EMBO/sd-character-level-ner huggingface dataset:
Figure 4. Mmi1 drives Rrp6-dependent heterochromatin gene silencing at pericentromeric regions. (C) RT-PCRs monitoring the accumulation of nam7 lncRNAs and nam7-L read-through transcripts, in the same cells and conditions as in (B). Red arrow heads point to the expected PCR products while the other bands correspond to non-specific PCR products. From the scheme and the agarose gels: black arrows, primers used for the three different reverse transcriptions; black lines and red numbers, expected PCR products of the three different RT-PCRs; M, DNA ladder Markers; tub1, tubulin control.
Interaction between HR1 and HR2 of SARS-CoV-2 and SARS-CoV.a-d. The 6-HB structure of SARS-CoV-2 and SARS-CoV is shown in cartoon representation. The HR1 domain is shown in green for SARS-CoV-2 and forest for SARS-CoV, while the HR2 domain is shown in cyan for SARS-CoV-2 and orange for SARS-CoV. Important residues are shown in sticks and labeled.
(S) A construct to express wild type YY1(WT), or two mutants lacking DNA binding activity, YY1(1-394) and YY1(S365D) were transfected into YY1iKO cells; the expression of mitochondrial gene was measured. Error bars represent s.d.'s of the mean. Student's t-test (two-sided): N.S = non-significant, *P<0.05, **P<0.01, ***P<0.001.
(B) Durations of dox addition at different time points during chemical induction (upper) and assessment of AP+ colonies (bottom). Error bars, mean ± S.D.; n = 3 biological replicates per condition; **P < 0.01, ***P < 0.001 as compared with condition-1, by two-tailed Student's  t-test. n.s., not significant.
(I) Quantification of the lipid droplet size in (H). Data were analyzed by one-way ANOVA with a post hoc Turkey's multiple-comparison test. Each point represents data from one fat body. At least 30 cells were analyzed from each fat body. Error bars represent ± SEM. ***: P < 0.001; NS: non-significant.
5C or mES medium was used during reprogramming. On day 15, Oct4GFP+ colonies were counted (A).
A, B Abolishments of force-induced binding residues by 3A mutations (MICA 3A, n = 526) and TAT mutations (MICA TAT, n= 580) impairs NKG2D's catch bond with MICA WT (n = 1505) (A) and IFN-γ release (B). Data information: Bond lifetimes in (A) of MICA WT and mutants with NKG2D came from at least 21 pairs of cells and beads of at least three repeated experiments. IFN-γ release of NK cells in (B) was one representative experiment of three total independent experiments. Data points represent mean values. Error bars represent mean ± SEM for biological triplicate experiments. **P < 0.01, ***P < 0.001, **** P <0.0001 (two-tailed unpaired t-test).
First derivative of tryptophan fluorescence upon thermal unfolding of recombinant OTULINcatWT and OTULINcatG281R (1.0 mg/mL) measured by nano differential scanning fluorimetry (nanoDSF). Apparent melting temperatures (Tm) are indicated (dashed lines). Data are representative to two independent experiments.­
Immunoprecipitation of SPATC1L in the testis using an anti-SPATC1L antibody. IgG was used as a control. In, input; IgG, normal rabbit serum.
(E) Time course of glycogen levels in Dmef2-Gal4 carcasses (muscle+body wall). Animals were fed for 24 h in high-nutrient food, then starved on low-nutrient food +/− 2.5 mg/ml CQ for 0-24 h. Starvation caused reduction of glycogen levels in both untreated and CQ-treated larvae over time. However, after 6 h of starvation, CQ treatment significantly increased glycogen levels compared to controls. SEM is indicated for n = 5-8 samples (*p<.05, **p<.01).
(c) The relative affinity of the GST-fusion proteins for phosphoinositides was quantified by densitometric analysis of the GST immunoblots in b from five independent experiments (n = 5). Error bars represent standard deviation (s.d.). A.U., arbitrary units.
(E) Enrichment of astrocyte specific genes (from scRNA-seq) and genes bound by SOX3 and SOX9 in GPCs in the four different astrocyte gene clusters (A1-A4), shown in Fig. 3C. Three-star significance equals p-value e-13 (prop.test, R) based on the size of the gene groups (A1=740, A2=1019, A3=882, A4=1236) (F) Enrichment of oligodendrocyte specific genes (from scRNA-seq) and genes bound by SOX3 and SOX9 in the four different oligodendrocyte gene clusters (O1-O4), shown in Fig. 3D. Three-star significance equal p-value e-7 for cluster O3 and e-16 for cluster O4 (prop.test, R) based on the size of the gene groups (O1=1371, O2=1451, O3=839, O4=141)
G MAGE-A10 and MAGE-A11 are not altered upon nutrient deprivation or DCAF12 deletion. A375 parental or DCAF12 KO cells were treated with EBSS for the indicated times. Cell lysates were probed for the indicated proteins.
TST activity in cerebrum (D), kidneys (E) and muscle (F) of Coq9+/+, Coq9R239X and Coq9Q95X mice.
(B, C) MCF10A-eGFP-LC3 cells left untreated or treated with 500 ng/ml TRAIL, 50 ng/ml TNF or 10 ng/ml IL‐1β for 24 h were analysed for LC3 translocation (B) and the DNA content (C). The values represent mean±s.d. for two independent experiments. **P‐value 0.01 and ***P‐value 0.001 as compared with untreated cells.
Quantification of tumor size in tibiae at 5-6 months of age (n=23 wa/+, 15 wa/wa)
F U2OS:GFP‐parkin cells transfected with siRNA targeting Drp1 (siDrp1) were treated with 25 μM antimycin A for 90 min prior to fixation. Samples were immunostained against TOM20 (blue) and the indicated mitochondrial marker (red). TOM20‐negative MDVs containing the specified cargo and colocalizing with GFP‐parkin (arrows) or not (circles) are indicated. Arrowheads show lack of colocalization between GFP‐parkin and the indicated mitochondrial marker. Scale bars, 20 μm (first two columns of panels) and 2 μm (remaining columns of panels).
(B) Anti-GA response in vaccinated mice measured by ELISA using GST-(GA)15 antigen. Tukey-style box plot shows 25th, 50th and 75th percentile and whiskers extend to ±1.5 interquartile range. Outliers depicted as dots. Two-way repeated-measure ANOVA (group x time) revealed a significant main effect of treatment (F(5, 57) = 478.2, P <0.0001) and time (F(6,342) = 296.1, P <0.0001) and a significant interaction between factors (F(30,342) = 119.5, P <0.0001), followed by Tukey's post hoc test.
(B-C) Schematic representations of TFE3 binding region in the ATF4 promoter in MEF cells treated with Tunicamycin (0.1 µg/ml) for 16 h (B) or starved in EBSS for 2 h (C). The distance upstream from the Transcription Start Site (TSS) of a potential E-box sequence is indicated in base pair (bp).
H. Permeability assay using vibratome section showing the leakage of retro-orbital injected cadaverine dye (red) in the brain of WT (top panel) and Glut1iECKO (bottom panel) mice. DAPI staining (white) shows the boundary of brain section.
C-I The same Age-TF regulates different sets of Age-DEGs and Age-BPs across tissues. The distribution of Age-DEGs and Age-BPs modulated by the Age-TFs shared in three tissues is shown by Venn diagrams.
Immunohistochemistry of paraffin embedded sections from the lungs, brain meninges, brain striatum, spleen and liver of the vector-injected mouse M16 (CAR) and a control mouse injected with PBS (PBS) stained against CD8 (αCD8), the CAR (αCAR), CD4 (αCD4), or CD19 (αCD19). Black arrowheads point at infiltrated lymphocytes, red arrowheads at histiocytes, and the yellow line indicates B-lymphocyte rich zones.
(a) Representative 2D μ-CT scans of RasV12; ctrl at day 6, RasV12; scrib-/- larvae at day 6 and day 10. Muscle (green), fat body (blue) and eye-antennal discs/tumor (red) are outlined.Scale bar: 1mm. Anterior (A), Right (R).
(E) Immunoblotting analysis for MIG-6 expression in BT549 cells with MIG-6 inducible knockdown (iMIG-6-shRNA) and the non-targeting shRNA control (iNT-shRNA) upon doxycycline treatment.
D. Schematic depiction of glucose-6-phosphate dehydrogenase (G6PD, UniProtKB ID P11413) cross-referencing both identical and non-identical sequences of Ensembl Stable IDs from five releases.
(e) MCF-7 cells treated with or without LY294002 for 48 h following RNAi. p27 siRNA pool-1, siRNA1, 2 and 3; arrows, membrane blebbing; triangles, apoptotic bodies. The scale bar represents 50 μm.
C Pull-down (PD) assay of lysates from P1.HTR cells with unphosphorylated (YENM) and tyrosine-phosphorylated IDO1 peptides (pYENM, pITIM1, and pITIM2), analyzed by sequential immunoblotting with anti−p85, −SHP-1, and -SHP-2 antibodies.
(A-B) SIRT5 expression is not affected by chemical oxidant treatment. HEK293T cells were treated with Paraquat (A) or H2O2 (B) for the indicated periods. The mRNA and protein expression of endogenous SIRT5 were determined by quantitative real-time PCR and western blot analysis, respectively. The results are average ± SD of 3 independent experiments. n.s.: not significant (two-tailed unpaired t-test).
E Munc18-1 mutants retain interaction with Syx1 shown by GST pull-down experiment. A representative gel displayed is from one of three independent replicates.
H. Schematic illustrating the Hsc70/DnaJC5/tau complex can be disrupted by both chemical (YM-01) inhibition.
(E) FISH of poly(A)+ RNA distribution in shScr, sh1 and sh2 stained with 5´Cys3-oligo dT(50). Nuclei were counterstained with DAPI. Scale bar, 20 µM.
WT and Mfn1/2 double KO were transfected with: GFP and the indicated plasmids. After 24 h, the transfected cells were sorted and plated for another 24 h. (I) RCR were calculated (n= 3 independent experiments). Data are presented as mean ± SEM.
B. Differences between NDC binding sites in GTP/GDP.Pi- and GDP-MT lattices. Deviations in backbone (Cα) atom positions are illustrated with colour-coded spheres.
MIAPaCa-2 cells were transfected with TGIF1 or TGIF1.2TD expression vectors, selected with G418 and resistance colonies were pooled (n=6). Binding of TGIF1 to the Twist1 promoter was examined by ChIP and agarose gel.
Percentage of headless sperm from cauda epididymides of WT (black), Spatc1l+/- (gray), and Spatc1l-/- (white) mice. The average percentage of headless sperm in WT, Spatc1l+/-, and Spatc1l-/- males was 6.3, 52.9 and 100, respectively. Each dot represents percentage of headless sperm in an individual mouse, and medians are represented as horizontal lines. (n=13, *P < 0.001, Student's t-test).
B. Quantification of the CD13-positive pericytes coverage in the brain vasculature of Glut1iECKO mice with (orange) or without (blue) lactate administration. (n=16 from 5 independent experiments. Error bars indicate the standard error of the mean (SEM) from 1-way ANOVA.) C. Quantification of the number of cadaverine 555 positive brain parenchyma cells in the brain vasculature of Glut1iECKO mice with (orange) or without (blue) lactate administration. (n=24 from 5 independent experiments. Error bars indicate the standard error of the mean (SEM) from 1-way ANOVA.) D. Quantification of CSF lactate level in WT and Glut1iECKO mice with/without lactate supplementation. (n=3 from independent experiments. Error bars indicate the standard error of the mean (SEM) from 1-way ANOVA.)
Immunoblot showing that the ~80kDa band disappears upon treatment of HEK293T and HaCaT cells with siRNAs against exon 3 and exon 15 of JARID2 RNA. Non-silencing control (NSC) was used as a transfection control.
HEK293 cells were transfected with siCTL or siDDX5. The next day, the cells that received siDDX5 were subsequently transfected with empty pcDNA3 vector (-), expression vectors encoding Flag-DDX5 (WT), or Flag-DDX5-RK (RK). The cells were subjected to Western blotting to visualize Flag-DDX WT and RK expression. Pol II ChIP and DRIP analysis of β-actin gene were performed. For the ChIP analysis, the Y-axis shows the signal-to-noise ratio of RNA Pol II IP relative to control IgG IP. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and ****: p < 0.0001.
A) Egg chambers expand and elongate through development. This is acheieved through collective migration (A") of t
A) AlexaFluor488-tagged (green) hnRNPA2 LC undergoes LLPS, while AlexaFluor555-tagged (red) TOG D1 does not. However, TOG D1 partitions into hnRNPA2 LC droplets when mixed at a 1:1 ratio. Conditions: 20 µM indicated protein (~1% fluorescently tagged), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 10 µm. hnRNPA2 LC control duplicated from Figure 1A as hnRNPF PLD and TOG D1 samples were made concurrently. B) Similar to hnRNPF PLD, AlexaFluor555-tagged TOG D1 does not undergo LLPS at 300 µM or partition into AlexaFluor488-tagged FUS LC droplets with both proteins at 300 µM. Conditions: 300 µM proteins (~1% fluorescently tagged), 20 mM MES pH 5.5 150 mM NaCl, 150 mM urea. Scale bar: 10 µm.
(C) Neurons from gba+/+ and gba−/− incubated with 5 mM methyl pyruvate 5 min prior to imaging. Cells bathed in 25 nM TMRM and 5 mM methyl pyruvate containing recording solution and fluorescence intensity analyzed via confocal microscopy. After 90 s, 1 μM oligomycin was added, followed by 1 μM FCCP (n = 3, three cells analyzed/experiment).
(B) Complex I-NADH: Ubiquinone reductase activity from gba+/+, gba+/−, and gba−/− brains expressed as a ratio to citrate synthase (n = 3).
E. Effect of NEK6 co-expression on ERα gene activation. Relative luciferase activity of ERα in the presence and absence of 1 nM E2 (17β-estradiol) was measured. A dose‑dependent reduction (ng plasmid transfected) in E2-Erα‑dependent luciferase expression was observed in HEK293T, MCF-7 and US-OS cancer cell lines. This effect was not observed with the NEK6(K74M&K75M) kinase‑dead version. Graphs represent ≥ 5 biological replicates, each derived from averaging 6 technical replicates. Error bars indicate SD across the biological replicates.
(F) ssDNA repair efficiency of the indicated mutants as determined by ChIP against Rfa1-YFP at the ARS305 replication origin. Cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour, treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium. The amount of Rfa1 at ssDNA was determined at 1 and 7 hours after MMS release. RPA enrichment was calculated as the ratio between immunoprecipitate and input values. All values were normalized to the wild type at 7 hours, taken as 1. Data information: The mean and SEM of 3 independent experiments are shown. Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test. One, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively. In (F) all values were significant relative to the untagged controls (not shown for clarity).
D. Drug specificity from high (++++) to low (+) of the different CDKs as described by Selleckchem, and Sec24C recruitment to SGs as a percentage of control.
B p53+/+ and p53-/- HCT116 cells were analyzed by chromatin immunoprecipitation (ChIP) assay using normal IgG and anti-p53 antibody as indicated.
(G) Identity of the clusters with the most significant differentially expressed gene(s)
Volume-rendered visualizations of the average structure of RyR1 fit with the RyR1 atomic model (PDB: 5TB2). The left panel is a thin section through the center of the volume.
(H) Immunoblotting analysis for MIG-6 and HIF1α expression in Luciferase and MIG-6 knockdown 293 without MG132 treatment.
(D) Mutant alleles used in this study. CRISPR-Cas9 strategy for creation and screening of catalytically inactive alleles of nsun-1 (mj473), nsun-2 (mj458) and nsun-4 (mj457). A homologous recombination template bearing a point mutation to convert the catalytic cysteine into alanine whilst creating a restriction site for HaeIII was co-injected. For the study of nsun-5, a 928 bp deletion allele (tm3898) was used. Image not to scale.
(A) Lysine 48 linked polyubiquitinated SCAP levels in parental and PKM2 KO MDA-MB-231 cells.
(D) qRT-PCR analyses of selected SREBPs downstream genes in shCtrl and shRNF5 PKM2 KO MDA-MB-231 cells (n=3). Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.
Figure 5 - Immediate late genes (ILGs) have long mRNA half-lives, are transcribed immediately and have GC-rich promoters. A: Boxplot comparison of mRNA half-life estimates based on modelling of gene induction (model-derived), transcriptional shutdown (ActD-derived) and metabolic labelling (4SU-derived). Estimates from 4OHT-pretreated HEK293∆RAF1:ER cells (ON panel) are more appropriate to characterise induced genes than estimates from unstimulated cells (OFF panel). Genes not assigned to any cluster are shown in grey.
A Echocardiographic assessment of percent fractional shortening in mice subjected to 5 weeks of TAC; n = 19 Wt sham, n = 22 LXRα-Tg sham, n = 24 Wt TAC, n = 26 LXRα-TgTAC. **P < 0.00001 versus Wt sham, *P = 0.0005 versus LXRα-Tg sham, #P = 0.009.
B) Planar division orientation in the follicular epithelium is dependent on apical tension in a Canoe-dependent manner, with Canoe-shRNA treated egg chambers showing a randomization of division orientation. Division angles were calculated relative to the elongating axis of the egg chamber using an unbiased analysis tool. Number of cells analyzed: w1118 n=52; Canoe-shRNA n=58; Random Number Generator n = 50. Mean division angle w1118= 31.6o, Canoe-shRNA= 43.3o, Random number generator = 46o; Mann Whitney test versus w1118: Canoe-shRNA p = 0.0345 ; Wilcoxon Signed Rank Test, Theoretical Mean of 45, w1118 p < 0.0001, Canoe-shRNA p = 0.7411, Random number generator p = 0.7779. Bars represent mean and standard deviation
G. Brigatinib treatment (0 or 150 nM) for 6h resulted in inhibition of ALK phosphorylation, and of activation of downstream signaling (ERK1/2), as well as MYCN expression. Cell lysates (Rosa26_Alkal2;Th-MYCN (#3540) and Alk-F1178S;Th-MYCN (#3456)) were immunoblotted with the indicated antibodies.
(c) Result of rapidly evolving genes (REGs). This calculates the χ2 critical value and P value for conducting the likelihood ratio test. The genes with P <0.05 were considered.
D Immunoblotting with anti-IDO1 and -p85 antibodies of IDO1 immunoprecipitates obtained from P1.HTR cells transfected with IDO1.WT or IDO1.Y149F. Mock-transfected P1.HTR cells were used as a control.
D Formation of LC3 puncta in GCN5 KO HeLa cells overexpressing Myc-tagged GCN5 or GCN5-E575Q (Scale bars, 10 µm).
Epifluorescent images of E. coli invaded by B. bacteriovorus HD100 with mCherry tagged Bd1971 at the times indicated throughout the invasive process during one round of synchronous infection. Fluorescence was acquired with a two second exposure and maximum sensitivity gain but different balance and contrast levels in the fluorescence channel were used to produce these images (see methods) as the fluorescence was fainter at later timepoints but we wished to show whether or not the cellular localization within the Bdellovibrio altered during predation. Fluorescence images and merges with phase contrast images are shown. Scale bars are 2 µm. Images are representative of three independent experiments.
After sorting Oct4GFP+ cells out (5C-Oct4GFP+ and mES-Oct4GFP+ cells), the expression of markers of Xist (C)
Ronin fl/del and Ronin del/del ESC stained for Ronin (left panel) or Oct4 (right panel). The nuclei are counterstained with DAPI.
(F) Whole lysates and cytosolic and membranous fractions were prepared from fat body samples of 3rd instar larvae expressing an RDH/CG2064-GFP knockin reporter. RDH/CG2064-GFP protein was detected by anti-GFP antibody in a western blot. RDH/CG2064-GFP is mainly present in the membranous fraction.
(P) Protein degradation curves were made after quantification and normalization of the bands from (O).
(A-C) Embryos co-expressing the optogenetic module and Sqh::mCherry mounted with the ventral epithelium facing the objective were subjected to cycles of photo-activation of the cell base in the anterior (left) half of the embryo and confocal recording of the whole embryo. The experiment was started at the end of cellularization when initial accumulation of apical myosin-II was detected and terminated at different time points to record the plasma membrane signal (CIBN::GFP-pm) in the whole embryo using two-photon illumination. Still frames visualizing the plasma membrane (CIBN::GFP-pm) at a sub-apical plane at the initial time point of the experiment (A), 3 min (B) and 10 min (C) after initial photo-activation.
(a) MEFs stably expressing EGFP-FKBP38(N402K) and FLAG-Parkin were incubated in the absence or presence of CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG (rabbit polyclonal). The fluorescence of EGFP-FKBP38(N402K) was also monitored directly. Higher-magnification views of the boxed areas are shown in the right panels of each set.
Mice were treated as described in Fig.1A. LP cells were isolated from each mouse. The proportions of IL-22+ cells in NKp46-RORγt+, NKp46+RORγt- and NKp46+RORγt+ were determined by flow cytometry.
Images of infected (right) or uninfected (left) ThP1 cells showing the vDNA (EdU) in red, the nucleus (DAPI) in blue, and selected nuclear body factors in green. (A) Green image shows immunolabeling of CPSF6.
C) Capture-C (MCF-7hypoxia - blue; 786-O - green) at the CCND1 locus from an enhancer (red highlight) bound by HIF almost exclusively in VHL defective 786-O and showing cell-specific cis-interactions, blue highlighted region indicates region of interaction at the TSS of CCND1, green highlighted region indicates region of interaction at an enhancer 3' to the MYEOV gene.
E. qPCR analysis of rab13 mRNA levels in individual 26-28 hpf clutch-matched sibling embryos (n≥9 embryos; ns: not significant; Kruskal-Wallis test with Dunn's correction). : +/+, +/∆ and ∆/∆ represent Tg(kdrl:EGFP) rab13+/+, rab13+/∆3'UTR and rab13∆3'UTR /∆3'UTR embryos, respectively
b-d, MEFs were cultured in nutrient-rich medium (fed) or Hanks' buffer (starved) for 1 h. Total cell lysates were prepared, and blotted with the indicated antibodies (b).
(E) Left image shows an observed, EdU-labeled vDNA focus. Right: simulations of a single linear or circular 10 Kb long chromatinized DNA polymer chain and corresponding predicted images in diffraction-limited (~300 nm resolution) microscopy. (F) Boxplots show the distribution of sizes (FWHM, full width at half maximum) of n=40 measured vDNA foci (for MOI 100) compared to the sizes predicted for linear (left) and circular (right) chains based on n=100 simulated configurations each. Blue lines in boxes define medians, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. All differences are highly significant (Wilcoxon test data vs. model: ***p ≈ 3 ⋅ 10 − 20, circular vs. linear model: ***p ≈ 10 − 17).
D283 cell lines were treated with PBS (vehicle, black) or with ActivinB (green). Cell cycle analysis by FACS measuring BrdU incorporation and 7AAD labelling at 48 hours upon ActivinB stimulation. The percentage of cells in the different phases of the cell cycle is represented (G0/G1, S and G2/M phases). Bars represent the mean ± SD. Number of replicates is n≥3.
B) Two human primary lung fibroblasts (HLF) and three primary tumor cell lines derived from patients suffering from either lung squamous carcinoma (LSC) or lung adenocarcinoma (LAD) were transfected with RNP complexes with scrambled or POLR2A targeting gRNA. 5 days post transfection, cell viability in each well was measured. Results are from at least 3 independent experiments containing 3 technical replicates. For all cell lines, P values (scrambled vs POLR2A) < 0.005.
G In vivo suppression of the activity of HY-pulsed CD8− DCs in combination with a minority fraction (5%) of pDCs transfected with siRNA as in (F) and conditioned in vitro with TGF-β (or medium) for 24 h. Analysis of skin reactivity of recipient mice to the eliciting peptide at 15 days is presented as change in footpad weight (experimental versus control footpads).
(C) Percentage of cells polarized for septin 7 among young, aged and aged LT-HSCs treated with CASIN (5µM). At least 3 biological repeats, at least 50 cells were scored per sample. Error bars indicate mean±SEM, unpaired t-test, **P<0.01, ***P<0.001.