EMBO/sd-character-level-ner huggingface dataset:
pax2amKO2 knock-in line faithfully reports pax2a expression and knock-down (A) Schematic of the knock-in strategy used to generate the pax2amKO2 line.Figure 7(A) Schematic of the knock-in strategy used to generate the pax2amKO2 line. Double strand break was induced between the penultimate codon and the STOP codon of pax2a gene using CRISPR/Cas9. DNA repair integrates the donor construct at the site of double strand break, resulting in a pax2a reporter line. The donor construct contains T2A-mKO2 reporter cassette flanked by left homology (LH) and right homology (RH) arms. (B) Whole mount immunofluorescence of 9.5hpf pax2amKO2 embryos stained with anti-mKO2 antibody (red) and anti-PAX2A antibody (green). Anterior is to the left, and dorsal side is to the top. Scale bar: 0.1 mm. (C) Whole mount immunofluorescence of 55 hpf pax2amKO2; Tg(tg:nls-EGFP) stained with PAX2A antibody (PAX2A-Ab) displays an overlap of mKO2 and PAX2A-Ab signal. The otic vesicle (OV), mid-hindbrain barrier (MHB), interneurons (IN) and thyroid gland (THY) is labelled. White dashed line represents the outline of the zebrafish larvae. Scale bar: 100 µm. Anterior to the right. (D - F) Confocal microscopy imaging of a sagittal section of a 55 hpf pax2amKO2 (red); Tg(tg:nls-EGFP) (green) embryos immunostained with PAX2A antibody (cyan) showing co-localization of mKO2 and pax2a in the pronephros (D), thyroid gland (E) and mid-hindbrain barrier (F). In the thyroid gland, mKO2 (red), PAX2A-Ab (cyan) and thyrocyte-specific GFP (green) show co-localization. Scale bar: 50 µm. (G , H) Snapshots from live imaging of 55 hpf pax2amKO2; Tg(tg:nls-EGFP) embryos injected with sgRNA targeting pax2a coding sequence. The anterior part of a representative control embryo (G) is shown alongside a representative crispant (H). Crispants display a strong reduction of mKO2 fluorescence, as well as an absence of GFP signal suggesting absence of thyroid (THY) tissue. White dashed line represents the outline of the zebrafish larvae. Scale bar: 100 µm. Anterior to the right.
Cell-Autonomous Degeneration of PCs in Chimeric Mice.Figure 3(A) Genotyping of chimeric mice. Upper gel: PCR to amplify a fragment of the npc1 gene from npc1−/−↔GFP, npc1+/−↔GFP, and npc1+/−mice results in npc1− (1,067 bp) and npc1+ (947 bp) bands. Lower gel: The three genotypes can be distinguished by ApaLI digestion of the PCR products. The npc1− allele gives rise to 937- and 130-bp bands. The wild-type allele varies depending on the source: the BALB/c allele is digested to 477-, 340-, and 130-bp bands while the allele from the GFP mouse is digested to 817- and 130-bp bands. Note that the 477- and 340-bp bands are absent from the homozygous mutant chimera. (B) Quantification of npc1−/−PC density in chimeric mice. For each mouse, the actual density observed (the average of the mean densities counted in each lobule) is compared to the number expected if no PC loss occurs (calculated from the density at 30 d in npc1−/−mice and the chimerism percentage). Five sections separated by at least 100 μm were analyzed for each mouse. For the three mice sacrificed at 70 d, only lobules I-IX were included in the analysis because of the lack of degeneration in lobule X in npc1−/−mice at this age. A value for the number of npc1−/−PCs expected if loss does occur (calculated from the density at 70 d in npc1−/−mice and the chimerism percentage) is also included for these three mice. No rescue of mouse−/−PCs was observed for any of the chimeric mice (p < 0.0001 for each mouse comparing observed versus expected if no loss occurs). (C) Quantification of npc1−/− PC density by lobule in C1.4 at 70 d. Observed and expected densities are shown for npc1−/− PCs in each lobule. No clear rescue of npc1−/− PCs was observed in any lobule (p > 0.05 for all lobules comparing observed versus expected if loss occurs, except II and III, where p-value is between 0.01 and 0.05; p < 0.001 for all lobules comparing observed versus expected if no loss occurs, except lobule X, where p > 0.05). D and E) Images of lobules II and X taken from C1.4 at 70 d. GFP is green and Calbindin staining is red. In lobule II, the number of PCs is clearly reduced and of the five remaining PCs, four are wild-type (GFP-positive). In lobule X (where no degeneration occurs during the normal npc1−/− life span), PC density is normal and the majority of PCs (23 of 32; 72%) are mutant, as expected based on npc1−/− contribution to this mouse (67%). (F) Quantification of wild-type PC density in chimeric mice. Observed and expected densities are shown for wild-type PCs. For mice with more than 15% wild-type contribution, the density of wild-type PCs is similar to the number expected if these cells have not degenerated (p > 0.05 for each mouse comparing observed versus expected if no loss occurs, except C5.2, where p = 0.02, and C5.6, where p = 0.0005). (G) Quantification of wild-type PC density by lobules in C1.4 at 70 d. No loss of wild-type PCs was observed in any of the lobules (p > 0.05 for all lobules comparing observed versus expected if no loss occurs; p < 0.001 for all lobules comparing observed versus expected if loss occurs, except for lobule X, where p > 0.05).
SUF acts in cisA A gametangiophore of MpEF1pro:SUF/suf-30ge [Y], indicating the inability of transgenic SUF overexpression in rescuing the feminization phenotype of sufge [Y].Figure 6A gametangiophore of MpEF1pro:SUF/suf-30ge [Y], indicating the inability of transgenic SUF overexpression in rescuing the feminization phenotype of sufge [Y]. Scale bar, 2mm. B Real-time RT-PCR analyses confirming SUF transcript accumulation in SUF-overexpressing lines. Constitutively expressed MpEF1α was used as control. Measurements of six biological replicates for WT [Y], and three biological replicates for each SUF-overexpressing line are plotted. Data information: bars represent mean ± SD. Asterisks indicate significant differences from WT [Y] (B) (p <0.05, two-tailed Student's t-test). C Structures of the gMpFGMYB-S and gMpFGMYB-L transgenes without (-S) or with (-L) the putative promoter and the TSS of SUF. D Real-time RT-PCR measurement of MpFGMYB transcript levels. Three biological replicates are analyzed for each line. Data information bars represent mean ± SD. Asterisks indicate significant differences from WT [X] (D) (p <0.05, two-tailed Student's t-test). E-H Gametangiophores (E, G) and gametangia (F, H) of gMpFGMYB-S [Y] (E, F) and gMpFGMYB-L [Y] (G, H). Scale bars, 1 mm (E, G), 50 μm (F), 100 μm (H).
The FYVE domain from FYCO1 containing the dimerizing CC is recruited to late endosomal/lysosomal structures.Figure 6.(A) FYCO1990-1,233 is the smallest FYVE domain-containing deletion mutant of FYCO1 consistently recruited to cytosolic vesicles. HeLa cells transiently transfected with FYCO1990-1,233 were imaged 24 h after transfection. (B) FYCO1990-1,233 is the smallest FYVE domain-containing deletion mutant of FYCO1 that can efficiently interact with the full-length FYCO1. Full-length myc-FYCO1 was cotranscribed and cotranslated with the indicated deletion mutants of GFP-tagged FYCO1 in rabbitreticulocyte lysate. S35-labeled FYCO1 complexes were immunoprecipitated with anti-SDS-PAGE antibody, separated by SDS-PAGE, and visualized by autoradiography. IP, immunoprecipitation. (C) FYCO1990-1,233 strongly colocalizes with Rab7, LysoTracker red, and Alexa Fluor 647-dextran (10,000 D) but only weakly with PXp40phox. HeLa cells transfected with the indicated constructs or labeled with LysoTracker red for 60 min and Alexa Fluor 647-dextran (10,000 D) for 4 h were imaged 24 h after transfection. (D) FYCO1 construct containing a CC region followed by a tandem repeat of two FYVE domains from FYCO1 (FYCO1990-1,233+1,156-1,233) localizes to the ER and perinuclear vesicles and only weakly colocalizes with PXp40phox. HeLa cells were imaged 24 h after transfection with the indicated constructs. (A, C, and D) Insets show an enlarged field of interest. Bars, 10 µm.
Figure 8Illustration of how CA10 may control HS specifically on neurexins. CA10 binding to neurexin in the ER along the secretory pathway to the cell surface, which sterically hinder the priming xylose addition by xylosyltransferases in the cis-Golgi. This interaction provides a mechanism to regulate glycan addition at a specific site, to regulate the balance between non-HS- and HS-neurexin in a specific cell type without affecting biogenesis of other HSPGs. Control of neurexin-HS by CA10 affects the affinities of neurexin to postsynaptic ligands.
CAR microclusters activate Gads-SLP76-actin pathway in the absence of LAT.Figure 5CAR (3rd generation) T cells were plated on supported lipid bilayers coated with CD19 and ICAM-1 and imaged by TIRF microscopy. A. Gads was recruited to CAR microclusters in both the wild-type and LAT-deficient (Jcam2.5) cells. n = 33 or 34 cells. Shown are the means ± SE. n.s., p = 0.58 by unpaired two-tailed t-test. As a control, Gads is not recruited to LAT-deficient cells activated by OKT3. CAR (3rd generation) T cells were plated on supported lipid bilayers coated with CD19 and ICAM-1 and imaged by TIRF microscopy. B. Phosphorylated SLP76, recognized by an anti-SLP76 pY145 antibody, was enriched in CAR microclusters in both the wild-type and LAT-deficient cells. In contrast, phosphorylated SLP76 was not enriched in TCR microclusters in LAT-deficient cells. n = 50 cells. Shown are the means ± SE. n.s., p = 0.95 by unpaired two-tailed t-test. CAR (3rd generation) T cells were plated on supported lipid bilayers coated with CD19 and ICAM-1 and imaged by TIRF microscopy. C. Actin remodeling was induced in LAT-deficient cells by CD19-, but not OKT3-coated SLB. No ICAM-1 was supplemented to SLB in this experiment to rule out the costimulation of actin via the LFA-1/ICAM-1 pathway. Actin network was revealed by F-tractin-mScarlet. n = 6 cells. Shown are the means ± SD. n.s., p = 0.68, and 0.98 by unpaired two-tailed t-test.
Figure 7The cleavage sequence of indicated proteins. Western blot analysis of HEK293T cells expressing GFP-tagged wild-type (WT) or an uncleavable mutant (diGly to GA) full-length (FL) version or the N-terminal domain (NTD) of indicated proteins. Full-length and NTD proteins are marked by * or # on the blot, respectively. Cycloheximide (CHX)-chase analysis of GFP-tagged FL and NTD versions of indicated proteins. GPS analysis of wild-type and mutant forms of FL or the NTD of indicated proteins with or without dominant-negative KLHDC2 (DNKLHDC2) treatment. A C-terminal Leu was added (+L) to NTDs to cap the diGly degron. GST pull-down assay using cells expressing GST or GST-tagged wild-type or mutant KLHDC2 and HA-tagged SDE2, FAU or TUG.
Figure 2(A) Experimental design of T cell depletion by αCD4 and αCD8a antibody treatment. (B) Representative flow cytometry blots of blood samples from mice of both groups 3 weeks after treatment start showing successful depletion of CD45+CD3+ T cells in the αCD4+αCD8 treatment group. (C) Representative IHC images of CD3+ T cells and Iba1+ macrophages/MG in the isotype and αCD4+αCD8 group. Scale bar; 100 μm. (D) Quantification of Iba1+ and CD3+ cells in IHC sections of BrM from the isotype (n=9) and αCD4+αCD8 group (n=7). (E) Representative MRI pictures of BrM in early, medium and late stage of both groups. (F) Kaplan-Meier curves depict BrM free survival of mice in the isotype and αCD4+αCD8 group (Isotype n=19, αCD4+αCD8 n=19). (G) Tumor growth curves depict the increase of absolute BrM volume for each mouse in both groups over time (Isotype n=19, αCD4+αCD8 n=19). (H) Heatmap displays the relative expression level of T cell effector and exhaustion marker in TIL-CD4 and TIL-CD8 compared to blood lymphocytes. Values display the average per group based on vst values (BL-CD4 or BL-CD8 with n=3 for each group) vs. BrM-associated TIL-CD4 or TIL-CD8 (n=3 for each group).
Amelioration of proteostasis by 4-PBA treatment improves the establishment of intrinsic polarity in cochlear HCs.Figure 7Intrinsic polarity is disrupted upon MG-132 treatment of cultured HCs and restored by the chemical chaperone 4-PBA. (A) LGN and F-actin immunostainings of E16.5+1DIV OCs reveal that MG-132 perturbs LGN distribution at the apical surface of HCs, while co-treatment with 4-PBA ameliorates its enrichment at the lateral edge. Scale bars=5µm; magnified views=2µm. Data information: #IHC; *OHC1; **OHC2; ***OHC3 Intrinsic polarity is disrupted upon MG-132 treatment of cultured HCs and restored by the chemical chaperone 4-PBA. (B) Acetylated alpha-tubulin immunostaining reveals that the kinocilium is centrally shifted by MG-132 and partially restored by 4-PBA in E16.5+1DIV HCs (yellow arrowheads = kinocilium base; scale bar=2µm). Intrinsic polarity is disrupted upon MG-132 treatment of cultured HCs and restored by the chemical chaperone 4-PBA. (C) Kinocilium position plotted as a fraction of HC diameter for each HC row (DMSO/MG-132/MG-132+4PBA IHC: n=57/67/53, ***p<0.001, F=34.52, DF=2; OHC1: n=61/66/53, ***p<0.001, F=57.88, DF=2; OHC2: n=55/73/58, ***p<0.001, F=124.7, DF=2; OHC3: n=48/66/43, ***p<0.001, F=67.16, DF=2; pooled from 4 explants per condition; mean±SD; One-way ANOVA with Tukey's multiple comparisons test). In vivo administration of 4-PBA improves LGN localization and kinocilium position in Elp3cKO cochlear HCs. Pregnant mice were treated daily with saline or 4-PBA from E13.5 and the cochleae of WT and Elp3cKO embryos were analysed by immunostainings at E17.5. (D) The arc of lateral LGN enrichment remains in many Elp3cKO HCs after 4PBA administration. Scale bars=5µm; magnified views=2µm. Data information: #IHC; *OHC1; **OHC2; ***OHC3 In vivo administration of 4-PBA improves LGN localization and kinocilium position in Elp3cKO cochlear HCs. Pregnant mice were treated daily with saline or 4-PBA from E13.5 and the cochleae of WT and Elp3cKO embryos were analysed by immunostainings at E17.5. (E) Acetylated alpha-tubulin immunostaining showing kinocilium position in WT or Elp3cKO OCs in the presence or absence of 4-PBA (yellow arrowheads = base of kinocilium; scale bar=2µm). In vivo administration of 4-PBA improves LGN localization and kinocilium position in Elp3cKO cochlear HCs. Pregnant mice were treated daily with saline or 4-PBA from E13.5 and the cochleae of WT and Elp3cKO embryos were analysed by immunostainings at E17.5. (F) Kinocilium position plotted as a fraction of HC diameter for each HC row (WT/WT+4PBA/Elp3cKO/Elp3cKO+4PBA IHC: n=35/31/35/36, p=0.6812, F=4.510, DF=3; OHC1: n=36/32/37/40, ***p<0.001, **p<0.01, F=27.60, DF=3; OHC2: n=35/33/38/42, ***p<0.001, F=76.62, DF=3; OHC3: n=36/32/38/39, ***p<0.001, *p<0.05, F=27.86, DF=3; pooled from 3 animals per condition; mean±SD; One-way ANOVA with Tukey's multiple comparisons test).
Figure 1(A) Unlabeled or Cy5-labeled dsRNA corresponding to the first 200 nucleotides of either the Renilla luciferase (dsRNA-RL) or the GFP (dsRNA-GFP) coding sequences visualised on gel stained with ethidium bromide (EtBr-stained, top) or by in-gel fluorescence (bottom). (B) Gating strategy used in the flow cytometry-based assay. First, cells were selected (side scatter (SSC) vs. forward scatter (FSC)) and gated for singlets (SSC-Width (W) vs. FSC) to hence exclude doublets or cell aggregates. The single cell gate was then further analysed for DAPI, gating on the live cells (DAPI negative). DsRNA-transfected cells (Cy5-positive) were then gated and the level of GFP expression analysed within that population. Flow cytometry counting beads (gated on SSC vs. FSC plot) were added to quantify cell number in each sample. (C) mESCs stably expressing GFP (mESCs-GFP) were mock transfected or transfected with the indicated Cy5-labeled dsRNA and the level of GFP in Cy5+cells was analysed 3 days later and is represented as histograms (left). Right panel, bar graphs displaying the percentage of GFP mean fluorescence intensity relative to mock control (D) mESCs stably expressing d2GFP (mESCs-d2GFP) were mock transfected or transfected with the indicated Cy5-labeled dsRNA. The level of d2GFP was analysed at the indicated timepoints post-transfection and displayed as in (C). (E) L929 cells stably expressing d2GFP were mock transfected or transfected with the indicated Cy5-labeled dsRNA and d2GFP level was measured as in (D).
Figure 7 (A) Confocal analysis of control C2, WHS LETM1+/- S4 and WHS LETM1+/+ S5 cells, immunolabeled with anti-TOM20 (red), DNA (green) and DAPI (Blue). Scale bar: 8 µm. (B) Confocal images of control C1 and WHS LETM1+/- S3 labeled with TOM20, DNA and DAPI, showed as merge. Scale bar: 8 µm  (C) Quantification of cells in (A) displaying mtDNA aggregates. At least 50 cells per cell line were counted from three independent experiments. Data are expressed as means ± SEM  (D) qPCR estimated mtDNA copy number in WHS and control cells. Values are expressed relative to control cells (C1-C3). Data are expressed as means ± SEM of n=3 independent experiments  (E) Relative abundance of DRP1 and DRP1S616 in whole-cell extracts of control and WHS fibroblasts, with GAPDH shown as a loading control. Immunoblot signals were quantified using ImageJ and are expressed as mean ± SEM of n=5 independent experiments
Figure 6Schema of the in vivo analyses of tumor growth in mice (n=10/group) injected with FaDu cells. After tumor appearance, mice were injected intratumoral with high titer viruses encoding for control or antimiR-9 sequences. After two injections of virus mice were treated with Cetuximab (IP injections) and RT (4Gy dose) as indicated and then sacrificed 36 days after injections. Graph reports the tumor volume of tumors described in A (n=10 mice/group). Data represent the mean (±SD) and two-way ANOVA was used to verify the statistical significance. Typical images of Hematoxylin&Eosin (H&E) (C) and Ki67 expression (Ki67) (D) evaluated by immunohistochemistry (IHC) in tumors explanted from mice treated as in A/B. Graph reports the percentage of Ki67 positive cells in tumors, as described in D. Data are expressed as mean (±SD) of Ki67 percentage counted in five randomly selected fields per tumor, in at least 4 tumors per group. Two-way ANOVA with Sidak's multiple comparison test was used to verify the statistical significance. TUNEL assay performed in tumors described in C. On the left, typical immunofluorescence images (blue - nuclei, green - TUNEL). On the right, graph reports the percentage of TUNEL positive cells in tumors as described in C. Each dot represents a tumor. Data represent the mean (±SD) and two-way ANOVA with Sidak's multiple comparison test was used to verify the statistical significance. Kaplan-Mayer curve evaluating the overall survival of HNSCC patients treated with RT+Cetuximab (CTX) combination included in the TCGA dataset, segregated on the expression of miR-9 in the primary tumor (Low expression <75819 reads n=8; High expression ≥75819 reads n=23). Number of evaluated samples (n) and p value are reported in the graph. Statistical significance was calculated with long-rank test. Kaplan-Mayer curve evaluating the progression free survival of HNSCC patients treated with RT+Cetuximab (CTX) combination at the CRO-Aviano National Cancer Institute and at the University Cattolica segregated based on miR-9 expression in primary tumors, defined as the expression in above (high expression n=18) or below (low expression n=17) the median expression, as defined by ddPCR. Hazard Ratio (HR) and statistical significance were calculated with long-rank (Mentel-Cox) test and are reported in the graph.
Figure 5Time courses of swarmbots treated with varying concentrations of carbenicillin and glucose. Solid lines indicate cell densities inside swarmbots; dotted lines indicate those in the chamber. Safeguard was established for lower nutrient levels (0.004% or 0.04% glucose) along with carbenicillin treatment. At a high nutrient level (0.4% glucose), the safeguard was lost due to either growth in the chamber (panels 3 and 6) or gradual killing of the population inside swarmbot (solid line in panel 9). These results are qualitatively consistent with the model predictions (Fig 4). Snapshots of safeguard performance at the end time point of (A). The red arrow in panel 6 indicates significant growth in the chamber; the dimmer fluorescence of the swarmbot in panel 9 indicates killing of the population.
α1-tubulin is essential for sporozoite formation A.Figure 1Cartoon of a simplified Plasmodium life cycle indicating in red the stages important for our work. Note that transfection is performed in blood stage parasites. Schematic cartoon of a sporozoite with an indicated apical cross section. Subpellicular (cytoplasmic) microtubules (green) can be found below the three membrane layers (plasma membrane and inner membrane complex) of the pellicle (bottom). Isolated sporozoites from the hemolymph (HL) and salivary glands (SG) of infected mosquitoes stained with Hoechst and SiR-tubulin. Note the similar staining of WT and α1-tubulin(-) complemented lines. Scale bars: 5 µm. SiR-tubulin staining (green, I and II) and TEM (III) show that microtubules shrink after wild type sporozoite formation. During budding microtubules can be up to 15 µm long (I), while after formation they measure on average only 6 µm (graph). Inset in II shows sporozoite cross-section at the sporoblast membrane; note the SiR-tubulin fluorescence (green, arrowheads) next to the nuclear Hoechst stain (blue). (III) TEM longitudinal section of budding sporozoites with an indicated subpellicular microtubule (white line, length). Green arrowheads point to microtubules close to the sporoblast. Rh: rhoptry; N: nucleus; ER: endoplasmic reticulum; Mi: mitochondrion; Sb: sporoblast. **** indicate p<0.0001; Kruskal-Wallis-test. Scale bars: I&II: 5 µm, magnification box in II: 1 µm, III: 1 µm, magnification box in III: 0.1 µm. Comparison of sporozoite development in oocysts of WT, α1-tubulin(-) and complemented lines. Hoechst (blue) and SiR-tubulin (green) staining reveal that the complemented line generates sporozoites indistinguishable from WT controls, while SiR tubulin staining is absent from α1-tubulin(-) oocysts. mCherry stains the cytoplasm of the parasite. Scale bars: 5 µm. Transmission electron micrographs of developing sporozoites in WT and α1-tubulin(-) parasite lines. Note the slender sporozoites in the WT oocysts, which are absent in the mutant. Sb: sporoblast; scale bars: 5 µm. TEM cross sections of WT and α1-tubulin(-) parasites showing the aberrant structure of the latter. Subpellicular and centriolar plaque (spindle pole body) associated (hemispindle) microtubules are readily recognizable in the WT sporozoites (green arrowheads in insert) but absent in the mutant, while the inner membrane complex is formed below the plasma membrane of both parasites (white arrowheads). Note the different scale chosen to highlight the malformed shapes in the mutant. N: nucleus; scale bars: 0.2 µm, magnification boxes: 0.1 µm,.
Figure 9ZR-75-1 cells were treated for 5 days with 1µM MK-2206 (A), 1 µM AZD5363 (B), 1µM GDC0941 (C) or DMSO. Human mRNAs were hybridised to NanoString human kinome and control code sets, then subjected to quantification using NanoString software. Results are presented as mRNA change of each kinase relative to mRNA isolated from control sample treated with DMSO. To permit data compaction, and simple kinome-wide comparisons, the fold changes are log2 transformed. The kinase mRNAs exhibiting prominent changes are annotated, with SGK3 highlighted in red. Similar profiles were obtained in one independent experiment.