EMBO/sd-character-level-ner huggingface dataset:
Fluorescent intensity measurements show the levels of two ISR-related proteins, PERK and relative phospho-eif2α (phospho-eif2α / total eif2α), are increased following different excitotoxic stress treatments in cortical neurons. Representative images indicate fluorescent intensity differences for phospho-eif2α and phospho-PERK levels 24 h post treatment with different excitotoxic stressor paradigms. Blocking of AMPA and NMDA receptors using known antagonists significantly reduces ISR-related protein levels, which parallels reduced DPR levels (Figure 3B). Changes in ISR proteins are relative to non-stressed cells (CTRL).
C) Two representative snapshots showing (left) a ZEB1 peak upstream of the MIR200A/B cluster and immediately downstream of the ADAM8 gene (right).
(c) A HeLa cell expressing EGFP-LC3B and lamp1-RFP, and labelled with AlPcS2a, was 635-nm illuminated within the white square region, resulting in LC3 recruitment onto photodamaged lysosomes, as shown in d. (d) Magnified view of the 635-nm illuminated cell region in c. LC3 is initially distributed away from lysosomes (lamp1, t=0 min, top panels), but appears on lamp1-positive structures 40 min after light activation (bottom panels). Scale bars, 10 μm (a,c); 3 μm (b,d).
G TUNEL assay in MB‐SCs. MB‐SCs isolated from Ptch1+/−miceMBs were treated with GlaB (5 μM) and compared to DMSO‐treated sample. Bottom panel shows a Western blot of caspase‐3 in GlaB‐treated versus control cells.
(B) Pyruvate and lactate enrichment were analyzed by GC/MS, after cultured with medium containing 10 mM [U-13C]glucose and 10 µM CL316,243 for 2 hours.
(G, H) C2C12 cells were transfected for 48 h with control (sc) (lanes 1, 2, 5, 6) or Atg5 siRNA (lanes 3, 4, 6, 7) followed by a second transfection for 48 h with same siRNA and control (lanes 1, 3, 5, 7) or Jumpy siRNA (lanes 2, 4, 6, 8). Cells were incubated for 1 h with or without 100 nM Baf A1, 10 μg/ml E64d and 10 μg/ml pepstatin in full media, lysed and analysed by immunoblotting with anti‐LC3 or anti‐actin (G). (H) Densitometric LC3‐II/actin ratios for samples treated with BafA1 and protease inhibitors from G (lanes 5-8). Inset shows Atg5 knock‐down by immunoblotting.
(B-C) Average amplitude (B) and average frequency (C) of IPSCs from wt, R6/2 ACSF, and R6/2 chol-high MSNs. Data information: The data are shown as scatterplot graphs with means±standard error. Each dot corresponds to the value of each cell recorded (B, C) Statistics: one-way ANOVA with Newman-Keuls post-hoc test (**p<0.01; ***p<0.001).
Skin tumor outcomes in K14-IL33tg,ST2KO mice treated with DMBA/TPA carcinogenesis protocol in combination with SB431542 (test, n=6) or acetone (control, n=5). (G) skin tumor counts per mouse over time,
Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions. strains were grown in 96-well plates and exposed to various degrees of GlcN6P depletion elicited by Nva-FMDP. Cells were harvested at indicated times and the β-galactosidase activities were determined. Complementation experiment analyzing the requirement of rapZ for glmY expression in cells grown to exponential phase under standard conditions in flask cultures. Strains Z197 and the ∆rapZ mutant Z225 were used. Tested plasmids were pFDX4291 (vector control for pFDX4324 and pYG82 = VC1), pFDX4324 (rapZ), pYG82 (rapZquad), pBGG237 (vector control for pBGG164 = VC2) and pBGG164 (strep-rapZ).
C. Quantitative RT-PCR was performed to identify transcriptional changes of the IFN-γ response gene expression (n=3). Data are shown as the relative fold change (RFC, color coded bar).
(A) Profiling of bioactivities of serum GM3 species in LPS-mediated monocyte activation (schematic).
(e) Immunoblot analysis of p62 in LPS-stimulated B cells (bottom). Above, quantification of band intensity (as in c).
Lyzs-Cre and p38γ/δLyzs-KO mice were fed a ND or a MCD diet for 3 weeks. (B) Liver triglycerides (C) and plasma ALT at the end of the diet period.
(D, E) Time course of days (one, three, and six days) post infection (dpi) using liver spheroids and SARS-CoV-2 at 0.1 and 1.0 multiplicity of infection (MOI) (n=1).
(D) Left panel: Absorption spectra of dark-adapted XccBphP after illumination with red, far-red, fluorescent white lamp light, sunlight or sunlight filtered through green leaves (LF). Pr/Pfr equilibriums scheme derived from absorption spectra is depicted in the bottom panel. Right panel: The XccBphP-C13S mutant was purified in the presence of BV and its absorption spectra recorded upon illumination with red, far-red and white light or dark adaptation.
F. Lats1 -/-; Lats2 fl/fl; SV40 mouse embryonic fibroblasts complemented with vector, LATS2 WT or ΔUBA mutant were infected with empty (control) or Zeocin-Cre retroviruses and selected with 400 μg/ml of Zeocin for 4 days. Harvested cells were analyzed by Western blotting for the indicated proteins.
(A) Co-expressing myc-Tau and eGFP-β-catenin increased nuclear translocation of β-catenin, while co-expressing myc-Tau and eGFP-β-cat K49R abolished the nuclear translocation of β-catenin in HEK293 cells. The nuclei were stained using DAPI (n=3 from three independent experiments, scale bar, 5 μm).
Flow cytometric assay of the SARS-CoV-2 RBD and its mutants (K417R, Q498H and K417R-Q498H) binding to human, mouse, rat and European hedgehog ACE2s expressed on the cell surface of BHK21 cells. The SARS-CoV-2 NTD was used as negative control.
HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). K TO-PRO-3 uptake Data are means ± SEM, n=3 biological replicates, ****P<0.0001 (ANOVA). The presented data are representative of at least three independent experiments.
survival rate (H) of NMT1-Myc overexpression transgenic plants. Two-week-old Super:NMT1-Myc plants were treated at −5°C (NA) or −8°C for 0.5 h (CA). In (H), each bar represents the mean ± SEM of three biological experiments, and asterisks indicate significant differences compared with the wild type for the same treatment (*P < 0.05, two tailed t-test).
B. Nox4-depleted cells had lower levels of phospho-eIF2α and ATF4 than control cells but these were restored in the presence of guanabenz (Gbz).
(B) The negatively-charged ions of RNase T1 fragment is decomposed in the instrument by collision-induced dissociation (CID) using helium gas. The product ions produced by CID are assigned on the sequence illustrated on the top right inset panel. Nomenclature of the product ions are described in the literature (McLuckey et al, 1992). Product ions of c and y series derive from the 5' and 3' termini of the fragment, respectively.
B-D. 3T3-L1 stable cells expressing vector control (EV), Flag-tagged WT or mutant WDTC1 proteins were adipogenically induced. Adipogenesis was assessed by Oil Red O staining (ORO) (B); plate (top) and microscopic (bottom) views, 0.1 mm scale bar.
(C) Gel retardation assays show the formation of the complex between RsaI and RsaG. The 5' end-labeled wild-type RsaI (RsaI), RsaI mutant 2 (RsaI mut2), RsaI mutant 4 (RsaI mut4), and RsaI mutant 1 (RsaI mut1) were incubated with increasing concentrations of RsaG given in nM on the top of the autoradiographies.
A-C Calcium flux measured by FACScan for splenic B cells isolated from Lyn-deficient B1-8 transgenic mice after stimulation with (A) NIP15-BSA (30 pM) or 1NIP-pep (80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells.
E2C- and S100A9-expressing HEK293T cells were infected with the indicated MOI of HIV-GFP for 48h and GFP+ cells were analysed by flow cytometry. Mean values ± SD from four independent experiments (n=4) were normalized to the E2C-HEK293T control and represented in graph.
MoLC from two different donors (donor #26 and #30) were left untreated (NT) or incubated with 1µg/ml of LPS for indicated time points before infection with HIV-GFP vectors for 48h. The percentage of GFP+ cells was analysed by flow cytometry (left dot-plots) and a graph representing the fold change of GFP+ cells, normalized to untreated infected MoLC is shown on the right.
(C) The FEZ1-GFP and Myc-ULK1 interaction is unaffected by amino‐acid starvation. Anti‐Myc and ‐GFP blots after HEK293 cells were co‐transfected with FEZ1-GFP and Myc-ULK1, incubated in FM or ES for 2 h, harvested and the complex was immunoprecipitated using anti‐GFP antibody.
A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of 5-AzadC or 5-AzaC. At 72 h post-treatment, viral production was measured by quantifying p24 antigen production in culture supernatants (A)
A) pSTAT1 translocation in mock and SARS-CoV-2-infected bronchioalveolar-like cells visualized in green. Scale bars indicate 50 μm. Nucleoprotein (NP) stains viral capsid (red). Nuclei are stained with Hoechst (white).
Arp2 localization in REP1 WT, hCDC14APD and DBN1 KO cells. Signal intensity was measured 30 min after serum starvation. The encircled area in MERGE is shown 4-fold enlarged on the right hand side of the figure
(C) Graph depicts the Mean Fluorescence Intensity measured for each condition in ERAI mice (6 dpi). Two-way ANOVA Tukey's multiple comparisons, ns: p>0.05; ***: p<0.001; ****: p<0.0001; mean ± S.E.M (n=5 mice per genotype and condition, one representative experiment from 2 independent experiments is shown).
(B) 5-day-old seedlings of WT and rgi5x were treated with 10 nM flg22 or co-treated with 1 μM GLV2 for 7 days before measuring fresh weight. Shown is the mean of relative fresh weight compared to the MS medium control -/+ SD, n=36-57 biological replicates pooled from 4 experiments (one-way ANOVA, Tukey post-hoc test, a-b p<0.001; a-c, p<0.01).
(E) Correlation analyses between STXBP4 and YAP in human normal kidney and clear cell carcinoma samples are shown as tables. Statistical significance was determined by chi-square test. R, correlation coefficient. N, nuclear localization. C, cytoplasmic localization.
Number of Us11-GFP expressing neurons at 3 days post-LY294002-induced reactivation in P6 SCG neuronal cultures transduced with shRNA targeting PML at 1 day post-infection with HSV-1 in the presence or absence of IFNα (150 IU/ml). n=9 biological replicates. Statistical comparisons were made using a Mann-Whitney test (ns not significant, * P<0.05).
B, Quantification of the nuclear localization of recombinant TFEB in ARPE-19 cells treated with 250 μM NaAsO2 for 1 h or EBSS for 4h and incubated in complete medium (refeed) for the indicated times. Data are presented as mean ± SD using two-way ANOVA, (ns) not significant as compared to the same treatment in TFEB-WT overexpressing cells, with >200 cells counted per trial from three independent experiments.
(d) Autophagosome (denoted by arrowheads) generation on amino-acid withdrawal was analysed by electron microscopy. Cell lines and conditions from c were used and representative images from the indicated amino-acid-starved lines are shown; scale bars, 0.4 μm.
Figure 1. Breast cancer cells derived from tumor-draining lymph nodes gain aggressive malignancy (A and B) Inguinal LNs were harvested from 4T1-injected mice at the indicated week after initial injection. LN tissues were fixed and paraffin-embedded. Immunoflurorescence was performed with anti-GFP antibody to visualize GFP+ 4T1 cells. (B) Histograms illustrated that the dynamic changes of GFP+ 4T1 cells metastasized to the inguinal LN. Cells were counted in whole sections (2 non-consecutive sections per LN per mouse). Each group was composed of at least two mice.Data information: In (A-G), all experimental data were verified in at least two independent experiments. All values are presented as mean or mean ± SD.
(B) Cryo-EM reconstruction of the native eEF3-80S complex with segmented densities for the 60S (grey), the 40S (tan) and eEF3 (pastel green).
Nontoxic dose of Cpd A enhances bendamustine cytotoxicity in cancer cells. Images were taken at 48 h posttreatment.
C, 31P NMR spectrum of the thioPO3 signal from phosphocysteine in PRL2. The broad peak is TCEP in the buffer.
RT-qPCR validation of m6A-RIP of FAM214A mRNA. The positions of primers are indicated above. Relative pulldown efficiency of each experiment was calculated as the ratio of pulldown efficiency (pulldown/input) in HSATIII KD cells to that in control cells. Data are shown as the mean ± SD (n=3); p-value (Welch's t-test) is shown above.
Integration of heterogeneous data sets identifies LOX as a therapeutic target in pancreatic cancer. Components of the hazardous PC-1 signature are overlaid with genes found to be overexpressed in a microarray and hits that cause a reduction in viable cell number in an RNAi functional screen. Overlap is shown as a proportional Venn diagram.
B. Cα trace of SPIN90(351-722) superposed onto the structure of SPIN90(306-722). His580 is shown in stick representation, as are residues on the H5-2 helix that contribute to the conserved surface patch. H5-1/2 is flipped away from the C-terminus of the ARM domain in the inactive 351-722 structure
(B) Surface view of P1 actin-stained organ of Corti confirms defective establishment of polarity in Elp3cKO as numerous stereociliary bundles are misaligned along the medio-lateral axis (M-L) or misshaped. Scale bars=5µm. Data information: #IHC; *OHC1; **OHC2; ***OHC3
(f) MMP7 staining of control and Huwe1 deleted small intestine. Note MMP7 staining is restricted to crypt base in Huwe1 deficient intestines. Scale bars = 100µm.
Chimerism among BM HSPCs fractions transplanted to HO-1-/- recipients are lower than from HSCs transplanted to HO-1+/+ recipients. Data are shown as mean ± SEM, n = 5-12/group
A) Ventral nests expansion at 0 min (onset); 245 min, when ventral and spiracle nests fuse; 515 min, when intersegment nests fuse; and 750 min, when most LECs are gone. We employed for these analyses the following stock: w; If/CyO; ZCL2207::hh-DsRed/MKRS. The green signal corresponds to ZCL2207 (Septate junction GFP marker) outlining all apical cell membranes and the red signal to nuclei from the posterior compartment expressing RFP under the control of the hedgehog promoter. Arrows point to the spiracular and ventral nests on the top panel and to the junction between these two nests in the second panel. Images of confocal sections have been rotated to maintain consistent alignment of the dorso-ventral axis (dorsal is up). The outlines of the confocal images are marked with a yellow dotted line. Scale bar = 40 µm.
Quantitative RT-PCR analysis of auxin signaling gene expression in roots of wild type (Col-0), uvr8, myb73 myb77, and myb73 myb77 uvr8. (D) Results under white light and (E) under white plus UV-B light. Seedlings grown in continuous white light for 12 days were kept in white light or transferred to white light plus UV-B for 1 day with the addition of 0.4 μM NAA, and roots were collected to analyze auxin signaling gene expression. Error bars represent SD of three biological replicates.
Mice were injected with the PPARα antagonist GW6471 (10µg/g) or vehicle (DMSO) 3h pre- and 3h post-CLP. Survival was monitored during 9 days, after which no further deaths occurred. Survival curve was analyzed via a Log rank test. Combined data of 2 independent experiments, n = 16/group.
(b) HeLa cells were transfected with EGFP-Vps16 together with Flag-UVRAG mutants as indicated and stained with anti-Flag (red), followed by confocal microscopy. Scale bars, 5 μm.
D. Representative currents of indicated WT and mutants of PC2 in a bath solution containing 70 mM Ca2+.
Box plots showing levels of genes up‐ and downregulated following let‐7 addition in Dgcr8Δ/Δ GPCs during astrocyte differentiation in vivo. P‐values were calculated using Fisher's exact test.
(K) Mean quantitation of TRAP-stained osteoclasts differentiated with M-CSF ± RANKL in co-culture with RM1 BD Irf- BV and RM1 BD Irf7 OE cells with representative wells shown (n = 3 per condition M-CSF; n = 6 per condition M-CSF + RANKL; also shown in Appendix Fig S4J). Scale bar represents 100 μm.
FoxD1-reporter mice (Foxd1::tdTomato) confirmed the expansion of mesenchymal cells in 14 weeks old Foxd1Cre::Pdgfrb+/J mice both in glomeruli and interstitium. Circles outline glomeruli. Scale bar = 50 µm. Quantification of FoxD1-Tomato positive cells in the cortical interstitium in 14 weeks old Foxd1Cre::Pdgfrb+/J and wt mice confirmed the significantly increased expansion of mesenchymal cells by 42% in Foxd1Cre::Pdgfrb+/J mice. Cells were counted in six viewfields at 40x magnification. Bar graphs show means ± SD of n = 3 mice per group.
Spatial distribution of the different TME populations across the analyzed five zones. Values are the estimated proportions based on de-convolution of the transcriptome of each zone with the average transcriptome of the cell types (Methods). Y axes show the estimated fractions of the cells, empty circles are the medians over all repeats (n=8 for tb, 11 for tc, 15 for ftb, 14 for ti and 15 for fz)
A. Primary data - polyacrylamide gel - a representative result. For description of bands/fragments see next panel legend. Asterisks indicate STUBS (16-19 nt). B. A schematic representation of quantitation of fragments. OUTSIDE (16-30 nt) are RNA fragments that originated by digestion of the full length RNA by RNases that were stopped either before reaching or at the point of reaching RNAP; STUBS (16-19 nt) are RNA fragments that originated by digestion of the full length RNA that were stopped at the point of reaching RNAP; INSIDE (< 16 nt) are RNA fragments (oligonucleotides) that could be only generated after the disassembly of the complex by the torpedo mechanism. The red color indicates parts of RNA that were digested. C. Quantitation of three independent experiments. "Full length" indicates the remaining undigested RNA. "OUTSIDE", "INSIDE" are fragments as explained in (B) and indicated in (A). The bars represent 100 % (all fragments). The black-grey-white boxes indicate the percentage of each fragment group (in %). The error bars indicate ± SEM for each group of fragments calculated from three biological replicates.
C) Results of a competitive uptake assay showing inhibition of 0.2 µM L-[3H]Trp or L-[3H]Leu uptake in MhsT-WT-expressing MQ614 cells, measured for 30 seconds in the absence (-) or presence of 12 µM of the natural amino acids (the one-letter code is used) or 4-fluoro-phenylalanine (4FP). Data are the mean ± S.E.M. of three independent experiments each performed as technical triplicates.
Atg36 is required for pexophagy. Validation of Pex11-GFP as a marker for pexophagy in WT, atg1, and atg36 cells by (A) microscopy and (B) western blot including pex14Δ cells. The fluorescence assay shows that like HcRed-PTS1, Pex11-GFP accumulates in the vacuole in starvation medium (A), which corresponds to the accumulation of GFP on a western blot (B).
D Co-immunoprecipitation of HEK293 cell lysates expressing myc-tagged gephyrin together with GFP-tagged gephyrin or G375D using myc-specific antibodies. Control IgG were used to show specificity of the assay.
C, PTPN3 and PTPN3 (D811A), but not PTPN3 (L232R), enhance TGF-β-induced growth inhibitory effect. HepG2 cells stably expressing PTPN3, PTPN3 (D811A) or PTPN3 (L232R) were treated with or without TGF-β (1 ng/mL) for indicated days. Cells were analyzed for cell proliferation by MTS assay. Data are shown as mean ± SEM; n = 3.
(B) 293T cells were transduced with the stated shRNAs and cells harvested 4 days after puro selection for Western Blot to verify KAP1 and SETDB1-depletion (left) and ChIP (right). Cell numbers were normalized per treatment group before sonication and IPs were performed in triplicate. Results show IP relative to total input and GAPDH normalized. IgG control IPs gave only background enrichments shown. One representative experiment of two is shown. Unpaired t tests were performed. P values: ZNF239: KAP1, 0.0292; SETDB1, 0.0137; HERVK14C_1: KAP1, 0.0227; SETDB1, 0.0082; HERVK14C_2: SETDB1, 0.0462; SVA D VNTR, SETDB1, 0.0064. The inlay shows qRTPCR expression analysis of HERVK14C in the same experiment, normalized to B2M or GAPDH as stated. All error bars show SD.
Micro-CT analyses of trabecular bone from Grounded and Tail suspended model (n=5 independent biological replicates). Scale bar=1 mm. Data were compared using an unpaired t‐test (* indicates P < 0.05, ** indicates P < 0.01), error bars are standard deviations.
B) RAW264.7 cells were co-transfected with control or miR-155 mimic and a wild-type (WT-Rheb) or mutated Rheb 3′UTR (mut-Rheb) luciferase reporter plasmid and assessed for luciferase activity at 24 h after transfection. Data are shown as the mean ± SEM of three independent experiments. ***, p<0.001; NS, not significant.
Knockdown efficiency of Larp1 shRNAs in 2iL (mean values ± SD). n = 3 biological replicates.
5-7.5 x 104 end labelled 100 bp double-stranded oligonucleotide was incubated with no protein (lanes 1 and 6) or 4 μΜ BSA (lanes 2 and 7) or 2 μΜ His-AosR + 2 μΜ BSA (lanes 3 and 8) or , 2 μΜ His-SigH + 2 μΜ BSA (lanes 4 and 9) or 2 μΜ His-AosR + 2 μΜ His-SigH (lanes 5 and 10). The reactions were either incubated in the presence of 10 mM reduced DTT or 10 mM diamide to reduce or oxidize thiols, respectively, and resolved on 8% PAGE gel. Protein-DNA complexes (bound) and free probe are indicated by arrows. To confirm the specificity of DNA-protein interaction, EMSA reaction was performed in the presence of 5, 10, 25, 50, and 100 molar excess of unlabelled 100 bp double-stranded oligonucleotide.
A) RNAscope in situ hybridisation analysis of TEAD1/2/4 mRNA expression from control (wild-type) and ApcMin mutant tumours from the small intestine (top) and colon (bottom). All three genes were strongly induced within the ApcMin mutant tumours, with TEAD2 being most strongly induced in colon ApcMin tumours (n=3 animals with multiple tumours within the small and large intestines).
G) Bar graph representing the percentage of protruding Caco-2 cysts in control conditions (non-treated, NT) or treated with NSC23766, Y27632 or H1152 (Means ± SEM of at least 3 independent experiments, unpaired t-test, ****p<0.0001, ***p<0.001, *p<0.05
(N) RT-PCR quantification of M2 (Arg1 and Cxcr4) and M1 (Cxcl9 and Nos2) markers in vehicle and glufosinate (20mg/kg) treated mice (n=6).
Bar graph represents the frequency of IAHCs containing recombined cells per embryo under the indicated conditions. Quantification of confocal images. Mean±SE (n=15 for untreated, n=7 for IgG and αDll4 treated).
Localization o visual-arrestin (v-arrestin) in semi-thin sections of retinas from either MOG/CFA-injected mice (EAE mice) or CFA-injected control mice (9 days after injection). Semi-thin sections were double-immunolabelle with antibodies against v-arrestin and RIBEYE (C-D) V-arrestin, visual arrestin; OPL, outer plexiform layer; INL, nuclear layer; IPL, inner plexiform layer. Scale bars: 20μm
(d) Midguts dissected from animals at puparium formation that contain an Atg1Δ3D loss-of-function mutant cell clone (lacking GFP) and analysed by fluorescence and differential interference contrast microscopy. Wild-type (+/+) control cell possesses stronger GFP and heterozygous Atg1Δ3D/wild-type (Δ3D/+) cells have weaker GFP. Representative images are shown. (e) Quantification (μm2) from d, n = 7 (+/+), n = 8 (Δ3D/+) and n = 8 (Δ3D/Δ3D) animal intestines per genotype with 1-5 cells measured per intestine.
(B) EMSA of retinoic acid receptor (RAR)/RXR binding to the p19ink4d ER8, and the rarβ DR5 retinoic acid response element, as a control.
D Sequence comparison between unicellular AMSIN and multicellular AITDs. Only the top BLASTP hits obtained by AMSIN as query searching against the GenBank database were chosen for alignment. The completely conserved cysteines are shaded in yellow and the nonconserved underlined once. Identical noncysteine residues are shaded in different colors based on their side-chain nature (red, acidic; purple, aromatic; blue, basic; cyan, polar and uncharged; green, hydrophobic). Residues in the N-terminus and the C-loop of AMSIN-1, which form a continuous hydrophobic surface in its dimer, are underlined twice in red.