EMBO/sd-character-level-ner huggingface dataset:
K Western blot analysis and quantification of NB-3 in cultured pyramidal neurons and astrocytes. NB-3 expression levels were normalized by b-tubulin and expressed relative to that of neurons. ***p < 0.001; one sample t-test. Data were analyzed from 3 independent experiments.
B) Volcano plot showing genes compared after two-days cytokine activated NK cells. Genes with significant changes (p value<0.05) and >1.5 log2 fold change between IL-2 and IL-15 activated NK cells are labeled in red. Genes belonging to cell surface (GO:0009986) are annotated with gene name.
Treatment with the inhibitor of actin polymerization Latrunculin B statistically significantly increased number of kugeln per embryo (100nM 1 hour; **p = 0.0041; control n=21 embryos 8.05 ± 1.76 (mean ± s.e.m.); Latrunculin n=21 embryos 17.19 ± 2.93 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Mann-Whitney U test).
Schematic diagram of the MIC19 protein. MIC19 contains an N-terminal myristoylation motif followed by a DUF37 domain (domain of unknown function) and a CHCH (coiled-coil helix-coiled-coil helix) domain, and is terminated by a diGly motif. Myristic acid was co-translationally attached to the N-terminal Gly2 residue by N-myristoyltransferases (NMTs) after removal of the initiating Met by methionine aminopeptidases. The N-terminal sequence of MIC19 is indicated at bottom with the myristoylation site, Gly2, and residues crucial for myristoylation labeled turquoise or underlined, respectively.
ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LE-expression constructs and autophagy assessed during amino acid starvation (AA starve) followed by immunoblotting or immunofluorescence analyses. Immunofluorescence analyses of amino acid starved cells (2 hr) using antibodies against WIPI2 (red) or ATG16L1 (green). Scale bar: 9 μm. Cells as in (A) with the addition of 3'MA (and pretreatment with the drug for 30 min). Scale bar: 9 μm.
(H) As in (G) showing promoter transcription. Indicated P-value by Kruskal-Wallis test. All pairwise comparisons are significantly different (P-values < 2.2e-16, two-sided Mann-Whitney U-test).
b) Caco-2 cells were infected with SARS-CoV-2 as per Figure 1A. Cells were washed, fixed and stained with DAPI (blue) to visualise cell nuclei and incubated with anti-N protein (red) to visualise SARS-CoV-2 infected cells. Scale bar denotes 10 µM.
(b) Western blot analysis for Atg5, LC3I and LC3II and p62 in purified primary lung tumour cells. Representative data are shown for tumour cells isolated from KRas;Atg5fl/+ and KRas;Atg5fl/fl mice 18 weeks after AdCre inhalation. β-actin is shown as a loading control.
(A) Western blot analysis after treatment with Ku70 or control siRNA, followed by incubation with DMSO, etoposide (ETO) or bleomycin (BLEO) for 16 h (i). p53 levels after Ku70 depletion normalized to siRNA control for each condition (ii, n=3). All the experiments were performed with HCT116 cells. GAPDH: loading control in A, B, C. Statistical analysis by unpaired t-test (*P<0.05; **P<0.01; *** P<0.001; NS, non significant). All error bars reflect SEM.
Figure 4: The limited enzyme supply defines yeast metabolic strategies in batch growth under different carbon sources. (A-B) Model simulations of maximum specific growth rate under different aerobic media conditions. (A) Yeast 7 simulations, with an average relative error of 100%. (B) ecYeast7 simulations, with an average relative error of 8%. (C-D) First two components of a PCA for (C) Yeast7 and (D) ecYeast7 flux simulations.
D. Phos-tag band-shift analyses of Hog1 phosphorylation. The yeast strain KT234 carrying the single-copy expression plasmid YCplac22I'-Pbs2 S514D/T518D was stimulated with the indicated concentrations of NaCl for 5 min.
Top: Data generated as described above in Figure 2 was compared to a separate microarray analysis in which the same cell line was exposed to 1uM palbociclib instead of veliparib. Cut-offs for comparison were a p value<0.05, and fold change of 1.5. Venn diagrams show the overlapping and non-overlapping genes of both down- (top) and up-regulated (bottom) genes in response to either treatment modality. Statistical significance was determine using the Chi-squared statistical test. Bottom: Genes found to be exclusively regulated by palbociclib, commonly regulated by palbociclib and veliparib, or exclusively regulated by veliparib were used for Gene Set Enrichment (GSEA) KEGG pathway analyses. Data indicate both FDR q value, where the darker colors indicates higher confidence (lower q). Numbers indicate q values. Blue arrow highlights the Homologous Recombination KEGG pathway
(D) Anti-FLAG immunoblot blot of DMSO and SRPIN340 pre-treated HEK293T cells transfected with in vitro transcribed CGG-nLuc-3xFLAG reporter RNA. β-Actin is used as a loading control. Error bars represent mean +/- SD (n=6 biological replicates). Statistical analysis was performed using Student's t test with Welch's correction. ∗p < 0.05
(G) Chs3 GFP GPD-CHS6 strain was sensitive to calcofluor to a similar extent as the WT strain. Plates were incubated at 30°C for 2-3 days. A representative drop test of three independent biological experiments is shown.
Immunoblotting analysis of the canonical and noncanonical branches of the NF-κB pathway and mediators of caspase-dependent cell death. Lysates of HCT116 VPS4B-/- cells were collected 66 h after transfection with siRNA (siCTRL#1 or different siVPS4A duplexes: #2, #4 or #5). Lysates of HCT116 VPS4B+/+ and non-transfected HCT116 VPS4B-/- cells were used to monitor the basal pathway activity. Representative blot from 10 experiments is shown. NT- non-transfected; p-RelA - phospho-RelA; p-IκBα - phospho-IκBα; cl - cleaved caspases or PARP-1. GAPDH or vinculin served as loading controls.
(F) Immunoblots of cell supernatants to analyze secreted MMP9, MMP10, MMP12 and MMP13 by mouse peritoneal macrophages stimulated with AG (1 μg/mL) for indicated times in absence or presence of inhibitor targeting ERK (PD98059) at the concentration of 10 μM; GADPH of cell lysates served as the loading control.
(H) Co-IP analysis of the interaction between two differently tagged TRIM16 variants.
(D) Representative images of cells after transfection with the double‐tagged mRFP‐GFP‐LC3. Yellow foci represent phagosomes (both GFP and mRFP signals visible), red foci represent lysosomes (only mRFP signal is insensitive to the acidic pH in lysosomes). Bar 10 μm.
(E) K437R‐Beclin 1 mutant abrogates the enhanced interaction of Beclin 1 with Vps34 during autophagy. HeLa cells expressing 3XFlag-Beclin 1 or 3XFlag-K437R‐Beclin 1 were treated with EBSS for 1 h, and harvested for immunoprecipitation.
Immunofluorescence (IF) staining against endogenous ∆Np63 in KP and KPL cell lines (blue: DAPI [staining of nuclei] and red/magenta: ∆Np63), Scale bar = 32µm.
A, B In vitro ubiquitination of Flag-TRAF2 (A) and Flag-TAK1 (B) in the presence of Ub-conjugated UbcH7 (catalyzed by PknG) and PknG or its mutants, with or without ATP as an energy source. The reaction products were analyzed by immunoblotting with antibodies against Flag-tagged TRAF2 and TAK1.
(D) HeLa cells were transfected with EGFP-ATG16L1 and mCherry-RAB11, and 5 min movies were recorded. Representative images from movies are shown.
(a-d) BCi-NS1.1 cells were stably infected with pLKO control or shEMP1.1. Data are representative of n=3 independent experiments. (b) Filter grown cells were fixed and stained for ZO-1 and DNA and analyzed by confocal imaging.
SUMO2 conjugates from HeLa/His10-SUMO2 cells subjected or not to formaldehyde or heat stress for 1 h were purified and immunoblotted with indicated antibodies.
Analysis of cell surface SCF revealed lower frequency of SCF+ ECs in HO-1-/- while no differences among CARs.
(C) Immunofluorescence confocal microscopy showing the subcellular distribution of TFEB-(1-30)-GFP in ARPE-19 cells coexpressing active RagB/D heterodimers (antibodies against GST were used to detect Rags). Bar, 4 µm.
(J, K) Representative track plots (J) generated from the open-field test from wt (N=16 at 9 weeks, N=22 at 11 weeks), R6/2 ACSF (N=26 at 9 weeks, N=23 at 11 weeks), R6/2 chol-low (N=11 at 9 weeks, N=7 at 11 weeks), R6/2 chol-medium (N=11 at 9 weeks, N=7 at 11 weeks), and R6/2 chol-high mice (N=18 at 9 weeks, N=16 at 11 weeks) and relative quantification (K) of the time spent in the center and in the periphery (%) of the arena. Data information: The data are from five independent trials and shown as scatterplot graphs with means±standard error. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).
(A) Northern blot experiments show the expression of RsaI during growth phase in the HG001 strain in MHB medium with or without the addition of 1,5 g/L of D-glucose. Glucose was added either at the beginning of growth (+ glucose, left panel) or after 3h of growth (right panel).
A Left panel: Immunoblotting of placensin content in HTR-8/SVneo, but not BeWo, JAR, and JEG3, cells. Right panel: Time-dependent secretion of placensin by HTR-8/SVneo, but not JAR, cells monitored using an ELISA (biological replicates, n=4-5).
(c) Percentage of huntingtinQ74-expressing cells with aggregates in CALM knockdown HeLa cells. Data depict one representative experiments performed in triplicate, out of three independent experiments and shown as mean ±s.d. (*P0.05; two-tailed t-test).
(F and G) Western blot analysis of free ATG12 and ATG5ATG12 complex levels with anti-HA antibody in HeLa cells transfected with HA-ATG12 and ATG5 and loaded with exogenous lipids. We quantified the ratio of ATG5ATG12 versus free ATG12 by direct infrared fluorescence detection on an Odyssey Infrared Imaging System to assess ATG5-ATG12 conjugation efficiency (mean ± SEM).
iTRAQ proteomics analysis (Appendix Fig. S2) resulted in 2,100 proteins consistently quantified at every point across the IDC. The heat map shows hierarchical clustering analysis of fold-changes (log2-transformed) in the levels of the proteins relative to an arbitrary average across the IDC. The 100 most up-regulated proteins at each time point were subjected to pathway enrichment analysis based on GO and MPM pathways annotated for P. falciparum 3D7. The significantly (P < 0.05) enriched pathways among the up-regulated proteins at each time point are summarized in tables for each time point
B) Q-PCR analysis of control and Apc5 mutant murine intestinal organoids in vitro, control and ApcMin mutant tumour samples in vivo, and control and Apcfl/fl knockout hyperplastic crypts in vivo. In all cases, TEAD1/2/4 mRNA expression was strongly elevated upon loss of Apc function, while TEAD3 mRNA expression was unaffected. (RNA was isolated from 6 individual tumours).
(A) 1.0 x 106 bone marrow mononuclear cells (BM-MNCs) from controls (Npm1F/FVav1Cre-), Npm1 cKO (Npm1F/FVav1Cre+), p53 KO (Npm1F/Fp53-/-Vav1Cre-) and DKO (Npm1F/Fp53-/-Vav1Cre+) mice were transplanted into irradiated recipient mice with 4.0 x 105 competitor (C57/BL6 Ly5.1) BM-MNCs.
A Morris water maze test of groups of 4−month−old WT or Tau mice without or with TFEB (+TFEB) treatment, showing longer latency (time to find hidden platform) in Tau mice compared to WT controls at any given day of training. TFEB has no effect on WT mice, but significantly reduced the latency in Tau mice. n = 14/group. ***P < 0.001 (2 way ANOVA).
(E) Immunoprecipitation analysis of HEK293T cells transfected with Flag-MAVS or its 4KR mutant together with Myc-NDP52. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results.
A. Mapping of enrichment across the positions targeted in dapF. The gray regions in the gene cartoon at the bottom highlights the windows containing targeted residues, with the enrichment map shown above for increasing AEC concentrations. Enrichments are color coded according the legend at the bottom. A histogram plot of enrichment is shown at the right
C. Yb Y23A and F129A mutants (green) impaired Yb body formation. Nuclei are shown in blue. Scale bar: 5 μm.
(b) EBV-ΔBRLF1-harboring HNE1 cells were transfected with control vector, BRLF1 or BRLF1 L578A in the absence or presence of BZLF1 for 48 h. The total RNAs were extracted and subjected to real-time PCR analysis.
A Immunoblotting of protein extracts from 1205Lu cells transfected with a siRNA control (CTRL) or two different sequences of siRNA (#1 and #2) directed against DDR1 or DDR2 alone or in combination prior being cultivated on MAF-derived ECM and treated or not with 5 µM BRAFi for 96 h, using antibodies against the indicated proteins. HSP60, loading control.
Fluorescent microscopy images shows Glutamate-induced (Glut) excitotoxic stress increases DPR levels in rat cortical neurons expressing the C9 DPR reporter and in iPS sMN derived from C9orf72 NRE patients. Glutamate-induced DPR levels are reduced by pretreating cells with the AMPA and NMDA specific antagonists NBQX and MK801, respectively. Relative fluorescent intensity quantification demonstrates there is a significant increase in DPR levels when primary cortical neurons and sMNs are challenged with the excitotoxic stressors: glutamate, AMPA, NMDA, and HC. Blocking either the AMPA or NMDA receptors reduces DPR levels in the presence of excitotoxic stressors. NES-mIFP or MAP2 were used to mark transfection positive rat cortical neurons or iPS sMNs, respectively, for quantification. DPR fluorescent intensity in iPS sMN was measured using DPR-specific antibodies. Changes in DPR levels are relative to non-stressed cells (CTRL) Cortical neuron: n = 6 with m > 40 cells per n. IPSC sMN: n = 4 with m > 25 cells per n.
(B) Gradient bars (green) signify the ranking of downregulated genes by fold-change, with genes ordered from most downregulated (gene #1) to least downregulated. Fold-change was determined by RNA-seq. Separate rankings were performed for each mutant (Foxn1 null or Aff4 cko). Pie charts indicate whether genes of a given rank in one mutant were downregulated in the other mutant. Percentages indicate the overlap in the mutants' downregulated gene populations.
(A) Schematic for EVs-based adjunctive immunotherapy and moxifloxacin-based chemotherapy in M.tb-infected mice.
Formaldehyde survival of C. elegans grown on L4440 control (CTRL) or smo-1 RNAi bacteria (mean±SEM; n=2 independent experiments).
(D) Time-lapse 3D-SIM imaging of sheath dynamics (TssB-sfGFP) in capsulated A. baylyi BD4 cells lacking TagX. Data information: Scale bars and time intervals are indicated.
Boxplots of 5´UTR (A), 3´UTR (B) lengths for mRNAs whose abundance decreased ('DOWN', dark green) or increased ('UP', light green) in PABP-depleted cells as compared to cells expressing PABP. Solid horizonal middle lines in box plots denote the median; lower and upper box limits correspond to first and third quartiles; whiskers extend from box limits to the most extreme values. Wilcoxon-Mann-Whitney tests (two-sided) were used to determine differences between upregulated mRNAs and downregulated mRNAs relative to each other and to the background (mRNAs whose abundance did not change in the absence of PABP relative to PABP-expressing cells).
16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium in presence of NCT502 (10 μM). Representative images showing the filling of the spheroid lumen with cell nuclei. The spheroids were stained for DNA (Hoechst 33342 in blue) and F-actin (Phalloidin in red). Scale bar: 50 μm. Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.
(E) Venn diagram and heatmap of metabolites whose expression increased in both HFD feeding and HF-R at ZT12 (n = 5, Cyber-T test p<0.05).
(D) Percentages of NK cells or neutrophils in the lungs of LV::S- or sham-vaccinated and SARS-CoV-2-challenged B6.K18-hACE2IP-THV transgenic mice at 3 dpi (n = 6/group). Percentages were calculated versus total lung live CD45+ cells. Data information: Statistical significance was evaluated by Mann-Whitney test (* = p<0.05, ** = p<0.01).
(C) FLAG-tagged TFEB-WT or TFEB deletion mutants were immunoprecipitated with the anti-FLAG antibody and analyzed by immunoblotting with antibodies against FLAG, 14-3-3 binding motif, or 14-3-3.
(L) Quantification of Psat1 mRNA expression in sgNf1.3 LUAD tumor sections analyzed by tumor grade. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.
(B-E) G1-arrested cell cultures (αf) were released in YEPD supplemented with CPT (50 μM). Sample collected at the indicated time points wer stained with propidium iodide to analyze the kinetics of nuclear division (C, E)
(D) Upper panel: a representative picture of the urogenital tract of a WT male and an ARLmon/Y mouse. Lower panel: a representative picture of the testis of a WT male and an ARLmon/Y mouse. Scale bar = 1 cm.
D-E Survival (D) and body-weight changes were monitored daily after challenge. (The values represent mean ± sd in each group) (E). Data information: For statistical analysis, two-way ANOVA analysis were used Log-rank test was performed *p<0.5, **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant
phase.Young and aged mice were infected with PR8. A. Bulk RNAseq was performed on young or aged lungs at 60 d.p.i., data is displayed as a heatmap (purple = relatively low expression, orange = relatively high expression) of 658 differentially expressed genes (549 up-regulated in aged relative to young, 109 up-regulated in young relative to aged) with p < 0.001.
CRAC/ORAI channel functions down stream of TCR activation, and plays important roles for iNKT cell differentiation and activation. NKT2 cells show higher and prolong Ca2+ signals than NKT1 cells after CRAC/ORAI activation. We propose this is regulated by two mechanisms: Mitochondria in NKT2 cells sequester Ca2+ at the vicinity of the channel to reduce Ca2+ dependent inactivation of CRAC/ORAI and help maintain the channel activity for Ca2+ entry; Lower PMCA expression and slower Ca2+ exportation in NKT2 further contribute to higher and sustained Ca2+ signals in NKT2 cells. Ca2+ in mitochondria is an important co-factor for key metabolic enzymes (PDH: pyruvate dehydrogenase; IDH: isocitrate dehydrogenase; OGDH: 2-oxoglutarate dehydrogenase). Consistently, NKT2 cells show more robust respiration and higher cellular ATP. Note: ER is an important regulator of Ca2+ homeostasis, and is not shown for purpose of simplicity. Figure was generated using BioRender.
(F) HEK 293 and HeLa cells were transfected with the indicated siRNAs. After 72 h, the newly transcribed RNA in cells were metabolically labeled and isolated by Click-iT, and then subjected to qRT-PCR analysis. The level of newly transcribed raptor RNA was quantified. The data from three independent experiments are presented as means ± S.E.M., ns, not significantly different, one-way ANOVA.
A, At 2 days post vaccination with NoVΔB2, UV- inactivated NoVΔB2 (UV- ΔB2) or culture medium DMEM, BALB/c mice were challenged with WT NoV. The survival data were analyzed by the log rank test, **** indicates p<0.0001, n=7 per group.
A) Schematic representation of kinesin constructs used in the inducible trafficking assays. Kinesin constructs contain a motor domain and a coil (MDC).
a. Uniform Manifold Approximation and Projection (UMAP) of sequenced single cells from inflammatory pseudotumor (IPT) CSF (n=1 sample, n=4,324 single cells, left), control CSF (n=6 samples, n=15,467 single cells, middle) and multiple sclerosis (MS) patients-derived CSF (n=6, n=18,412 cells, right). Indicated immune cell subsets as identified by Seurat v4 reference (ref) mapping.
H. Protein samples (20 μg) from Jurkat cells treated as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Where indicated, lysosomal proteases inhibitors E64D and PepA were added. WB are representative of at least three independent experiments.
D Scheme of sampling references for analysis of the phosphorylation states of SRSF9 in (F). The CLK1 inhibitor KH-CB19 (10 μM) or DMSO as a control was administrated during the recovery period. The numbers correspond to the lane numbers in (F).
D, CSF-1 protein (pg/mg total protein) measured by ELISA in the sera of naïve mice, mice bearing mock-irradiated tumours, and mice bearing irradiated tumours analysed at time points as indicated (n=4/group). Data are presented as mean ± SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison.
(A) COUP-TFII mRNA levels in human kidney tissues of control (n=8) and CKD (n=53) subjects from GSE66494. ***p<0.001 by t-test.
t-SNE maps highlighting the expression of marker genes for each of the cell types. Transcript counts are given in a Log2 scale.
D Lack of Irc5 decreases formation of recombination intermediates during TS. Wild type (W303-1a), irc5-Δ1 (IL012), sgs1Δ (MC012) and irc5-Δ1 sgs1Δ (TB051) cells were synchronized in G1 with alpha-factor and released in media containing 0.03% MMS. The amount of replication intermediates at ARS305 was analyzed by 2D gel. Schematic representation of major 2D gel signals and DNA fragment analyzed are presented. Measurement of DNA content by FACS and X-molecule quantification are displayed (n=2)
Kaplan Meier survival curves illustrate the overall (OS) and progression-free (PFS) survival of patients with high or low GPRC5A. In primary tumors, mean OS for GPRC5Ahigh was 39 months vs 56 months for GPRC5Alow (C). In patients with metastatic tumors, mean OS for GPRC5Ahigh was 37 months vs 60 months for GPRC5Alow. Mean PFS in metastatic tumors was 20 months for GPRC5Ahigh vs 35 months for GPRC5Alow (D). No significant difference in mean PFS in primary tumor.
(B Proportions and numbers of (B- Kb-SIINFEKL+ CD8+ T cells were enumerated, or (E- IFN-γ-secreting CD8+ T cells following restimulation ex vivo with SIINFEKL peptide were quantified. Flow cytometry plots show representative percentages of CD8+ T cells co-stained with CD11a and (B) Kb-SIINFEKL or (E) IFN-γ.
Knockdown of USP36 reduces SUMOylation in cells. H1299 (C) cells infected with scrambled (scr) or USP36 shRNA encoding lentiviruses were assayed by IB.
(G) Representative H&E images of lung metastasis in mice used in (F). Scale bars, 100μm. (H, Number of metastatic nodules per lung (H) in mice used in (F). Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. **P < 0.01.
B) NMR-1D targeted metabolic profiling in age-1; gas-1 mutants (normalized to gas-1). Dashed lines indicate fold change cutoff (|log2(fold change)| <1.23). Red bars: not significant, Light blue bars: FDR<10%, |log2(fold change)| <1.23, Dark blue bars: FDR <10%, |log2(fold change)| >1.23. Error bar: mean + SEM, n=7.
A. Structure of 5-azacytidine and formation of a covalent RNA methyltransferase adduct.
(A) Experimental design of transcriptome analysis. Primary preadipocytes and differentiated adipocytes ((w/o) rosiglitazone), isolated from inguinal white adipose tissue (iWAT) of five inbred mouse strains of varying browning propensity (evaluated based on Ucp1 transcript levels in RPKM (reads per kilobase per million mapped reads)). Mean values ± SD, n=3 (biological replicates) (Data ref: Array Express E-MTAB-8344, 2019) (Li et al., 2019) were subjected to RNA sequencing to identify regulated long noncoding (lnc) RNAs.
Weight of tibialis anterior (D), quadricep (E) and soleus (F) muscle normalized . to initial body weight (n=15).
G p(S22)-Lamin A levels were analyzed by HTM in si-RNA transfected HeLa cells (n = 3, 1000 cells examined per experiment and condition). Data information statistical significance was determined by Wilcoxon rank-sum test; *, p < 0.05; ***, p < 0.001.
(C) Six week-old female BALB/c cohorts of mice were pre-treated with single and combination BI6727 and AZD6244 inhibitors as indicated in panel A for 4 days, followed by 4T1.2 cells were injected in the 4th inguinal mammary fat pad. Survival of the mice was then monitored over the indicated period of time and the significant of data was analyzed by log-rank test (P=0.0005); n=6 mice/group.