EMBO/sd-character-level-ner huggingface dataset:
Figure 2A. Schematic structure of pRb, location of 14 CDK sites and the seven Ser/Thr-to-Ala substitutions (denoted by *) in Rb∆K7 mice. Phosphorylation of upstream sites is indicated: arrow, phosphorylated; blocking line, not phosphorylated in Rb∆K7 thymocytes. B. BrdU immunohistochemistry analysis of sagittal sections of E13.5 Rb∆K7 and control littermate embryos. Arrows point to lateral ventricle and liver showing similar intensity of staining/cell proliferation. C. Left, representative flow cytometric analysis for CD3, CD4 and CD8, showing near normal T cell development in 10-day-old Rb∆K7 mice. Right, average of CD3, CD4 and CD8 single- and double-positive cell populations in Rb∆K7 vs control thymocytes with no significant difference (n.s.) by student's t-test. Results are presented as mean ±SD, n=4 biological replicates). D. Left, images showing apparent normal division of retinal progenitors in Rb∆K7 mice. Mice were labeled with BrdU for 2 hrs; retinal sections from indicated genotypes were stained with DAPI (blue), Ki67 (green), PH3 (red) or BrdU (red) to mark nuclei, all dividing cells, mitotic or S-phase cells, respectively. Right, quantification of staining showing no significant difference among the genotypes. Bars denote mean ±SD; n=3 biological replicates). Scale bar: 20 μm. E. Left, top, western blot analysis of Rb∆K7 thymocytes with an antibody that recognizes both hyper-phosphorylated (ppRb) and hypo-phosphorylated pRb species. Right, phospho-specific immunoblots showing absence of phosphorylation at upstream Thr350 and Ser601 or the substituted Ser773Ala site in Rb∆K7 thymocytes. Right, immuno-precipitation (IP)-Western blot analysis demonstrating phosphorylation of pRb∆K7 on Ser243/Thr246 (top) and Ser605 (bottom). n.s., non-specific band. F. Phospho-specific immunoblots showing absence of phosphorylation at upstream Thr350, and reduced but not complete loss of phosphorylation at Ser601 (arrow) in Rb∆K4 thymocytes.
Cac is required on the lysosomes in neurons to promote lysosomal fusion.Fig 8Cac and its accessory subunits are present on the lysosomes. Under normal conditions of activity requiring basal levels of autophagy, Cac allows for calcium efflux from endo/lysosomes to provide for SNARE-mediated fusion of autophagic vacuoles to lysosomes and subsequently efficient degradation of unwanted cellular material and to maintain neuronal homeostasis. In the absence of Cac, there is a block in the autophagic flux and lysosomal fusion, leading to an accumulation of fusion-primed autophagic vacuoles. This subsequently results in an accumulation of improperly formed proteins or damaged organelles, such as mitochondria in the neurons, and causes neurodegeneration.
All panels show immunofluorescent staining.figf6All panels show immunofluorescent staining. Blue shows nuclear DAPI staining and green shows cones marked with PNA. (a-f) Staining for HIF-1α (red signal). (a) Wild type (PW10) stained for HIF-1α (inset shows higher magnification). (b, c) Cross sections in wild type stained for HIF-1α (PW10). DAPI overlap is shown in c. (d-f) During cone degeneration in Pde6b−/− (PW10) mice, we found increased levels of HIF-1α in cones (inset in d shows higher magnification with DAPI overlap). Cross sections indicated that the increase of Hif-1α occurred mainly in cones (arrows point to cones that are located in the top layer of the inner nuclear layer at this stage; e). DAPI overlap is shown in f. (g) GLUT1 expression in wild type (PW10) (red). Most of the signal in between the cones reflects expression in rods. (h-j) Increased expression of GLUT1 in cones during degeneration seen in flat mounts (h) and sections (i, j). PNA overlap of j is shown in i. Retinal flat mounts are shown in a, d, g and h. Retinal sections are shown in b, c, e, f, i and j.
Figure 1A. Schematic representation of SDH composition (catalytic subunits: SDHA, SDHB and membrane anchor subunits: SDHC, SDHD) and SDH activity. B. ARPI (CSS, ENZA, ODM-201, 14449) decreased, while R1881 increased, SDHA and SDHB subunit protein levels in both LNCaP and LAPC4 cells. PSA is used as a positive control. C, D. ARPI treatment (CSS or ENZA) for 12 h in LNCaP cells decreased SDH activity (C) and increased intracellular succinate levels (D) compared to vehicle treated control. Data information: ENZA: enzalutamide, CSS: charcoal stripped serum, ODM-201: darolutamide. Data shown as mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey's test *, p<0.05, **, p<0.01, ****, p<0.0001 compared between groups. E. ENZA decreased (left panel), whereas R1881 increased (right panel), transcript levels of SDHA and SDHB in LNCaP cells. Data information: ENZA: enzalutamide, CSS: charcoal stripped serum, ODM-201: darolutamide. Data shown as mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey's test *, p<0.05, **, p<0.01, ****, p<0.0001 compared between groups. F. Quantitative PCR on DNA immuno-precipitated with AR antibody in ChIP assay showed increased signal for SDHA, SDHB along with PSA after treatment with R1881 compared to androgen deprivation in LNCaP cells. PSA is used as positive control. Data information: ENZA: enzalutamide, CSS: charcoal stripped serum, ODM-201: darolutamide. Data shown as mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey's test *, p<0.05, **, p<0.01, ****, p<0.0001 compared between groups. G. EMSA shows dose-dependent interaction between SDHA and SDHB AREs and AR-DNA binding protein, which was disrupted with mutated ARE probes (SDHA-M, SDHB-M). Quantification of binding of three independent experiments is shown at the bottom panel. Data information: ENZA: enzalutamide, CSS: charcoal stripped serum, ODM-201: darolutamide. Data shown as mean ± SD of three independent experiments. Statistical analysis was performed using two tailed unpaired Student's t-test *, p<0.05, **, p<0.01, ****, p<0.0001 compared between groups.
Figure 6(A) HCT116 p53+/+ or p53-/- cells treated with 50 µM etoposide were assayed by Western blot with antibodies for p53, γH2AX, H3K9Ac, panH3, and GAPDH at times 0, 8, 16, or 24 hr post-treatment. (B) ChIP-qPCR for γH2AX (upper panels) or IgG control (lower panels) for cells treated as indicated in panel A. (C-D) RT-qPCR analysis for cells treated as shown in panel A. C-D) RT-qPCR analysis for cells treated as shown in panel A. (C) RT-PCR for the p21 (top), or PARD6G mRNA (lower). (D) RT-PCR for primer pairs spanning subtelomere18q as indicated below. Lower panel shows the No-RT control for 18qsubtelomere primers. RT-qPCR was quantified as relative to Actin with error bars indicating SD and * indicating p value <.05 using student t-test comparing etoposide (24 hr) relative to untreated for specific primer pairs. (E) Meta-data analysis of published GRO-Seq and ChIP Seq data sets. Gro-Seq Minus (red/maroon) or Plus (purple/pink) RNA strand from Control or Nutlin -3a treated MCF7 cells was mapped to human 18q subtelomere. Histone H3K27ac, H3K4me1, and H3K4me3ChIP-Seq data sets from untreated MCF7 are also shown for these subtelomeric regions. Right panel shows a zoom in of approximately 1000bp (light blue on left panel) surrounding the p53 response element within the subtelomere. (F) Summary of GRO-Seq and ChIP-Seq metadata analyses from human subtelomeres with p53 binding sites. Each p53 response element is located upstream of a region of bidirectional transcription which is modulated in a p53 dependent manner along with local bidirectional transcription at the p53 response element itself.
Figure 4In TIFA‑KO cells transiently expressing TIFA‑tdTomato, co-localization of TRAF6 (A), TRAF2 (B), cIAP1 (C) and TRAF3 (D) with TIFA-tdTomato, which formed TIFAsomes upon infection with H. pylori P1 wild‑type strain, were detected by immunofluorescence staining. The nuclei were counterstained with DAPI. Scale bar = 10 µm. TIFA‑KO cells transiently expressing TIFA‑tdTomato were infected with H. pylori P1 strain mutated in the gmhA gene (∆HP0857). Scale bar = 10 µm.
Colocalization of DPCK-mCherry with the apicoplast marker api-SFG A.Figure 2A.) Genotyping PCR confirming the insertion of a 40bp attB element in the p230p gene, with amplification demonstrating integration at the Δ5' and Δ3' loci and lack of wild type parasites (as indicated by the failure to amplify at the wild type 5' and 3' loci) as compared to the parental control. B.) Genotyping PCR confirming the insertion of the PfDPCK-mCherry construct using attP/attB integration. PCR reactions demonstrate integration at the Δ5' and Δ3' integration sites and lack of parental parasites (P) as compared to the parental control (P230p-attB). C.) Live epifluorescence microscopy of PfMev parasites that are expressing api-SFG (green) and the P. falciparum DPCK-mCherry protein (red) with DAPI marking the nuclei (blue). The borders of the red blood cell and parasite are marked with dotted blue and white lines, respectively. Microscopy images represent fields that are 10µm long by 10µm wide.
Complex I assembly kinetics in ∆4-CYB cells.Figure 6(A) SDS-PAGE resolving the radioactively labeled mitochondrial translation products after a 2-hour pulse (P). The 35S-Met and the cycloheximide were removed from the medium and cells were collected at the indicated chase times (2, 5 and 24 hours). (B) First dimension (1D) BNGE analysis of the same cells prepared with digitonin. (C) Denaturing second dimension (2D) BNGE analysis of the same samples allowed following the incorporation of the individual labeled subunits inside their corresponding complex and supercomplex species. (D) Complex I IGA (cI-IGA) analysis of digitonin-solubilized samples after inhibiting mitochondrial translation with doxycycline for 6 days (0h). After removing the drug and restoring synthesis of the mtDNA-encoded subunits, the cells were collected at the indicated times to follow the appearance of cI reactivity with time. The gels were incubated in the reaction mixture for 24 hours. SS = steady state. The asterisk indicates the presence of a high molecular weight cI-containing band of unknown nature (E) 2D BNGE, Western blot and immunodetection analysis of WT and ∆4-CYB mitochondria from cells collected at the same times after doxycycline treatment to follow the incorporation kinetics of the indicated cI nuclear-encoded subunits, belonging to different structural modules. The blots shown were either exposed for 16 sec. (low exposures) or 160 sec. (high exposures) in order to visualize the qualitative signals in the ∆4-CYB samples.
Fig.Figure 4(A) Real-time PCR analysis of the interfering efficacy and linear LMP2A mRNA levels after transfection of three circLMP2A shRNAs. (B) Tumour sphere culture analysis of the tumour sphere formation in SNU-4th cells transfected with sh-circLMP2A-2, sh-circLMP2A-3 or sh-control. The central horizontal lines represent the median, top and bottom positions of the box represent upper and lower quartiles, and error bars represent the mean ± SD. The average number and maximal diameter of spheres were calculated under a microscope in five randomly chosen fields in each independent experiment, n=3 biological replicates. Scale bars =100µm. EMT-related markers, stemness markers and drug resistance genes were evaluated by real-time PCR (C) in SNU-4th cells transfected with sh-circLMP2A-2, sh-circLMP2A-3 or sh-control. EMT-related markers, stemness markers and drug resistance genes were evaluated by WB (D) in SNU-4th cells transfected with sh-circLMP2A-2, sh-circLMP2A-3 or sh-control. (E-F) Transwell assay (E) and colony formation assay (F) analysis of the migratory and invasive capability and colony-forming ability in SNU-4th cells transfected with sh-circLMP2A-2, sh-circLMP2A-3 or sh-control. Scale bars =100µm. (G-H) Flow cytometric analysis of the rate of SP cells (G) and apoptosis (H) in SNU-4th cells transfected with sh-circLMP2A-2, sh-circLMP2A-3 or sh-control. Subcutaneously established tumour xenografts in NOD/SCID mice. Tumour volume (I) of the xenografts were remarkably inhibited by down-regulation of circLMP2A. Six mice per group. Subcutaneously established tumour xenografts in NOD/SCID mice. tumour weight (J) of the xenografts were remarkably inhibited by down-regulation of circLMP2A. Six mice per group. (K-L) Representative images of bioluminescence (K) and lung metastases (L) are presented to measure the metastatic colonies. Six mice per group, scale bars =50µm.
Figure 5A. Schematic representation of human Nup358-IR region and the constructs used in this study. IR, internal repeats; SIM, SUMO-interacting motif; 1, 2, SIM1 and SIM2. Amino acids substituted in Ubc9 mutant are indicated with red asterisks. B. HEK293T cells were transfected with indicated constructs and immunoprecipitation (IP) was performed using GFP specific antibodies and probed for HA-AGO2 by western blotting (WB) using HA antibodies. Endogenous RanGAP and Ubc9 were probed with specific antibodies. C. Lysates prepared from cells expressing the indicated constructs were subjected to IP and WB using indicated antibodies. Presence of endogenous RanGAP and Ubc9 was determined by WB. D. GFP-control, GFP-IR1+2 wild type or mutants were co-expressed with HA-AGO and IP and WB analysis were performed to detect the interaction using indicated antibodies. E. Lysates prepared from cells expressing the indicated proteins were immunoprecipitated with GFP specific antibodies and the presence of specific proteins in the immunoprecipitates was detected by WB with indicated antibodies.
Figure 1(A) Representative EPSP and mEPSP electrophysiological traces from larval NMJs at Muscle 6 in which a motoneuron specific driver was used to overexpress either a control transgene (DVGlut-GAL4 > UAS-RFP), dNmnat (DVGlut-Gal4 > UAS-dNmnat), or Wnd (DVGlut-Gal4 > UAS-Wnd). (B) Quantification of mean (±SEM) EPSP amplitudes, in which 75 consecutive evoked events were averaged per cell, and then cell amplitudes were averaged per genotype. [UAS-RFP n = 13 cells, UAS-dNmnat n = 14 cells, UAS-Wnd n= 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=36, F=12.66, p<0.0001. UAS-RFP vs UAS-dNmnat p < 0.0001 (****), UAS-RFP vs UAS-Wnd p = 0.0093 (**)]. ]. (C) Quantification of mean (±SEM) mEPSP amplitudes, in which 75 consecutive spontaneous events were averaged per cell, and then cell amplitudes were averaged per genotype. [UAS-RFP n = 15 cells, UAS-dNmnat n = 14 cells, UAS-Wnd n= 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=38, F=11.66, p = 0.0001. UAS-RFP vs UAS-dNmnat p=0.994 (NS), UAS-RFP vs UAS-Wnd p = 0.0003 (***)]. (D) Quantification of quantal content, which was calculated individually per cell by dividing the mean EPSP amplitude by the mean mEPSP amplitude, and then averaged per genotype. [UAS-RFP n = 15 cells, UAS-dNmnat n = 14 cells, UAS-Wnd n= 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=38, F=9.286, p = 0.0006. UAS-RFP vs UAS-dNmnat p=0.0015 (**), UAS-RFP vs UAS-Wnd p = 0.0003 (***)]. ]. (E) Representative images of the NMJ synaptic terminal at muscle 4 in third-instar larvae. The genotypes in this experiment were identical to those tested in the electrophysiology experiments in A-D. NMJs were stained for the presynaptic bouton marker DVGLUT (green) and nerve membrane marker HRP (red). (F) Quantification of the mean (±SEM) number of DVGlut+ boutons per muscle 4 NMJ in each genotype. [UAS-RFP n = 23, UAS-dNmnat n = 21, UAS-Wnd n= 20 NMJs. 1-way ANOVA w/Tukey's multiple comparisons, DF=63, F=68.31, p < 0.0001. UAS-RFP vs UAS-dNmnat p=0.9818 (NS), UAS-RFP vs UAS-Wnd p < 0.0001(****)].
cleaved RNA and drives Siwi-piRISC biogenesis (A) Analysis of Siwi-piRNA and Ago3-piRNA production in DDX43- or Vasa-knockdown BmN4 cells.Figure 3(A) Analysis of Siwi-piRNA and Ago3-piRNA production in DDX43- or Vasa-knockdown BmN4 cells. Left: The expression levels of DDX43 and Vasa following RNAi treatment. Middle and right: Comparison of Siwi-piRNA and Ago3-piRNA levels following DDX43 or Vasa knockdown by RNAi. The piRNAs were isolated by the immunoprecipitation of Flag-tagged proteins from knockdown cells transfected with Flag-Siwi or Flag-Ago3 and then 32P-labeled. (B) Artificial Siwi-piRNA production from a reporter plasmid encoding the PiggyBac-piRNA target sequence. (C) Analysis of the artificial piRNA levels in DDX43-knockdown BmN4 cells. Left: Artificial piRNA and Flag-Siwi were detected by northern blotting and western blotting, respectively. Right: Quantification of artificial Siwi-piRISC formed by the ping-pong cycle pathway. Data represent the mean+/- standard deviations, n = 3 independent experiments. *p < 0.05 as determined with the t-test. (D) RNA unwinding assays upon Ago3-piRISC-dependent target RNA cleavage Recombinant Vasa or DDX43 was added and incubated in the reaction mixture upon cleavage and the materials were separated using a magnet to supernatant (Sup.) and bead (Beads) fractions. RNAs cleaved by Ago3 are shown as "Cleaved RNA". DDX43 but not Vasa released the cleaved target RNA from Ago3-piRISC. (E) Time dependency of the DDX43 unwinding activity. Sup: supernatant. (F) RNA unwinding assays upon Ago3-piRISC-dependent target RNA cleavage as in (D). Recombinant DDX43 was added and incubated in the reaction mixture upon cleavage for 0 min (i.e., no cleavage), 15 min, 30 min, and 60 min, and the materials were separated using a magnet to supernatant (Sup.) and bead (Beads) fractions. RNAs cleaved by Ago3 are shown as "Cleaved RNA". DDX43 fails to unwind cleaved RNAs from Siwi-piRISC. − DDX43 means no DDX43 added in the reaction mixture.
Figure 4The schematic of therapeutic treatment of the TAC model using miRNA mimics followed by phenotypic characterizations. HW/TL ratio is measured (n=4-10 per group). Ctrl-miR: negative control miRNA mimics. (B) H&E and WGA staining of murine hearts in the therapeutic models. Scale bar: 1 mm for H&E and 50 μm for WGA. Cardiomyocyte surface area was quantified from WGA staining in the right panel. >120 CMs are quantified in each group. The black line shows medium value for the group and the dotted lines represent two quartile lines in each group. (C) Picrosirius red staining of murine hearts in the therapeutic models. Scale bar: 1 mm. The fibrotic area was quantified in the right panel (n=4-10 per group). (D) Echocardiography analysis of the cardiac function of mice in the therapeutic models at the end-point of 6 weeks post-surgery (n=4-10 per group).
Figure 6A. KV10.1 localizes to pericentrin-positive structures during ciliary resorption. After short (4h) reintroduction of FCS in serum starved hTERT RPE1 cells, the signal of anti-KV10.1 antibody co-localized with pericentrin. Scale bar: 25µm. B. Cells expressing a KV10.1*mVenus fusion under the control of the endogenous promoter to render a mild overexpression were fixed and immunostained for pericentrin. Venus fluorescence colocalized with pericentrin-positive structures. Scale bar: 5µm.
Figure 4(A) Western blot analysis of HIF1A and FTO protein levels in 3T3-L1 cells overexpressing Vec or HIF1A plasmid. (B) Western blot analysis of HIF1A and FTO protein levels in siCtrl or siHif1a 3T3-L1 cells. (C) The O2 consumption rate (OCR) in differentiated beige adipocytes from 3T3-L1 cells overexpressing Vec or HIF1A plasmid. The average basal and maximal respiration rates are shown in right panel. Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (D) The O2 consumption rate (OCR) in differentiated beige adipocytes from siCtrl or siHif1a 3T3-L1 cells. The average basal and maximal respiration rates are shown in right panel. Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (E) Western blot analysis of UCP1, PPARGC1A and PPARG in differentiated beige adipocytes described in (C). (F) Western blot analysis of UCP1, PPARGC1A and PPARG in differentiated beige adipocytes described in (D). (G) qPCR analysis of genes involved in thermogenesis in differentiated beige adipocytes described in (C). (H) qPCR analysis of genes involved in thermogenesis in differentiated beige adipocytes described in (D). ( Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (I) Immunofluorescent staining of UCP1 in differentiated beige adipocytes described in (D). Scale bar, 20 μm. (J) ChIP-qPCR analysis of the association of HIF1A with the promoter of thermogenic genes in 3T3-L1 cells differentiated into beige adipocytes. IgG was used as control. Consensus HIF1A binding sites were obtained from JASPAR database (a database of transcription factor binding profiles). Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Figure 5(A) Western blot of CathB levels (for both the precursor (pro-CathB) and the cleaved-mature forms) in the culture media from ASMko and wt BMDMs. Staining of Ponceau-S is shown as loading control. (B) Mean ± SEM fold increase in CathB levels in the culture media from wt and ASMko BMDMs (n = 5 independent cultures, Student´s t-test). (C) Immunofluorescence staining against LAMP-1 (yellow) and Iba-1 (red) in Cb of ASMko and wt mice. Scale bar, 50 μm, zoomed images 20 μm. (D) Immunofluorescence staining against LAMP-1 in non-permeabilised BMDMs from ASMko and wt mice. Scale bar, 50 μm. (E) Mean ± SEM of LAMP-1 levels on the surface of ASMko and wt BMDMs (n = 4 independent cultures, >40 cells per culture, Student´s t-test). (F) Mean ± SEM microvesicle and exosome numbers in the culture media from ASMko and wt BMDMs (n = 6 independent cultures, Student´s t-test). (G) Western blot of CathB and Flotillin1 levels in the secreted exosome fractions from ASMko and wt BMDMs. (H) Western blot of CathB levels in the exosome depleted culture media from wt and ASMko BMDMs. (I) Mean ± SEM MTT absorbance reflecting cell viability in primary neurons from wt mice treated with conditioned media from ASMko and wt BMDMs in the presence or absence of the CathB inhibitor Ca074 (n = 4 independent cultures Two-way Anova, Bonferroni post hoc). (J) Immunofluorescence staining against Iba-1 (green) and CathB (red) in lobes of the Cb sensitive or resistant to Purkinje cell death. Asterisks indicate Purkinje cells in the resistant lobes. Scale bar, 50 μm. (K) Mean ± SEM percentage of CathB positive microglia in resistant and sensitive Cb lobules of ASMko mice (n = 5 mice, χ2 test). (L) Western blot of Cath B and β-Actin levels in cerebellar extracts of wt and ASMko mice treated or not with PLX. (M) Mean ± SEM of CathB levels normalized by β-Actin in cerebellar extracts of wt and ASMko mice treated or not with PLX (n= 6 mice Two-way Anova, Bonferroni post hoc). (N) Diagram showing the experimental procedure to obtain media from cerebellar organotypic cultures dissected from the brain of wt and ASMko mice orally treated or not with PLX. (O) Western blot of CathB levels (for both the precursor (pro-CathB) and the cleaved-mature forms) in the culture media from ASMko and wt cerebellar organotypic cultures from mice fed with vehicle (veh) or PLX (PLX). Below, Western blots of organotypic culture lysates against Iba-1, to confirm microglia depletion, and against GAPDH as loading control. (P) Mean ± SEM increase in pro-CathB (left) and CathB (right) levels in the culture media from cerebellar organotypic cultures from wt and ASMko mice treated or not with PLX (n = 3 mice per group Two-way Anova, Bonferroni post hoc).
Figure 3A-F. Kaplan-Meier curves of DFS (A) and OS (B) in 249 patients in the training set. Kaplan-Meier curves of DFS (C) and OS (D) in 122 patients in the internal validation data set. Kaplan-Meier curves of DFS (E) and OS (F) in 180 patients in the external independent validation data set. Hazard ratios (HRs) were calculated with a univariate Cox regression analysis, P-values were calculated with the log-rank test.