EMBO/sd-character-level-ner huggingface dataset:
G-K Co-immunostaining of NB-3 (green), PDGFR-β (red), and GFAP (blue) on serial sagittal sections from the same NB-3+/+spinal cord 14 days post-injury. (G1-K1) High-magnification images of boxed areas in (G-K), respectively.L Co-immunostaining of NB-3 (green), PDGFR-β (red), and GFAP (blue) on sagittal sections from the NB-3−/− spinal cord 14 days post-injury. (L1) High-magnification image of the boxed area in (L). The arrows indicate the lesion sites.
A. Frame series shows the off-axis movement of an IFT-particle (#1). A neighboring particle (#2) serves as a fiduciary marker.
Example images of IHC for PH3+ cells in 60d old CTRL and ECE2 KO COs. Quantification of PH3+ cycling cells in 60d old COs, normalised to length of the apical surface of ventricle-like structures, shows an increase upon ECE2 KO (Data shown as box plots with median as black line, mean as red line; n=number of analysed ventricles from 2 batches of COs; One Way ANOVA: **P = 0.004).
B. mtDNA replication, as illustrated by EdU incorporation, in wt and larp mutant (larp) ovarioles. mtDNA replication is dramatically reduced in the larp ovariole. Arrows: mtDNA; arrowheads: nuclei.
(a) Beclin-1 alone (lanes 1-4) or Beclin-1 and ATG14L (lanes 5-8) were overexpressed in HEK293 cells. Beclin-1 was purified either by direct immunoprecipitation (lanes 1-4) or by ATG14L immunoprecipitation (lanes 5-8). Immunoprecipitated samples were subjected to an in vitro ULK1 kinase assay with increasing amounts of ULK1. Reactions were immunoblotted with the indicated antibodies. Black line denotes discontinuous lanes from the same gel. Two unique experiments were performed.
A Western blot analysis of Hwa in HEK293T cells co-transfected with Hwa-Flag, and ZNRF3-HA (5 μg) or ZNRF3ΔRING (2.5 μg). Lysate was immunoblotted with Flag, HA, and GAPDH antibodies. Blots are representative of 3 independent experiments. B Densitometry analysis of Hwa bands from the western blot in (A) for corresponding lanes normalized to GAPDH. n = 3 independent experiments, mean ± SEM, ns p > 0.05, **p < 0.01 by unpaired t test.
(E) STING co-localized with EGFR after cGAMP stimulation. STING-/- MEF cells were transfected with STING-GFP (Green) for 24hours and stimulated with cGAMP for 1h, cells were fixed and labeled with EGFR (Red) and Calnexin (Purple) antibodies. Co-localization was analyzed by Fuji and co-localized dots are shown in white. Scale bars represent 10 µ m.
(G) Upper, representative western-blots of EAAC1 and Smo from HEK293 cells transfected with empty vectors (Ctrl), constitutively active form of Smoothened (SmoA1), EAAC1, or EAAC1 plus SmoA1. Lower, 3H-glutamate uptake by cells transfected with the indicated vectors. n=9.
Primary BMDMs were stimulated with recombinant murine TNF (100 ng/ml) in combination with TAK1i for 6 or 4 h respectively.
(J) Representative confocal images of HeLa and TRIM16KO cells treated with MG132 (20 µM, 2h) and processed for IF analysis with Ub and LC3B antibody.
WT and C2βD1212A/D1212A mice were subjected to tMCAO followed by 24h of reperfusion. (B) Protein expression of TNFα, IL-1β and IL-6. Data represent mean ± SEM (n=4 mice per group; *p=0.05; Mann-Whitney test). Data represent mean ± SEM (n=5-7 mice per group; **p<0.01; Mann-Whitney test).
A Schematic diagram showing organization of mouse major and minor satellite repeats. Major and minor satellite sequences comprise six megabases of a 234 bp- repeats and six hundred kilobases of a 120 bp repeats, respectively. The terms "sense (S)" and "antisense (As)" refer to T- and A-rich transcripts, respectively. The length of major and minor satellite sequences used to perform in vitro transcription is indicated.
A The simulated period of ASPD models is shorter than that of the WT model in DD. Thus, they have a ~4 h more advanced phase than the WT model in LD 12:12 (inset). PER2 protein concentrations in each mutant are normalized to the maximum PER2 concentration of the WT model (dashed gray line; inset).
(C) Immunoblot analysis of BAT from AdRiKO and control mice housed at either 22°C or 4°C for 2h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice).
E. Confocal microscopy images of RFP, RFP-PNRC1 or RFP-PNRC1W300A-expressing cells stained for DCP1α, DDX6 and LSM1. RFP-PNRC1WT or RFP-PNRC1W300A-transfected cells are indicated with arrowheads. Scale bar: 10µm
E, F Percentages of leading-edges amino acid genes in 6 weeks (E) or 9 weeks (F) KIC mice as compared to age-matched KI mice that contribute to the GSEA enrichment. Red spots represent the p-value of the deregulated amino-acid pathways in KIC mice as compared to KI mice. Red line corresponds to a p-value of 0.05.
D. Sperm chromatin treated with 2000 J/m2 of UV-C was incubated in non-replicating egg extracts in the presence or absence of the indicated PARP inhibitors (PARPi; Veliparib (50 µM), Olaparib (50 µM), Talazoparib (10 µM)). At the indicated time points, chromatin was recovered via chromatin spin-down and samples were immunoblotted with indicated antibodies.
Healthy mice were tail vein injected with 10 nmol TLR7-54 (circles) or FA-TLR7-54 (squares), and peripheral blood was collected at indicated time points after drug injection. (D-F) Effect of drug concentration on plasma levels of IL-6 (D), IFNα (E), and TNFα (F) at 1.5h, 1h, or 1h after treatment, respectively (n=2).
(B) LATS1 and LATS2 were knocked down using siLATS1 and siLATS2 SMARTPools in MCF10A cells. MCF10A control cells were treated with control siRNA (siControl) against fire fly luciferase. Cells were stained for TRIP6 and LATS1 as in (A) (quantification of TRIP6 localization at cell-cell junctions is shown in Figure EV3E). Scale bar=20µm.
Quantification of the numbers of senescent cells in the presence or absence of the inhibitors, as shown in (A). Error bars represent SEM (n = 3). *P 0.05; **P 0.01.Quantification of the numbers of spheres in the presence or absence of the inhibitors, as in (A). Error bars represent SEM (n = 3). *P 0.05; **P 0.01
(A) Time-lapse confocal imaging of the in vitro nucleation of soluble ASC-mTurquoise (red) by ASC-TagGFP (ASC-GFP) specks (green), that were left untreated (None), or pre-incubated with VHHASC or mutVHHASC (200 µg ml−1 for 15 min). Scale bars: 10 μm. (B) Median fluorescence intensity (MFI) of mTurquoise graphed over time showing its polymerization seeded by ASC-TagGFP specks. Each line shows the mTurquoise MFI around a seeding ASC-TagGFP speck. Data is one representative of three independent experiments.
B. Gene ontology (GO) analysis of differentially expressed genes in each region.
Time courses of swarmbots treated with varying concentrations of carbenicillin and glucose. Solid lines indicate cell densities inside swarmbots; dotted lines indicate those in the chamber. Safeguard was established for lower nutrient levels (0.004% or 0.04% glucose) along with carbenicillin treatment. At a high nutrient level (0.4% glucose), the safeguard was lost due to either growth in the chamber (panels 3 and 6) or gradual killing of the population inside swarmbot (solid line in panel 9). These results are qualitatively consistent with the model predictions (Fig 4).
a, Correlative light and electron microscopy analysis of STX17 knockdown HeLa cells expressing GFP-LC3 (white arrows) infected with GAS (red arrows). DAPI, 4′,6-diamidino-2-phenylindole.
(A) Left Panel: Representative ABR to supramaximal click stimuli revealed stable response at the characteristic peaks I-V in 3-week old WT pigs at 100 dB SPL, but not in age-matched USH1C pigs up to 120 dB SPL (left) (1 WT and 2 USH1C pigs, both ears, 2 traces each). Middle Panel: Hearing deficits measured by ABR were more pronounced at higher frequencies in 8-week old USH1C pigs. Differences between WT and heterozygous USH1C+/- (het) were not significant (t-test) for the respective frequencies, but a 2-sided ANOVA test indicated a <0.01 significance (**) overall. p-values for differences between USH1C pigs and WT or heterozygous pigs were <0.0001 (****) at all tested frequencies and <0.01 (**) for a click test. Data points represent mean values (MV) ± standard deviation (SD). Right Panel: Behavior tests supported impaired hearing in USH1C pigs. In their first days of life, reaction times of sleeping piglets upon calling by the mother sow were measured. Piglets waking up immediately entered the box within 15 seconds (Video S7). If not waking up within 60 seconds, piglets were stimulated by nudging (#). Mean values are indicated.
E) and F) Respiration was measured in control and Mfn2KD C2C12 myotubes untreated or treated with NSC-134754 and ATP-coupled respiration (E) and proton leak (F) were calculated (n=8 independent experiments). Data were expressed as a fold change compared to control C2C12 myotubes.
Western blot analyses of EV preparations. (D) EVs isolated by UC under glutamine-depleted conditions as in (B) and then subjected to Proteinase K (Prot K) digestion in the presence or absence of Triton® X-100 (Triton-X). Only membrane-associated CD81 is digested in the absence of Triton® X-100, while CD63 is resistant to digestion, even in the presence of Triton® X-100.
(A) PC3 cells were incubated for migration (16h) and invasion (24h) assay. DMEM/10% FBS, together with various concentration of NaHS (10nM-1mM) acting as a chemoattractant Data information Data shown represent the means ± SD (n=3 biological replicates) ANOVA followed by Tukey's post-hoc tes was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001)
D cGAS, G3BP1 and Cy5-dsDNA were incubated. A quantitative analysis of a representative cGAS-DNA droplet is shown. Along the white line on the merged image, the fluorescence intensity of G3BP1, cGAS and dsDNA channels were recorded. Data information: Representative images are shown Scale bars, 10 μm 45 μM cGAS-mCherry, 10 μM G3BP1-mEGFP, 2 μM dsDNA were used in the assays.
A lineage tracing strategy was employed to determine the fate of CBCcells that lack Snai1. Tissue from Lgr5‐EGFP‐IRES‐creERT2Snai1+/+ (control) and Lgr5‐EGFP‐IRES‐creERT2Snai1fl/fl (Snai1KO) that also contained the ROSA26LacZCre reporter allele was stained with X‐Gal 5 days after tamoxifen injection. Quantification of X‐Gal‐positive foci per cm shows Snai1 knockout tissue failed to lineage trace indicating that no progeny are produced from Snai1 knockout cells. Bars represent mean ± SD (n = 3, P = 0.0001). Scale bars: 0.50 mm. Two‐tailed Student's t‐test was used to assess significance. See also Supplementary Figure S2.
quantitative RT-PCR (qRT-PCR) measurement of Prelid1, Tamm41, Pgs1, Ptpmt1, and Cls1 expression in Opa1Crispr and WT MEFs. Fold change is indicated relative to WT control. Data represent mean ± SD of three independent experiments, One-way ANOVA.
(B) First dimension (1D) BNGE analysis of the same cells prepared with digitonin.
(G) RT-PCR showing the downregulation of ASCL1, NEUROD1, BCL2, INSM1, MYC, MYB, AURKA and CDK7 genes after 50nM lurbinectedin treatment (light blue bars), 50nM or 20uM Cisplatin (CsPt; Light yellow and dark yellow bars, respectively) as compared to untreated samples (Ctl- Dark blue bars). Data is represented as Relative mRNA Expression normalized to Ctl. Data is presented as Mean ± SD. (n=2 biological replicates).
(D) Classification of temperature-dependence in genes with CHX-dependent splicing events. Number of genes and percentages are indicated.
Mice (N=4 per group) treated as in (A) and 6h after challenge, ileum was taken and RT-qPCR performed to study gene expression of cell death ISRE genes Mlkl and Zbp1
F Percentage of HK2 dots positive for SPLICSL in HeLa, COLO 741, MDA-MB-231, PN 04.4 and S462 cells (n≥6 cells for each cell line; mean±SD).
(d) eIF2α phosphorylation in WT or p53−/− HCT116 cells treated with PFT-α (n = 5).
Representative images of Masson's trichrome staining of heart sections from F-LARP6 KO and F-DKO mice 21 days after ISO challenge; Scale bar, 50 μm. Quantification of collagen content in mouse heart sections after ISO challenge. #P < 0.05 vs saline group (two-way ANOVA); saline groups, n = 3 each, ISO groups, n = 7 each. , Hydroxyproline content in hearts from F-LARP6 KO and F-DKO mice treated with ISO. #P < 0.05 vs saline group (two-way ANOVA); saline groups, n = 4 each, ISO groups, n = 8 each.
F. Representative Hematoxylin Eosin (Hamp;amp;E) and Masson Trichrome (MT) staining of heart sections in mice with indicated chelator treatment undergone sham or I/R. Scale bar = 1100μm. Bar graph represent quantification of tissue fibrosis. ANOVA followed by post-hoc Tukey test was performed. * P=0.048. 2-3 sections from each mice were quantified, N=4 mice for PBS, N=5 mice for DFO and N=6 mice for BPD.
Example of a region annotated "LTR" in RepeatMasker and overlapping with a PE and with a CAGE peak from the FANTOM5 consortium data, indicative of a site of transcription initiation.
B Western blot analysis of sciatic nerve protein extracts (10 μg) from female mice after PNGaseF treatment shows a similar hierarchy of PrP expression in Tg lines to that of brain (see Fig3). The asterisk indicates a greater than full-length fragment present in TgPrP(S3.F88W) mice, possibly corresponding to incomplete removal of the N-terminal signal peptide.
(A) Single Particle Tracking using PALM (SPT-PALM) was performed on α3-NKA-Dendra expressing primary neurons. A representative image shows an epifluorescence image and trajectories (strands of different colors) of minimum 5 points in length. Scale bars: 1 µm in panel (A)
J HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 were treated with Baf A1 for 6 hours and immunostained for endogenous LAMP1. Scale bars are 5 μm for the confocal microscopy images and 2 μm for the airyscans.
(c) Quantification of liposome-associated Atg3. Densitometric plot of the amount of Atg3 recovered in the top fraction in flotation assays as in b. Error bars indicate s.e.m. from three independent experiments. P values calculated as compared with putative negative control (0% PE, 400 nm). *P 0.05. Uncropped versions of all gels are included in Supplementary Fig. 7.
(H) Serial z-projections of ~4 nm thickness from one cartwheel repeat unit of C. reinhardtii. Left-most z-projections display the central hub, right-most projection shows the microtubule wall. Blue circles delineate one ring-pair. White arrows mark individual emanating spokes. White arrowheads mark fused spokes. White dashed line follows the tilt of the spokes. Black arrow indicates the final merged spoke (D1 density). Dark orange arrow marks the D2-rod. Scale bar, 50 nm.
(C and D) Thioglycolate-elicited peritoneal immune cells harvested from the mice were immediately fixed for transmission electron microscopy to visualize the appearance of macrophages (C) and DCs (D). Scale bar indicates 1 μm.
A. LukAB octamers (4TW1) highlighting the location of where the deleted residues would be in the pore structure (left- LukABmut1, right- LukABmut2), as shown from the side view of the pore. The octameric LukAB structure was obtained from the Protein Data Bank and the motifs labeled using PyMOL.
(F) IP of HTT with hydrazide-linked beads shows that both unexpanded and expanded HTT 586 aa phosphomimetics (S13, 16D-586 aa) are modified by SUMO-2. Arrows indicate SUMO-conjugated HTT. Note that all experiments including the Y2H assay were done in triplicate; representative experiments are shown. Arrows indicate SUMO-2-modified HTT.
C, Normalized spectra of liver, kidney, iBAT, and rpWAT in vivo and ex vivo. The two spectra in each plot are from the same animal. Data information: In the figure, A.U. = arbitrary units.
(e) Confocal photomicrographs of mt-Keima-transduced WT and KO MEFs treated with CCCP or DMSO vehicle for 12 h (six individual cells from each group were scanned and representative cells are shown); Intracellular mt-Keima excited at 450 nm was shown in green colour, while red colour indicated the excitation by 550 nm in the same cell. Scale bar=20 μm.
E. Z-stack maximum image projection of the EB1b-GFP plus-end reporter tracked over 30 sec, in epidermal cells at different growth zones. Scale bars 10 µm.
B Representative photographs of T2-weighted MRI of WT and C2βD1212A/D1212A mice and graph quantification of ischemic lesion after 24 hours permanent MCAO (electrocoagulation model). Data represent mean ± SEM (n=9-10 mice per group; ns, unpaired t-test).
(C) ape1Δ cells containing P22L API on a plasmid were pulse labeled for 5 min, harvested by centrifugation, and subjected to a nonradioactive chase in either nitrogen-containing (SMD) or nitrogen starvation (SD-N) medium. Samples were collected at the times indicated and immunoprecipitated with API antiserum. The resulting SDS-PAGE gels were quantified using a Molecular Dynamics STORM phosphorimager.
A Left, time lapse confocal images of LimE in single cells following RacGEF1ΔN recruitment, induced by rapamycin addition at time 0. Right, temporal profile of normalized mean LimE intensity at cell cortex (mean±s.e.m., n=20 cells).
Quantification of PML puncta at time of infection in P6 SCG neurons treated with IFNα (600 IU/ml) for 18 hours prior to infection or for 18hours at 3 days prior to infection. n=60 cells from 3 biological replicates.
Representative images of H&E-stained lung sections from KrasG12D mice treated with either vehicle (control) or the ADAM17 prodomain inhibitor, A17pro (1mg/kg, 3 times a week), for 6 weeks starting on the day after Cre inhalation. Scale bars, 3mm. Quantification of lung parenchyma area occupied by tumor lesions in the indicated groups (n = 6 per group)
(C) WT and EGFR-/- BMDMs were transfected with cGAMP; 5h post transfection, IFNα mRNA induction was measured (n=4).
B, D. Calbindin (green) and 4',6-diamidino-2-phenylindole (DAPI)(blue) staining of micecerebellum indicates that there are swollen PC axons (arrows) in the granular layers of the Cacna1atg-lamicecerebellum at day 25 (B) and the Cacna2d2du-2Jmice at day 50 (D). Scale bar, 20 μm
F miR-150 levels in HFL1 fibroblasts transfected with a control plasmid (Ctrl) or a miR-150 expression plasmid (miR150). * P = 0.038.
Figure 7c. Down-regulated genes tend to be longer than not-regulated genes, while up-regulated genes show little difference. Box plots show length of genes with no differential expression (-0.10.01), up-regulated (log2 fold-change≥0, p<0.01) or down-regulated (log2 fold-change ≤0, p<0.01) as determined by DESeq2. P-values from two-sided Wilcoxon rank sum tests comparing median lengths for up- and down-regulated genes, respectively, to non-regulated genes are indicated on top. n=3 replicates.
(D) Flag pull-down assays were performed with Flag-EV or Flag-fused CaMKK2 protein and His-CARS purified from E. coli.
Biochemical enrichment of PTPδ protein in the hippocampal SLM layer relative to the SR layer. Micro-dissected SLM and SR lysates (2 months) were immunoblotted for PTPδ (C-terminal antibodies) and α-tubulin (control). The asterisk indicates a non-specific band. Analysis of immunoblots reveal significant enrichment of PTPδ in the SLM compared to the SR. (n = 4 mice for SR and SLM, mean ± SEM, ***p < 0.001, Student's t-test; See Dataset EV2 for statistical details for these and all following results).
A. Schematic diagram of constructing chromosomal mutations in rpoC gene in E. coli based on a CRIPSR/Cas9 system. A 20-nt guide RNA targeting a region between K74 and K87 on β′ subunit is shown in purple. Double-strand DNA fragments, which contains synonymous mutations (yellow background) in sgRNA targeting region to facilitate clone selection and aimed mutations at K74 or K87 or both residues (only example for constructing the Ec-K74A-K87A strain is shown), were used as donor templates.
A. Representative traces of Ca2+-currents and 4-AP insensitive K+-currents evoked by step depolarizations (from −50 mV to +10 mV in 10 mV increments), recorded from the calyx terminals in acute slices from littermate control and Nrxn123 TKO mice at P12-P14. B. Same as (A) but after addition of 200 nM iberiotoxin (IbTx) by perfusion. C. Representative traces of presynaptic BK currents , which are calculated by subtracting currents shown in (B) from currents shown in (A). D. Summary graphs of the capacitance, Ca2+-current density, and BK-current density.
B The immunoprecipitation analysis. Cytoplasmic ENTREP co-precipitated with either ITCH C830A, delWW12 or delWW34 but not with delWW14. Data shown are representative of at least two independent experiments. C The immunoprecipitation analysis. HA-CXCR4SS-DsRed hardly co-precipitated with either ITCHC830A or delWW mutants. HA-CXCR4DD-DsRed co-precipitated with ITCH C830A and delWW12 but not with delWW14 and delWW34. Data shown are representative of at least two independent experiments.
Genes repressed by ERS in MPH (3 independent experiments) (J) were ranked based on their Log2 FC ERS/Control and divided into quartiles (increased repression from Q1 to Q4). The fraction of LIVER-ID genes in the 4 quartiles was defined and is displayed relative to that obtained for Q1 arbitrarily set to 1. Chi-square test with BH correction for multiple testing was used to assess statistical significance, *P < 0.05.
F-I. Traction force microscopy analysis of wt and Sharpincpdm MSFs on collagen-coated matrices. F. Representative images of traction forces observed in wt and Sharpincpdm cells (colour scale units: Pa; cell boundaries are indicated by dashed lines). G. Quantification of strain energy per cell (n = 8 − 11 cells from two independent experiments).
Representative images of combined EdU staining (Click-it) and DNA FISH for telomeres. HuO9 or SaOS2 cells were transfected with 20nM siRAD54#2 (representative images for siRAD54#1 not shown) for 48 hours followed by a 1.5 hr pulse of EdU. Pan-nuclear EdU stain represents S-phase cells. White arrows indicate non-S-phase cells containing EdU colocalizing with telomeres. Scale bars = 10 µm Quantification of data shown in A. HuO9 or SaOS2 cells were mock transfected or transfected with 20nM siRAD54#1 or 20nM siRAD54#2 for 48 hours followed by a 1.5 hr pulse of EdU. Cells with pan-nuclear EdU signal or greater than 8 EdU foci were excluded from analysis as S-phase cells. Non-S-phase cells were considered positive if they contained at least one EdU foci colocalizing with telomeres. Data were normalized to mock condition for each repeat. At least 100 non-S-phase cells were counted per condition per repeat, n=3. Values shown are mean±sd. Data were analyzed using two-way ANOVA followed by Dunnett's multiple comparison test.
(D) The steady-state accumulation of endogenous Ubp6 and Ubp6 derivatives with short (19 amino acid) or long (54 amino acid) tails at their C-termini. The 19 amino-acid tail destabilized Ubp6 and the 54-amino-acid tail lead to complete depletion of Ubp6. Protein levels were determined by western blotting for the HA-tag, Ubp6 or Scs2 in SDS-PAGE gels of S. cerevisiae protein extracts. The proteasome was inhibited by 100 µM bortezomib as indicated. Protein loading levels in each lane were normalized in all lysates by western blotting for the integral endoplasmic reticulum membrane protein Scs2.
(D) Steady-state accumulation of endogenous Mdy2 in S. cerevisiae, with deleted (get4∆) or endogenous (wt) Get4 in the presence or absence of the proteasome inhibitor bortezomib. Protein levels were determined by western blotting for the HA-tag, Flag-tag, Mdy2 or Scs2 of SDS-PAGE gels of S. cerevisiae protein extracts. Protein loading levels in each lane were estimated in all lysates by western blotting for the integral endoplasmic reticulum membrane protein Scs2.
(D and E) RNAi against SH3PX1 inhibits formation of LTR (D) and mCherry-Atg8a-positive structures (E) at early stages of developmental autophagy in fat bodies of 110-h-old larvae. Genotypes: (B and D) hs-flp; UAS-dicer/+; Act>CD2>GAL4 UAS-GFPnls/UAS-SH3PX1 RNAi; (C and E) hs-flp; UAS-dicer/+; r4:mCherry-Atg8a Act>CD2>GAL4 UAS-GFPnls/UAS-SH3PX1 RNAi;
The role of skeletal muscle miR-183 and miR-96 in fuel metabolism. The effects of miR-183 and miR-96 deficiency (genetic ablation) and downregulation (HFD feeding), and low levels of miR-183 and miR-96 (more oxidative myofibers) on gene expression, enzyme or transcription factor activity, and metabolic pathways involved in fuel usage are shown by up/down arrows or cross in red.