EMBO/sd-character-level-ner huggingface dataset:
Increased FAK activation in human pancreatic adenocarcinoma-associated fibroblasts is an independent prognostic marker.Figure 2(A) Representative images of Definiens® Tissue studio multi-resolution segmentation algorithm on two PDAC TMA examples (X, Y). From left to right: IHC for pY397 FAK; ROI detection of tumour cells (dark blue), stroma (orange) and other (light blue/grey); Nuclear/ cytoplasm detection in blue and green respectively; IHC staining classification for pY397 FAK intensity signal (dark red for high signal, orange for medium signal, yellow for low signal and white for negative signal). (B-C) Disease Free Survival (B) and Overall Survival (C) from 120 patient cohort according to pY397 FAK expression in CAFs; HR: Hazard Ratio. Survival curves were estimated with the Kaplan-Meier method and compared using log-rank test, non-parametric tests (chi2-test) to compare independent groups for categorical data. (D-E) Disease Free Survival (D) and Overall Survival (E) from 120 patient cohort according to pY397 FAK expression in tumour cells. Survival curves were estimated with the Kaplan-Meier method and compared using log-rank test, non-parametric tests (chi2-test) to compare independent groups for categorical data.
Figure 1A Immunoblot analysis of the knockdown of exogenous USP19 in 293T cells expressing Flag-USP19 (top) or endogenous USP19 in HeLa cells (bottom) treated with USP19-specific siRNA or scrambled (Scr) siRNA. B Representative images of GFP-LC3B puncta in HeLa-GFP-LC3B cells transfected with USP19-specific siRNA or scrambled siRNA during growth in normal medium (DMEM) or EBSS medium for 3 h. Arrows denote representative autophagosomes. Scale bar, 200 μm. C Quantification of GFP-LC3B puncta in HeLa-GFP-LC3B cells transfected with USP19-specific siRNA or scrambled siRNA. Bars represented mean ± standard error of the mean (SEM) of triplicate samples (20 cells per sample). *p < 0.05, **p < 0.01 and ***p < 0.001. D Immunoblot detection of the relative accumulation of LC3 I and LC3 II in HeLa cells transfected with USP19-specific siRNA or scrambled siRNA with rapamycin (250 nM) treatment for 12 h. E HeLa cells were transfected with USP19-specific siRNA or scrambled siRNA and treated with indicated concentration of rapamycin for 12 h, p62 levels were detected by immunoblot. F Human peripheral blood mononuclear cells (PBMCs) transfected with control or USP19-specific siRNA were treated with rapamycin (250 nM) for 18 h, the lysates were analyzed with each antibody. G Flag-USP19 stably expressing 293T cells were incubated with DMEM or EBSS medium for 3 h in the absence or presence of 50 μM of chloroquine (CQ). Cells were then assayed for the relative accumulation of LC3-I and LC3-II by immunoblot analysis using anti-LC3 antibody. H Flag-USP19 inducible HeLa cells were treated with 200 ng/ml doxycycline (Doxy) for overnight to induce the expression of Flag-USP19. The protein levels of p62 were analyzed after rapamycin treatment for 12 h at indicated concentrations. I GFP-LC3B stably expressing HeLa cells were transfected with pcDNA3.1 empty vector or plasmid expressing the wild-type and the mutant forms of USP19 (CS or CS/HA). Pictures in (B) and (I) were taken using Leica DMI3000 B microscope with a×100 oil-immersion objective. Scale bar, 200 μm.J Average GFP-LC3B puncta per cell were calculated. Bars represented mean ± SEM of triplicate samples (20 cells per sample). *p < 0.05, **p < 0.01 and ***p < 0.001.
a) p62 vesicle.f2(a) p62 vesicle formation in CALM knockdown HeLa cells. Confocal pictures are shown. #CALM-downregulated cells where p62 vesicles accumulate. Data are representative of three independent experiments and shown as mean ±s.e.m. (n≥500 cells; *P0.01; two-tailed t-test). Scale bars, 5 μm. (b) Western blot analysis of p62, actin and GAPDH in HeLa cells and HEK cells (basal conditions, BC, or without serum for 1 h) where CALM was knocked down using shRNA or siRNA, as indicated. Data are mean ±s.d (n=3 experiments for HeLa cells and HEK cells; *P0.05; two-tailed t-test). (c) Percentage of huntingtinQ74-expressing cells with aggregates in CALM knockdown HeLa cells. Data depict one representative experiments performed in triplicate, out of three independent experiments and shown as mean ±s.d. (*P0.05; two-tailed t-test). (d) Tau-positive tangle formation in CALM knockdown cells. HeLa cells transiently expressing DsRed-tau 4R were treated with Baf A1 for 4 h as indicated. Cells were fixed and analysed by confocal microscopy after immnunostaining for phosphorylated tau using PHF1 antibody (green). Data represent the number of cells with phosphorylated tau-positive tangles as mean ±s.e.m. (n=3 experiments; *P0.01; two-tailed t-test). Scale bars, 5 μm. (b) Western blot analysis of p62, actin and GAPDH in HeLa cells and HEK cells (basal conditions, BC, or without serum for 1 h) where CALM was knocked down using shRNA or siRNA, as indicated. Data are mean ±s.d (n=3 experiments for HeLa cells and HEK cells; *P0.05; two-tailed t-test).
Analysis of autophagy in TCHP-depleted endothelial cells A, Immunofluorescent staining for LC3 and p62 in TCHP knock-down and control cells.Figure 3Immunofluorescent staining for LC3 and p62 in TCHP knock-down and control cells. Scale bars, 25μm. Lower panel: quantification. (n=90 cells, unpaired t-test; LC3: **p<0.0001 vs control; p62: **p<0.0001 vs control). Western blot of p62 and LC3 during under normal culture condition or starved condition (HBSS) or the presence of BafA1 (2 hours) in TCHP knock-down or control cells. Lower panels: p62 quantification. (one-way ANOVA; **p=0.0003 vs control, ##p=0.0093 vs shTCHP); LC3 quantification. (one-way ANOVA; **p=0.0008 vs control, ##p=0.0011 vs shTCHP). HUVECs were transduced with the tandem mCherry-EGFP-LC3 and with shRNA TCHP or control vectors. Left panels: Representative picture of mCherrry-EGFP-LC3 reporters. Scale bars, 25μm. Right panel: Quantification of the number of mCherry-only (red bars, autolysosomes) or double-positive (mCherry+/EGFP+; yellow bars, autophagosomes). (n=80 cells, one-way ANOVA; *p=0.0447 vs control; ##p=0.0007 vs shTCHP). Ratiometric flow cytometric analysis of mCherrry-EGFP-LC3 reporters as in C. (one-way ANOVA; *p=0.0247 vs control; ##p=0.0088 vs shTCHP). Quantification of long-lived protein degradation assay in TCHP knock-down and control cells by flow cytometry (one-way ANOVA; **p=0.0018 vs control, ##p<0.0001 vs shTCHP). HUVECs were transduced with TCHP-V5 and control vectors. Western blot for anti-V5 and anti-p62 antibody during under normal culture condition or HBSS or BafA1 or Torin-1 treatment. Lower panel: quantification. (one-way ANOVA; **p=0.0044; #p=0.0117 vs TCHP-V5).
Visual comparison of [18F]FDG- and [68Ga]Pentixafor PET scans.Figure 4Visual comparison of [18F]FDG- and [68Ga]Pentixafor PET scansNumber of patients with visual positivity for the indicated PET tracer (total: n = 14).Number of patients (total n = 14) for whom imaging with [18F]FDG PET (FDG, n = 2) or [68Ga]Pentixafor PET (Pentixafor, n = 7) was superior, with comparable positivity (comparable, n = 3), and with dual imaging providing complementary visual information (complementary, n = 2).
Figure 6Infected RBCs can interact with both endothelial cells and neutrophils. Left panel, iRBC adhere to the endothelium as a strategy for escaping removal by the spleen. The binding of iRBC to the endothelial cell is mediated by PfEMP1 and different endothelial receptors e.g., ICAM-1, CSA, CD36, EPCR. Right, neutrophils surface express ICAM-1 and binds to PfEMP1 on iRBC. This result to extracellular killing or the phagocytosis and killing of the iRBC (not shown)
Figure 6(A, B) Co-immunoprecipitation of endogenous APC11 with endogenous p65 in HEK293T cells. (C) Western blot analyses of the degradation of p65 protein in NC and APC11si HEK293T cells. (D) Western blot analyses of the degradation of p65 protein by APC11. HEK293T cells were transfected with Vector and HA-APC11 plasmids for 36 h, cells were treated with or without 10 μM MG132 for 6 h before collection. (E) Co-immunoprecipitation of APC11 with endogenous p65 in NC and CDC20sh HEK293T cells. (F) Western blot analyses of p65 expression in NC or CDC20sh HEK293T cells after the overexpression of Vector or HA-APC11 plasmids. (G) The efficiency of APC11 knockdown determined by western blot in hBMSCs. (H, I) The ALP staining (H) and ALP quantification (I) of NC and APC11si hBMSCs after 7 days osteogenic differentiation. (n=6)
Figure 5Specific interaction of CLK1 with HSATIII during thermal stress recovery. HSATIII-ChIRP at various time points during and after thermal stress exposure. Endogenous CLK1, SRSF7, and SRSF9 were detected using anti-CLK1, anti-SRSF7, and anti-SRSF9 antibodies, respectively. HSATIII was detected by ChIRP-RNA purification, followed by RT-PCR. Input: 0.1% for CLK1, 1% for SRSF7 and SRSF9, and 100% for HSATIII. The domain structures of wild-type (WT) CLK1, the catalytically inactive mutant (CLK1 KR), and the N- and C-terminal partial fragments (CLK1∆C and CLK1∆N). Specific interaction of HSATIII with CLK1 proteins during stress recovery. HSATIII-ChIRP/western blotting was performed using HeLa cells expressing FLAG-CLK1 WT or the KR mutant. FLAG-tagged CLK1 and SRSF9 were detected by western blotting using an anti-FLAG and an anti-SRSF9 antibody, respectively. HSATIII was detected by ChIRP followed by RT-PCR. Input: 1% for western blotting, 100% for RT-PCR. Re-translocation of FLAG-CLK1 to nSBs after thermal stress removal. HeLa cells were transfected with a FLAG-CLK1 expression vector, cultured for 16 h, and then exposed to thermal stress (42°C for 2 h) with or without recovery (37°C for 1 h). FLAG-CLK1 was visualized with an anti-FLAG antibody. Nuclear stress bodies and nuclei were visualized by HSATIII-FISH and DAPI staining, respectively. Scale bar: 10 μm. Box plot of colocalization correlation coefficients between HSATIII and FLAG-CLK1 WT. The first and third quartiles are the ends of the box, the median is indicated by the vertical line in the box, and the minimum and maximum are the ends of the whiskers. Recovery phase-specific interaction of HSATIII with the CLK1 N-terminal region. HSATIII-ChIRP/western blotting was performed on HeLa cells expressing FLAG-CLK1∆C or FLAG-CLK1∆N Asterisk: an unidentified partial fragment of FLAG-CLK1∆N. Dependency of SRSF9 on the interaction of CLK1 (G) with HSATIII. HSATIII-ChIRP was performed using control and SRSF9-depleted cells expressing FLAG-CLK1 proteins (42°C for 2 h and recovery for 1 h at 37°C). Input: 1%. Graphs represent coprecipitation ratios of FLAG-CLK1 proteins to HSATIII in the control and SRSF9-depleted cells. H Dependency of SRSF9 on the interaction of CLK1∆C (H) with HSATIII. HSATIII-ChIRP was performed using control and SRSF9-depleted cells expressing FLAG-CLK1 proteins (42°C for 2 h and recovery for 1 h at 37°C). Input: 1%. Graphs represent coprecipitation ratios of FLAG-CLK1 proteins to HSATIII in the control and SRSF9-depleted cells.
Membrane position of TREM2-TMH and the K186A variant.Figure 4(a) Paramagnetic relaxation enhancement (PRE) with a free-radical-modified stearic fatty acid in DPC micelles. Low values indicate location inside whereas values close to one suggest a location outside the detergent micelle. Orange bars: TREM2-TMH wt, green bars: K186A variant. The N-terminal residues of TREM2-TMH-wt are located deeper inside the micelles as in the K186A case, whereas the C-terminal end is positioned at the outside for wild-type and inside for the K186A variant. Negative bars indicate missing assignments. (b) NOE-cross peaks between backbone amides of TREM2-TMH wt (top) or the K186A variant (bottom) and selected regions of DPC (see color coding of the structure of the DPC molecule on the right). The K186A variant shows strong contacts to the hydrophobic aliphatic regions of the detergent throughout the transmembrane helix whereas no such contacts can be seen at the kink site of the wild-type helix, indicating a location outside of the micelle for this part. (c) Model of the position of TREM2-TMH wt and K186A within the membrane. The amino acids in the kink region are exposed to the solvent in the wild-type case but buried in the membrane for the K186A variant.
Inhibition of HIV and M.Figure 8Inhibition of HIV and M. tuberculosis by 1,25D3 is CAMP and autophagy dependent.MDM were transduced with non-specific scrambled shRNA (shNS) or CAMP shRNA (shCAMP) and selected using puromycin resistance. Five days later, cells were incubated with 100 pmol/L 1,25D3 or vehicle control for 4 h before infection with HIV and/or M. tuberculosis (TB) for 3 h. Cells were then washed and incubated with 100 pmol/L 1,25D3 or vehicle control for 7 days. (A) Immunoblot analysis performed using antibodies raised to CAMP or β-actin after initial pathogen exposure (Day 0) or after 7 days. (B) Day 7 HIV-1Ba-L and TB co-infected cells were harvested and stained for saponin-resistant LC3B-II at day 7. A representative histogram from three donors are shown. C) ELISA performed for HIV p24 antigen release over 7 d. (D) Intracellular mycobacteria were harvested and assayed for mycobacterial growth by cfu enumeration at day 0 and day 7. All bar and line graphs are reported as mean ± s.e.m. of three independent experiments performed in triplicate. * P<0.001.
Figure 4(A) Schematic diagram of the kinase-dead (KD) Pax5-Jak2 fusion protein, which is encoded by the Pax5Jak2-KD allele and contains the K272E mutation known to abolish the Jak2 kinase function. (B, Flow-cytometric analysis of bone marrow and spleen from Pax5Jak2-KD/+ (black; n = 11) and control Pax5+/+ (grey; n = 7) mice at the age of 6-8 weeks. The frequencies of the indicated B cell types are shown for each organ (B). The data are presented as mean percentages with SEM and were statistically analyzed by multiple t-tests (unpaired and two-tailed with Holm-Šídák's correction); ns (P > 0.5), *P < 0.05, **P < 0.01. C) Flow-cytometric analysis of bone marrow and spleen from Pax5Jak2-KD/+ (black; n = 11) and control Pax5+/+ (grey; n = 7) mice at the age of 6-8 weeks. The expression of CD19 and B220 on bone marrow B cells of the indicated genotypes is shown (D, Tumor progression in mice, transplanted with Pax5Jak2-Luc/+ tumor cells, in the presence or absence of the JAK1/2 inhibitor ruxolitinib. At day 14 after cell transfer, the transplanted mice were treated twice daily with ruxolitinib or vehicle, and the tumor mass was monitored by bioluminescence measurements. Images of three representative mice with or without ruxolitinib treatment are shown The bioluminescence measurements of transplanted mice treated with ruxolitinib (black) or vehicle (grey) at the indicated days after cell transfer are shown as mean radiance values with SEM (E). n, number of mice analyzed. Statistical data are shown as mean value with SEM and were analyzed by the mixed-effect model REML with Geisser-Greenhouse's correction and Tukey's multiple comparison test: *P < 0.05, ***P < 0.001. (F) Kaplan-Meier survival analysis of the transplanted mice upon prolonged treatment with ruxolitinib (black) and vehicle (grey). Statistical analysis of the survival curves (F) was performed with the log-rank (Mantel-Cox) test; **P < 0.01.
Figure 1Expression pattern of Rgcc in the embryonic day 14.5 (E14.5) mouse cortex as detected by in situ hybridization. The right panels showed the expression patterns of NSC markers, including Pax6, Sox2, and Ki67, along the ventricular zone (VZ) of the E14.5 mouse cortex. Scale bar: 20 μm. Analyzing single cell transcriptomic database of the E14.5 mouse cortex, showed the specific expression of Rgcc in NSCs. Data were downloaded form http://zylkalab.org/datamousecortex. RGCC expression and distribution in developing human fetal neocortex. RGCC microarray data were downloaded from BrainSpan Atlas of the Developing Human Brain (Available from: human.brain-map.org; Donor id: 14751, donor name H376.IIIB.02, donor age 16pcw). Two independent probes NO: A_23_P204937 and NO: A_23_P10137 were used. Heatmap was further prepared by MeV. Only the expressions along neocortex were shown. Single cell RNA-seq analysis of human fetus cortex (https://cells.ucsc.edu/?ds=cortex-dev). Cell-type clusters expressing RGCC was circled by red dash line. Expression patterns of NSC markers were showed in the right panels. Immunofluorescent staining of hCOs of day30. RGCC and PAX6 were co-stained. Scale bars: 100 μm. Immunofluorescent staining of hCOs of day30. RGCC and TBR1 were co-stained. Scale bars: 250 μm.
Mitochondrial injuries induce a PLS3-dependent externalization of CL to the outer surface of mitochondria.figf6Mitochondrial injuries induce a PLS3-dependent externalization of CL to the outer surface of mitochondria. Cargo incorporation into autophagosomes is facilitated by interactions of CL with the N-terminal domain of LC3. Clearance of CL-exposed mitochondria by mitophagy (green arrows) would be predicted to prevent CL oxidation and accumulation of pro-apoptotic signals (red arrow).
Intravitreal gene supplementation with AAV2.Figure 6(A) Representative photopic ERG flicker series at 1, 5 and 10 Hz with 3 cd.s/m2 illumination at 10 WPI. Arrowheads indicate light stimulus. (B) Representative photopic single flash ERG signal with 1, 3 and 10 cd.s/m2 illumination. The vertical dotted line indicates light stimulus. (C) Quantification of photopic B-wave amplitude from single flash responses to 1, 3 and 10 cd.s/m2 illumination of wildtype C57BL6/J x 129/sv eyes (WT, n = 3) compared to treated eyes (IVT/GL, n ≥ 3) and untreated eyes (UN, n ≥ 3) at 10 weeks post injection. (D) Graphical representation of mouse fundus (d=dorsal, n=nasal, v=ventral, t=temporal) divided into quadrants. Q2 indicates locus of intravitreal injection. (E-F) Confocal images of sections proximal to the injection site in Q2 (E) and distal to the injection site in Q4 (F), showing Cnga3 expression in PNA-positive cone outer segments. OS, outer segment layer; ONL, outer nuclear layer; INL, inner nuclear layer. (G-I) Confocal images of Cnga3-/- treated (G), wildtype (H) and Cnga3-/- untreated (I) retinal sections stained for Cnga3. Note some non-specific signal detected in INL and adjacent synaptic layer. Scale bar: 50 μm. OS, outer segment layer; ONL, outer nuclear layer; INL, inner nuclear layer.
SUMO-2 Proteins Accumulate in HD Brain.Figure 7(A) Western blot analysis of HeLa cells over-expressing exogenous PIAS1 in the presence of mutant HTT (97Q) when separated into detergent-soluble and detergent-insoluble fractions. No difference is detected in monomeric HTT (top panel, Soluble), but HMW HTT levels increase with PIAS1 overexpression. Anti-PIAS1 antibody (Invitrogen) was used to detect PIAS1. (B) Acute knockdown of PIAS1 decreases HMW HTT species in the detergent-insoluble fraction. PIAS1 knockdown is detected in detergent-soluble and -insoluble fractions using anti-PIAS1 antibody. (C) Drosophila melanogaster expressing mutant HTTex1p (93Q) in a reduced Su(var) 2-10/dPIAS genetic background exhibits statistically significantly reduced photoreceptor neuron degeneration (left panel, p = 0.033) when comparing dPIAS/+ to +/+ flies and increased overall survival (right panel, p = 0.047) when comparing dPIAS1/+ to +/+ flies. Significance was measured by Student's t test. (D) HMW SUMO-2 accumulates in postmortem HD striata. Western blot analysis of the insoluble fraction from three control and three HD postmortem striata as described (Experimental Procedures).
C-circles bind to the NPCs.Figure 3A Senescence curves of four est2∆ and six est2∆ NIC96-RFP independent clones used to generate type II survivors for C-circles co-IP experiments. The time when the cells were collected for the Co-IP is indicated by an arrow. The results are shown for one of the 3 independent experiments with similar outcomes. B Telomere length and recombination were analyzed by TG1-3 probed Southern blot of XhoI-digested DNA prepared from samples of the replicative senescence. Two representative clones are shown. C Detection of the t-circles co-immunoprecipitated with Nic96-RFP from the extracts obtained from several type II survivors. The DNA co-immunoprecipitated with Nic96-RFP was amplified by RCA assay, loaded on a dot blot and detected with a telomeric TG1-3 probe. The numbers above the blot refer to the growth curves in (A) and correspond to type II survivors produced in independent cultures. The lower panel shows the efficiency of Nic96-RFP IP that was evaluated by western blot with anti-mCherry antibody. D Quantification of the RCA product after co-IP of the C-circles with Nic96-RFP showing the effect of ADA2 deletion. Dot signal intensities were quantified using the Fiji software and were calculated as the percentage of INPUT that was immunoprecipitated normalized to est2∆ signals. Relative signals were calculated by setting the mean of est2∆ signals from each independent experiment to 1. Means and s.e.m. of the six independent co-IPs experiments each with 2-6 clones per genotype are shown. Outliers were removed using ROUT robust outlier test. The p-values from one-way ANOVA were adjusted using the Bonferroni's correction for multiple comparisons. est2∆ versus est2∆ NIC96-RFP (p=0,000007), est2∆ NIC96-RFP versus est2∆ ada2∆ NIC96-RFP (p=0,00012).***P<0,0001. E Quantification of the RCA product after co-IP of the C-circles with Nic96-RFP showing the effect of SUS1 deletion. The graph shows means and s.e.m. of the three independent co-IPs with 2-5 clones per genotype. Outliers were removed using ROUT robust outlier test. The p-values from one-way ANOVA were were adjusted using the Bonferroni's correction for multiple comparisons. est2∆ versus est2∆ NIC96-RFP (p=0,002), est2∆ versus est2∆ sus1∆ NIC96-RFP (p= 0,0006). **P<0,001, ***P<0,0001.
Figure 3(A) (Left) Representative western blot analysis of PGC-1α protein expression in the gastrocnemius muscle from the indicated genotypes, heart (Ht) lysate from WT mice and PGC-1α mKO (MCK-Cre) mice as positive and negative controls, respectively. (Right) Quantification of the PGC-1α/Tubulin signal ratios normalized (=1.0) to the NTG control (n = 5 mice per group). *p = 0.00017. (B) Expression of the Ldhb, Ldha, Mb (myoglobin), and Cox5a genes (RT-qPCR) in the gastrocnemius muscle from the indicated genotypes (n = 5 mice per group). *p < 0.001 (vs. NTG), ‡p < 0.01 (vs. 499Tg). (C) (Left) Representative western blot analysis performed with gastrocnemius muscle total protein extracts prepared from the indicated mice using cytochrome c, myoglobin, and α-tubulin (control) antibodies. (Right) Quantification of the Cyt c/Tubulin signal ratios normalized (=1.0) to the NTG control. NTG, n = 5; 499Tg, n = 6; PGC-1α mKO, n = 5; 499Tg/PGC-1α mKO, n = 5. *p < 0.01 (vs. NTG), ‡p = 0.00018 (vs. 499Tg). (D) Cross-section of gastrocnemius muscle from the indicated mice stained for SDH activity (Top) as well as MHC1 immunofluorescence (IF) (Bottom), Representative images were shown. Scale bar: 500 μm. (E) Respiratory exchange ratio (RER) during a graded exercise regimen as described in Materials and Methods. Notably, no significant increase in exhaust speed was observed in 499Tg/PGC-1α mKO mice compared to PGC-1α mKO mice. PGC-1α mKO, n = 5; 499Tg/PGC-1α mKO, n = 8. (F) The bars represent the mean blood lactate levels from the indicated mice following a 25 min run on a motorized treadmill. NTG, n = 9; 499Tg, n = 7; PGC-1α mKO, n = 6; 499Tg/PGC-1α mKO, n = 9. *p = 0.0009 (NTG vs. 499Tg), p = 0.316 (NS, not significant).