EMBO/sd-character-level-ner huggingface dataset:
(c) Intravascular endothelial cell interactions and transmigration of neutrophils (Ly-6G+ CX3CR-1-; red) and cMOs (Ly-6G- CX3CR-1low; green) to the perivascular tissue as assessed 6 h after intrascrotal stimulation with recombinant murine uPA, PAI-1, or uPA-PAI-1 in postcapillary venules of the cremaster of CX3CR-1GFP/+ mice by multi-channel in vivo microscopy. Representative still images (scale bar: 50: µm) and quantitative data are shown (mean±SEM for n=4 mice per group; #p<0.05 vs. saline; *p<0.05 vs. uPA; §p<0.05 vs. PAI-1; One-way ANOVA).
A. The expression of Prom1, radixin, AKAP7, AKAP8, AKAP8I, AKAP9, AKAP10, AKAP12 or AKAP13 was silenced in WT hepatocytes using siRNAs. Hepatocytes were then serum-starved for 18 hours and stimulated with glucagon (10 nM) for 10 minutes. The phosphorylation of PKA substrates was determined by immunoblotting.
(C) Active BAX is present in a subset of LC3‐positive cells. Sections from P7 and P14 MCL‐1Δ/Δ brains were stained for LC3 (Green) and active BAX (6A7; Red). Confocal images were taken using × 63 objective. Arrows point to BAX‐positive, LC3‐negative cells. Representative images are shown. Scale bars=50 μm.
A. Screening of 707 cell lines from 47 tumor types for sensitivity against IBL-302. Tumor forms with n≥5 cell lines screened are displayed. Comprehensive screening data are available in Appendix Supplementary Materials.
D PCDH7 and PCDH1 enriched on endoplasmic reticulum fraction. Whole‐cell lysate, cytoplasmic, endoplasmic, and mitochondrial fractions were blotted against an endoplasmic reticulum protein, KDEL, a cytoplasmic protein, actin, a mitochondrial protein Cox‐2 and PCDH7, PCDH1.
C Mitochondrial translation products in osteosarcoma 143B control and Rho0 cell lines as described in A. Scale bars, 25 µm.
J Heatmap showing expression levels (Z-score) of the 12 cognition-related DEGs with node size ≥ 6 in the PPI network.
(B) 3D rendering of injected brains generated based on post-surgery MRI scans showing the injection sites and the brain volume containing the vector suspension in the different treatments groups. EC, External capsule; Thal, thalamus; CR, corona radiata; ant, anterior; post, posterior.
Functional inference of the predicted SEPs (N=11,238) as determined using BlastP against NCBI-annotated SEPs having an associated function (N=5,175). The color code associated with each category is the same as in panel A.
H, CNE1-vector and CNE1-INSL5 cells were grown in the presence of the fluorescent glucose analog 2-NBDG (Invitrogen) for 15min, and glucose uptake was then quantified using flow cytometry (FACS) (left panel). Statistical analysis of the glucose uptake was showed in the right panel.
e-g) NPC cells either with or without exogenously expressing ARID1A were examined by anchorage-independent colony formation assay (e), migration assay (f) and WB assay with the indicated antibodies (g).
Figure 3: Hippocampal FMN2 is essential for extinction learning and cognitive flexibility. C. Similarly, hippocampal FMN2 protein levels were decreased in mice injected with Fmn2 siRNA when compared to the scramble control group (*P < 0.05).Error bars indicate SEM.
H BMMΦ of wt or MyD88-/- mice were transduced with retrovirus expressing MAL (pMIGR1 vector) and stimulated with LP for 6 days. MGC formation was quantified as in (B). Depicted are means ± SEM from 3 independent experiments.
MDA-MB-436 cells were treated with TNFα and indicated DR5 agonists for indicated times. Lysates were analyzed for PD-L1, CSN5, phosphorylated p65 and PARP. GAPDH is loading control in G.
(K) Tumor regression efficacy of MD5-1 (50μg), GSK269962A (<2mg/kg) and MD5-1+ GSK269962A (50μg + 2mg/kg) 4T1 tumors (n=4-7). 6-8 weeks old BALB/c mice bearing 4T1 tumors (~100mm3) were intraperitoneally (i.p.) injected with 50μg of indicated antibody every third day (n=4-6). GSK269962A (ROCK1i) was injected directly into the tumors. Tumor volumes were quantified at indicated days by caliper measurements.
(a) Immunoblot analysis of the WIPI4 protein (apparent mobility at ∼35 kDa) in LCLs (top). Truncated forms were not detected (middle). HSP90 was used as a loading control (bottom). An asterisk indicates nonspecific immunoreactive bands.
Scattering difference (ΔI(q) = I(q)PICK1+Tat-P4-(C5) 2 - I(q)PICK1) between PICK1 in presence and absence of Tat-P4-(C5)2 at indicated concentration shows a clear difference in scattering below 0.1 Å-1.
D. Chromosome segregation in gcn5-47 or cut3-477 mutant cells. Cells growing at 32°C were fixed and processed for immunofluorescence microscopy against Mis6-HA and Cdc11-GFP to reveal centromeres (Cen) and Spindle Pole Bodies (SPB), respectively. DNA was stained with 4',6-diamidino-2-phenylindole (DAPI). Left panel: single mutant cells cut3-477 or gcn5-47 exhibiting chromatin bridges (b) or trailing chromatin (t). Bar: 5 µm. Right panel: frequencies of chromatin bridges and trailing chromatin as a function of spindle length (SPB-SPB distance, n> 40 for each category).
E Immunofluorescence staining for several naïve specific markers in 2a2iL-RH6. Scale bar: 100 µm.
Effect of PEBP1 on the Raf1/ERK/IκB and IKK/IκB/NF-κB signaling pathways. The levels of indicated proteins in total protein lysates (C) were analysed by Western blot in C11-PEBP1-KO cells and mock C11 cells.
(E) Model of Cand2-dependent stabilization of Grk5 by Cul1 neddylation inhibition in mTOR-dependent manner.
A, B Real-time PCR (qRT-PCR) analyses of WTAP mRNA expression in PBMCs (A) or BMDMs (B) upon challenge with VSV-eGFP, poly(I:C), HSV-1 or HSV60 at indicated time points. n = 3 independent biological replicates, and error bars represent standard deviations.
b) PIN1 dimer stability induced by NPA after expression in HEK cells. Relative distribution between monomer and dimer after NPA treatment is given relative to distribution of untreated samples after solubilization with 50% (v/v) CL27. Each measurement given (three for each concentration) represents the mean of three gel lanes for wild type (circles) or triple S2523, S253E, S261E phosphomimetic sequences (crosses) (example images are given in the figure inset).
(A) PJ69‐4A cells were co‐transformed with each pGBD and pGAD plasmid as indicated. Transformants were selected on Trp− Leu− plates, and then two‐hybrid interaction (+ or −) was assessed for growth on Ade− Trp− Leu− plates.
High and constitutive expression of CXCR4 in the membrane of SW1417 CRC cells as measured by flow cytometry.
(F) Schematic of immunoprecipitation assay shown in G and H. Cultured MDA-MB-436 cells were treated with KMTR2 (IgG1-Fc) or IgG1 control ± ROCK1i (GSK269962) for 6 hrs. After 6 hrs, 500nM anti-PD-L1 avelumab (IgG4-Fc) was added to the media for additional 1 hr. Cellular lysates were pulled down with anti-hu-IgG4 specific beads.
D-E. Unlike deletion of Atg11, deletion of other known selective Atgs required for the CVT pathway (Atg19), mitophagy (Atg32) and pexophagy (Atg36), does not result in increase of GFP-Snc1-PEM protein level (D), intra-cellular accumulation of GFP-Snc1-PEM, (E),
Representative apoptosis FACS plots of cell lines treated with DMSO or the DOT1L inhibitor SGC-0946 for two days. Annexin V staining and DAPI staining were performed on unpermeabilized cells to distinguish live (Annexin V low; DAPI low), apoptotic (Annexin V high; DAPI low) and dead (Annexin V high; DAPI high) cells. Quantification of several independent FACS experiments, including the experiment shown in panel (A). Mean with individual data points of 2-4 replicates each of two independent lines per genotype. p<0.001 (***) by two-way ANOVA, comparison to corresponding DMSO control.
E) The p53-mediated transcriptional activity is measured by a luciferase reporter under the control of a p53-response element promoter, which was transfected into HEK293T cells 72-96 h after the initiation of the UBE4B and LSD1 single or double knockdowns (n = 6).
(e, f) Transcriptional change (obtained by Affymetrix array) by spermidine treatment of all autophagy-related genes categorized into GO terms of macroautophagy (GO:0016236), microautophagy (GO:0016237) or autophagy in general (GO:0006914) at day 3 (e) and day 10 (f) of chronological ageing yeast. Data represent means of two independent arrays from independent biological samples.
D. Normalized expression of selected genes (Atoh1, Neurod1) were mapped onto the UMAP dimension.
D Quantification of the experiment presented in (C). Both SKMEL28 and WM3248 cells were analyzed.
E, F BMMΦ were stimulated with LP and DNA-methylation of CpG sites -192 and -196 (D) or in the promoter/enhancer region and control site -845 (E) were analyzed by pyrosequencing at indicated timepoints. Depicted are means ± SEM of 4 independent experiments. *p < 0.05, ***p < 0.001, ****p < 0.0001 (two-tailed unpaired t-test).
Two-tier analysis strategy and design for the confirmation of processing areas in Alu RNAs in human brain combining different sequencing approaches and patient cohorts (CBB cohort , n=24; MAP cohort , n=241).
Proliferation curves of wt and 2 independent smc5 clones grown at 39.5ºC, with the estimated doubling time on the right. Data represents means ± SD of 4 experiments. Asterisks indicate p value ≤ 0.05, as derived from unpaired t-test type 3, two-sample unequal variance, carried out to check statistical significance between smc5 clones and wt. smc5 clone 1 versus wt, p=0.016, smc5 clone 2 versus wt, p=0.04.
IF staining of week 6 COs for astrocytic markers in the surrounding of VZs (WT: n=6 from 2 batches, KO: n=6 from 2 batches). Scale = 100µm (left) , 50µm (right).   Staining for the DNA damage markers γH2AX and 53BP1 in the nuclei of cells in the VZ of week 6 COs at physiological conditions, together with the pan-radial glia marker SOX2 (WT: n=8 from 3 individual batches, KO: n=12 from 3 individual batches, W-AAV: n=4 from 1 batch). Scale = 50µm (left), 25µm (right).   Quantification of γH2AX (top) and 53BP1 foci (bottom) in the nuclei of cells composing the innermost layer of the VZ, normalized to the total number of nuclei in this layer. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WT: n=8 organoids from 3 batches, KO: n=12 organoids from 3 batches, W-AAV: n=4 from 1 batch).  Data information: n.s (non-significant), *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001.
B) Apoptosis was assessed using a TUNEL/apoptosis detection kit, according to the manufacturer's protocol. Cells were then examined under a laser-scanning confocal microscope (LSM 510; Zeiss). Percentages of TUNEL-positive, PI-positive, and TUNEL-/PI-double-positive cells were calculated. Data are representative of three separate experiments.
(E and F) THP-1 cells transfected with mock, Eis-WT, or Eis-ΔAT constructs were pretreated with SP600125 (SP; 20 µM) or SB203580 (SB; 5 µM) for 45 min before infection with Mtb-Δeis for 30 min (E) or 18 h (F). E, Intracellular superoxide production was analyzed by flow cytometric analysis. F, ELISA analysis for TNF-α and IL-6 levels. Data are presented as the mean±SD of three experiments. **p<0.01, ***p<0.001, vs. SC (A, C, E, and F); mock control (B and D). UI, uninfected; SC, solvent control (0.1% DMSO).
ELISA of human soluble and insoluble brain (C) Aβ1-42 and (D) Aβ1-40 levels in 8-month old Tg-APPmice injected IP daily for 3 weeks with 5 mg/kg Bosutinib (N = 9).
The nanoconjugate contains a fusion protein [T22-GFP-H6 -composed of the peptide T22 as a CXCR4 ligand, a green fluorescent protein and a Histidine tail - bound to the payload drug
A- Histogram showing the distribution of a number of putative eRNAs paired to genes in RPB1-52R and RPB1-25R cells upon 15 and 30 min TPA treatment (200 nM) as well as in the DMSO controls.
Sample recordings from 7-week-old hESC-derived Cerebral Organoids (COs). WWOX's coding sequence was re-introduced into week 6 WWOX-KO COs using lentiviral transduction (lenti-WWOX) (F) Immunofluorescent staining showing WWOX expression in different populations in WWOX-KO organoids following infection with lentivirus. NT= non-treated. Scale = 50µm .
A. The expression of PEBP1 was analysed by Western blot in TZM-bl cells infected with mock, Lv-PCDH-empty or Lv-PCDH-PEBP1 plasmids.
G DNA content of synchronized cells described under (F) measured by flow cytometry. Green overlays indicate the fraction of E2F8-EGFP positive cells within each doxycycline-treated cell line.
E, OTUB1 induces membrane RAS re-localization. Confocal imaging of HeLa cells expressing the indicated constructs. For each sample, z-stacks obtained by scanning the sample from the apical to the basal layer. Step-size, 2 micron.
(A) Photographs of Fus+/+ and FusΔNLS/ΔNLS pups immediately after birth (P0 animals).(B-C) FusΔNLS/ΔNLS mice showed significantly reduced body weight (B) and length (C). Weight and length values normalized to wild type (Fus+/+) are presented. N=11 Fus+/+, N=26 FusΔNLS/+ and N=14 FusΔNLS/ΔNLS; (*) p<0.05 (**) p<0.01 vs Fus+/+, (#) p<0.05 vs Fus∆NLS/+; One way ANOVA followed by Tukey post hoc test.
B mRNA expression of adipogenesis-related factors of SVF cells isolated from iWAT of male mice. D mRNA expression of PPARγ, C/EBPα and FABP4 expression of 3T3-L1 cells treated with curcumin.
A. Representative images of dressed bodies of the adult male and CtM Hanwoo (n = 10 in each group).
L Immunoblot analysis of TAP-tagged Ctk1 proteins in extracts prepared from the strains Anti-PGK blotting shows similar loading.
A C57BL/6N mice pups at P3 received 1 μg of mouse Sema3C-Fc (right eye) or control IgG-Fc (left eye). The retinal vasculature was examined at P5 by whole-mount CD31immunofluorescence staining. Representative images show the total retinal vasculature and sprouting tips at the vascular front (iii and iv, arrows) and the enlarged intercapillary space (ii and iv, asterisks). Scale bar, 100 μm and 20 μm (iii and iv).B,C (B) Retinal vessel outgrowth measured as the total area from the central optic disk to the vascular front normalized to control IgG-Fc treatment, ***P = 0.0009. (C) Vessel density was quantified by CD31-stained vessels and normalized to the total vessel area, ***P = 0.0002. (Mean ± s.e.m; n = 23 retinae in each group from four independent experiments; unpaired Student's t-test.)D,E (D) Number of sprouting tips per retina, *P < 0.0001. (E) Branch points in the capillary plexus represented as the ratio to the control IgG-treated retinae, ***P < 0.0001. (Mean ± s.e.m; n = 18 retinae in each group from three independent experiments; unpaired Student's t-test.)
Expression of CD31 (F, G) at day 5 and 7 by immunostaining on tissue sections. The dashed lines indicate the border of the wound bed and epidermis or eschar. e, epidermis; es, eschar; wb, wound bed. Quantification data are expressed as mean ± SEM, n = 3 wounds per group
F Genomic sequencing confirming the origin of pups from Stx2repro34 spermatocytes. Arrows indicate the expected point mutation of Stx2repro34. Y indicates a hybrid status with T and C bases.
(a) Flowthrough from nickel-agarose column, separated by SDS-PAGE and stained with Coomassie blue.
E EMSA titration experiment, with increasing concentrations of either Myc bHLH-LZ variant (WT or HEA) in presence of a fixed amount of the Max bHLH-LZ (2 μM) and of the fluorescently-labeled IRD-CACGTG probe (500 nM). The plot shows the mean and standard deviation of the quantification of the shifted Myc/Max bands as a function of the Myc concentration (the experiment was performed in triplicate). % Bound probe: fraction of the IRD-CACGTG probe associated with the Myc/Max complex. HEA/WT Kd ratio: Kd value for MycHEA/Max/CACGTG divided by the Kd value for MycWT/Max/CACGTG. The apparent Kd values were 3.78 μM for MycWT/Max and 5.57 μM for MycHEA/Max. Data information: Note that the differences in apparent Kd values between panels E and F-H can be explained by the different thresholds required to detect quantifiable complexes in the protein titration experiment (E), as opposed to a loss of signal intensity from an already detectable complex in the presence of unlabeled competitor (F-H): as such, competition experiments provide a better approximation of actual binding affinities. (F-H): The plots show the mean and standard deviation. The experiments were performed in triplicate. Unpaired Student t-test was used to compare IC50 values and expressed as p-values.
(B) Simulations from model M4 to show the percentage of variance attributed to time as a function of the number of cells in each sample.
(B) Schematic representation of HMCES domain organization and deletion mutants used in this study.
A Structural view of UBE2SHTH (green) interacting with APC2 for diUb synthesis (Brown et al., 2016). Residues of interest at the UBE2SHTH-APC2 interface are indicated as red spheres.
A. P5 or P10 mouse cerebella were electroporated with H2B-GFP plasmids and immunostained with anti-Atoh1 (magenta) the following day (P6 or P11). Nuclei of electroporated cells were detected by GFP (green). Arrows indicate the cells of interest where GFP signals are co-localized with ATOH1. The proportions of ATOH1-positive cells(AT+GCPs, A'). Bars and error bars are representing as Mean ± SEM. Student T-test was performed between P6 mice electroporated at P5 (P5-6) and P11 mice electroporated at P10 (P10-11). ***p < 0.001. Number of cells: 108, 72 and 104 cells from N=3 mice (P5-6), 182, 45 and 61 cells from N=3 mice (P10-11).
A-H LM2-4luctumor cells were implanted intravenously into SCIDmice, generating predominantly lung-specific metastases in one experiment (n = 5-6; A-D) and predominantly lymphatic-specific metastases in another experiment (n = 12; E-H). (C and G) Photographs of typical metastases in the lungs and caudal lymphatics, respectively. Kaplan-Meier survival analysis showed that Vasculotide (VT) significantly delayed mortality due to lungmetastases (A) but not lymphaticmetastases (E). Quantitative analysis of in vivo bioluminescent images taken at 24DPI (B) showed that concurrent VT treatment effectively reversed the sunitinib (SU)-induced acceleration of lungmetastases (D). With regard to lymphaticmetastases (F, H), SU treatment did not accelerate their progression. Geometric means ± 95%CI are depicted in (D and H); P values were derived by one-way ANOVA (D) and Kruskal-Wallis test (H). The same trends were reproduced in confirmatory experiments summarized in Supplementary Fig S5.
B Quantitation of sEPSC frequency mPFC from pyramidal neurons in mPFC. C Quantitation of sEPSC amplitude of mPFC pyramidal neurons. Mock, n=14 neurons from 4 mice; ZIKV, n= 15 neurons from 5 mice in B and C. Data information: Data were collected from PL and IL regions of mPFC, and were presented as mean ± SEM. *p<0.05, **p<0.01. Two-tailed unpaired t-test unless described otherwise.
Hcp release assay. FLAG-tagged Hcp (HcpFL) release was assessed by separating cells (C) and cell-free culture supernatant (S) fractions from 109 cells of the indicated strains. Proteins were separated by 12.5%-acrylamide SDS-PAGE and TolB and HcpFL were immunodetected using anti-TolB (upper panel) and anti-FLAG (lower panel) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in triplicate and a representative result is shown.
E, OTUB1 levels correlates with increased levels of phosphorylated-ERK1/2 in wt KRAS lung adenocarcinomas. Association between OTUB1 and pERK1/2 expression levels was assessed in the same tumor samples by IHC.
A, UV-Vis spectra of the photosensory domain of A. nidulans FphA (PGP) after co-expression together with bacterial HO (BphO) or A. alternata HoxA, HoxB or HoxA plus HoxB overnight at 20°C in the dark. PGP was purified via the Strep-tag system and concentrated before recording the spectra. Inset, left lane: Molecular mass markers stained with Coomassie Blue. Middle lanes: Zinc-induced red fluorescence (labeled zinc, Zn2+) of equal amounts of PGP expressed in E. coli along with bacterial BphO (O) or A. alternata HoxA plus HoxB (A+B). right lanes: Coomassie Blue staining (C.B.) of the polyacrylamide gel used for the Zn2+ fluorescence.
(E) Scatter plot representation of cardiolipin lipidomics. The average (n = 4, biological replicates) normalized peak intensity of each of the 11 detected cardiolipin species, log10 pareto-scaled is visualized. The horizontal lines represent the mean peak intesity of all 11 caridolipin species in each cell line. ­­(F) Scatter plot representation of a subpopulation of the phosphoethanolamine (PE) lipidomics. The average (n = 4, biological replicates) normalized peak intensity of each of the 20 phosphoethanolamine (PE) species (decreased in PEX3-/-), log10 pareto-scaled, is visualized. The horizontal lines represent the mean peak intesity of all 20 PE species in each cell line. (G) Scatter plot representation of ether-phospholipid lipidomics. The average (n = 4, biological replicates) normalized peak intensity of each of the 21 detected ether-phospholipid (plasmalogen) species, log10 pareto-scaled, is visualized. The horizontal lines represent the mean peak intesity of all 21 plasmalogen species in each cell line.
Normal, si-control-transfected, or si-PTEN-transfected SGC7901 cells were treated with CDDP (1 µg/ml) and M2-si-control-exo or M2-si-CRNDE-exo. Si-control, the negative control of si-PTEN. The cell apoptosis in SGC7901 cells. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. one-way ANOVA. **P<0.01.
D: Induction of the Uba2~Ubc9 disulfide is a conserved mechanism. HeLa, MCF7, U2OS or RPE cells were treated with increasing concentration of H2O2 for 15 min, lysed in non reducing buffer supplemented with 20 mM NEM, and analyzed by immunoblotting with Uba2 antibodies.
(A) YTS KIR2DL1 cells were mock-transfected or transfected with N.S siRNA or Cbl-b siRNA, c-Cbl siRNA and SHP-1 siRNA, using Amaxa electroporation. After 48 hours, cells were lysed, and subjected to western blot with anti-Cbl-b, anti-c-Cbl or anti-SHP-1 antibodies. Analysis by ImageJ densitometry is summarized in the graph below. Data are means ± SEM of three independent experiments.(n=3) P values were calculated by One-sample t-test, and are indicated by asterisks *P < 0.05.
Dynamic ranges of corresponding Ca2+-influx (gray) and glutamate release (color-coded) with V1/2 depicted. Note that the synapses from cluster 1 (purple), employing Ca2+-nanodomain-like control of release (m = 1.3), exhibit wider dynamic range than the other synapses.
C The volcano plot shows a differential expression between 2a2iL- versus primed hPSCs lines. X-axis shows the log2 fold-change, and the y-axis shows the negative log10 scaled adjusted P-value (Benjamini-Hochberg). Red and blue points represent up-regulated genes in 2a2iL- and primed cells, respectively (absolute log-2 fold-change >1, adjusted P-value <0.05). Gray points indicate non-significant genes between these states.
(F) SPR experiment showing recombinant HSPA8 protein interaction with recombinant hAhR on a SPR chip. Data shown are from three biological replicates.
C, D. Scatter plots showing individual recordings of pairs of uninfected and infected neurons in slices expressing shRNA against GSK3α at −60 mV (AMPAR-mediated EPSCs; C) or at +40 mV (NMDAR-mediated EPSCs; D). Error bars represent SEM. Statistical significance was calculated according to the Wilcoxon signed-rank test. E. Quantification of individual (open circles) and average (grey bars) AMPA/NMDA ratios of neurons from uninfected and GSK3α shRNA lentivirus infected slices, where AMPAR-mediated current is taken as the peak response at −60 mV, and NMDAR-mediated current is measured at +40 mV at a latency of 60 ms following stimulation. Error bars represent SEM. Statistical significance was calculated according to the Mann-Whitney U test.
(A) CD86 and CD40 expression was quantified on WT (blue dots) and Irf3‑/‑Irf7‑/‑ (orange dots) DCs in non‑draining (ndLN) and draining (dLN) popliteal lymph nodes of WT and Irf3‑/‑Irf7‑/‑ mice injected with poly(I:C) or PBS. Data are pooled data from 2 independent experiments.