EMBO/SourceData huggingface dataset:
Figure 2Co‐localization of Hsp70 with Siglec‐5 and Siglec‐14 ligands in lung bronchiolesImmunofluorescence of human lung and prostate sections probed with Siglec‐5 R119A Fc or Siglec‐14 R119A Fc (red) with an anti‐Hsp70 antibody (green). The co‐localized signals for the Siglec‐Fc probes with an anti‐Hsp70 antibody suggest that Hsp70 is one of the possible sialic acid‐independent ligands of Siglec‐5 and Siglec‐14 in the lung bronchiole. White dashes mark the lung bronchiole epithelium. All images are representative of at least 2-3 replicates.
Figure 3Immunoblot probed with Siglec‐5 Fc and Siglec‐14 Fc to detect direct binding to Hsp70. B6N domain of GBS β‐protein was used as a positive control, and clusterin was used as the negative control.ELISA demonstrates direct binding of Siglec‐5 Fc and Siglec‐14 Fc, but not Siglec‐7 Fc, to Hsp70. Bovine serum albumin (BSA) was used as the negative control.Siglec‐5 or Siglec‐14 Fc immobilized on magnetic beads was incubated with heat‐shocked HEK293A cell lysates. A Western blot of the captured proteins using an anti‐Hsp70 antibody demonstrates Hsp70 endogenously produced by HEK293A cells interacts with Siglec‐5 and Siglec‐14.HEK293A cells were transfected with expression plasmids for Siglec‐7, Siglec‐5 or Siglec‐14, and then subsequently incubated with (red) or without (gray) recombinant Hsp70 (bottom panels). Flow cytometry analysis reveals Hsp70 binding to cell surfaces of HEK293A cells expressing Siglec‐5 and Siglec‐14, but not Siglec‐7. Histogram of Siglec expression after transfection is shown as blue lines (top panels).Using flow cytometry, undifferentiated or PMA‐differentiated THP1 cells with empty vector (EV), Siglec‐5 (Sig5), or Siglec‐14 (Sig14) overexpression were assessed for the presence of endogenously secreted Hsp70 bound on the cell surface. The mean fluorescence intensity (MFI) of signals from an anti‐Hsp70 antibody on unpermeabilized THP1 cells is shown.
Figure 4A comparison of the relative binding affinities between Siglec‐5 WT versus R119A Fc, and Siglec‐14 WT versus R119A Fc to Hsp70 by ELISA. The arginine‐mutated Siglecs display lower affinity for Hsp70 in comparison with their wild‐type sequence.A lysine‐to‐alanine mutation in residue 134 in Siglec‐14 Fc (K134A) also abrogates its affinity for Hsp70, as determined by ELISA. Lysine 134 was identified as a possible residue that changes binding affinity by aligning the sequences of Siglec‐5 and Siglec‐14 with Siglec‐7. The Siglec‐14 R119A and K134A double mutant (DM) Fc reduces the interaction with Hsp70 to background levels.Antibodies against Siglec‐5 and Siglec‐14 neutralized the interaction between Siglec‐14 Fc to Hsp70, as determined by ELISA.Co‐incubation of B6N peptide with Siglec‐5 and Siglec‐14 Fc decreased binding of each Siglec to Hsp70 in an ELISA.
Figure 5Secretion of pro‐inflammatory cytokine TNFα by THP1 cells transfected with an empty vector (EV) or Siglec‐5 (Sig5) expression plasmid was evaluated by ELISA after concurrent exposure with LPS and 10 μg/ml Hsp70 or LPS alone. Secretion of TNFα was reduced only in THP1 Sig5 cells exposed to both LPS and Hsp70 but not LPS alone.Similarly, IL‐8 secretion from THP1 EV and Sig5 cells was evaluated by ELISA after concurrent exposure with LPS and 10 μg/ml Hsp70. Secretion of IL‐8 was also reduced in only THP1 Sig5cells exposed to both LPS and Hsp70 but not LPS alone.THP1 Sig5 cells were incubated with Hsp70, and the levels of SHP‐1 recruitment to Siglec‐5 were evaluated by immunoprecipitation of Siglec‐5 and Western blot for SHP‐1. Higher levels of SHP‐1 were co‐immunoprecipitated with Siglec‐5 when the cells were incubated with Hsp70.Western blots demonstrate phosphorylated p38 (p‐p38) was reduced in THP1Siglec‐5cells when exposed to both LPS and Hsp70 in comparison with LPS alone. This reduction was also greater in THP1Siglec‐5cells in comparison with the control THP1cells. Numbers below immunoblots indicate densitometric analysis of each band normalized to the respective cell line's unstimulated control group, divided by the respective loading control (total p38).Secretion of pro‐inflammatory cytokine IL‐8 by PMA‐differentiated THP1 EV or THP Sig5 cells was evaluated by ELISA after concurrent exposure with 10 ng/ml LPS with an anti‐Hsp70 antibody or isotype‐matched antibody. Secretion of IL‐8 was increased in only THP1 Sig5 cells exposed to LPS and an anti‐Hsp70 antibody in comparison with the isotype control antibody, but this increase was not replicated in THP1 EV cells.
Figure 6Secretion of TNFα by THP1 cells transfected with a Siglec‐5 or Siglec‐14 expression vector was evaluated by ELISA after exposure to Hsp70 at the various concentrations in the presence of endotoxin‐neutralizing agent polymyxin B. Only THP1 Siglec‐14 cells exhibited significant secretion of TNFα in an Hsp70 dose‐dependent manner.IL‐8 secretion was also evaluated after THP1Siglec‐5 and Siglec‐14cells were exposed to Hsp70. Similarly, only THP1Siglec‐14cells exhibited significant secretion of IL‐8 in an Hsp70 dose‐dependent manner.Western blot analysis demonstrates a greater increase in phosphorylated p38 (p‐p38) from THP1 Siglec‐14 cells after exposure to Hsp70 in comparison with control THP1 EV cells in a time‐dependent manner. Graph below immunoblots indicates densitometric analysis of each band normalized to the respective cell line's unstimulated control group, divided by the respective loading control.Exposure to Hsp70 also yielded greater degradation of the NF‐κB inhibitor, IκBα, in THP1 Siglec‐14 cells in comparison with control THP1 cells, augmentation of the pro‐inflammatory response. Graph below immunoblots indicates densitometric analysis of each band normalized to the respective cell line's unstimulated control group, divided by the respective loading control.Secretion of pro‐inflammatory cytokine IL‐8 by PMA‐differentiated THP1 EV or THP Sig 14 cells was evaluated by ELISA after concurrent exposure with an anti‐Hsp70 antibody or isotype‐matched antibody. Secretion of IL‐8 was decreased in only THP1 Sig 14 cells exposed to LPS and an anti‐Hsp70 antibody in comparison with the isotype control antibody, but this change was not observed in THP1 EV cells.
Figure 7TNFα secretion from SIGLEC14+/+ monocytes was measured after exposure to LPS or Hsp70 in the presence or absence of endotoxin‐neutralizing compound polymyxin B. Hsp70 was able to induce secretion of TNFα even in the presence of polymyxin B, suggesting a mechanism independent of endotoxin contaminants. DnaK, the E. coli homolog of human Hsp70, did not induce significant secretion of TNFα above background levels.TNFα secretion from SIGLEC14+/+ monocytes by Hsp70 was reduced when the cells were exposed to an anti‐Siglec‐5/Siglec‐14 blocking antibody.Human monocytes isolated from SIGLEC14+/+, SIGLEC14+/−, and SIGLEC14−/− individuals were exposed to varying concentrations of Hsp70, and TNFα secretion was evaluated. The amount of TNFα secreted was normalized to the respective cells' response to 1 ng/ml LPS to minimize the impact of other possible polymorphisms and factors that contribute to variation between individuals.TNFα secretion from SIGLEC14−/− monocytes stimulated with 15 ng/ml LPS and various concentrations of Hsp70. TNFα secretion was reduced with increasing concentrations of Hsp70.
Figure 1(B) B-RafE586K signals independently from Ras-GTP, but requires an intact AL. The MEK-ERK activation potential of the indicated HA-tagged B-Raf mutants was assessed by Western blotting using TCLs from transiently transfected Plat-E cells.(C) B-RafCAAX but not the B-RafV600E oncoprotein signals independently from Ras-GTP, but requires an intact AL. Same experimental set-up as in (B). (D) Quantification of experiments shown in (B) and (C). The signal elicited by the individual reference proteins (B-Rafwt, B-RafE586K, B-RafV600E and B-RafCAAX) was set in each analysis to 100%. n = 3, mean + SEM, t-test, * p < 0.05, ** p < 0.01
Figure 2(A) Braf-/-;ERTmHRasG12V MEFs were reconstituted with HA-tagged hBRAF constructs or empty vector (EV). Infected cells were either treated with 4-HT (1µM) to induce oncogenic Ras activation or with ethanol (OH) as a control. (B) Quantifications of pMEK (left) and pERK (right) normed to WT level. n = 3, mean + SEM, t-test, ** p < 0.01.(C) The AVKA mutation has no discernible influence on Ras-induced paradoxical ERK activation triggered by the D594A mutation. Experimental set-up as in (A), shown are lysates from 4-HT treated MEFs. (D) Quantification of pMEK and pERK levels normed to HA expression. n = 3, mean + SEM, t-test, ** p < 0.01.(E) Top: Braf-/-;ERTmHRasG12V MEFs were reconstituted with the indicated B-Raf constructs. Following induction of Ras signaling, HA-B-Raf proteins were purified using anti-HA antibodies and subject to Western blotting to analyze immunoprecipitation of Raf-1. Bottom: Quantification of co-immunoprecipitated Raf-1 normed to HA-B-Raf levels. n = 3, mean + SEM, t-test, * p < 0.05.
Figure 3(B) Genomic PCR with the primers indicated as red arrows in (A) showing 3 out of 9 clones being positive for homologous recombination.(C) Southern blot analysis of genomic DNA of parental W4 ES cells, wildtype MEFs and the ES cell clones #286 and #273 (with recombined B-raffloxAVKA allele). Genomic DNA was digested by AseI and analyzed using the external probe indicated in (A).(D) Genomic PCR showing Cre-mediated recombination of the BraffloxAVKA allele in vivo using the primer pair E14fwd and E15rev (indicated as blue arrows in A).
Figure 4(A) Weight at weaning of BrafAVKA mice from two different founders showed no significant differences. 215: n = WT = 24/ Heterozygous = 32/ AVKA = 19; 273: n = WT = 114/ Heterozygous = 117/ AVKA = 52, mean + SEM, t-test(B) Homozygous BrafAVKA mice show reduced splenic cellularity. n = 7, mean + SEM, t-test, * p < 0.05.(C-E) Relative number of CD8+ T cells. FACS analysis shows an increase in naïve cells (C), whereas differentiated TEM (D) and Tregs (E) reveal a reduction in BrafAVKA mice compared to WT. n = 8, mean + SEM, t-test, * p < 0.05, ** p < 0.01.(F) Primary B cells of BrafAVKA mice show reduced CD69 surface expression upon LPS and anti-IgM stimulation. n = 3, mean + SEM, test, ** p < 0.01.(G) Primary B cells also show reduced proliferation upon stimulation compared with WT B cells. n = 3, mean + SEM, test, ** p < 0.01
Figure 5(A) Facial appearance of BrafAVKA mice.(B) Top: In vitro kinase (IVK) assay of B-Raf complexes purified from either WT or BrafAVKA knock-in brains from both founder strains revealed a reduced MEK phosphorylation potential regardless of their purification from NLB or RIPA lysates. Botttom: Quantification of IVK differentials: #215 samples indicated in black, #273 samples in grey.(C) Western blot analysis showing reduced pMEK and pERK levels in BrafAVKA brains. n = 5, mean + SEM, t-test, ** < 0.01.(D) Immunohistochemistry showing reduced pERK levels in Purkinje cells of homozygous BrafAVKA mice. Scale bar indicates 10x magnification.
Figure 6(A) Total brain lysates of WT and BrafAVKA mice were subject to immunoprecipitation (IP) using either anti-B-Raf antibodies or non-specific IgG as negative control. The co-purified proteins were visualized using the indicated antibodies and quantified. Ksr1 is significantly decreased in B-RafAVKA complexes while the other proteins are equally bound to B-RafWT or B-RafAVKA. n = 3, mean + SEM, t-test, * p < 0.05.(B) Anti-Myc IP of Plat-E cells transfected with Myc-tagged Raf-1 and different HA-B-Raf constructs. Western blot analysis showed that B-RafAVKA is not impaired in its ability to interact with Raf-1.(C) Anti-Myc IP of Plat-E cells expressing different Myc- and HA-B-Raf proteins. Note that B-RafAVKA is not impaired in its ability to interact with other B-Raf proteins, while homo-dimerization of B-RafEVKD is enhanced.(D) Top: Exemplary Western blot analysis of EGF stimulated primary astrocytes. Mutant astrocytes show drastic reduction in basic and stimulated levels of pMEK and pERK. Bottom: Quantification of all Western blot samples. WT: n = 7, AVKA: n= 6, mean + SEM, t-test, * p <0.05, ** p < 0.01.
Figure 7(A) MEFs expressing B-RafWT or B-RafAVKA were stimulated with EGF for the indicated time points (min) and analyzed by Western blotting.(B) IP of EGF stimulated MEFs expressing B-RafWT or B-RafAVKA. Sorafenib was used to increase complex build up.(C) Proliferation of two different MEF pools expressing either B-RafWT or B-RafAVKA. MEFs expressing B-RafAVKA show a strong reduction in growth rate. n = 3, mean + SEM, t-test, ** p < 0.01, *** p < 0.001.(D) Cell morphology of B-RafAVKA expressing MEFs are enlarged and present with abnormal nuclei(E) MEFs expressing B-RafAVKA show a significant increase in caspase-3 activity. n = 3, mean + SEM, t-test, ** p < 0.01.(F) Decay of TAg expression in MEFs following dox withdrawal.(G) Morphology of MEFs expressing B-Rafwt or B-RafAVKA in the presence or absence of TAg.
Figure 8(A) MEFs were then infected with retroviral vectors encoding the indicated B-Raf proteins. Giemsa staining revealed transformed and hyper-proliferated cells by dark purple colour. (B) Quantification of Focus formation potential of the B-Raf mutants used in (A). n = 3, mean + SEM, t-test, *** p < 0.001.
Figure 1A. Example of littermate males of the indicated genotypes 26 days post partum. Additional examples are provided in Appendix Fig S2.B. Weights of mice of the indicated genotypes from 18 to 120 days post partum. Boxplots indicate 5-95 percentile. A minimum of 8 animals is plotted at each age. Asterisks denote statistical significance (NS, not significant, *P value <0.05, **P value <0.01 and ***P value <0.001) determined by the unpaired two-way Wilcoxon rank-sum test.C. Example of dissected brains (upper panel) and coronal sections (left and right panels) stained with H&amp;amp;E of the indicated genotypes. Note the severe hydrocephaly and ventricular dilation of the lateral ventricle (LV) and third ventricle (TV) in a Gemc1-/- mouse (bottom right panel) compared to a Gemc1+/+ littermate animal (bottom left panel). Periventricular malacia (asterisks) and hippocampal atrophy (arrow) is also evident in the Gemc1-/- sample. Scale bars = 500 µm.D. Kaplan Meyer survival curve of a cohort of mice of the indicated genotypes and numbers (n). The study was terminated at 16 months and mice were examined for histology. 97% (31/32) of the Gemc1-/- mice examined to date exhibited hydrocephaly (additional histological examples in Appendix Fig S2).E. RT-QPCR analysis of Gemc1 expression in murine tissues from Gemc1+/+ mice using a Taqman probe. Data is compiled from duplicate samples of 3 male mice per tissue and mean and standard deviation are plotted.F. RT-QPCR analysis of Gemc1 in trachea, oviduct and ovary tissue from Gemc1+/+ and Gemc1-/- mice shows that the knockout reduces Gemc1 mRNA to undetectable levels. Mean and standard deviation of duplicate samples from 2 female mice are plotted. Additional examples from other Gemc1-/- tissues are shown in Appendix Fig S1.
Figure 2. Example of dissected testes from littermate mice of the indicated genotypes at 2 months of age.B. Testes from Gemc1-/- mice have strongly reduced cellularity compared to Gemc1+/+ or Gemc1+/- animals, as measured by disaggregation of the tissue and cell counting with a Neubauer Chamber (n=3). Mean and standard deviation are indicated.C. Histological analysis of tubules from 6-week-old Gemc1-/- mice revealed thinning of the spermatogenic cell layer and decreased numbers of elongated spermatids. Scale bars = 100 µm (left panels) and 50 µm (right panels).D. Sperm counts from the cauda epididymis of wild type and Gemc1-/- mice revealed no sperm in mice lacking GEMC1 (n=3). Mean and standard deviation are indicated. E. Examples of sperm morphology from spermatocyte spreads from 2 month old mice. Scale bars = 10 µm.F and G. Histological evaluation of the caput epididymis in sagittal (F) and coronal (G) orientation from 2 and 4 month old animals, respectively. Note the absence of luminal spermatozoa in the Gemc1-/- mice compared to wild type. Scale bars = 100 µm (F, left panels) and 50 µm (F, right panels and G).
Figure 3A. Examples of ovaries from Gemc1+/+ (top panels) and Gemc1-/- mice (bottom panels) at 6 weeks of age. Ovaries were smaller in Gemc1-/- mice and contained primarily degenerated antral follicles. A histologically normal antral follicle is indicated in the Gemc1+/+ ovary (top right, black arrow) and degenerated antral follicles with high levels of cell death are indicated in the Gemc1-/- ovaries (bottom right, red arrows). Scale bars = 200 µm (left panels)) and 100 µm (right top panels and right bottom panel respectively). B. Morphological abnormalities in the oviduct of 3-week-old Gemc1-/- mice (right panels) compared to Gemc1+/+ (left panels). A clear lack of cilia (bottom panels) in Gemc1-/- oviducts is observed by H&amp;amp;E staining. Scale bars = 50 µm (top panels).C. Staining of frozen sections of oviduct with antibodies for Ac-tubulin (cilia) and γ-tubulin (basal bodies) reveals normal cilia organization in the wild type oviduct (left) while basal bodies and cilia are not detectable in Gemc1-/- oviducts (right). D. FOXJ1 and E. RFX3 staining that is characteristic of MCCs was clearly evident in Gemc1+/+ oviducts and absent in Gemc1-/- mice. Scale bars = 50 µm (C-E).
Figure 4A. H&amp;amp;E staining of the nasal mucosa of E17.5 embryos of the indicated genotype. Normal respiratory pseudostratified columnar ciliated epithelium is shown in Gemc1+/+ embryos compared to the attenuated, flat columnar to squamous epithelium of Gemc1-/- embryos. Scale bars = 20 µm.B. PAS staining (purple) of the nasal mucosa of E18.5 embryos of the indicated genotype. Normal PAS positive goblet cells (inset, red arrows) are interspersed between MCCs (inset, black arrows) with their characteristic goblet or cup-like shape in Gemc1+/+ embryos. The Gemc1-/- mucosa has abundant PAS positive materials that accumulate in the apical borders of almost every columnar epithelial cell (inset, red arrows) and lacks typical goblet cells. Scale bars = 20 µm.C. Transmission electron micrographs of cross-sections of adult trachea from Gemc1+/+ animals revealed the presence of secretory cells (SC) and multiciliated cells (MCC). The apical membrane of MCCs accumulated basal bodies (BB) nucleating multiple cilia (C) and microvilli (MV). In the Gemc1-/- airway epithelium, no MCCs are identifiable and only secretory-like cells with MV at the apical surface are detectable. Scale bars = 5µm. Higher magnifications of the boxed regions are shown below, scale bars = 0.5µm.D. Lack of Ac-tubulin (top panel) and RFX3 (second panel from top) staining in the trachea of 1 month old mice.E. Cilia (Ac-tubulin staining) are not detectable in the ependymal cells of 2 month old Gemc1-/- mice (right panel). Choroid plexus (CP) and lateral ventricles (LV) are indicated. Scale bars = 20 µm
Figure 5E and F. RT-QPCR analysis of individual genes related to MCC formation in the oviduct (E) and trachea (F) of Gemc1-/- mice compared to wild type littermates, using 18S rRNA as a reference (n=2). Genes examined generally fall into 3 classes: centriole duplication (Cep152, Plk4, Cetn2), Deuterosome mediated centriole expansion (Deup1, Ccdc78, Ccno) and transcription of genes required for MCC differentiation (FoxJ1, Myb, Mcidas).
Figure 6A. GEMC1 interacts with E2F4 and DP1. Western blot of HA or FLAG tagged protein expression in the input lysates are shown in the top panel. FLAGimmunoprecipiation (IP) of GEMC1co-IPs DP1 (α-HA, lane 5) and E2F4 (α- HA, lane 6). The interaction is enhanced upon co-expression of HA tagged E2F4 and DP1 (α-HA, lane 7).B. GEMC1 interacts with E2F5 but not E2F1. Co-IP of E2F5 (α-HA, lane 3) and E2F5-DP1 (α-HA, lane 6) but not E2F1 or E2F1-DP1 (α-HA, lanes 2 and 5). In the FLAG-IP panels, a short (top) and long (second from top) exposure of the Western are shown.C. Identification of the domain required for E2F4 interaction. GFP tagged GEMC1 and mutant forms were expressed and IPs were performed against GFP. Wild type GEMC1 (lane 5) or GEMC1 lacking the CC domain (lane 6) pulled down E2F4-DP1. A GEMC1 protein lacking the TIRT domain or GFP alone did not IP E2F4-DP1 (lanes 7, 8).D. Mutation of the single amino acid analogous that identified in MCIDAS in human RGMC patients (G313D) impairs the interaction with E2F4-DP1 (compare land 5 and 6).E. GEMC1 interacts with Multicilin through its CC domain. Wild type GEMC1 or a mutant lacking the TIRT domain pull down Myc-tagged Multicilin (anti- Myc blot, lanes 4 and 6) while a CC domain mutant does not (anti-Myc blot, lane 5).
Figure 7A. Transient overexpression of GEMC1 leads to increased levels of FOXJ1 and MCIDAS expression (RT-QPCR) in HEK293T cells. Results for MCIDAS are graphed on a smaller scale in the right panel: V = vector and G = GEMC1. The mean and standard deviation of 3 individual experiments in HEK293T cells are graphed. Similar induction of FOXJ1 was observed in U2OS and T98G (Fig EV3).B. Both the CC and the TIRT domain of GEMC1 are required to activate FOXJ1 (RT-QPCR). A corresponding Western blot showing relative expression levels of the mutant GFP tagged GEMC1 variants is shown from HEK293T cells. The mean and standard deviation of 3 individual experiments in HEK293T cells are graphed. Similar results were also observed in U2OS (Fig EV3).C. Activation of FOXJ1 by GEMC1 is inhibited by the co-expression of Geminin (compare 3rd and 4th lanes). Example Western blot is shown below. Results graphed are the mean and standard deviation of 3 individual experiments in HEK293T cells.D. Co-expression of GEMC1 and Multicilin results in similar levels of FOXJ1 expression (measured by RT-QPCR) as Multicilin alone in HEK293T cells. The mean and standard deviation of 4 individual experiments in HEK293T cells are graphed.E and F. The activation of FOXJ1 by GEMC1 is partially dependent on Multicilin. HEK293T cells were transfected with a control siRNA (siC) or 2 different siRNAs against MCIDAS (si1 and si2) prior to being transfected with GFP or GFP-GEMC1 expression vectors. Relative MCIDAS (E) and FOXJ1 (F) expression levels were assessed by RT-QPCR. The mean and standard deviation of a representative experiment performed in triplicate is graphed. An additional experiment is presented in Fig EV3.
Figure 1IL-1β ELISA of supernatants from LPS-primed (1 μg/mL, 4hrs.) MDMs pre-incubated with 0.1% DMSO or P22077 (2.5 µM) 15 mins. before treatment with either nigericin (10 μM, 45 mins.) or CPPD crystals (250 μg/mL, 2 hrs.). Bars represent the mean, ± S.D. n = 13 and 11 independent blood donors for nigericin and CPPD, respectively. IL-18 ELISA of supernatants from MDMs treated as in A. Bars represent the mean, ± S.D. n = 11 independent blood donors.LDH-release from MDMs treated as in A. Bars represent the mean percentage of LDH release relative to the total cells lysed, ± S.D. n = 10 and 11 independent blood donors for nigericin and CPPD, respectively.Western blots of supernatants and cell lysates from MDMs treated as in A. Bands in the figure represent: Pro-IL-1β (31 kDa); mature IL-1β (mIL-1β, 17 kDa); pro-IL-18 (24 kDa), mature IL-18 (mIL-18, 18 kDa), pro-caspase-1 (pro-Casp-1; 45 kDa) and mature caspase-1 (mCasp-1; 20 kDa). β-actin is shown as a loading control. Blots are representative of at least 3 independent blood donors.