EMBO/sd-character-level-ner huggingface dataset:
Figure 5A. Experimental design. Thirty days after the last pIpC injection, PKCδfl/fl and PKCδ cKO mice were treated with a single dose of 5-FU (150mg/kg, i.p.) and analyzed subsequently by FACS and BrdU incorporation studies. B. Kinetics of LSK cell recovery. Representative FACS profiles of BM LSK cells from WT and cKO mice at the indicated time points after 5-FU treatment. Numbers in the contour plots indicate the percentages of LSK subsets among Lin- live cells. C. Average absolute numbers of BM LSK cells at day13 and day28 after 5-FU treatment. Data are pooled from two independent experiments. (total of n=6-8 mice per time point and genotype). Data information: Data presented as mean ± SEM. The statistical significance was assessed using repeated-measures two-way ANOVAs with Sidak's multiple comparisons tests (C) *p<0.05, **p<0.01 and ***p<0.001. D. Quantification of apoptotic HSPCs at the indicted time points after 5-FU treatment of control and cKO mice (n=6 per genotype). Data information: Data presented as mean ± SEM. The statistical significance was assessed using repeated-measures two-way ANOVAs by two-tailed Student's unpaired t-test analysis (D and E). *p<0.05, **p<0.01 and ***p<0.001. E. Representative FACS plots showing analysis of in vivo BrdU incorporation assays. Mice were received 2mg of BrdU (i.p.) at d13 (left) or d28 (right) after 5-FU treatment. 20hrs later, BrdU incorporation in BM LSK cells was assessed using anti-BrdU antibodies. Bar chart at right indicates percent of BrdU+ LSK cells at d13 and d28 after 5-FU treatment (n=6 per genotype and time point). Data information: Data presented as mean ± SEM. The statistical significance was assessed using repeated-measures two-way ANOVAs with two-tailed Student's unpaired t-test analysis (D and E). *p<0.05, **p<0.01 and ***p<0.001. F. Kaplan-Meier survival cures of control (n=12) and cKO (n=11) mice after sequential 5-FU treatment. Mice were treated with 5-FU (150mg/kg, i.p.) once each week for three weeks. Arrows indicate time points of 5-FU injection. Data information: Data presented as mean ± SEM. Survival data (F) were analyzed using a log-rank nonparametric test (Mantel-Cox test) (p=0.0141).
Figure 2MtDNA heteroplasmy analyzed by PCR/RFLP, 24 hours post mitoTev-TALE and mitoTALEN transfection. The RFLP analysis shows increased %WT mtDNA in sorted cells when compared to the the untransfected. mitoTALEN monomers positive for both eGFP and mCherry were isolated as "Yellow". The "Black" cells represent the mitoTALEN sorted population of cells negative for eGFP and mCherry and the "GFP-+" population represents mitoTev-TALE sorted cells with low levels of eGFP fluorescence. GFP++ were cells positive for GFP after mitoTev-TALEs transfections Quantification of the % of WT load in cybrid sorted populations after transfection Representative RFLP analysis showing the change in heteroplasmy at 1 day and 15 days, after sorting Quantification of the heteroplasmic shift over time Determination of the levels of mtDNA (ND1/ACTB) by qPCR of mutant cybrids transfected with MERRF mitoTev-TALE mtDNA levels in wild-type sorted cells transfected with the MERRF mitoTev-TALE. Data is expressed as percentage of the Untransfected cells (%UNT)
aluRNA overexpression induces an increase in nucleolar volume and activityHeLa cells were transfected with an empty vector or plasmids expressing aluRNA or L1‐repeat RNA and GFP from a separate promoter.Figure 5HeLa cells were transfected with an empty vector or plasmids expressing aluRNA or L1‐repeat RNA and GFP from a separate promoter. Transfected cells were identified by GFP fluorescence, and nucleoli volumes were evaluated based on the immunofluorescence signal of the nucleolar marker NPM. The graphs show that the volume of nucleoli is increased by 30% in the presence of ectopic aluRNA (± 95% CI, n = 20). **P‐value 0.01, t‐test. Scale bar, 10 μm. CLSM live‐cell imaging of RFP‐NCL in cells transfected with a plasmid expressing aluRNA and imaged for 10 h. The arrows indicate nucleolar domains displaying time‐dependent increase in the nucleolar size or fusion of two domains into one. Scale bars, 10 μm. HeLa cells were transfected as in (A) and incubated for 24 h. Nascent RNA was pulse‐labeled for 30 min, and nucleoli were visualized by immunofluorescence of NPM. Scale bars, 10 μm. Graphs represent the intensity of nucleolar fluorescence signals as percentage of the intensity of nuclear signals and reveal a 27% increase in the presence of ectopic aluRNA (± 95% CI, n = 30). **P‐value 0.01, *P‐value = 0.01, t‐test.
Figure 3(A) In vitro quantification of EVs isolated from conditioned medium of LPS-stimulated primary CPE cells grown in a transwell system in the absence or presence of the exosome inhibitor GW4869 (n=3). (B-D) TaqMan assay quantification of the miRNAs miR-9 (B), miR-146a (C) and miR-155 (D) in supernatant of LPS-stimulated primary CPE cells grown in a transwell system and either left untreated or pretreated with GW4869 to inhibit exosome secretion (n=3). miR-1a levels were below detection limit. (E-H) TaqMan assay quantification of the miRNAs miR-1a (E), miR-9 (F), miR-146a (G) and miR-155 (H) in cell lysate of LPS-stimulated primary CPE cells grown in a transwell system left untreated or treated with GW4869 to inhibit exosome secretion (n=3). Data are displayed as mean ± SEM and analyzed by Student's t-test. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01.
Jumpy siRNA increases recruitment of PI3P‐binding Atg factor WIPI‐1 (Atg18) to autophagic membranes.Figure 5(A-C) C2C12 were transfected for 48 h with Jumpy siRNA, followed by a second transfection overnight with Jumpy siRNA and GFP‐WIPI‐1, incubated for 4 h in starvation media, fixed and immunostained with anti‐Atg16 antibody (red). GFP‐WIPI1‐transfected cell (A), endogenous Atg16 immunostaining (B), colocalization of GFP‐WIPI‐1 and Atg16 is indicated in yellow in merge image (C). Boxed areas are shown at higher magnification as inset. Scale bars, 10 and 5 μm in insets. (D-I) C2C12 cells were transfected for 48 h with control (scramble) (D, E) or Jumpy siRNA (F-H), followed by a second transfection overnight with corresponding siRNA and GFP‐WIPI‐1. Cells were incubated for 4 h in full (D, G) or starvation media (E, F, H) in presence (F) or absence (E-H) of 100 nM wortmannin, fixed and analysed by confocal fluorescence microscopy. Quantitation of WIPI‐1 puncta per cell (I). Bars, s.e.m. (n=3 independent experiments, with an average of 30 cells per experiment). *P0.05, **P0.01 (t‐test).
Figure 5A Schematic of the cyp-34A4 gene. Large boxes indicate coding sequences. Small boxes indicate UTRs. The location and nature of individual mutations is shown. Sequence deleted in od107 is shown with a horizontal black bar. B Relative abundance of mRNA for the cyp-34A4 gene. Values are based on qRT-PCR analysis, with the relative levels for each gene normalized to the value observed in the wild-type control. ***P<0.001, ANOVA with Dunnett's posthoc comparison to wild type. N=3 trials. Error bars indicate SEM. C Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals of the indicated genotype at L4+48 hours. ****P<0.001, ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point. N=20 animals per genotype and time point. Error bars indicate SEM. D Quantified fluorescence of UbG76V-GFP normalized to mRFP, as in (C). ****P<0.001, ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point. N=20 animals per genotype and time point. Error bars indicate SEM. E Expression of the cyp-34A4::gfp transgene. Scale bar, 100 microns. F Expression of the cyp-34A4::gfp transgene (green). Intestinal autofluorescence (red) can be distinguished. Scale bar, 100 microns.
a,b) Fluorescent microscopy of (a) NDP52 or (b) P62 in cells expressing LINE-1-MS2 and MS2-GFP.f4(a,b) Fluorescent microscopy of (a) NDP52 or (b) P62 in cells expressing LINE-1-MS2 and MS2-GFP. (c,d) Fluorescent microscopy of endogenous LINE-1 RNA and (c) NDP52 or (d) P62. (e) Four-colour confocal microscopy of NDP52, P62, GFP-DCP1A and mCherry-TIA-1. (f) Percent of foci of NDP52, P62 or neither co-localizing with GFP-DCP1A or mCherry-TIA-1. *P=0.007 GFP-DCP1A, P=0.00005 mCherry-TIA-1, analysis of variance (ANOVA). (g) Percent of foci of NDP52, P62 or both co-localizing with GFP-DCP1A or mCherry-TIA-1, n=674 stress granules, n=53 P-bodies. *P=0.043 GFP-DCP1A, P=0.028 mCherry-TIA-1, ANOVA. (h) Venn diagram of the percent of P-bodies (GFP-DCP1A) or stress granules (mCherry-TIA-1) co-localizing with NDP52, P62, both or neither. . (i) Relative number of P-bodies per cell (detected with 18033 serum-recognizing GW182) treated with indicated siRNAs. n=3, *P=0.0002 NDP52, P=0.004 ATG5. j) Relative number of stress granules (detected with antibody recognizing TIAR) 1 h after arsenite treatment and after 30 min recovery from 1 h treatment with arsenite. n=3, *P=4x10−6 p62, P=0.019 NDP52, P=2 × 10−5 ATG5, ANOVA. (k) Percent of cells expressing GFP when co-transfected with LINE-1-RP retrotransposition GFP reporter and either of two independent siRNA (black and grey bars) targeting NDP52, P62 or control. Results are normalized to a retrotransposition-incompetent control (n=3, *P=0.017, P=0.002 NDP52; P=0.001, P=0.013 p62, ANOVA). (l) Relative number of G418-resistant colonies in cells transfected with siRNA-targeting NDP52, P62 or control, Alu-retrotransposition reporter and retrotransposition-enhancer LINE-1 ORF2p (n=3, *P=0.012, P=0.011 NDP52; P=0.008, P=0.011 p62, ANOVA). All error bars represent s.e. of the mean. All experiments were performed with a minimum of three biological replicates.
Figure 2A RIP experiments were performed using anti-myc antibodies and extracts from Trt1-myc expressing strains followed by qRT-PCR to detect G-rich TERRA, polyA+ TERRA, ARRET, TER1 and ACT1. Values represent fractions of input RNA detected in immunoprecipitated material expressed as fold increase over untagged (unt) wt strain. Bars and error bars are averages and SD from three independent experiments. * P < 0.05 (relative to unt; two-tailed Student's t test). B RIP experiments as in (A) where, prior to RNA extraction, washed beads were resuspended in DNaseI buffer (DNbuff) and incubated for 1 hour at 30° C in presence or absence of DNaseI. Values represent fractions of input polyA+ TERRA detected in immunoprecipitated material expressed as fold increase over an untagged (unt) wt strain. Bars and error bars are averages and SD from three independent experiments. * P < 0.05 (relative to unt; two-tailed Student's t test). C RIP experiments were performed using anti-myc antibodies and extracts from Trt1-myc expressing strains (either ter1+ or ter1Δ) cultured in YES medium for ~ 43 pds. Values represent polyA+ TERRA detected in immunoprecipitated material expressed as a fraction of the input (graph on the left) or after normalization to ACT1 RNA in the corresponding input (on the right), and expressed as fold increase over an untagged wt strain (unt ter1+). Bars and error bars are averages and SD from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 (relative to unt ter1+; two-tailed Student's t test). D Western blot analysis of Trt1-myc in cells used in (C). Ponceau S staining serves as a loading control.
Effect of TLR activation on the induction of the autophagic machinery.Figure 3(A) (I) MDC staining in neutrophils treated with LPS, LTA, peptidoglycan (PGN), loxorubin (LOX) and R848 (400×). (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. LPS (50 ng/mL) (bar 2: 69.67±8.21%), LTA (bar 3: 75.17±9.80%), peptidoglycan (bar 4: 77±6.96%), loxorubin (bar 5: 66.67±5.61%), R848 (bar 6: 69.5±7.45%), poly I:C (bar 7: 16.33±3.14%) and flagellin (bar 8: 19.83±5.16%)−treated cells compared with control conditions (bar 1: 14.5±4.97%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched−pairs test, *p<0.05, n.s.=not significant. (B) LC3B lipidation status of cells treated with LPS (lane I), LTA (lane II), peptidoglycan (lane III), loxoribin (lane IV) and R848 (lane V), detected by immunoblotting. One representative out of six independent experiments is shown. (C) LC3B−II/LC3B-I ratio as obtained from integrated optical density measurements. LTA (bar 2: 1.75±0.84), peptidoglycan (bar 3: 1.82±1.17), LPS (bar 4: 1.9±1.7), loxorubin (bar 5: 1.32±0.49) and R848 (bar 6: 1.4±0.77) compared with control neutrophils (bar 1: 0.76±0.30). Data are representative of six independent experiments and are presented as mean±SD. Wilcoxon matched−pairs test, *p<0.05. (D) Analysis of LC3B gene expression levels in neutrophils treated with TLR agonists. LPS (bar 1: 2.27±0.43), peptidoglycan (bar 2: 2.07±0.42), LTA (bar 3: 2.1±0.49) and R848 (bar 4: 1.89±0.35) compared with medium−treated control cells. Dotted line represents LC3B mRNA levels of medium-treated control cells. Data are representative of six independent experiments and are presented as mean relative expression±SD. Wilcoxon matched−pairs test, *p<0.05. (E) Assessment of ATG gene expression levels in PMN after LPS stimulation (50 ng/mL). ATG3 (bar 1: 2.94±0.52), ATG4 (bar 2: 1.72±0.42), ATG5 (bar 3: 1.84±0.34), ATG6 (bar 4: 1.8±0.3), ATG7 (bar 5: 3.13±0.64) and LC3B (bar 6: 2.27±0.43) compared with control cells. Dotted line represents mRNA levels of medium-treated cells. Data are representative of six independent experiments and are presented as mean relative expression±SD. Wilcoxon matched−pairs test, *p<0.05.
Auto-inhibition of Mif2 is crucial for chromosome segregation and cell viability.Figure 8Phenotypic analysis of Mif2 mutants using a galactose inducible overexpression system. Strains harboring the indicated Mif2 expression constructs on pESC plasmids were plated in serial dilution on doHIS-Glucose or doHIS-Raffinose/Galactose plates and incubated at 30 °C. Serial dilution assay of chromosomally integrated Mif2 variants under the control of the pGAL promoter. Strains were plated on YEP-Glucose and YEP-Raffinose/Galactose plates. Analysis of chromosome segregation in strains from (B). Distribution of fluorescently labeled Chromosome III was analyzed in large-budded cells 16 hours after switching the medium to Galactose. Scale bar represents 2 μm. Microscopic analysis of kinetochores in Mif2 overexpressing cells visualizing Mtw1-GFP and Spc42-mCherry. Images of representative large budded cells after 16h in Galactose are shown. One kinetochore cluster is enlarged in the white box for each strain. Scale bar represents 5 µm. Brightfield images show the morphology of the corresponding yeast cell. Only large budded cells were included in the analysis. Quantification of (D) in which large budded cells were classified according to their Mtw1-GFP signal into the four indicated groups. A total of 100 cells was quantified for every strain. Mean values of the three indicated groups were calculated and plotted in a bar chart. Error bars indicate the SEM of 2 independent experiments. Co-IP experiment of indicated Mif2-Flag variants and Mtw1-Myc from yeast cell extracts. Phenotypic analysis of Mif2-∆sigAT mutant using the same galactose inducible overexpression system as in (A). Strains harboring the indicated Mif2 expression constructs on pESC plasmids were plated in serial dilution on doHIS-Glucose or doHIS-Raffinose/Galactose plates and incubated at 30 °C.
Figure 6(A) Isoform-specific subcellular localization shown in cultured hippocampal neurons (DIV14) co-expressing DsRed-CA7 (left) and EGFP-CA2 (middle). (B,C) Representative confocal images of precocious in vivo expression of (B) EGFP-CA2 and (C) EGFP-CA7 in P40 mouse cortical layer 2/3 pyramidal neurons. Neurons were transfected at E14.5 with EGFP-CA2 or EGFP-CA7 using in utero electroporation and images were taken from fixed slices. Right panels in (B) and (C) show higher magnification of the primary apical dendrite marked with a box. (D) The expression of EGFP-CA7 disrupted the normal spine morphology and induced the formation of thick, filopodia-like protrusions.
Tetrapod homologues of Atg4 share several conserved cysteine residues.Figure 6(A) Amino‐acid alignment of the region surrounding the active cysteine residue among members of the Atg4 family. Sequences of the following accession numbers are aligned: BAB83890, BAB83889, NP_777364, NP_777363, NP_001001171, CAG32326, AAH73017, AAH82660, AAH95617, XP_393739, NP_608563, XP_661074, NP_014176 and NP_191554. The alignment was preformed using the ClustalX multiple alignment program and is depicted by the ESPript 2.0 program, representing identity by a black frame and homology by a gray background. Numbering is according to the HsAtg4B sequence. α‐Helices and a β‐sheet found in this region according to the recently solved structure of HsAtg4B ( Sugawara et al, 2005; Kumanomidou et al, 2006) are marked as black ellipses and a gray arrow, respectively. The catalytic cysteine residue is marked by a wide black arrow; other conserved cysteines are marked by thin black arrows. (B) A phylogenetic tree was created based on the alignment presented in (A) using the NJplot program ( Perriere and Gouy, 1996). Bootstrap values indicating the reliability of the tree branches (with 1000 being the maximal value) are shown.
a) Proliferation rates.figf7(a) Proliferation rates. HCT116.vector, HCT116.UVRAG, HCT116.UVRAGΔC2, HCT116.UVRAGΔCCD and HCT116.UVRAGCCD cells (1 × 105 per well) were seeded into 6-well microtitre plates. Cell numbers were counted on the days indicated. The results represent means of three independent assays. (b) Anchorage independence. HCT116.vector, HCT116.UVRAG, HCT116.UVRAGΔC2, HCT116.UVRAGΔCCD and HCT116.UVRAGCCD cells (1 × 104) were incubated in soft agar as described in the Methods. After ten days, colonies were photographed and counted. The scale bars represent 50 μm. (c) Tumorigenicity. HCT116.vector, HCT116.UVRAG and HCT116.UVRAGΔCCD cells (1 × 107) were subcutaneously injected into 5 nude mice, separately. At 16 days postinoculation, mice and tumours were photographed and tumour weights were measured. Arrows indicate location of tumours.
The effects of AGO4 and AGO6 on siRNAsHeatmap depiction of the dependence of AGO4‐ and AGO6‐bound siRNAs on Pol IV.Figure 7A Heatmap depiction of the dependence of AGO4‐ and AGO6‐bound siRNAs on Pol IV. Identities of siRNAs were retrieved from Havecker et al (), and siRNAs were grouped into 1,210 AGO4‐bound clusters and 1,486 AGO6‐bound clusters (see Materials and Methods for details), each of which corresponds to a colored horizontal bar; the bars are stacked numerically into a column (y‐axis). Information on siRNA levels in Pol IV mutant and wild‐type plants was retrieved from Zhang et al (). B Heatmap depiction of the dependence of AGO4‐ and AGO6‐bound siRNAs on Pol V. C Quantification of individual 24‐nt siRNAs by TaqMan small RNA assays. Double: ago4‐6 ago6‐2 double mutant. snoR101 was used as an internal control. RNA levels in the mutants were relative to those of the wild‐type (Col‐0). Error bars indicate SD, n ≥ 3.
Figure 5A DNA replication in mitochondrial replisomes was detected in mouse cortical neurons after a 3-hour pulse of the thymidine analog, EdU. Cells were labeled additionally with the mitochondrial marker, Mitotracker CMXRos, and with an antibody to the neuron marker, MAP2 B Quantification of EdU uptake into mouse cortical neuron replisomes showed that nutrients inhibit ­mtDNA replication by ~60%, an effect that was blocked by AβOs. Error bars represent ± s.e.m. Statistical analyses were performed using Student"s two-tailed unpaired t-test and are representative of 3 independent a C WT mouse cortical neurons expressing either Flag-Raptor H-Ras25 or Flag-Raptor Rheb15 were serum-starved in AβO-containing Hank"s balanced salt solution for 2 hours. Next, the cells were pulse-labeled for 3 hours with EdU. Note that forcing mTORC1 to the plasma membrane (Flag-Raptor H-Ras25) yielded an ~80% increase in EdU incorporation into mtDNA in the presence of AβOs regardless of whether or not the neurons had been stimulated with insulin+R+L. In contrast, EdU uptake into mitochondrial replisomes was not stimulated by AβOs in neurons whose mTORC1 was forced to lysosomes (Flag-Raptor-Rheb15). Error bars represent ±
Figure 2A. Typical fluorescence intensity trace collected over 10 seconds. B. Typical normalized FCS curves of Smc3-GFP at different cell cycle phases. Dotted lines indicate fitting of the one-component anomalous diffusion model to the respective FCS curve. C-F. Diffusion times of Smc3-GFP, Scc3-GFP, and Scc1-GFP at the G1 phase (C), S phase (D), G2 phase (E), and mitosis (F). Data shown as mean ± s.e.m. from at least three independent experiments. *: p<0.01, #: p<10-13 with the Mann-Whitney test.
Figure 6A, B Immunoblot analysis of indicated cell lines, starved for 6 hours (HBSS) as indicated, and treated with MG132 or Baf A1 as indicated. Numbers below the blots in A represent relative intensity of the bands in the shown blots normalized against GAPDH loading control. In B, the bars represent the mean±sd of band intensities of three independent experiments as quantified using ImageJ. Statistical comparison was analyzed by one-way ANOVA and significance displayed as **p ˂ 0.005, *p ˂ 0.01; ns is not significant. C Immunoblot analysis of HeLa cells transfected with the indicated siRNAs. D, E Immunoblot analysis of HeLa cells transfected with the indicated siRNAs and treated with Baf A1 as indicated. In D, the panels are from more than one western blot experiment but only a single GAPDH loading control is shown. In E, the bars represent the mean±sd of band intensities of three independent experiments as quantified using ImageJ. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as ***p ˂ 0.001, **p ˂ 0.005; ns is not significant. F HeLa KO CALCOCO1 cells were transiently co-transfected with EGFP-CALCOCO1 and Myc-VAPA or Myc-VAPB, and then immunostained with anti-Myc and anti-LC3B antibodies. Arrows indicate dots of co-localization of all three proteins. Scale bars, 5 μm for large merged images and 1 μm for zoomed images.
Figure 1(A) The expression pattern of FOXM1 in fetal human cortex at GW15. VZ, ventricular zone; ISVZ, inner subventricular zone; OSVZ, outer subventricular zone. Right panels show higher magnification images. Arrows show that FOXM1+cells were colocalized with SOX2+ cells. Scale bars, 100μm (left); 50μm (right). (B) Staining for NESTIN and FOXM1 in human neural progenitor cells cultured in vitro. Down panels show higher magnification images. Scale bars, 50μm(up); 10μm(down). (C) Staining for SOX2 and FOXM1 in human neural progenitor cells cultured in vitro. Down panels show higher magnification images. Dashed shapes show that FOXM1+cells were colocalized with SOX2+ cells in human neural progenitor. Scale bars, 50μm(up); 10μm(down). (D) Staining for Pax6 in E15.5 mice after IUE at E13.5. Arrowheads indicate Pax6+ cell in enlarged SVZ. Right panels show higher magnification images. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bars,50μm(left); 20μm (right). (E) Graph shows the percentage of Pax6+ cells among GFP+ cells (n = 5 each group; two-tailed (F) Graph shows the percentage of Pax6+ cells among GFP+ cells in enlarged SVZ (n = 5 each group; two-tailed unpaired t-test, control vs. hFOXM1, P<0.0001; mFoxm1vs. hFOXM1, P<0.000 1). (G­) Staining for upper neuron marker Cux1 in P3 mice after IUE at E13.5. Arrowhead and asterisk indicate deformation of the cortical surface at regions containing electroporated cells. Right panels show higher magnification images. Arrows show GFP+ Cux1+ double-positive cells. Scale bars,100μm(left); 20μm (right).