EMBO/sd-character-level-ner huggingface dataset:
(F) 293T cells were co‐transfected with expression vectors for HA‐tagged IRE1α (IRE1-HA), TRAF2-FLAG, and MYC‐tagged BI‐1 (BI1-MYC). After 48 h of transfection, HA‐tagged proteins were immunoprecipitated and the possible interaction with TRAF2 was analysed by western blot. Right panel: the percentage of TRAF2 dissociation from IRE1α by the presence or absence of BI‐1 was quantified and normalized with the expression levels observed in the inputs. For comparison, the co‐IP signal observed in the absence of BI‐1 was normalized as 100% co‐IP in each independent experiment (N=3). Mean and standard deviation are presented, *P0.05. Figure source data can be found with the Supplementary Information.
Reduction of cell wall synthesis gene expression causes a tolerance defect. (C) Survival of a ∆pbp1b ∆pbp1a Piptg:pbp1a strain after 6 hours of PenG exposure. Pup indicates conditions of high IPTG concentrations (100 µM), Pdown low IPTG concentration (2 µM). Survival fraction is cfu/ml of PenG-treated cultures normalized to untreated cultures grown in and plated on medium containing the same IPTG concentration.
(E) ChIP followed by PCR confirmed that PBX1 binds to genomic regions in close proximity to the TSS of Pitx3 (Pitx3-Gbf1), Onecut2and Nfe2l1 genes. No co-immunoprecipitation was detected for the unrelated gene, Lrp1b (Low Density Lipoprotein Receptor-Related Protein 1B).
A Luciferase assay of NF-κB activation in HEK293T cells transfected with WT or mutant Mtb PknG. The graph shows the mean ± SEM of three independent biological replicates, n = 3. *P < 0.05, **P < 0.01. Data were analyzed according to one-way analysis of variance (ANOVA) using GraphPad Prism 7.
(b) Immunofluorescence of LC3 in control MEFs (Ctr) or those knocked down (KD) for three different Cx maintained in the presence or absence of serum. Left: representative fields and higher magnification insets. Nuclei are highlighted with DAPI. Right: Quantification of the average number of LC3-positive puncta per cell (n = 3 wells, 3 independent experiments, >50 cells per experiment).
U2OS CALR-RFP were injected subcutaneously (s.c) in the flank of nu/nu mice. Tumors were further injected with PBS (Ctr) (n=5), 0.5 mg/kg tunicamycin (TM) for 6 h (n=3) or 0.5 mg/kg DACT for 6 h (n=4) or 24 h (n=3). Tumor slices were cut and stained with a peIF2α antibody followed by an AlexaFluor-647 secondary antibody and counterstained with Hoechst 33342. Two slices per tumor were imaged for their DAPI, RFP and Cy5 signals. Out-of-focused images were removed from the dataset leading to the analysis of the following conditions: 8 x Ctr, 5 x TM, 8 x DACT 6 h and 6 x for DACT 24 h. PeIF2α was quantified measuring Cy5 intensity in the cytoplasm (K, L) and CALR translocation by measuring the coefficient variation (CV) of the RFP signal in the cytoplasm (M, N). Representative images of peIF2α are shown for Ctr, TM and DACT at 6 h (K), whereas images of CALR are shown for Ctr and DACT at 24 h
C: Representative immunoblots of FATP2, CD36, FABP1 and GADD45β from liver whole tissue lysate (W) as well as fractionated organelles/intraceuular structures including nuclei (N), mitochondria (MT), microsomes (MS) and cytoplasm (C), from GADD45β+/+ (WT) and GADD45β-/- (KO) mice. D: Quantified band densities of FABP1 enrichment from fractions in C (n = 4/group).
SFA/UFA-ratio of WT and indicated mutant strains overexpressing SCT1 cultured without (data taken from Fig 5A) and with 1 mM ethanolamine (etn) or 1 mM choline (cho) as indicated. Data is presented as average ± SD (n≥3). * p < 0.05, ** p < 0.01, *** p < 0.001, unpaired t-test of the indicated bar compared to the corresponding condition of WT. Underlying data for this figure can be found in Appendix Table S1.
F) Graph indicating the inter-axis distances measured in wild-type pachytene bivalents; pachytene-like Rec8−/− and zygotene-like Stag3 mutant and Smc1β−/− univalents. Each measurement in wild-type and Rec8−/− corresponds to the median of 3 distances measured along one homolog (n=60). Each measurement in Stag3 mutant and Smc1β−/− mutant axes correspond to 1 distance measured at sites of local separation of AEs (n=40 and n=20, respectively). Horizontal lines indicate median.
E. β-catenin-mediated reporter activity in HEK293T cells co-expressing oncogenic RNF43 (R519X) and dominant negative ∆N-TCF4. Cells were treated with control medium (no Wnt3a) or Wnt3a-conditioned medium (Wnt3a) overnight. Average β-catenin-mediated reporter activities ±s.d. in n = 2 independent wells are shown.
Scheme of the experimental setup. Σ1278b MATa yeast cultures were grown and treated in biological triplicates with the indicated stimuli in a three-point time course. Proteins were purified and digested using R2-P2 with Fe3+-IMAC or Ti4+-IMAC. The spectrum library was generated by DDA measurement of pooled time points of Fe3+-IMAC and Ti4+-IMAC enriched samples. The chromatogram library was generated by measuring a pooled sample from Fe3+-IMAC enrichment of all conditions and time points in seven staggered narrow-window DIA-MS runs. The Fe3+-IMAC fractions of each sample were measured in a single injection wide window DIA-MS experiment. The time used for sample preparation and measurement is indicated on the top and numbers of identified phosphopeptides on the right.
(E) Immunoblot of DMSO and SPHINX31 pre-treated HEK293T cells expressing AUG-nLuc-3xFLAG control or CGG-nLuc-3xFLAG RAN translation reporters. (n=3 biological replicates). (F) Anti-FLAG of DMSO and SPHINX31 pre-treated HEK293T cells expressing GGGGCC-nLuc-3xFLAG (GA70) and +2CGG-nLuc-3xFLAG (FMRpolyA) RAN translation reporters (n=3 biological replicates). Data information: Error bars represent mean +/- SD. Statistical analysis was performed using two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.
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). F Measurement of TO-PRO-3 uptake as in (B). Dead TO-PRO-3high cells were discarded. Data are means ± SEM of three independent experiments. *P<0.1, ****P<0.0001 (ANOVA).
(C) Cathepsin B (CtsB) activity in RPE lysates from miR-211-/- and WT mice sacrificed 3h after light on at 10 AM (diurnal condition). RPE was isolated 1 week after DMSO or NSC668394 treatment from 2-month-old miR-211-/- mice. CtsB was rescued in NSC668394-treated miR-211-/- compared to DMSO-control miR-211-/- mice. Bar graphs represent percentage of CtsB activity ± s.e.m. of independent experiments (n=3 mice) Mann and Whitney test (DMSO-miR-211-/- vs WT; NSC668394-miR-211-/- vs DMSO-miR-211-/-), *p ≤ 0.05. (D) GUSB activity from retina from miR-211-/- and control mice sacrificed 3h after light on at 10 AM (diurnal condition). Retina was isolated 1 week after DMSO or NSC668394 treatment from 2-month-old miR-211-/- mice. GUSB was rescued in NSC668394-treated miR-211-/- compared to DMSO-control miR-211-/- mice. Bar graphs represent percentage of GUSB activity ± s.e.m. of independent experiments (n=3 mice). Mann and Whitney test (DMSO-miR-211-/- vs WT; NSC668394-miR-211-/- vs DMSO-miR-211-/-).
C, D. Bu-1a fluctuation analysis of wildtype and primpol cells in which the endogenous +3.5 G4 has been deleted (ΔG4) or with (GAA)10 and (GAA)20 sequence orientated such that it is replicated as the leading (C) or lagging (D) strand template for a fork entering from the 3' end of the locus as shown in panel (A). At least two independent fluctuation analyses were performed. Circles represent the percentage of Bu-1a loss variants in at least 24 individual clones from these experiments, with mean ± SD reported. ****p < 0.0001, * p ≤ 0.05, ns = not significant; one-way ANOVA.
mice.a-e, Transcriptomic analysis from Ly6efl/fl (WT) and Ly6eΔHSC (KO) mice. a-b, Heat maps displaying significant changes (mean RPKM > 0.5; fold change > 2; FDR ≤ 0.05) in liver (a) and spleen (b). Dendrograms are normalized read data (row z-score) clustered with complete linkage method employing Spearman Rank correlation distance measurement.
LC-MS-MS analysis of in-gel-digested HEK293 cells transfected with hNICD-GFP, and subjected to immunoprecipitation using GFP antibody, identified multiple phosphorylation sites in NICD, highlighted in green.
B. Knockdown of LRP6 induced the phosphorylation of YAP even under nutrient-rich condition. HEK293T cells were transfected with GFP siRNA as a control and LRP6 siRNA under nutrient-rich conditions. The ratio of p-YAP/total-YAP of three independent immunoblot bands was quantified (biological replicates, right panel). Error bars indicate standard deviation of biological triplicate measurements. *P < 0.05. Student's t-test was used for statistical analysis.
D, E. Immunoblotting shows significantly increased H3K9 acetylation after 966 vs vehicle. Data information: Data expressed as mean fold change N= 3-9 biological replicates. (*p<0.05, **p<0.01, ***p<0.005) indicate significant difference of 966 versus vehicle Student's t-test)
(F) Representative confocal images of Stat3+/+ cells subjected to proximity-ligation-assay (PLA) by using anti-Stat3 and anti-ATAD3 antibodies (See Appendix Figure S2 for ATAD3 localisation in nucleoids). Only when the two proteins are close to each other an enzymatic reaction takes place, producing discrete fluorescent red dots in the nanometer range (bottom). Anti-Stat3 or anti-ATAD3 alone were used to assess the assay specificity (top and centre respectively). DAPI serves as a nuclear counterstain. Scale bar, 10µm.
A. HEK293 Tet-off experiments showing the degradation of REL-OPT and REL-WT transcripts (left) as well as IL6-OPT, IL6-WT and IL6-DE transcripts (right), post-doxycycline addition. Data information: data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***). The half-lives of the respective transcripts are indicated in brackets.
F ΔAS proximal CTCF binding sites with reciprocal 5hmC/5mC were examined for the impact on alternative splicing. Sites with relative increased 5hmC and decreased 5mC (shown in orange) were considered as potential sites of CTCF gain, whereas sites with decreased 5hmC and increased 5mC (shown in blue) were considered as sites of CTCF loss. Exons were sub-categorized based on CTCF location (upstream or downstream) and assessed for directional changes in splicing as either activation-included (stippled boxes) or activation-excluded (solid boxes). Pie charts show the relationship between affected exon splicing categories and reciprocal methylation at upstream or downstream CTCF binding sites as percent of exons (bottom left). Median +/- standard error of ΔPSI values for exons with up- or downstream CTCF and increased or decreased relative 5hmC are plotted (right), mean values are given above.
(D) Equal aliquots from either DRMs or soluble fractions were pooled, the protein content determined and displayed as percentage of total protein in DRM and soluble gradient regions.
(G) Table shows the quantification of the percentage of asynchronous migration (n≥23 neuroblasts for each condition, n≥3 independent experiments) and of aneuploid cells respectively. For the quantification of aneuploidy, the whole brain was analyzed and prometaphase and metaphase neuroblasts were scored for chromosome number. An aneuploid cell was considered when >8 chromosomes (chr) were visualized (n≥360 neuroblasts from at least 7 larvae brains for each condition, n≥3 independent experiments). ND, not determined.
(B) Specific detection of the pY41-peptide by the antibody produced by the hybridoma cell line 8B2-C1. The plate-bound pY41-peptide, in contrast to the corresponding non-phosphorylated peptide, is specifically detected by antibodies in the serially diluted supernatant of the hybridoma cells by ELISA assays, as measured by absorption at the wavelength of 450 nm. Mean values with SEM are shown for two independent experiments.
C. Immunofluorescence staining of BrdU or H3P and cTnI or EGFP and quantification in the heart tissues after MI. The arrow head indicates cTnI+/BrdU+, cTnI+/H3P+ or EGFP+/BrdU+, EGFP+/H3P+proliferated CMs. The scale bar represents 50 µm.
(H) Module-trait relationship of WGCNA analysis showing correlation and corrected P values (in brackets) for each gene module (indicated by colors) as related to the different external traits selected (disease condition, brain region, age and sex). Correlation test p-value calculated by WGCNA package.
(f,g) glp-1(e2141) animals expressing (f) LMP-1::GFP or (g) SQST-1::GFP were raised at the non-permissive temperature (25°C) and fed bacteria expressing control or hlh-30 dsRNA from hatching. Micrographs of the posterior intestine were taken on day 1 of adulthood, and LMP-1::GFP fluorescence (mean±s.d. of ~10 animals, **P0.01, Student's t-test) and SQST-1::GFP foci (mean±s.d. of ~30 animals, **P0.01, Student's t-test) were quantified. Experiments were performed at least three times with similar results. See Supplementary Fig. S2a,c for images of whole animals and Supplementary Fig. S2b,d for replicates. Magnification, × 200; scale bar, 100 μm.
Quantification of Vegfr3Luc emission in the liver and lung of mice in experiment (A), respectively. N= 4 mice per condition. Statistical significance was determined by Two-Way-ANOVA.
A Scatter plot comparing the transcripts of GV oocytes and zygotes from WT and Pabpn1l-/- females. Transcripts that increased or decreased by more than 3-fold in Pabpn1l-deleted GV oocytes or zygotes are highlighted in red or blue, respectively.
(I) Bioluminescence imaging quantification of lung colonization by 40,000 MDA-MB-231 cells overexpressing PLCβ1, PLCβ1H331Q, or control vector. For Cntrl and PLCβ1H331Q OE, N = 6 mice/group. For PLCβ1 OE, N = 5 mice. Right, H&amp;amp;E staining of representative lung sections.
A, Schematic illustration of the nuclear envelope, depicting the link between the lamina, SUN proteins, giant nesprins and actin.
(B-D) Maximum intensity z-stack projections of IF images in control and importin β(71-876)-injected cells were used to quantify the spindle vs. whole-cell area fraction (B) and the ratios of the average signal in the spindle vs. cytoplasm for the tubulin (C) and TPX2 (D) staining. Means ± SDs, with t-test in (B, C) and Mann-Whitney test in (D), oocyte numbers indicated in brackets.
A Ccno‐deficient mice develop severe hydrocephalus resulting in characteristic head deformation at P21 (arrow).
(b) Clasping scores. n = 11 for all three groups (scale: 0, no clasping; 1, clasping of the forelimbs only; 2, clasping of both fore and hind limbs once or twice; 3, clasping of both fore and hind limbs more than 3 times or more than 5 s).
A) Left: comparison of the number of transposon insertions per gene (dots) for libraries with PE synthesis targeted to the MIM and PC synthesis targeted to peroxisomes, in KennedyON and OFF conditions. CSF1 and VPS13 (highlighted in red) are more required in KennedyOFF conditions. Right: Transposon insertion profile displayed in the UCSC genome browser for the indicated libraries for the genomic region of CSF1. Bars correspond to transposons, rows to libraries. The boundaries of the full-length Csf1 protein, the truncated Csf1ΔC protein, the Chorein-N motif and the transmembrane (TM) domain are indicated.
(j,k) MEFs of the indicated genotype were starved for 5 h in the presence of 2.5 μg ml−1 oligomycin where indicated. Viability was determined as in e,f. Data represent mean ± s.e.m. of five independent experiments.
B. Representative electron microscopy images of middle corpus callosum sections from control mice (a), cuprizone treated-mice for 5 weeks in the absence (b) or in the presence of D-Asp (c), and from mice 2.5 weeks after cuprizone withdrawal in the absence (d, vehicle) or in presence of D-Asp (e). Scale bars in a-e: 1 μ
HST genomic sequence spanning exons 11-to-13 and position of the hst-1/-25/-26 mutations. EMS-induced nucleotide transitions (arrows) in hst-25 and hst-26 are in red; upper-/lower-case fonts: exonic/intronic sequences.
Representative snapshots from CFPAC1 cells transduced with the HOXB8-expressing lentivirus.
(A) Stretched DNA was incubated in the HSS. Endogenous Smc3 (green) and Mcm2 (magenta) were detected by immunofluorescence staining. DNA was counterstained with SYTOX (gray). Colocalization of Smc3 and Mcm2 was quantified (bottom, n = 3, mean ± s.e.m.). Scale bar, 2 μm.
(b) Principal component analysis for relatedness between CHH DMRs at TEs in meiocytes (data from Walker et al. 2018) and stem (+) and non-stem (-) cell nuclei of 7, 14, or 35 day-old plants (D7/14/35).
, Sections from the experiment in 7A were quantified for , number of TUNEL positive cells
B. HeLa cells were transfected with indicated siRNAs, followed by RL-3xBulge construct. Immunoprecipitation (IP) was performed with control mouse IgG (IgG IP) or anti-AGO2 antibody (AGO2 IP) using lysates prepared from these cells. Bottom left panel; RNA isolated from IgG control or AGO2 immunoprecipitate samples was reverse-transcribed using oligo(dT) primer. RL-3xBulge mRNA association with AGO2 was quantified by q-PCR and normalized to the level of GAPDH mRNA associated with AGO2.
Flow cytometry to detect annexin V-FITC and PI staining to determine the percentage of LN229 and GBM#P3 cells undergoing apoptosis after exposure to 0 - 20 μM 24OHC for 72 h. Data are shown as the mean ± SEM (n = 3).
A. Fold change (FC) and significance for transcripts in Kdm6apKO vs. control pancreas. Genes significant at q<0.05 are shown as colored dots above the horizontal line.
E ATF4, CHOP, sXBP1 and BiP mRNA levels were quantified by qPCR in MM.1s BzR cells treated as in (C). sXBP1 mRNA levels were normalized to total (spliced + unspliced) XBP1 mRNA level. Data are Mean RNA fold changes (2-∆∆CT) ± SD, relative to untreated controls. N = 5 (cumulative data of 2 independent experiments with technical duplicates and triplicates), pairwise compared by nested one-Way ANOVA with Tukey's multiple comparison test, exact p-values indicated. Thapsigargin (Tg, 2 µM, 6h) was used as a positive control.
VM-glial cultures were transduced with sh-Sgk1 (or sh-cont) lentivirus. Medium was condition in sh-Sgk1-glia (or sh-cont-glia), and the conditioned medium (CM) treatment effects on neuronal SNCA aggregation were assessed by WB
(G-H) Representative microscopic pictures and diameter of C2C12 myotubes stained for myosin heavy chain (G) or human myoblast-derived myotubes (H) after 48h treatment with C26 CM and ferric citrate (n=3 per condition). Scale bars =50 and 100 µm, respectively. Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by one-way Anova with Bonferroni's correction Data are mean ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001 compared to control and ##p < 0.01, ###p < 0.001 compared to C26 CM-treated group.
B. The gene-specific correlation between protein copy number (x-axis) and predicted protein copy number based on the RNA levels (RNA-based prediction) is shown for four tissues and four cell lines. The other seven tissues and five cell lines are also shown in Figure EV5 and predicted copy numbers can be found in Table EV9.
A. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for four hours with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs. The phosphorylation status of GSK3β (Ser9), ERK1/2 and EGFR (Tyr1173) was assessed by western blotting and normalized according to the total protein levels. RHOB overexpression was also monitored by western blotting.
(H) Mfn2 KO cells transfected with the pEGFP plasmid were incubated with 1 μM Tg alone or in combination with 2 μM CHX for 24 h (3 h of pre‐incubation with CHX). Florescence microscopy images show that CHX prevents cytoplasmic vacuolization. Scale bar: 10 μm.
(D) His-tagged Pdia4 was incubated with the recombinant proteins, Gst-TecSH3, Gst-Ndufs3, and Gst-p22phox. The immunoprecipitates (IP) and recombinant proteins underwent immunoblotting analysis with anti-Gst and anti-His antibodies.
Atf3 mRNA expression by qRT-PCR in vehicle, 6 hour, 24 hour, and 72 hour HDT treatment. Each data point represents the mean of triplicate technical replicates from a single mouse, n=3-4 mice in independent experiments. Significance determined by One-way ANOVA, Tukey. Error bars denote standard deviation.
Lysine 128 of Cideb is required for the interaction with Sec12. 293T cells transfected with HA-Sec12 and the indicated Flag-tagged Cideb constructs were subjected to immunoprecipitation with an anti-Flag antibody, and levels of the co-immunoprecipitated HA-Sec12 were detected with an anti-HA antibody.
HO-1 deficient and control C57BL/6xFVB mice were injected with CoPP three times, every second day and sacrificed 24 hours after last injection. Total leukocyte counts in PB (HO-1+/+: n = 5, HO-1-/- DMSO: n = 6, HO-1-/- CoPP: n = 4 mice per group). Granulocyte percentage among PB leukocytes.
F, Percentage of GFP+-acceptor photoreceptors connected via a donor cell protrusion across different imaging modalities (n=3 animals per imaging technique).
(G) Quantification of cystine levels (nmol/mg protein) by HPLC-MS/MS in control and cystinotic zebrafish (n = 3).
(A) Representative 5-day-old seedlings of wild-type Arabidopsis thaliana Col-0 and TF mutants gata12 and wri3 grown in the dark. Scale bar, 1 mm.
Quadriceps from saline or LPS treated, WT or iNOS KO mice, were isolated and used for western blot analysis with anti-iNOS (n=4). Ponceau S was used for total protein staining.
, D The reduced fraction of GFP+ tumor cells in the xenografts derived from shUPS33-expressing GSCs relative to the control xenografts indicated that USP33 disruption impaired hypoxia response in tumor cells. ***, p < 0.001 (n = 5 tumors; mean ± s.e.m.; two tailed unpaired t-test).
IdU track lengths and percentage of origin firing (mean+SD) from U2OS cells supplemented or not with nucleosides (NS). >195 (track length) and >350 (origin frequency) fibers obtained from 2 independent experiments were measured for each condition.
Overall survival stratified by tumor mutational burden (TMB) across treatment groups in 3,217 patients. Samples were classified as TMB-high if the amount of non-synonymous mutations per expressed MB (rnaMB) was ≥ the median number of non-synonymous mutations per rnaMB across the whole SCAN-B cohort (0.082 mutations per rnaMB) and TMB-low otherwise. In each Kaplan-Meier plot, TMB-low cases are plotted in blue, TMB-high cases are plotted in red, the log-rank P value is given, and the hazard ratio (HR) for TMB-high is given with a 95% CI and after uni-variable and multi-variable (MV) Cox regression adjustment. Covariables included in the MV analysis were age at diagnosis, lymph node status, tumor size, and the variables denoted by the following symbols: ¶, ER, PgR, HER2, and NHG; ¤, ER, PgR, and NHG; $, HER2 and NHG; #, NHG. Abbreviations: TMB, tumor mutational burden; ER, estrogen receptor; PgR, progesterone receptor; HER2, human epidermal growth factor receptor 2; NHG, Nottingham histological grade; HoR, hormone receptor; TNBC, triple-negative breast cancer.
H Bar graph showing GSEA of genes altered in both mouse and human EpCAM+ cells upon R5020 stimulation of tissue microstructures. Bars define the averaged NES between ex vivo R5020-treated human and mouse tissue, ticks indicate averaged P-values given by GSEA.
The structure of a mitoTev-TALE. It consists in a mitochondrial localization sequence (MLS) and a TALE-DNA binding domain which is fused to a monomeric endonuclease (I-TevI), through a flexible linker (linker)
(E and F) double mutants for atg8a and ref(2) P loss-of-function alleles lacking the PB1 (E) or UBA (F) domain do not form ubiquitinated protein aggregates. Ubiquitin exhibits a cytoplasmic staining pattern (arrows). Genotypes: (E) atg8aKG07569/Y; ref(2) Pod2/ref(2) Pod2, and (F) atg8aKG07569/Y; ref(2) Pod3/ref(2) Pod3.