regrowth defect severity and statistics were calculated as in Fig. 1; Wilcoxon-Mann-Whitney test; ***p<0.001; n,s, not significant.
Comparison of SARS-CoV-2 N-NTD with related viral N-NTD structuresFigure legend: A.Figure 3:A. Mapping the conserved surfaces of four CoV N-NTDs in SARS-CoV-2 N-NTD structure. The multiple sequence alignment used for mapping is shown in Fig. 1C. Blue denotes conserved residues among 4 CoVs N-NTD; green denotes one variation among 4 CoVs N-NTD; pink denotes two variations among 4 CoVs N-NTD; red denotes three variations among 4 CoVs N-NTD B. Electrostatic surface of the SARS-CoV-2 N-NTD, SARS-CoV N-NTD, MERS-CoV N-NTD, HCoV-OC43 N-NTD. Blue denotes positive charge potential, while red indicates negative charge potential; C. Overall structural comparison of SARS-CoV-2 N-NTD with related viral N-NTD structures. Left: superimposition of SARS-CoV-2 N-NTD (blue) to SARS-CoV N-NTD (green); middle: superimposition of SARS-CoV-2 N-NTD (blue) to MERS-CoV N-NTD (brown); right: superimposition of SARS-CoV-2 N-NTD (blue) to HCoV-OC43 N-NTD (orange).
Figure 8The cartoon illustrates how too low membrane fluidity triggers large-scale lipid phase separation associated with both segregation and confined diffusion of membrane-integral proteins (left part). Too low membrane fluidity also results in dissipation of membrane potential likely due to a reduced electron transport chain activity (Budin et al, 2018). The core diffusion barrier function of the membrane, however, is maintained (right part).
OPCML represses oncogenic Gas6/AXL signalling.Figure 3 SKOV3 (A (EMPTY and OPCML transduced) cell lines were serum starved and stimulated with Gas6 over a 24 hour time course. GAPDH was used as a loading control Data in (A, B, D, F) are representative blots of at least three experiments. RPPA analysis was performed in triplicate PEA1 (B) (EMPTY and OPCML transduced) cell lines were serum starved and stimulated with Gas6 over a 24 hour time course. GAPDH was used as a loading control Data in (A, B, D, F) are representative blots of at least three experiments. RPPA analysis was performed in triplicate (C,E) RPPA data showing phospho-RTK and downstream signalling moieties activated by Gas6/AXL stimulation in SKOV3-EMPTY and SKOV3-OPCML. Data is represented as Log2 normalised and median centred (D) Western Blotting of SKOV3-EMPTY and SKOV3-OPCML upon AXL activation. GAPDH was used as loading control Data in (A, B, D, F) are representative blots of at least three experiments. RPPA analysis was performed in triplicate (C,E) RPPA data showing phospho-RTK and downstream signalling moieties activated by Gas6/AXL stimulation in SKOV3-EMPTY and SKOV3-OPCML. Data is represented as Log2 normalised and median centred (F) Western blotting of Non-Targeting siRNA (NTrg), or siOPCML (si 6, si 7) transfected FT190 cells stimulated with Gas6 over a 24 hour time course. GAPDH was used as loading control. Data in (A, B, D, F) are representative blots of at least three experiments. RPPA analysis was performed in triplicate
Conserved Elp2 loops are important for Elongator function.Figure 3A. Schematic of the conserved residues within four Elp2 loops that were mutated to alanine within an ELP2 plasmid. B. Plasmids containing ELP2 and the four loop mutants were transformed into elp2Δ yeast. The strains were then plated onto -Ura media containing caffeine. The pRS416 vector is used for these experiments. C. Growth curve analysis of loop mutants in -Ura media with and without K. lactis toxin zymocin. Shaded regions correspond to the SEM at each time point. N=3. D. Lysates of Elp4-FLAG-containing yeast expressing the Elp2 loop mutants were incubated with α-FLAG resin and eluted with 3xFLAG peptides then analyzed by Western Blot using α-HA and α-FLAG antibody
Comparative deuterium-exchange experiments identify structured segments and flexible elements of Okp1 or Ame1 in COMA-Nkp1-Nkp2.Figure 3A,B) Plots showing hydrogen-deuterium exchanged peptides, after 10 sec of deuterium exchange of Okp1 (A) each in COMA or COMA-Nkp1-Nkp2. For this representation, and all following of this type, detected peptides are colour-coded according to their measured deuterium-exchange in % of the maximum measured exchange (1-25 %: dark blue; 26-50 %: light blue; 51-75 %: yellow; 76-100 %: red). Peptides are represented as bars. Each bar is plotted row-wise corresponding to its position in the amino-acid sequence of the protein it derives from. Amino-acid sequence scheme is illustrated at the top with thin or thick lines; thin line: flexible element: thick line: structured segment. Positions of predicted coiled coils are shown as helices. Plots for full time courses are in Appendix Fig S3C,E. A,B) Plots showing hydrogen-deuterium exchanged peptides, after 10 sec of deuterium exchange of Ame1 (B), each in COMA or COMA-Nkp1-Nkp2. For this representation, and all following of this type, detected peptides are colour-coded according to their measured deuterium-exchange in % of the maximum measured exchange (1-25 %: dark blue; 26-50 %: light blue; 51-75 %: yellow; 76-100 %: red). Peptides are represented as bars. Each bar is plotted row-wise corresponding to its position in the amino-acid sequence of the protein it derives from. Amino-acid sequence scheme is illustrated at the top with thin or thick lines; thin line: flexible element: thick line: structured segment. Positions of predicted coiled coils are shown as helices. Plots for full time courses are in Appendix Fig S3C,E.
Fig.Figure 1(A) Diauxic growth of WT E. coli (NCM3722) in minimal media containing glucose and acetate, bacterial density measured as optical density (OD600). Growth on glucose is captured by the exponential fit (blue dashed line), and proceeds until glucose runs out (black dashed line) at time = 0 h. This is followed by a period of growth-lag lasting ~3.5h before exponential growth resumes on acetate (red dashed line). (B) The central carbon metabolism pathways are illustrated along with the nodes branching out into amino acid precursor synthesis (green arrows). Glucose to acetate diauxie requires switching from the pathways facilitating glucose consumption (represented in blue) to those responsible for acetate consumption (represented in red). More details in Fig. EV1B. (C) The mRNA fractional abundances for aceB, aceA and the gluconeogenesis genes (summed abundance of maeA, maeB, pck, and ppsA genes) were estimated by RNA sequencing performed at various time points during the diauxic transition as indicated on the x-axis. The x-axis is truncated 2.3 hours into the shift and the "post shift" (pink) regime represents transcript levels when growth fully resumes on acetate, measured under steady state growth on acetate minimal medium. The series of RNA-seq through the growth transition was performed once. (D) Lag-times for controlled titration of aceBA expression using an inducer construct in strain NQ1350 (inset). Addition of chlorotetracycline (cTc) removes the tetR repression and induces aceBA expression. As the expression of aceB/aceA during the response (inducer added at time = 0h) is increased, lag times decrease. Bar plot shows mean lag-times (N=3 biological repeats) for different inducer concentration with error bars denoting the Standard Deviations (SD). (E) Of the overall transcription and translation fluxes (arrows), a strong allocation towards the expression of shunt and gluconeogenesis genes (red) increases the novel synthesis of required enzymes and should thus lead to faster growth recovery.
Figure 3Aberrant accumulation of mitophagosomes co-labeled by antibodies against LC3 and cytochrome c (Cyto c) in the hippocampal mossy fibers of mutant hAPP Tg mouse brains. Note that mitophagosomes indicated by white arrows are not readily observed in WT mouse brains The average number of mitophagosomes per section (320 μm × 320 μm) was quantified from the total number of brain slice sections (n) indicated on the top of the bars C Clustering of mitophagosomes (white arrows) co-labeled by antibodies against LC3 and Cyto c or p62 and HSP60 within swollen/dystrophic presynaptic terminals indicated by synaptophysin (SYP) surrounding amyloid plaques in AD mouse brains. Quantitative analysis showing presynaptic retention of mitophagosome-like structures in the hippocampal regions of mutant hAPP mouse brains. Data were quantified from the total number of the imaging fields (n) indicated in parentheses representative TEM images showing presynaptic retention of mitophagosome-like structures in the hippocampal regions of mutant hAPP mouse brains. White arrow-labeled mitophagosomes are not readily detected in WT mouse brains. Black arrows marked amphisomes or degradative autophagic vacuoles (AVd). F,G Mitophagic accumulation at Aβ-enriched synapses in AD mouse brains. Note that the levels of mitophagic/autophagic proteins (red box) along with Aβ (green box) are increased in synaptic fractions isolated from AD mouse brains. Equal amounts (10 μg) of synaptosomal preparations (Syn) and post-nuclear supernatants (PNS) from WT and mutant hAPP mouse brains were sequentially immunoblotted with antibodies against hAPP/Aβ, Rheb, LC3, p62, and Rab7, as well as mitochondrial proteins TOM20 and SOD2 on the same membrane after stripping between each antibody application. The purity of synaptosomal fractions was confirmed by relative enrichment of SYP and less abundance of p115 and GAPDH (F). Relative protein levels in the synaptosomal preparations from mutant hAPP mice were compared to those in WT littermate controls. Data were quantified from five repeats (G). H,I Representative blots (H) and quantitative analysis (I) showing increased levels of Rheb along with LC3-II, p62, and Rab7 (red box) in Mito fractions purified from AD mouse brains. The purity of Mito fractions was confirmed by enrichment of mitochondrial proteins VDAC and TOM20 and by the absence of GAPDH and p115, relative to PNS fractions. Data were quantified from four repeats.
Figure 1(A) IC50s for baricitinib (orange) in cytokine-stimulated human CD4+ T cells, CD8+ T cells, NK cells, and monocytes are shown for IL-2/pSTAT5, IL-4/pSTAT6, IL-3/pSTAT5, IL-6/pSTAT3, IL-10/pSTAT3, IL-15/pSTAT5, IL-21/pSTAT3, G-CSF/pSTAT3, GM-CSF/pSTAT5, and IFNγ/pSTAT1. Average daily percent STAT inhibition was calculated using in vitro concentration response curves and published human exposure data. Cytokine treatments that did not result in sufficient pSTAT stimulation are denoted as "NS" in the radar plots. (B) Fold-change ± standard error in baseline IL-6 plasma levels at week 12 from RA patients treated with placebo (n=47), baricitinib 1-mg (n=23), 2-mg (n=24), 4-mg (n=23), and 8-mg (n=23) in the phase 2b randomized, placebo-controlled study NCT01469013. p values are for comparisons of baricitinib 1-mg, 2-mg, 4-mg, and 8-mg change from baseline compared to the placebo change from baseline. Treatment effects were estimated using a mixed effects repeated measures model (SAS Proc Mixed) using log10 transformed IL-6 levels with an unstructured covariance matrix.
Figure 7Immunoprecipitation and biotinylation of SUMM2NB-FLAG (A), RPP4NB-FLAG (B), or CHS1NB-FLAG (C) by SNIPER1 H129Y-HATurboID in N. benthamiana. Two biological repeats were carried out with similar results. Interaction of SNIPER1 H129Y-CLuc and SUMM2NB-NLuc (D), RPP4NB-NLuc (E) or CHS1NB-NLuc (F) as tested by split-luciferase complementary assay in N. benthamiana. Three biological repeats with four technical repeats each were carried out with similar results.
Figure 3(A) Decrease in the fraction of chromosome arms exhibiting conservative (Consrv) DNA replication of the entire telomere length or part of the telomere (E+P) in U2OS cells after depletion of POLD3 or POLD4 by siRNA. si Ctrl, control siRNA; NE, cells expressing normal levels of cyclin E; OE, cells overexpressing cyclin E for four days. Bars represent means and standard errors of the mean from two independent experiments. For statistical comparisons the effects of PolD3 and Pold4 depletion were examined after grouping together the data from the NE and OE cells. PolD3 depletion reduced the fraction of conservatively replicated telomeres at a level of P=0.072; whereas for PolD4 depletion the decrease was significant at a level of P<0.025, as calculated using the paired t test.(B) Decreased telomere length following PolD3 or PolD4 depletion in U2OS cells expressing normal levels of cyclin E (NE) or overexpressing cyclin E (OE). The cells were transfected with control siRNA (si Ctrl) or siRNAs targeting POLD3 or POLD4 and, five days later, fixed and examined for telomere length by Q-FISH. Bars indicate means and standard error of the mean from more than 2,600 telomeres examined per condition. The differences between control and PolD3 or PolD4-depleted cells were significant (P<10-6) for both NE and OE cells, as determined by unpaired t tests. (C) Decreased telomere length following PolD3 or PolD4 depletion in parental U2OS cells. The cells were transfected with control siRNA (si Ctrl) or siRNAs targeting POLD3 or POLD4 and, five days later, fixed and examined for telomere length by Q-FISH. Bars indicate means and standard error of the mean from more than 2,800 telomeres examined per condition. The differences between control and PolD3 or PolD4-depleted cells were significant (P<10-6), as determined by unpaired t tests. (D) Increased frequency of chromosome end-to-end fusions following POLD3 or POLD4 depletion. U2OS cells expressing normal levels of cyclin E (NE) or overexpressing cyclin E for four days (OE) were transfected with control siRNA (siCtrl) os siRNAs targeting POLD3 or POLD4. For each condition, 25 metaphases were examined (representing about 1,900 high quality chromosome arms per condition); the percentage of chromosome fusions was determined for each metaphase and used to calculate means and standard errors of the mean. For statistical analysis, the number of chromosome end-to-end fusions were compared by chi square tests among the various conditions. Cyclin E overexpression led to a higher number of fusions (P<0.0005); PolD3 and PolD4 depletion also led to a higher number of fusions (P<0.001 and P<0.025, respectively).
NPC2 reduces intracellular cholesterol accumulation in IMCs.Figure 7Intracellular un-esterified cholesterol distribution as detected by filipin staining in freshly isolated Npc2+/+ and Npc2+/hypo IMCs. Scale bars, 25 μm. Filipin-stained cells were graded from 0 to 3+ as indicated in the microphotographs (see Materials and Methods for criteria), and % of each grade in IMCs from Npc2+/+ (+/+) and Npc2+/hypo (+/hypo) BVE mice is shown in the bar chart. The data were obtained by analysing 176 Npc2+/+ and 242 Npc2+/hypo IMCs from three independent Npc2+/+ and Npc2+/hypo pairs. CLSM imaging of fresh Npc2+/hypo IMCs stained with filipin (cyan) and LAMP2 immunofluorescence (purple). Scale bars, 5 μm. Coarse structures strongly stained with filipin are surrounded by cytoplasmic membranous staining of LAMP2. Single-colour greyscale images are also demonstrated in the middle (filipin) and right (LAMP2). Intracellular un-esterified cholesterol distribution as detected by filipin staining in Npc2+/hypo IMCs cultured with or without 50 μg/ml bNPC2 for 48 h. Scale bars, 25 μm. Quantitative data in the bar graph were obtained by analysing 265 bNPC2-untreated (-bNPC2) and 252 bNPC2-treated (+bNPC2) cells from three independent cultures using the method described in (A).
a, COS7 cells co-transfected with GFP–ATG14 and DsRed–ER or RFP–SEC61&bgr (an ER marker) were immunostained for the mitochondria marker TOMM20.f1a, COS7 cells co-transfected with GFP-ATG14 and DsRed-ER or RFP-SEC61b; (an ER marker) were immunostained for the mitochondria marker TOMM20. Scale bars, 2 µm. b, An immunogold electron micrograph of starved HeLa cells expressing GFP-ATG14 probed with anti-GFP antibodies (red arrows). Scale bar, 200 nm. c, Immunoblots of subcellular fractions isolated from HEK293 cells stably expressing GFP-DFCP1. Cyto, cytosol; micro, microsome; mito, mitochondria; PNS, post-nuclear supernatant.
Measuring translation initiation and translation termination signals across the E.Figure 2Genetic design of the LacZ reporter construct whose expression is activated by the inducer IPTG. Normalized RPF count profile averaged for all E. coli transcripts. Profiles generated for cells grown in the absence and presence of IPTG (1 mM). Start and stop codons are shaded. Bar chart of all measured RBS initiation rates ranked by their strength. Strong RBSs with initiation rates >1 ribosome/s are highlighted in red. (D) Bar chart of all measured translation termination efficiencies at stop codons ranked by their strength. Stop codons with translation termination efficiency >0.99 are highlighted in red. (E) Distribution of initiation rates for cells grown in the absence and presence of IPTG (1 mM). (F) Distribution of translation termination efficiencies for cells grown in the absence and presence of IPTG (1 mM).
Alvocidib or THZ1 treatment is concordant with CDK9 or CDK7 inhibition based on RNA polymerase II ChIp-seq.Figure 5A) CrizR1 cells were treated with vehicle control, 200nM alvocidib or 100nM THZ1 for 6 hrs, then chromatin was precipitated with an anti-RNA polymerase II antibody and sequenced. Plotted is the average number of peaks per condition. GSEA analysis using the HALLMARK gene collection for the differentially enriched peaks around the TSS (±1kb) with (B) alvocidib Shown on the right is the plot of the "MYC targets"-enriched signature. GSEA analysis using the HALLMARK gene collection for the differentially enriched peaks around the TSS (±1kb) with (C) THZ1 treatment. Shown on the right is the plot of the "MYC targets"-enriched signature. D) Gene tracks of RNA polymerase II occupancy at the MCL1, BIRC5 (Survivin), CCND1 and MYC genes. E) qPCR of MYC and CCND1 upon treatment of CrizR1 cells with Alvocidib or THZ1 for 6h. The graph represents the mean fold change ± SD. MYC: alvocidib vs DMSO P =0.03, THZ1 vs DMSO P <0.0001; CCND1: alvocidib vs DMSO P =0.001, THZ1 vs DMSO P =0.003. F) BIM is upregulated upon MYC silencing in CrizR1 cells. G) Annexin V assay showing the percentage of apoptotic CrizR1 cells upon MYC downregulation using siRNA. Early apoptosis P =0.002, late apoptosis P=0.04, alive P =0.0008. H) Annexin V assay showing the percentage of apoptotic CrizR1 cells after treatment with trametinib, rapamycin or both. Trametinib: early apoptosis P =0.002, late apoptosis P =0.05, alive P =0.009; rapamycin: early apoptosis P =0.04, late apoptosis P =0.8, alive P =0.003; tram+rapa: early apoptosis P =0.0001, late apoptosis P =0.04, alive P =0.01.
Figure 6(A) Schematic of the NAD+ synthesis pathway in Drosophila. Red text indicates enzymes tested within the context of synaptic release at the larval NMJ. (B) Representative EPSP and mEPSP electrophysiological traces from larval NMJs at Muscle 6 in which a motoneuron specific driver was used to overexpress either a control transgene (DVGlut-GAL4 > UAS-RFP), or one of the catalytic dNmnat mutants (DVGlut-Gal4 > UAS-WR-dNmnat, DVGlut-Gal4 > UAS-H30A-dNmnat). (C) Quantification of mean (±SEM) EPSP amplitudes, in which 75 consecutive evoked events were averaged per cell, and then cell amplitudes were averaged per genotype. [UAS-RFP n = 8 cells, UAS-WR-dNmnat n = 7 cells, UAS-H30A-dNmnat n= 7 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=21, F=0.8596, p = 0.4383. UAS-RFP vs UAS-WR-dNmnat p = 0.8810 (NS), UAS-RFP vs UAS-H30A-dNmnat p = 0.9997 (NS)]. (D) Quantification of quantal content (±SEM) [UAS-RFP n = 8 cells, UAS-WR-dNmnat n = 7 cells, UAS-H30A-dNmnat n= 7 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=21, F=1.154, p = 0.3365. UAS-RFP vs UAS-WR-dNmnat p = 0.8696 (NS), UAS-RFP vs UAS-H30A-dNmnat p = 0.9697 (NS)]. ]. (E) Representative EPSP and mEPSP electrophysiological traces from larval NMJs at Muscle 6. Recordings were taken from WT control larvae (Elav-Gal4>UAS-RFP), hiw mutants driving a control transgene (hiwND8;Elav-Gal4 > UAS-RFP), and hiw mutants expressing an RNAi to knockdown NAD+syn (hiwND8;Elav-Gal4 > UAS-NAD+syn-RNAi). (F) Quantification of mean (±SEM) EPSP amplitudes, in which 75 consecutive evoked events were averaged per cell, and then cell amplitudes were averaged per genotype. [WT n = 10 cells, hiw n = 10 cells, hiw > NADsyn-RNAi n = 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=29, F=14.02, p<0.0001. WT vs hiw p <0.0001 (****), WT vs hiw > NADsyn-RNAi p = 0.5147 (NS)]. ]. (G) Quantification of quantal content. [WT n = 10 cells, hiw n = 10 cells, hiw > NADsyn-RNAi n = 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=29, F= 6.635, p = 0.0047. WT vs hiw p = 0.0063 (**), WT vs hiw > NADsyn-RNAi p = 0.8942 (NS)]. (H) Representative images of the NMJ synaptic terminal at muscle 4 in third-instar larvae. The genotypes in this experiment were identical to those tested in panels E-G. NMJs were stained for the presynaptic bouton marker DVGLUT (green) and nerve membrane marker HRP (red). (I) Quantification of the mean (±SEM) number of DVGlut+ boutons per muscle 4 NMJ in each genotype. [WT n = 25, hiw n = 21, hiw > NADsyn-RNAi n=24. 1-way ANOVA w/Tukey's multiple comparisons, DF=67, F=141.3, p < 0.0001. WT vs hiw p < 0.0001 (****), WT vs hiw > Nadysn-RNAi p > 0.0001 (****)].
Figure 4(A) Primary CD4 T cells were nucleofected with a control (CRISPR Control) or anti-CD59 (CRISPR CD59) guide RNA/Cas9 complex. CD59 surface levels were analyzed by flow cytometry 7 days after nucleofection. Isotype control (grey) and CD59 stainings (blue). One representative experiment is shown on the left. Numbers indicate the % of CD59+ cells (middle) or the MFI of staining in CD59- cells (Right). Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells. (B) NLAD8∆Vpu-infected CD59 KO primary CD4 T cells were cultured with indicated antibodies and NHS or HIHS for 24h. Numbers indicate the percentage of infected (Gag+) cells. One representative experiment is shown. (C) CD59-KO (CRISPR CD59) or control (CRISPR Control) CD4 T cells infected with HIV WT (left) or HIVΔVpu (right) (strain NLAD8) were cultured with the indicated antibody, and NHS or HIHS for 24h. The % of CDC was calculated as the % of disappearance of Gag+ cells after culture with NHS compared to HIHS. Each dot represents a single donor of CD4 T cells. n= at least 6 donors of CD4 T cells. (D) CD59 surface levels on non-infected (NI) or HIV-1-infected (strain NLAD8) primary CD4 T cells were analyzed by flow cytometry. Results are expressed as the MFI of staining with an isotype control or the anti-CD59 antibody. n=4 donors of CD4 T cells.