EMBO/sd-character-level-ner huggingface dataset:
C3H mice were injected with CoPP, SnPP or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 hours after the last injection. Heme degradation reaction catalyzed by HO-1.
The oxygen consumption rates in adipocytes treated with NC, QKI siRNA or both QKI and PGC1α siRNA. (n=3 biological replicates)
(B) Viral infection assays determining virus titers by end point titration at 8 hours in HeLa wild-type and PI4KB knockout cells upon transfection of wild-type PI4KB, the complex-disrupting RL494EA PI4KB mutant or the kinase dead D674A PI4KB mutant. Left: Coxsackievirus A10 infection. Middle: Poliovirus-1 infection. Right: Coxsackievirus B3 infection. Values were statistically evaluated compared to the GalT control using a one-way ANOVA. **, P<0.01; *, P<0.05; N.S., P>0.05.
i) Flow cytometry of myeloid blood cells from Ccr2RFP/+ mice. Left: CD45+CD11b+ cells were further subdivided according to the expression of Ly6C and CD115 into CD45+CD11b+CD115-Ly6Cint granulocytes (gate 1), CD45+CD11b+CD115+Ly6Chi inflammatory (gate 2) and CD45+CD11b+CD115+Ly6Clo resident monocytes (gate 3), respectively. Right: representative histograms are shown (Ccr2RFP/+ solid red line, Ccr2+/+ controls dotted grey line). Four mice were investigated.
A. Scatter plot of the relative timing of cell constriction versus the relative timing of nucleoid separation. The gray scale indicates the density of dots in a given area of the chart. The dotted contours represent the 0.5, 0.75 and 0.95 probability contours of the 240 WT replicates. The Pearson correlation is ρ = 0.65, with a 95% CI = [0.63, 0.66]. The black dotted diagonal represents the line where a strain should be if both nucleoid separation and cell constriction happens at the same time. Red dots highlight strains
B) Inhibition of palmitoylation (via two independent rasp RNAis) rescues NB EdU incorporation defects induced by cortex glial (NP2222-GAL4>) htlACT overexpression, while knockdown of Rasp in cortex glial cells alone does not alter NB EdU index (n=10, 15; 10, 16; 18, 21; 26, 29 brain lobes).
(E) Cells depleted of ATP5ME were immunolabeled for Mic60 and recorded with STED. The arrows point to crossing points of the opposite Mic60 distribution bands.
(A) Proteomic profiles of different proteins, showing the relative abundance distribution across the 3x3 subfractions obtained from Jurkat cells. Proteins with very similar profiles (represented by the same colours) are likely part of the same EV subtypes. Each profile consists of three independent data triplets (F1- F2- F3A, F1- F2- F3B and F1-F2-F3C).
D: Transient PIKfyve suppression in thapsigargin-treated Gt1 cells with recovery after 6 hours. PERK phosphorylation confirmed ER stress.
A Representative images of 1205 cells cultivated on collagen-I (Coll-I) or FRC-derived ECM for 48 h prior treatment with vehicle or 5 µM BRAFi or 2 µM BRAFi plus 0.01 µM MEKi for 96 h. Immunofluorescence for Phospho-DDR1 (P(Y792)-DDR1) (red; left panels) and Phospho-DDR1/2 (P(Y796)-DDR1/P(Y740)-DDR2) (red; right panels), F-actin (green) and nuclei (blue) is shown. Enlarged images of P-DDR1 and P-DDR1/2 immunostaining are shown. White arrows indicate P-DDR1 and P-DDR1/2 cell membrane linear clustering. Scale bar, 25 µm (enlarged images: scale bar, 10 µm).
(H) The supernatants of liver lysates form WT or PAQR3 knockout mice were subjected to gel filtration analysis, followed by IB.
(E) Ultrastructure of autophagic vacuoles induced by ABT737. Transmission electron microphotographs are shown.
B, Reconstructed MSOT image (800 nm) with linear unmixing and difference value of lipid in lower abdominal section. Unmixing result: blue for Hb, red for HbO2, yellow for lipid, jet for 700 nm - 930 nm difference. The colour bar shows the colour coding of MSOT a.u. from 0 to maximum (bottom to top) (Maximum value control/steatosis: Hb: 1.1/0.5; HbO2: 1.9/0.8; Lipid: 21000/24000; 700-900nm: 3000/3000) Scale bar: 4 mm.
(B) cABN derived from a Pearson coefficient threshold of 0.832, consistent with constraints previously defined for ESC self-renewal (Dunn et al. 2014). Solid arrow, required interaction; dashed arrow, possible interaction; black arrow, activation; red arrow, inhibition. There is no regulation hierarchy associated with component positioning
E. Immunoblots of LDLR and Mpdz, with β-actin as loading control. The MPDZ immunoblot shows the efficiency of the shRNA-mediated knockdown. Bands from three immunoblots were quantified by normalization to β-actin bands (mean ± SD).
C Cardiac mitochondria were solubilized in 0.4% DDM and complexes were separated by BN-PAGE and analyzed by Western- blotting with the indicated antibodies.
e) Flow cytometric quantification of RFP+ cells in Ccr2-RFP mice among CD45+CD11b+CD115-Ly6Cint granulocytes (triangles, n = 4), CD45+CD11b+CD115+Ly6Clo monocytes (filled squares, n = 4 mice), CD45+CD11b+CD115+Ly6Chi monocytes (open squares, n = 4 mice), CD45loCD11b+ rMG (squares, n = 6 mice), CD45+CD11b+CD64+F4/80+ cbMΦ (triangles, n = 6 samples from twelve mice) and CD45+CD11b+CD64+F4/80+ cMΦ (filled circles, n = 12 mice, unpaired t test ***p < 0.0001). Data were obtained from one (rMG) or two independent experiments (blood, cbMΦ, cMΦ). Data are presented as means ± s.e.m.
F Immunoblot analysis of SHEP cells transfected with plasmids encoding wild-type CSPα-mCit-PA and mutant variants. Blot was developed with an anti-CSP antibody; loading control: anti-Histone-H3 antibody. Arrowhead and bracket indicate monomeric CSPα-mCit-PA and SDS-insoluble protein aggregates, respectively.
Amount of GS-MQ at indicated timepoints after incubation of GS-MQ +/- NAC (n = 2). See Fig. EV4 for chemical reaction. Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4.
Relative increase of mitochondrial proteins was normalized to mtDNA copy numbers and plotted (E).
D Day-18 tumor weights at the endpoint of (C) [n=11, PBS (-) injected tumor-bearing WT and KO mice; n=12, anti-Ly6g antibody (+) injected tumor-bearing WT and KO mice].
A The intrinsic fluorescence of NAD(P) H co-localizes extensively with mitochondria (marked by Mitotracker CMXRos)
(G) Schematic illustration of the novel object recognition test. After a 30 min habituation phase, Shank2+/+ and Shank2-/- mice were allowed to investigate two identical objects in the open field arena (training session). 10 min later, one of the objects was replaced with a novel object (test session). Lower panels show a representative tracking path for a mouse in each test session.
(A) PKH26 (PE)-labelled RM1 (V500/eCFP) cells were injected intracardiac (IC) into C57BL/6 mice and FACS-isolated from bones with evident tumor burden (8 individual mice across 5 independent experiments) from day 16 onward and individual dormant and proliferating cells were isolated for scRNA-seq. Representative tumor burden at the whole mouse level and in bone shown by bioluminescence with representative FACS plots of PKH+ (PE/V500) and PKH- (V500) RM1 cells and bone marrow cells (grey).
(G) Immunofluorescence (IF) assay was performed to determine the subcellular location of WT or mitochondrial signal peptide deleted (dMSP) GDH1. GDH1 localization was indicated by GDH1 antibody and MitoTracker (left). Signal intensities and distances were quantified (right). Scale bars: 20 μm.
Haematoxylin/Eosin (HE) staining and in situ hybridization of specific (uc.291) or not specific (Negative Control, NC) probes on human skin section. Scale bars denote 50 and 500 µm.
I) Effect of TRAIL (1µg/ml), COMBO and their combination on HCT DKO and TKO cell death at different time points normalized to control cells. Each dot corresponds to the average of 3 technical replicates from 4 independent experiments. *** p<0,001.
A. Live imaging of MCCs shows the distribution of Ccdc108 in MCCs cilia and basal bodies. Embryos expressing GFP-Ccdc108 (green) and Cetn4-RFP (purple) were imaged at stage 27. To better visualize ciliary structures, maximum intensity projections of images stacks for regions containing cilia and basal bodies are shown separately.
Vote counting of cancer tissue metabolites (reported in at least six cohorts) showed consistently deregulated metabolites. Blue bars: decreased metabolites; red bars: increased metabolites. * (asterisk) in front of the name of the metabolite indicates a statistical trend (p < 0.1); red arrows denote key metabolites mentioned in the main text (see Table EV5 for full annotation).
(A) IF for pan-cadherin, combined with DNA staining, to quantify asymmetric vs. symmetric division in VZ APs. Dashed white lines denote the cleavage plane, and solid lines mark the "cadherin hole". Asterisks mark the apical-most basolateral membrane flanking the "cadherin hole". Cell divisions were scored as symmetric or asymmetric when the cleavage plane bisects (left) or bypasses (right) the cadherin hole, respectively. Scale bar 2 μm.
C-E. Involvement of myosin motors in BiFC foci polarization. Time-lapse micrographs of agarose-embedded cells carrying the COPI•Dsl BiFC pairs Dsl1pVN•ε-COPVC or β'COPVN•Dsl3pVC were taken. Either wild type cells (E) or mutants carrying the myosin V mutations myo2-66 (C) or myo4∆ (D) were analyzed. Cells were grown at RT with fresh PM Glc+ura medium supply. In the myo2-66 cells, the BiFC signal polarization was hardly detectable even at permissive temperature, while it was unaffected in myo4∆ compared to the control cells.
serum FITC-dextran level of colon section from the pre-treated WT colitis mice on day 8 (n = 8).
E ChIP-qPCR analysis displaying the fold-enrichment of the indicated proteins and histone H3 acetylation at the TSS of CDK12-target genes in the indicated cell lines. Mean ± s.d. (n = 3). P-values by two-tailed Student's t-test. Cell line information: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells S, trastuzumab-sensitive.
A, Western blot of BT463NS showing phosphorylation of ATM (pATM) and Chk2 (pChk2), and accumulation of RAD51 at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + HGF) of HGF (10 ng/ml). Total ATM is also shown. H3 was used as loading control.
Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3, followed or not by infection with NL4-3 EGFP-Nef+ HIV-1 (red boxes). (B) Number of particles secreted were quantified by NTA. Results are expressed as a ratio to the number of particles secreted by uninfected luciferase shRNA cells.
F-G, shows flow cytometric analysis of CD206hi (F) TAMs in MC38 and KPC tumours 5 days following IR. Data are presented as mean ± SEM and analysed by one-way ANOVA with Tukey post-hoc adjustment (n=6 mice/group).
Viral titres in the supernatants were measured by the cytopathic effect method. The results represent the means ± SD from three independent experiments. Statistical significance was analyzed using Student's t test (**P < 0.01).
(A) Summary and representative analysis of NET formation of pPMNs stimulated by 50 nM PMA for 3 h, detected by fluorescent microplate reader and confocal microscopy, respectively. Scale bar 20 µm.
Cellular localization of ecGAMP in WT and CGAS KO THP-1 cells stimulated with FITC-ecGAMP (5 μg/ml, green) for 2 h, nucleus in blue (DAPI). Data are presentative of 5 independent experiments. Scale bar, 10 μm.
(A) After incubation of BMDCs with HK parasites or STAg, TLRs are stimulated and induce transcriptional activation of the three pathways of the UPR. The IRE1α/XBP1s pathway stimulates the secretion of IL-6 and IL-12 by BMDCs. Blue arrows indicate down-regulated pathways and red arrows, up-regulated pathways.
Model for TRAPPIII-mediated Rab1 activation in cells. Inactive Rab1-GDP/GDI exists in an equilibrium where Rab1-GDP samples various organellar membranes. TRAPPIII is recruited to specific membranes containing negative charges (red circles) and utilizes an amphipathic helix to bind stably to the membrane surface. Rab1 is locally activated by TRAPPIII in order to recruit downstream effectors to facilitate trafficking or autophagic events.
(f) Bif-1 knockdown HeLa cells were transfected with GFP-LC3 in combination with Bif-1 shRNA-resistant Bif-1-HcRed, Bif-1ΔBAR-HcRed or empty HcRed vector for 20 h. The cells were then cultured in EBSS or complete medium for 2 h and the number of GFP-LC3 dots per HcRed-positive cell was determined using a fluorescence microscope (mean ± s.d.; n = 35).
E. MD derived binding mode of praja2530-570 (green cartoon) to the C-terminal region of TBC1D31 (red cartoon).
(F) RNA levels of Tsc22d1 and the same three target genes is shown as average normalized tag counts derived from RNA-seq data from Ventricular Zone (VZ), Sub-Ventricular Zone (SVZ) and Cortical plate (CP) of E14.5 mouse Cortex.
C HEK293 cells previously transfected with EGFP-NAV3 (WT), EGFP-NAV3-D1047N or EGFP-NAV3-A459P were treated with cycloheximide (CHX; 50 μg/ml) or with the solvent, DMSO, for the indicated time intervals, followed by cell lysis and immunoblotting using anti-EGFP and anti-α-tubulin antibodies. The signals obtained were quantified and normalized to time zero (untreated cells; right panel).
(I,J) Quantifications showing almost 100% of co-localization of COX6A2+ cells and PV+ cells in the somatosensory (SS) and motor (MT) cortices, and the hippocampus (HI) of adult mice (n animals=4, 2 months old, n sections=24 SS, 16 MT, 8 HI).
(B, C) PPM1D-dependent dephosphorylation of Ulk1 at Ser637 and the induction of autophagy. PPM1D+/+MEFs and PPM1D−/−MEFs were treated with etoposide (10 µM), and the expression of each protein was examined by western blotting. Dead cells (%) indicates the population of apoptotic cells. (C) Semiquantitative analysis of protein expression is shown (n = 3; mean ± SD). *p < 0.05, **p < 0.01, NS: not significant. (D-I) Suppression of etoposide-induced autophagy in PPM1D−/−MEFs.
B. Overexpression of MITOL CS mutant fails to degrade Parkin. HeLa cells stably expressing HA-Parkin were transfected with the indicated vectors and treated with DMSO or CCCP (10 µM) for the indicated time. Lysates of cells were subjected to an IB assay with the indicated antibodies. MITOL CS mutant; lacking ubiquitin ligase activity.
M-O Number of Ki67-positive (M), number of γH2AX-positive (N), and number of active caspase-3-positive (O) cells per field in the lung tumors.
F Caco-2 cells were stimulated by OMVs (100 μg/mL) with or without apilimod (30 nM) for 24h. Proteins of lysates extracted from Caco-2 cells were subjected to immunoblots with the indicated antibodies.
(B) 25HC activates ACAT and stimulates the formation of lipid droplets. Calu-3 cells were cultured in medium containing 2% LPDS prior to the treatment of ethanol or 5 µM 25HC for 16h. Subsequently, cells were stained by LipidSpot to visualize intracellular lipid droplets. Scale bar, 50 μm.
(b) O2 consumption rates (OCR) in stable HeLa cells expressing irrelevant shRNA, clone shHe2, and clone shHe2 re-expressing MCUR1, exposed sequentially to oligomycin (i), FCCP (ii) and rotenone plus myzothiazol (iii).
Overlaying of the amplifications in chromosome 6 across multiple regions showing that all amplicons contained the cMet oncogene. Colour of the block corresponds to the segment mean (the degree to which a genome segment is lost (blue) or gained (red).
(B and C) A549 (B) lung cancer cell lines stably expressing the indicated constructs were counted with Trypan blue for several days following stress stimuli (over-confluency (O-C) for A549). Data was normalized against 1st day values. Δ160p53 shows similar pro-proliferative capacities as mutant R273Hp53. On the other hand R273Hp53 lost pro-proliferative functions when deficient for Δ160p53 expression (M160AR273Hp53). Also shown is the WB of A549 cells used in (B) and stably expressing the indicated constructs.
(H) IDH1 K224 deacetylation promoted GSH production in HCT116 cells. In HCT116 stable cells with IDH1 knockout and re-expressing the indicated proteins, the ratio of GSH/GSSG was assessed as followed by manufacture's instruction. 1 Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For H, statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01. n.s. = not significant.
Anti-nuclear-IgM (ANA) of control-immunized (CI, n=3) and complex InsA peptide (cInsA) immunized mice on day 7 (n=3) and day 85 (n=3) analyzed by indirect immunofluorescence. We used (E) total serum (dilutions: 1:20) or (G) Insulin-specific IgM (concentration: 500 ng/mL) purified from serum at indicated days on commercial HEp-2 slides. Scale bar: 10 µm. Green fluorescence indicates IgM bound to nuclear structures. Data are representative for three independent experiments with isotype control: n=3, day 7: n=3, day 85: n=3.
(D). Control fibroblasts, non-transfected or knocked-down for MIC60 and MIC19, were analysed by immunoblot for expression of SLC25A46, MIC60 and MIC19. SDHA was used as a loading control.
C) Representative immunofluorescence staining of cortical neuron cultures transduced at 8 DIV (days in vitro) with 10e9 VG/ml of AAVRh10-GFP or AAVRh10-∆N-DGKk and assessed after 5 days using anti-MAP2 and anti-HA for ∆N-DGKk or direct 488 nm excitation for GFP. DAPI was used to visualize nuclei on merged images.
C Fasting insulin levels of mice in (A) at 10 weeks of HFD. Blood samples were harvested after eight hours of fasting. n=5 for GFP and n=4 for PLTP. *P<0.05 relative to GFP by Student's t-test.
(B) Stable sheath foci formation in A. baylyi cells that are in contact with E. coli. A. baylyi cells lacking TagX (ΔtagX tssB-sfGFP) were mixed well with cells of E. coli (MG1655 Ptac-mRuby3) and incubated on an LB agar plate for at least 90 min before imaging. Yellow arrows indicate sheath foci that are stably present at the contact site with E. coli. Data information: Scale bars and time intervals are indicated.
I) Quantification of dystrophin western blot in panels F and G. Dup18-30 untreated, n = 2; Dup18-30 treated, n = 4.
C IFA analysis showed that the down regulation of PKAr-TyDD leads to the abnormal segregation of the apicoplast-associated thioredoxin family protein (ATrx1) 24 hours after Shld-1withdrawal.
D. Immunoblotting of ATF4 and ASNS in melanoma cells 72 hr after treatment with siASNS, siATF4, or both.
(B) Survival rates of mice treated with wtSPINK6 and mutSPINK6 was analyzed with Mantel-Cox test.
Representative images of open field test showing tracking pattern (open arena) of WT (females, n=7, males n=6) and AAV-mWwox-injected KO mice (females n=7, males n=5) at 8-10 weeks. Dot plot graphs represent the velocity (B), total distance travelled (C)
(H) Schematic model of PGC-1α-NRF1-FUNDC1 axis in mitochondrial quality and quantity control and adaptive thermogenesis.
The oxygen consumption rate (OCR) of 1×105 differentiated myeloid cells were monitored after the addition of oligomycin (OA) (1 µM), the uncoupler carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 1 µM), and the electron transport inhibitor rotenone and antimycin A (R/AA, 5 µM) at indicated time points. The basal OCR, basal extracellular acidification rate (ECAR), spare respiratory capacity, ATP production and maximal respiration based on the OCR value were quantified.
(B) Immunostained images of neonatal rat cardiomyocytes 4 hours after treatment with 0.01% DMSO (Control, upper row) or 2 μM MYK-461(MYK-461, bottom row). These cells were stained with an α-tubulin (green) and a plakoglobin (red) antibody.
(A) K-means clustering of gene expression data for Th0 cells and iTreg cells differentiated in vitro under the conditions of TGFβ alone, TGFβ + VitC, TGFβ + RA; TGFβ + RA + VitC using genes differentially expressed between the pairwise comparisons indicated by the arrows on top of the heatmap. The number of genes in each cluster is shown on the left.
(b) LC3 dot counting in CALM knockdown cells. CALM was knocked down in HeLa cells expressing GFP-LC3 and with or without an siRNA-resistant form of wild-type CALM (rescue), as indicated. The cells were starved in HBSS for 4 h, fixed and subjected to fluorescence microscopy to score the number of LC3 dots per cell. The knockdown efficiency and the level of the siRNA-resistant form of CALM are shown on the left on the western blotting. The number of LC3 dots per cell (shown as mean ±s.d.) is shown on the graph for each condition (n≥300 cells per condition; BC, basal conditions). The ratio of the number of LC3 dots per cell between starvation and basal conditions is shown on the right (*P0.01; NS, not significant, two-tailed t-test). Pictures obtained from automated microscope are shown. Scale bars, 20 μm.
(G, H) Quantification of the percentage of oocytes (G) with chromosome segregation errors in anaphase I and (H) with defective MTOC coalescence during metaphase I. Means ± SDs, oocyte numbers are shown in brackets.
Longitudinal tumor growth follow-up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination. n represents number of tumors, in A: vehicle n=16, PI3Ki n=8, ERKi n=16, Combination n=8 Pvalues represent the mean of all the time points
L: Wt and null neurons counterstained with an antibody against Tuj1 and DAPI at different time points. The binary mask is representative of a neuron used to study morphology. Scale bars: 30 µm. M-Q: Each graph represents a different morphological parameter analysed at selected time point. Two-way ANOVA indicated both a significant time effect ((F 4,20)=282,5 and p<0,0001 for the number of intersections; (F 4,26)=104,5 and p<0,0001 for the total dendritic length; (F 4,20)=58,51 and p<0,0001 for the maximal dendritic length; (F 3,22)=38,32 and p<0,0001 for polarization) and genotype effect ((F 1,20)=25,1 and p<0,0001 for the number of intersections; (F 4,26)=16,0 and p=0.005 for the total dendritic length; (F 1,20)=17,24 and p=0,005 for the maximal dendritic length; (F 1,22)=18,76 and p<0.003 for polarization). Bonferroni multiple comparison test was used to assess difference between wt and null neurons at each analysed time point. The dotted line in panel P represents the cut-off above which a culture is defined polarized (Horton et al, 2006). Values are represented as mean ± SEM; *: p-value<0.05; **: p-value<0.01; ****: p-value<0.0001. Sample size: n≥3 wells deriving from three independent preparations; for each well we measured at least 15 cells.
(H,I) HeLa cells were co-transfected with TDP-43ΔNLS-GFP and iRFP or GA175-iRFP for 2 days. For the final 16h, cells were treated with either vehicle or rolipram (30 µM). Filter trap assay of SDS-insoluble TDP-43ΔNLS-GFP aggregates quantified by densitometry. n=3 biological replicates. Scatter dot plot, mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.
D. Spermatocytes were immunolabeled against gamma-tubulin (green), alpha-tubulin (red), and stained with DAPI (blue). Plk1 and Aurka cKO spermatocytes arrest at a prometaphase stage. Scale bars = 5 μm.
E Quantification of DDX39B binding to different interaction partners. Quantification of V5-tagged DDX39B protein amount was normalized over HA-tagged interaction partner intensity and displayed as a fold change over the ratio of untreated cells. Data that represent the mean of 3 replicates are highlighted in red. A fold change > 0.5 is considered significant.
Cultured astrocytes engulf microglial debris. CAG-EGFP mouse-derived primary astrocytes were co-cultured with or without fluorescence-labelled apoptotic microglia. (A) Fluorescence images of GFP (green), microglial debris (red) and GFAP immunoreactivity (blue). Images were acquired using the same laser power and sensitivity, and image processing was the same in the two images (red). Scale bar, 50 µm.
IKKε expression was assessed semi-quantitatively using a 0-3 grade scale ("0": no staining; "1": weak; "2": moderate; "3": strong) on human breast cancer sections using immunohistochemistry. Representative images according to the IKKε staining grade are shown. Scale bar: 100 μm. Distribution table of IKKε expression in the cohort of 66 human breast carcinomas according to the staining grade.