EMBO/sd-character-level-ner huggingface dataset:
Figure 3Representative images of TRAP staining of femur from AdipoqCre; ranklfl/fl (Rankl-/-) and ranklfl/fl (Rankl+/+) mice at 8 weeks. Scale bar=200 μm. Panel a1 and a4 indicate trabecular bone, a2 and a5 stand for growth plate, a3 and a6 denote cortical bone. Numbers of Trap+ cells on trabecular bone (a1 vs. a4, see (A)) (n=6 independent microscopic vision fields from random mice samples). Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations. Numbers of Trap+ cells under the growth plate (a2 vs. a5, see (A)) (n=6 independent microscopic vision fields from random mice samples). Data were compared using an unpaired t‐test, error bars are standard deviations. Numbers of periosteal Trap+ cells (a3 vs. a6, see (A)) (n=6 independent microscopic vision fields from random mice samples). Data were compared using an unpaired t‐test, error bars are standard deviations. Immunofluorescence staining of osteocalcin from 8-week-old mice (n=6 mice in Rankl-/- group and 4 mice in Rankl+/+ group). Scale bar=50 μm. TB: trabecular bone. Statistical analyses of Ocn+ cells (n=6 independent microscopic vision fields from random mice samples). Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations. Calcein staining of trabecular bone at 8 weeks. Scale bar=50 μm. TB: trabecular bone. Images are representative of 6 independent biological replicates in Rankl-/- and 5 replicates in Rankl+/+ group. Mineral apposition rate (MAR) of trabecular bone at 8 weeks.(n=6 mice in Rankl-/- group and 5 mice in Rankl+/+ group). Data were compared using an unpaired t‐test ( ** indicates P < 0.01), error bars are standard deviations. Serum CTX-1, OCN and bone marrow RANKL levels from Rankl-/- and Rankl+/+ mice at 4 weeks (n=5 and 5), 8 weeks (n=5 and 6) and 16 weeks (n=5 and 5). Data were compared using an unpaired t‐test (* indicates P < 0.05, ** indicates P < 0.01), error bars are standard deviations..
Figure 6A Mice expressing nuclear‑localized GFP under the control of the Pdgfra promoter were maintained under normoxic (21% O2) or hyperoxic (85% O2) conditions, and lungs were harvested, processed and immunostained for Ki67 to determine proliferation status. DAPI staining revealed nuclei of all cells present in the section. Low-magnification images from individual channels are presented to the right of the merged (large) image first row of images. The area demarcated by the white box in the merge image of the first row is magnified in the second and third rows to allow for visualization of greater magnification of the demarcated region of the merged image, as well as visualization of a single Ki67+,GFP+ cell (white arrowhead) in all three channels separately. B The number of PDGFRα+ cells in four microscopic fields was assessed for co-staining with an anti-Ki67 antibody to reveal proliferating cells. P values were calculated by unpaired Student's t‑test (n=4 fields for each group, trends are representative of those observed in two other experiments). C The Ki67 staining and GFP fluorescence was controlled for by examining lungs from wild-type mice that were treated with an isotype-matched control IgG used for the Ki67 staining experiments. Sections were examined for GFP fluorescence as well as in the red channel used to detect the Ki67 staining. Scale bar: 100 μm. DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein.
Figure 4A Schematic structure of LAMTOR1 mutants. GCC are lipidation sites. Blue cans indicate α-helices. Dashed lines indicate the locations of amino acid deletions. Arrowheads indicate the locations of amino acid substitutions. WT, wild-type; ∆N, N-terminal deletion; ∆C, C-terminal deletion; ∆K1, K20R; ∆K2, K31R. Lysates from Hela cells co-transfected with LAMTOR1-Flag or its mutants ∆N and ∆C and TRPML1-YFP were immunoprecipitated with anti-GFP or control IgG antibodies and probed with the indicated antibodies. Left, input proteins; right, immunoprecipitated (IP) proteins. C Lysates from Hela cells transfected as described in B were immunoprecipitated with anti-Flag or control IgG antibodies and probed with Flag and GFP antibodies. Lysates from Hela cells co-transfected with LAMTOR1-Flag or its mutants , ∆N1 and ∆N2 and TRPML1-YFP were immunoprecipitated with anti-GFP or control IgG antibodies and probed with the indicated antibodies. Left, input proteins; right, immunoprecipitated (IP) proteins. Lysates from Hela cells co-transfected with LAMTOR1-Flag or its mutants , ∆K1 and ∆K2 and TRPML1-YFP were immunoprecipitated with anti-GFP or control IgG antibodies and probed with the indicated antibodies. Left, input proteins; right, immunoprecipitated (IP) proteins. Note one group (Starved) of Hela cells transfected with LAMTOR1-Flag and TRPML1-YFP in E were incubated in medium without amino acids and serum for 2 h. F Quantification of co-IP results. N = 3 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by one-way ANOVA with Dunnett's post-test (F). *P < 0.05, ***P < 0.001.
Ubiquitin and p62 positive structures accumulate in cells expressing CHMP2 mutants.Figure 7.Ubiquitin and p62 positive structures accumulate in cells expressing CHMP2 mutants. HeLa cells transfected with myc-tagged wild-type CHMP2B (A), CHMP2BIntron5 (B), or CHMP2BΔ10 (C) were labeled with antibodies against c-Myc (red), ubiquitin (green), and p62 (blue) and analyzed by confocal microscopy. Single channel images in black and white are shown. Bars,10 μm. (D) Total levels of p62 in these cells were quantified in 20 cells from three independent experiments using the Zeiss LSM 510 Meta software and the average normalized to the average p62 level in control (mock transfected) cells. Error bars = SEM. (E) Western blot analysis showing accumulation of p62 in cells transfected with myc-CHMP2BIntron5 compared with mock-transfected cells (control) and cells transfected with CHMP2Bwt (F) Immuno-EM showing p62-positive aggregates containing dense clusters of vesicular-tubular structures in cells transfected with myc-CHMP2BIntron5. Bar, 200 nm.
Figure 1Immunoblot analysis of ADAR1 p110, ADAR1 p150 and ADAR2 expression in various organs isolated from wild-type (WT) mice. The expression of GAPDH is shown as a reference. The relative expression of ADAR1 p150 mRNA (B) and total ADAR1 mRNA (C) in sorted DN, DP, 4SP and 8SP thymocytes isolated from WT mice. The relative expression of ADAR1 p150 mRNA (B) and total ADAR1 mRNA (C) were normalized to the level of expression in DN thymocytes (DN values set to 1). Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM (n = 5; Mann-Whitney U-test, *p < 0.05, **p < 0.01). The relative expression of type I interferon-stimulated genes (ISGs) in sorted DN, DP, 4SP and 8SP thymocytes isolated from WT mice. Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, *p < 0.05). The relative expression of ISGs was normalized against the level of expression in DN thymocytes (DN values set to 1).
Figure 1A. Schematic diagram illustrating the separation of GFP-expressing (+) and non-GFP-expressing (-) cells from stress-treated zebrafish embryos of transgenic line huORFZ using LMD methods followed by microarrays. B. Graphical representation of two microarray expression analyses. A total of 298 over-represented transcripts and 104 under-represented transcripts were obtained from both microarrays. C. Upper panels: top view of EGFP expressed in zebrafish embryos derived from zebrafish transgenic line huORFZ under non-stress and heat-shock treatment. Middle panels: lateral view of endouc expression patterns in embryos under non-stress condition and treated with heat-shock. Lower panels: top view of endouc expression patterns. D. Quantitative RT-PCR determined the relative expression level of endouc mRNA in non-stress condition and heat-shock-treated embryos. Values representing the non-stress and Heat-shocked conditions were calculated from three independent experiments and presented as mean ± S.D. (n=3). Student's -t test was used to determine significant differences between each group (***: P <0.001). E. The endouc and ela3l (negative control) mRNAs were separately microinjected into zebrafish embryos from huORFZ at a one-cell stage. Upper panels: observation of GFP signal expressed in huORFZ embryos at 72 hpf. Lower panels: magnification of pictures corresponding to the upper panels.
Figure 3PS decarboxylase activity in a psd1Δpsd2Δdpl1Δ mutant expressing Psd1, Psd1(ER) or Psd1(Mt) expressed as (A) percentage of the lipid-incorporated 13C15N-label recovered in PE, PS decarboxylase activity in a psd1Δpsd2Δdpl1Δ mutant expressing Psd1, Psd1(ER) or Psd1(Mt) expressed as (B) the molecular species composition of *PS and *PE after 20 min incubation with 13C315N-serine PS decarboxylase activity in a psd1Δpsd2Δdpl1Δ mutant expressing Psd1, Psd1(ER) or Psd1(Mt) expressed as ; (C) the corresponding molecular signatures of PSD activity. (D) Cartoon depicting the enrichment of di-unsaturated PE molecular species produced by Psd1(Mt) versus Psd1(ER), with the thickness of the arrows reflecting relative enzyme activities. Based on the data presented in panels A and B; DB:1 and DB:2, fractions of *PE molecular species with 1 and 2 double bonds, respectively.
Fig.Figure 1A. Schematic representation of Casp8 deletion in adult ECs indicating the time points for tamoxifen treatment, analysis of the phenotype (early (day 15) or late (day 18)), and the timeframe of disease progression. B-C. Graphs showing the relative weight (B; n= 15 WT, 12 ECko; curve comparison) and the survival (C; n= 15 WT, 11 ECko; Log-Rank test) of mice upon tamoxifen treatment. Data information: Data shown as mean ± SEM. **P <0.01, *** P <0.001, **** P <0.0001, ns: not significant. D. Representative images of the intestine of Casp8WT and Casp8ECko mice, with mild and severe hemorrhages at a late disease stage. E-F. Representative images of H&E staining (E) and quantification of intestinal pathology (F) of the small intestine at an early disease stage in Casp8WT and Casp8ECko mice (enteropathy score; n= 6 WT, 6 ECko; two tailed unpaired Student's t-test). Scale bars: 100µm; insets 50µm. Data information: Data shown as mean ± SEM. **P <0.01, *** P <0.001, **** P <0.0001, ns: not significant. G-H. Quantification of intestinal pathology (enteropathy score, G; n= 8 WT, 7 ECko; two tailed unpaired Student's t-test) and hemorrhages into the mucosa (H; n= 4 WT, 5 ECko; two tailed unpaired Student's t-test) at a late disease stage in Casp8WT and Casp8ECko mice. Data information: Data shown as mean ± SEM. **P <0.01, *** P <0.001, **** P <0.0001, ns: not significant. I. Overview of H&E stained swiss roll preparations of Casp8WT and Casp8ECko mice. Scale bars: 2mm. J. Representative images of H&E staining of the small intestine of Casp8WT (j') and Casp8ECko mice with mild (j'') and more severely (j''') affected areas at a late disease stage (white arrows point to hemorrhages, asterisk points to focal erosion of the epithelium). Scale bars: 100µm.
Figure 1DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. Representative confocal microscopy images of CMAC-stained conjugates formed between B lymphocytes (isolated from DICER-KO mouse splenocytes and pre-activated with a mixture of LPS plus IL-4) and CD4+ T cells (from OVA-specific OT-II transgenic mice); conjugates were formed either in the presence or in the absence of OVA. A representative experiment of at least 3 independent experiments is shown. DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. showing the percentage of B cells forming conjugates DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. the accumulation at the IS of the TCR Conjugates were assessed using the Image J plug-in for IS analysis DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. the accumulation of the actin at the IS Conjugates were assessed using the Image J plug-in for IS analysis DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. the distance from the CD4+ T cell MTOC to the contact site with the B cell MTOC-B-cell distance was measured with Imaris analysis software. DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. Flow cytometry analysis of B lymphocytes co-cultured with OT-II isolated T cells in the presence or absence of OVA, showing IgG1 expression after 24h of co-culture, DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. Flow cytometry analysis of B lymphocytes co-cultured with OT-II isolated T cells in the presence or absence of OVA B cell proliferation after 24h, 48h and 72h co-culture (Ki67 expression in live B cells and cell violet tracer dilution, respectively) DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. B cell proliferation after 24h, 48h and 72h co-culture (Ki67 expression in live B cells and cell violet tracer dilution, respectively), DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. Flow cytometry analysis of B lymphocytes co-cultured with OT-II isolated T cells in the presence or absence of OVA survival (staining with the live marker DAPI) DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. Flow cytometry analysis of B lymphocytes co-cultured with OT-II isolated T cells in the presence or absence of OVA apoptosis (cleaved Caspase-3 expression) 24h, 48h and 72h after IS formation.
Figure 1A Tissue distribution of RIPK2 determined by anti-FLAG immunoprecipitation. Organ homogenates from WT (wild-type) and KI (homozygous FLAG-RIPK2) mice were subjected to anti-FLAG immunoprecipitation and immunoblotting. B Inflammatory signaling in wild-type (WT/WT), RIPK2 CRISPR KO (KO/KO) and FLAG-RIPK2 heterozygous (WT/KI) and homozygous (KI/KI) BMDMs. BMDMs were primed with IFNy, stimulated with MDP for indicated times and analysed by immunoblotting. C Cytokine production of RIPK2 CRISPR KO (KO), wild-type (WT) and FLAG-RIPK2 heterozygous (WT/KI) and homozygous (KI/KI) BMDMs in response to MDP. BMDMs were left untreated or treated with IFNy or IFNy and MDP over-night and cytokines were measured by ELISA. N = 5-8 mice. Shown is average ± SEM. ns = P > 0.05; ** = P ≤ 0.01; **** = P ≤ 0.0001; two-way ANOVA. D Serum cytokines in RIPK2 CRISPR KO (KO), wild-type (WT) and FLAG-RIPK2 heterozygous (WT/KI) or homozygous (KI/KI) mice after i.p. MDP administration. Mice were injected i.p. with PBS or MDP, sacrificed after 4h and serum cytokines were measured by ELISA. N = 3-6 mice. Shown is average ± SEM. ns = P > 0.05; ** = P ≤ 0.01; two-way ANOVA.
Figure 6A. Representative immunofluorescence images of the nephron structures within kidney organoids derived from hiPSCs on day 18 of the differentiation; proximal tubules (LTL, green), parietal epithelial cells (PAX8, purple), kidney glomerular podocytes (NPHS1, red), nephron progenitors (WT1, purple and SIX2, green). Cell nuclei were stained with DAPI. Scale bars: 50 µm. B. Representative images showing morphological collapses in iPSC-derived human kidney organoids following Stx2a (10 ng/mL) treatment for 72 h in the presence or absence OSMI-1 (10 µM, final). C, D. ELISAs were used to analyze the inhibitory effect of OSMI-1 (10 µM, final) on Kidney injury molecule-1 (KIM-1) secretion (C) and IL-8 and CCL-2 production (D) in culture supernatants of iPSC-derived human kidney organoids exposed to Stx2a (10 ng/mL) for 72 h (n = 3 biological replicates). The effects of OSMI-1 were compared with those of the vehicle (DMSO) controls. Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001. E, F. Human apoptosis antibody array analysis of multiple proteins using pooled lysates from iPSC-derived human kidney organoids following Stx2a (10 ng/mL) treatment for 72 h in the presence or absence OSMI-1 (10 µM, final). (E) Antibody spots representing signal differences are indicated in red boxes. (F) The graph shows the average of relative spot intensities for each protein compared to those measured in the control in the absence of Stx2a exposure (n =2 biological replicates). Dashed line represents the reference point of the fold change. Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.
SGK1 protects against starvation‐induced muscle atrophy A.Figure 6A. H&amp;amp;E staining of tibialis anterior sections reveals no changes in muscle architecture (scale bar 90 µm). B. Morphometric analysis demonstrates a decrease in muscle fiber size of the wild−type control mice compared with sgk1tg transgenic littermates (p = 0.0015). C. Western blot and densitometry analysis shows that levels of phosphorylated Foxo3a at S315 and T32 are increased in sgk1tg.
Figure 6A. Rabl2D73GQ80L localized in the ciliary shaft and did not affect ciliogenesis efficiency. RPE1 cells infected with adenovirus to express GFP-Rabl2D73GQ80L were serum-starved for 48 h. The white arrow denotes the cilium shown in insets. Quantification results were from three independent experiments. Uninfected RPE1 cells served as control. At least 107 cells were scored in each experiment and condition. B. Rabl2D73GQ80L underwent bidirectional IFT. Ciliary GFP-Rabl2D73GQ80L in RPE1 cells was live imaged at 5 fps. The 1st frame and kymograph of a representative cilium (also see Movie EV6) are shown. Velocities were quantified from 10 cilia. Rabl2D73GQ80L did not induce ciliary accumulation of BBS proteins. Ciliated RPE1 cells expressing GFP-tagged Rabl2Q80L or Rabl2D73GQ80L were immunostained (C). The white arrows (C) point to positions of cilia. D. Rabl2D73GQ80L did not induce ciliary accumulation of BBS proteins. Ciliated RPE1 cells expressing GFP-tagged Rabl2Q80L or Rabl2D73GQ80L were immunostained (C). In quantification results (D), at least 100 cells were scored in each experiment and condition. E, F. Rabl2D73GQ80L did not repress ciliary translocation of SMO and GPR161. RPE1 cells expressing GFP-tagged Rabl2Q80L or Rabl2D73GQ80L were serum-starved for 24 h and treated with DMSO (mock) or SAG for an additional 24 h prior to immunostaining (E). The white arrows (E) point to positions of cilia. In quantification results (F), at least 100 cells were scored in each experiment and condition. G. Ciliary Rabl2D73GQ80L did not repress the BBSome export. FLIP assays were performed in RPE1 cells co-expressing 3NG-Bbs5 with RFP-tagged Rabl2Q80L or Rabl2D73GQ80L as depicted in Fig 5F. Photobleaching was executed at t = 0. Quantification results on half-life (t1/2) were from 10 cilia for each condition. H. Rabl2D73GQ80L displayed an attenuated association with IFT-B as compared to Rabl2Q80L. mEPCs infected with adenovirus to express GFP-Rabl2Q80L or Rabl2D73GQ80L were lysed and immunoprecipitated using anti-GFP resin. Gapdh served as the negative control. I. A model delineating the effects of Rabl2 and mutants on the TZ passage of ciliary BBSome and its cargo GPCR: (1) In physiological conditions, the GTP hydrolysis of Rabl2-GTP results in Rabl2-free IFT-B capable of mediating the TZ passage of the BBSome and its cargos together with the retrograde IFT machinery; (2) Rabl2D73GQ80L dissociates from IFT-B to allow similar TZ passage; (3) The BBSome sheds from IFT-B in the presence of Rabl2Q80L or Rabl2-GTP during the passing process and is therefore blocked in the ciliary shaft with its cargos.
Figure 7Representative immunohistochemical staining for VGLL4 and IRF2BP2 in human non-small cell lung cancer tissues. IHC score is indicated. Scale bar: 50 μm Staining of VGLL4 and IRF2BP2 on human non-small cell lung cancer tissue arrays containing 71 samples was quantified as described in the Materials and Methods section. Correlation of VGLL4 and IRF2BP2 expressions was significant (Pearson correlation test; R = 0.6341, P < 0.001) Correlation analysis of VGLL4 and PD-L1 expression on human non-small cell lung cancer tissue arrays containing 71 samples Kaplan-Meier survival analysis of patients with high (n=74) or low (n=130) VGLL4 mRNA levels from GSE31210, a lung cancer data set. Survival curves are calculated according to the Kaplan-Meier method Model for the regulation of PD-L1 expression by VGLL4 and YAP. VGLL4 regulates PD-L1 expression through controlling IRF2BP2 protein stability. YAP represses IFNγ-inducible PD-L1 expression partially through miR-130a mediated inhibition of VGLL4 and IRF1 expression
Fbxw5 is required for ciliogenesis in an MCAK-dependent manner A.Figure 7A. Polyclonal RPE-1 cells expressing mNG-MCAK under a doxycycline-inducible promoter were seeded on coverslips, induced with 20 ng/ml doxycycline and grown for 24 hours. Subsequently, cells were washed twice with PBS and incubated for another 24 hours in serum-free medium without doxycycline. Cells were then fixed in methanol and subjected to IF using the indicated antibodies together with Hoechst staining. Left: Representative images. Scale bar = 10 µm. Right: Quantification of ciliated cells. Error bars show standard deviation of 5 independent experiments covering more than 200 cells and asterisks indicate p-value of a two-tailed unpaired Student's t-test (**P < 0.01). B. Immunoblot of extracts of cells used in A with the indicated antibodies for detection. C. RPE-1 cells were treated with the indicated siRNA for 48 hours followed by serum-starvation for another 24 hours. Cells were then fixed in methanol and subjected to IF using the indicated antibodies together with Hoechst staining. Left: Representative images. Note: Left panel shows an example image of siFw5-1. Scale bar = 10 µm. Right: Quantification of ciliated cells. Error bars show standard deviation of 4 independent experiments covering more than 200 cells and asterisks indicate p-value of a two-tailed unpaired Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001). D. Immunoblot of RPE-1 cell extracts. Left: Representative blots. Asterisk indicates an unspecific band detected by the Fbxw5 antibody. Right: Quantification of MCAK/Uba2 signal ratio. Error bars show standard deviation of 6 independent experiments and asterisks the p-value of a two-tailed unpaired Student's t-test comparing each knockdown with the non-targeting control (**P < 0.01, ***P < 0.001). E. Model: MCAK is regulated by two distinct E3 ligases at different time points. While SCFFbxw5 targets MCAK for degradation during G2/M, the APC/C takes over after mitotic exit. Via the SCFFbxw5 pathway, cells ensure that levels of MCAK and its orthologs are kept low upon entry into G0 and thus permit ciliogenesis in the following cell cycle.
Figure 7A-D Percentage of EdU incorporation in T47D (A, C) or BT20 (B, D) depleted of EGFR or not, transfected with wt or T693A, and stimulated or not with FGF10 (A, B) or with TGFα (C, D) and pre-treated (C, D) or not (A, B) with FGF10 for 40 min. Values represent the median ± SD of N= 4. P value =<0.05 *, <0.01 **, <0.001*** (one-way ANOVA with Tukey test). E. Relative invasion of wt- or T693A-transfected BT20 cells into fibronectin-supplemented collagen I was quantified as described in Material and Methods. Graph depicts mean ± S.E.M. of N=8. *P value=<0.05 (Student's t-test). F. Representative images of E. Black indicates cells. Confocal depth is indicated between panels. Scale bar, 250 μm. G. Model of reciprocal priming between FGFR and EGFR, based on this study.