The EJC model for human NMD postulates that any translation termination event taking place >50–55 nucleotides upstream of an exon-exon junction should result in NMD. However, during our studies of the human NMD pathway, we observed that a β-globin mRNA, in which the adenovirus major late (AdML) intron was inserted into the 3′ UTR 175 nucleotides downstream of the normal β-globin mRNA translation termination codon, did not show enhanced mRNA decay as compared to the wild-type β-globin mRNA in human HeLa Tet-off cells (compare Figure 1A and 1B). Moreover, in contrast to a well-characterized β-globin NMD substrate, which contains a premature termination codon at position 39 (β39; Figure 1C), the AdML intron containing β-globin mRNA is not stabilized when the central NMD factor hUpf1 is knocked down (Figure 1A, middle panel and Figure S1) or when a point mutation causes translation termination to take place downstream of the inserted intron (Figure 1A, bottom panel). Sequencing of cDNAs derived from the mRNAs in Figure 1A revealed the expected splicing patterns (unpublished data). Thus, in contrast to the prediction from the EJC model for human NMD, the AdML intron is not sufficient for triggering NMD when positioned in the 3′ UTR of β-globin mRNA, even though the AdML intron has been previously demonstrated to recruit an EJC [13].

The observation in Figure 1A was surprising, because a β-globin mRNA in which the MINX-intron had been placed in the 3′ UTR was previously found to cause reduced mRNA steady-state levels [23]. Therefore, to rule out the possibility that the observations in Figure 1A represent an unusual property of the specific mRNA reporter, we tested two other substrates. As seen in Figure 1D and 1E, insertion of the triosephosphate isomerase (TPI) mRNA intron 6 or the AdML intron, 140 or 149 nucleotides downstream of the termination codons of β-globin or TPI mRNAs, respectively, failed to cause hUpf1-dependent mRNA decay, despite the previously demonstrated ability of each of these introns to recruit an EJC [13]. Sequencing of cDNAs derived from the tested mRNAs revealed the expected splicing patterns (unpublished data), although a minor fraction of the βTPIi6 mRNA fails to remove the TPI intron (see asterisk in Figure 1D). We attempted to test the β-globin mRNA with the MINX-intron in the 3′ UTR, which was previously found to accumulate at reduced steady-state levels as compared to wild-type β-globin mRNA [23] (construct generously provided by A. Kulozik and M. Hentze). However, the MINX intron (as well as a number of other introns tested in this study) failed to be spliced out of the β-globin mRNA 3′ UTR in the HeLa Tet-off cells used here (unpublished data). We conclude that a 3′ UTR intron is not sufficient to trigger NMD in human cells. This contradicts an EJC-centric model for human NMD.

Our observation that 3′ UTR introns are not sufficient for triggering human NMD spurred us to test whether cytoplasmic PABP may antagonize NMD in human cells, as it does in S. cerevisiae and D. melanogaster. We therefore first manipulated the position of the poly(A) tail relative to the termination codons of β-globin and TPI reporter mRNAs and tested the effect on mRNA decay. As seen in Figure 2A and 2B and Figure S2, artificial extension of the 3′ UTRs of β-globin or TPI mRNAs (from 292 and 447 nucleotides, respectively, to 846–1,112 nucleotides) through insertion of fragments of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Figures 2A and S2) or green fluorescent protein (GFP) (Figure 2B) mRNAs results in mRNA destabilization (compare with Figures 1B and S2, top panel). This is due to NMD because depletion of the NMD factors hUpf1 or hUpf2 stabilizes the mRNAs (Figure 2A and 2B and Figures S1 and S2). Moreover, the introduction of single point mutations in the termination codons that results in termination on the same mRNAs in proximity (180–357 nucleotides upstream) of the poly(A) tail, results in mRNA stabilization (Figure 2A and 2B and S2, bottom panels). Even though sequencing of cDNAs derived from the tested mRNAs revealed no cryptic splicing in the extended 3′ UTRs (unpublished data), depletion of the central EJC component eIF4AIII results in stabilization of the βGAP mRNA (Figure S3), possibly reflecting the ability of the EJCs in the β-globin mRNA open reading frame to stimulate translation as has been previously observed [24,25]. Successive shortening of the 3′ UTR of the βGAP mRNA revealed that a 3′ UTR as short as 422 nucleotides can trigger NMD (Figure S4). This is surprising because a large fraction of human mRNAs contain 3′ UTRs longer than 422 nucleotides [12]. We conclude that artificially extended 3′ UTRs trigger NMD in human cells. This is consistent with recent reports in which steady-state levels of PTCs containing TPI, β-globin, and Ig-μ reporter mRNAs lacking 3′ UTR introns were measured [26–28], and with observations using unspliced Rous sarcoma virus RNAs in chicken cells [29]. Thus 3′ UTR introns are neither necessary (Figure 2) nor sufficient (Figure 1) for human NMD.

Having observed that 3′ UTR introns are not required for NMD, we asked whether a completely intron-less mRNA can undergo NMD. It was observed previously that introduction of PTCs in the naturally intron-less Hsp70 and histone H2A mRNAs does not result in their decreased steady-state levels, which led to speculations that intron-less mRNAs are immune to NMD [30]. However, it has been pointed out that wild-type Hsp70 and histone H2A mRNAs are both highly unstable and may thus not be further destabilized by a PTC [5]. We therefore tested the stability of three naturally occurring intron-less mRNAs (encoding eRF3b, SFN, and TBCC) and found that both wild-type and PTC containing versions of these mRNAs were unstable (∼100- to 150-min half-lives, unpublished data). Thus, mRNA instability may be a general feature of natural intron-less mRNAs. However, when the only intron in the Glutathione Peroxidase 1 (GPx1) mRNA is removed, introduction of a PTC triggers NMD, although not as efficiently as in the presence of the intron (Figure 2C, compare lower and upper panels). Thus, neither 3′ UTR introns nor internal introns are essential for human NMD. However, similarly to a previous report [30] we have not been able to identify a natural human intron-less mRNA for which NMD could be observed, perhaps due to the observed inherent instabilities of the tested mRNAs.

To more directly test whether cytoplasmic PABP antagonizes NMD in human cells, we examined the effect of positioning cytoplasmic PABP in proximity of a PTC using two different approaches. First, as seen in Figure 3A and 3B, when an MS2-PABPC1 fusion protein (PABPC1 is one of five human cytoplasmic PABPs [31]) is artificially tethered downstream of a PTC in two different β-globin NMD reporter mRNAs, a partial rescue of NMD is observed. This rescue is due to tethered PABPC1, because similar levels of unfused PABPC1 (Figure 3A) or MS2 coat protein (Figure 3A and 3B) do not stabilize the mRNAs. Moreover, tethering of the nuclear poly(A)-binding protein PABPN1 does not rescue NMD (Figure 3A) even though it is expressed at levels similar to MS2-PABPC1 (Figure S5). The efficiency of the rescue from NMD by tethered PABPC1 decreases as the MS2 binding sites are moved more distal to the PTC in the β-globin PTC-39 mRNA (Figure S6).

As a second independent approach to ask whether PABP can antagonize human NMD, we tested the effect of inserting a binding site for PABP downstream of the PTCs. As seen in Figure 3C and 3D, inclusion of a poly-A₃₀-stretch, but not that of a random 30-nucleotide stretch, 182 or 230 nucleotides downstream of the PTC of two different NMD reporter mRNAs, results in partial rescue of NMD. Thus, similarly to S. cerevisiae [7] and D. melanogaster [5], cytoplasmic PABP can antagonize NMD in human cells when placed in proximity of a PTC. Recent observations suggest that while cytoplasmic PABP can antagonize NMD in S. cerevisiae, it is not required for discriminating a normal mRNA from an NMD substrate [11]. Attempts at testing whether PABPC1 is required for preventing NMD in human cells failed because HeLa Tet-off cells became detached from plates upon short interfering RNA (siRNA)-mediated PABPC1 depletion (unpublished data).

Our observations raise the question of whether naturally occurring mammalian mRNAs with long 3′ UTRs, which can be several kilobases in length, are normal targets of NMD or whether they have evolved mechanisms to evade the NMD pathway. We noted that mRNAs identified by microarray assays to be upregulated upon hUpf1 knockdown in HeLa cells [32] contain on average significantly longer 3′ UTRs than those mRNAs unaffected by hUpf1 knockdown (Figure S7). Moreover, the majority of these 3′ UTRs (75%) are longer than the ∼420 nucleotides observed here to trigger NMD in the βGAP reporter mRNA (Figures S4 and S7). It is possible that at least a subset of these transcripts undergo NMD due to an increased distance between the termination codon and the poly(A) tail. Indeed, when the 1,342-nucleotide 3′ UTR of one of these mRNAs, encoding hSmg5, is replaced for the β-globin 3′ UTR (βSmg5), the chimeric mRNA undergoes NMD (Figure 4, top two panels). Thus, the Smg5 3′ UTR stimulates NMD, and a subset of mRNAs may have evolved long 3′ UTRs to be regulated by the NMD pathway. However, numerous mRNAs with long 3′ UTRs are not upregulated upon hUpf1 knockdown [32] (Figure S7). When the 3′ UTRs from two such mRNAs, Cript1 and Tram1, were inserted into the β-globin mRNA, no NMD was observed (Figure 4, bottom panels). This is in sharp contrast to the observations using artificial long 3′ UTRs (compare to Figures 1 and S4) and suggests that the ability of a subset of endogenous long 3′ UTRs to evade NMD is an acquired property (see Discussion).

Our observations that 3′ UTR introns are neither necessary (Figure 2) nor sufficient (Figure 1) for human NMD raises the question of whether introns play any role in human NMD. We therefore tested the effect of inserting the AdML intron into the 3′ UTR of an mRNA, which already undergoes NMD due to an extended 3′ UTR. Interestingly, insertion of the AdML intron downstream of the termination codon of βGAP mRNA results in enhanced mRNA decay (Figure 5; βGAP-AdML mRNA). This effect is only observed when the intron is positioned in the 3′ UTR, as insertion of the same intron upstream of the termination codon (without disrupting the open reading frame) does not enhance mRNA decay (Figure 5; βAdML-GAP mRNA). Thus, while a downstream intron is neither sufficient nor necessary for triggering NMD in human HeLa cells (Figures 1 and 2) [33], it can enhance the degradation of an mRNA that is already a target of NMD due to an extended 3′ UTR (Figure 5A and 5B). Consistent with this, the presence of an intron appears to also stimulate NMD of a PTC containing GPx1 mRNA (Figure 2C).

How does cytoplasmic PABP antagonize NMD when positioned in proximity of the termination codon? Both cytoplasmic PABP and Upf1 have been previously observed to stimulate translation termination in yeast cells [8,34] and to associate with translation release factor eRF3 [35–40]. This raised the possibility that cytoplasmic PABP inhibits NMD by preventing Upf1 from interacting with eRF3 and the terminating ribosome. As seen in the co-immunoprecipitation (co-IP) assays in Figure 6A, endogenous hUpf1 and PABPC1 can both be observed in complex with eRF3 in RNase-treated HeLa cell extracts. However, PABPC1 co-IPs much more efficiently than hUpf1 with eRF3 (Figure 6A), in spite of comparable estimated number of molecules of cytoplasmic PABP (∼8 × 10⁶/cell) and hUpf1 (∼3 × 10⁶/cell) in HeLa cells [41,42]. Consistent with this, bacterially expressed GST-tagged eRF3 was found to associate much more efficiently with rabbit reticulocyte-lysate-translated PABPC1 (K_d ∼ 5 nM) than hUpf1 (K_d > 1 μM) (unpublished data).

To test whether PABPC1 can antagonize the interaction between eRF3 and hUpf1 in vitro, we immunopurified transiently expressed epitope-tagged eRF3, PABPC1, and hUpf1 proteins from HEK 293T cells and tested the ability of hUpf1 to associate with eRF3 in the presence of increasing amounts of PABPC1. As seen in Figure 6B, in contrast to the negative control protein hnRNP A1, increasing amounts of PABPC1 efficiently prevent the interaction between hUpf1 and eRF3, even when hUpf1 is present in 10- to 40-fold excess over PABPC1 (Figure 6B, compare lanes 5 and 6 with lanes 4 and 7). Thus, PABPC1 can antagonize the interaction between hUpf1 and eRF3 in vitro. However, no reduction in the co-IP efficiency between hUpf1 and eRF3 was observed upon transient over-expression of FLAG-tagged PABPC1 in HeLa or HEK 293T cells (unpublished data). Thus, either exogenous PABPC1 failed to express at adequate levels to observe a competition in cells, or the relation between hUpf1, PABPC1, and eRF3 is more complex in cells than it is in vitro.

To test whether amino acid residues of eRF3, which are important for cytoplasmic PABP interaction, are also important for the interaction with hUpf1, we constructed a eRF3 protein (eRF3 KAKA) mutated in four N-terminal residues that are conserved between cytoplasmic PABP-binding proteins [38,43]. As seen in the co-IP assays in Figure 6C, the exogenously expressed eRF3 KAKA mutant protein is equally impaired in interaction with PABPC1 and hUpf1 (Figure 6C, compare lanes 5 and 6 with lanes 2 and 3). As a control, the mutant eRF3 KAKA protein associates with eRF1 with similar affinity as wild-type eRF3, suggesting that these mutations do not cause gross structural alterations, although local changes cannot be ruled out. Thus, hUpf1 and PABPC1 interact with a similar, though not necessarily overlapping region of eRF3. The ability of PABPC1 to antagonize the association between hUpf1 and eRF3 in vitro could therefore be a result of a direct competition for eRF3 binding, or of a local structural alteration of eRF3 upon PABPC1 binding, which prevents hUpf1 association.

All plasmid sequences are available upon request. Plasmids expressing different β-globin reporter mRNAs were derived from the pcTET2-βwt plasmid that was constructed by inserting the human β-globin gene between HindIII and ApaI sites of a pcDNA3-based plasmid containing six copies of the Tet-operator sequences upstream of the TATA box. For extended 3′ UTR constructs, parts of the GAPDH mRNA coding sequence and the entire GAPDH 3′ UTR (pcTET2-βGAP) or the GFP ORF (pcTET2-βGFP) were inserted between NotI and XbaI sites of the pcTET2-βwt plasmid, thus replacing the β-globin 3′ UTR. The β-globin stop codon was mutated to UAC by site-directed mutagenesis to generate pcTET2-βGAP-UAC and pcTET2-βGFP-UAC. Plasmids expressing βGAP-UAC-696, βGAP-UAC-485, and βGAP-UAC-422 mRNAs were generated by site-directed mutagenesis of pcTET2-βGAP-UAC to introduce a stop codon (UAA) in the GAPDH sequence, respectively, 696, 485, or 422 nucleotides upstream of the polyadenylation site. The plasmid expressing βwt mRNA was described earlier [47]. To construct plasmids expressing β39–2xMS2-Ex2, β39–2xMS2-Ex3, and β39–2xMS2-3UTR, the 2xMS2 cassette from the previously described plasmid pcβ-2bs [47] was inserted into the BamHI, EcoRI, or NotI sites, respectively, of the pPC-β39 plasmid described earlier [62]. A stretch of A₃₀ (pPC-β39-A₃₀) or N₃₀ (pPC-β39-N₃₀) was inserted into the BamHI site of pPC-β39 plasmid using annealed DNA oligos. Similarly, A₃₀ (pcTET2-βGAP-A₃₀) or N₃₀ (pcTET2-βGAP-N₃₀) was inserted into the XbaI site of the pcTET2-βGAP plasmid. To construct the pcTET2-βGAP-4xMS2 plasmid, four MS2 binding sites were amplified from a previously described plasmid pcβ-4bs [47] and inserted into the XbaI site of pcTET2-βGAP. Plasmids expressing βAdML, βAdML-UAC, and βTPIi6 mRNAs were constructed by cloning the AdML intron or TPI intron 6 (TPIi6) and flanking exon sequences into the XbaI site in pcTET2-βwt or pcTET2-βwt-UAC plasmids. βGAP-AdML and βAdML-GAP mRNA– expressing plasmids were constructed by inserting the same AdML intron into XbaI and EcoRI sites, respectively, in the pcTET2-βwtGAP plasmid. Plasmids expressing chimeric β-globin mRNAs with 3′ UTRs from Smg5, Cript1, and Tram1 genes, the respective 3′ UTRs, were cloned into the NotI-XbaI sites of pcTET2-βwt.

Plasmids expressing TPI reporter mRNAs were constructed by inserting the entire human TPI gene between HindIII and XbaI sites of the pcTET2 plasmid. A NotI site was inserted into exon 6 (in a manner that preserved the encoded protein) by site-directed mutagenesis. Codon 189 was mutated to TGA using site-directed mutagenesis to generate pcTET2-TPI-189. To remove intron 6, a NotI-XbaI fragment containing exon6-intron6-exon7 was replaced by the same region amplified from TPI cDNA, to generate pcTET2-TPIΔi6–189. To extend the TPI 3′ UTR, a fragment containing part of the GAPDH coding region and 3′ UTR was inserted into the NotI site of pcTET2-TPIΔi6–189 to generate pcTET2-TPIΔi6–189-GAP, or into the NotI site of pcTET2-TPIΔi6 to give rise to pcTET2-TPIΔi6-GAP. TPI-AdML mRNA–expressing plasmid was constructed by inserting the AdML intron and flanking exonic sequences into the XbaI site of pcTET2-TPI.

The plasmid expressing intron-containing GPx1 mRNA with a PTC (pPC-GPx1-UAA) was described earlier [18]. GPx1 cDNA (HindIII-XbaI) sequence replaced the intron-containing sequence in pPC-GPx1Δi-UAA.

The constructs for knockdowns were based on the pSHAG plasmid (a gift from Dr. G. Hannon) and contained inserts expressing precursors to hUpf1, hUpf2, or eIF4AIII siRNAs described earlier [63,64].

Plasmids expressing FLAG-hUpf1, FLAG-PABPC1, FLAG-hnRNP A1, and Myc-hnRNP A1 were described earlier [47,62]. pcDNA3-Myc-eRF3 was constructed by inserting the ORF of eRF3 (longer isoform) between BamHI and NotI sites of the pcDNA3-Myc vector previously described [65]. pcDNA3-Myc-eRF3 KAKA was prepared using site-directed mutagenesis (the mutations are: L66K, N69A, A70K, F73A). pcDNA3-MS2-FLAG-PABPC1 or pcDNA3-MS2-FLAG-PABPN1 were obtained by inserting PABPC1 and PABPN1 cDNAs, respectively, into BamHI-NotI sites of pcDNA3-MS2-FLAG described previously [62].

NMD factor knockdowns were performed by co-transfecting cells with reporter mRNA plasmids and plasmids encoding small hairpin (sh)RNAs targeting hUpf1, hUp2, or eIF4AIII, 60 h before pulse-chase mRNA decay assays were carried out.

mRNA decay assays were performed in HeLa Tet-off cells in DMEM/10% FBS/tetracycline (50 ng/ml) transfected with β-globin mRNA expression plasmids. For each 2-cm well of HeLa Tet-off cells, 10 ng of pcβG or pcβwt (as an internal control) and 0.2 μg of tetracycline-regulated reporter mRNA expression plasmids were co-transfected using TransIT HeLa Monster reagent (Mirus). For knockdowns, 0.5 μg of pSHAG plasmids were co-transfected. In each transfection, empty pcDNA3 vector was added to 1 μg of total plasmid. 36–40 h after transfection, or approximately 60 h in the case of knockdowns, transcription of reporter mRNAs was induced by removal of tetracycline through washing cells with 1 ml of phosphate-buffered saline (PBS) and adding DMEM/10% FBS. 6 h later, transcription was shut off by adding tetracycline to a final concentration of 1 μg/ml. Cells were washed with 1 ml PBS and taken up in 500 μl of TRIzol (Invitrogen) starting 30 min after tetracycline addition (0 min time point), and subsequently at time points indicated in each figure. For analysis of knockdown of endogenous hUpf1, hUpf2, and eIF4AIII, 0.2 μg of the plasmid pSUPERpuro was co-transfected instead of the plasmids expressing β-globin mRNA, and cells were treated and harvested as described earlier [66]. Total cellular RNA was isolated and analyzed by Northern blots as described earlier [47]. The anti-sense RNA probe used for β-globin mRNA detection was described earlier [47]. Northern blots for exogenously expressed TPI mRNAs were probed using UltraHyb reagent following the manufacturer's protocol (Ambion), with a short anti-sense RNA probe complementary to the bovine growth hormone 3′ UTR sequence encoded from the pcDNA3 plasmid. GPx1 mRNAs were probed as described earlier [18].

Rabbit polyclonal anti-sera raised against eIF4AIII (amino acids 1–41), hUpf1 (amino acids 1–416), hUpf2 (C-terminal 206 amino acids), and hUpf3b (full-length) were described earlier [18,47]. Monoclonal mouse antibodies were commercially obtained (anti-FLAG M2, Sigma; anti-Myc 9B11, Cell Signaling). Monoclonal mouse anti-HuR antibodies were described earlier [67]. Rabbit polyclonal eRF3 (#ab-49878) and mouse monoclonal PABPC1 (#ab-6125–100) antibodies were from Abcam.

In immunoprecipitations shown in Figure 6B, HEK 293T cells were transiently transfected in 3.5-cm plates with plasmids expressing FLAG-hUpf1 (0.4 μg), FLAG-PABP1 (0.5 μg), or FLAG-MS2 (0.5 μg), 0.5 μg of plasmid expressing wild-type or mutant Myc-eRF3 and 0.1 μg of pcDNA3-Myc-hnRNP A1. Empty pcDNA3 plasmid was added to each transfection to a total of 2 μg. 36–40 h post-transfection, cells were lysed in 400 μl of hypotonic gentle lysis buffer (10 mM Tris-HCl [pH 7.5], 10 mM NaCl, 2 mM EDTA, 0.5% Triton X-100, 1.0 mM phenylmethylsulfonyl fluoride, 1 μg/ml of aprotinin, and 1 μg/ml of leupeptin) for 10 min on ice. NaCl was added to 150 mM, and RNase A was added to 125 μg/ml. The extracts were incubated on ice for 5 min and cell debris was removed by centrifugation. RNase-treated lysed cell extracts were incubated for 2 h at 4 °C with 40 μl anti-FLAG M2 agarose (Sigma). The beads were washed eight times with NET-2 (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.05% Triton X-100) and the FLAG-tagged protein was eluted off the beads by gently shaking the beads for 2 h at 4 °C in 20 μl of NET-2 containing 200 μg/ml of FLAG peptide. Immunoprecipitates separated by SDS-PAGE were probed with anti-Myc 9B11 monoclonal antibody (Cell Signaling) at a 1:1,000 dilution.

Co-IPs between wild-type or KAKA-mutant eRF3 and eRF1 were performed as described above from the cells co-transfected with 0.5 μg of plasmids expressing FLAG-tagged proteins (eRF3, eRF3-KAKA, or MS2 as control), 0.5 μg of plasmids expressing Myc-eRF1, and 0.1 μg of Myc-hnRNP A1 expressing plasmid. Endogenous eRF3 IPs (Figure 6A) were performed as described above except that ∼2.5 × 10⁷ HeLa cells were lysed in 1 ml hypotonic gentle lysis buffer, and the lysates were incubated with 10 μg of anti-eRF3 rabbit polyclonal antibody (Abcam), or rabbit pre-immune serum as control, pre-conjugated to 5 mg of protein-A sepharose beads (GE Healthcare).

Approximately 10⁷ HEK293T cells from a 10-cm plate expressing Myc-eRF3, or Myc-peptide as a negative control, were lysed in 1 ml hypotonic gentle lysis buffer as described above. The RNase A–treated, cleared extracts were subsequently incubated with 40 μl anti-Myc resin (Sigma) at 4 °C for 2–3 h, following which the beads were washed eight times with 1 ml of NET-2 buffer. The beads were divided into eight equal parts, and indicated amounts of FLAG-hUpf1, FLAG-PABP1, or FLAG-hnRNP A1 proteins, which had each been affinity-purified from RNase A–treated HEK293T cell extracts (protein concentrations estimated by comparison in anti-FLAG Western blot to a GST-FLAG fusion protein of known concentration), were incubated in 50 μl of NET-2 supplemented with 0.1 mg/ml BSA and 0.2 mg/ml FLAG peptide. The reactions were gently shaken at 4 °C for 2–3 h following which the beads were washed eight times with 1 ml of NET-2 buffer. The beads were resuspended in 25 μl of SDS-loading buffer (10 mM Tris-HCl [pH 6.8], 2% SDS, 10% glycerol, 0.5% bromophenol blue, and 50 mM DTT), and 10 μl of the protein sample was resolved on SDS-PAGE followed by Western blot analysis using anti-FLAG M2 antibody (Sigma, 1:1,000 dilution).