EMBO/sd-character-level-ner huggingface dataset:
Pacs1-/- B cells have reduced IP3R expression (A) Immunoblot of expression of all three IP3R isoforms and SERCA2 in primary splenic B cells from Pacs1+/+ and Pacs1-/- mice.Figure 5(A) Immunoblot of expression of all three IP3R isoforms and SERCA2 in primary splenic B cells from Pacs1+/+ and Pacs1-/- mice. (B) Real-time quantitative PCR of IP3R and SERCA2 transcripts in B cells. Data are presented as mean ± SD from three independent Pacs1+/+ and Pacs1-/- pairs of mice. (C) Pacs1-/- FOB cells were stimulated with 0.625 µM thapsigargin (black arrowhead) under Ca2+-free conditions to measure intracellular Ca2+ stores. Kinetic traces of four independent experiments are shown (Pacs1+/+ gray, Pacs1-/- pink) with the mean overlaid in bold (Pacs1+/+ black, Pacs1-/- red). (D) Plateau of cytosolic Ca2+ flux from intracellular Ca2+ stores in (A) calculated by the mean value over the last 30 seconds of analysis with baseline subtracted. Two-tailed paired t test, *P < 0.05. (E) AUC of cytosolic Ca2+ flux from intracellular Ca2+ stores in (A). Two-tailed paired t test, *P < 0.05.
Fig.Figure 2A-J The indicated Sox14 or Mical constructs were tested for their ability to suppress pruning defects induced by PP2A-29B dsRNA expression. C4da neurons were labeled by CD8::GFP expression under ppk-GAL4, and dendrite pruning defects were assessed at 18 h APF. Panels A - E show effects of Sox14/Mical pathway manipulations alone, panels F - J show neurons coexpressing PP2A-29B dsRNA. The scale bar in A is 50 μm. (A, F) C4da neurons (co-) expressing lacZ. (B, G) C4da neurons (co-) overexpressing Sox14. (C, H) C4da neurons (co-) overexpressing Mical. (D, I) C4da neurons (co-) expressing Mical∆redox. (E, J) C4da neurons (co-) expressing Mical∆CH. K Penetrance of pruning defects in panels A - J. * P<0.05, *** P<0.0005, Fisher's exact test. Numbers of neurons (animals) for each genotype are given in the figure the numbers of animals are given below in parentheses. L Severity of pruning defects in panels A - J as assessed by number of primary and secondary dendrites attached to soma at 18 h APF. Data are mean +/- s. d., * P<0.05, *** P<0.0005, Wilcoxon's test. Numbers of neurons (animals) for each genotype are given in the figure.
Immunoelectron microscopy of macrophages 10 h after infection with HSV-1.figf5 Immunoelectron microscopy of macrophages 10 h after infection with HSV-1. (a-c) Accumulation of LC3 (a,b) and gB (c). (d) Fusion of four-layered membrane structures and lysosomes preloaded with bovine serum albumin-gold (BSA-gold; black dots). Original magnification, × 54,800 (a,b), × 69,000 (c) or × 38,000 (d). Images are representative of three (a-c) or two (d) independent experiments.
Figure 7Representative confocal microscope images of skeletal muscle tissue sections immuno-stained against LC3B (Alexa 647) and LAMP1 (Alexa 555) Quantification of lysosomes (LAMP1 puncta) in skeletal muscles 24 h after iron injections. Representative TEM images of skeletal muscle 24 h after iron injections. Representative western blot images of phospho-UVRAG S550 and total UVRAG in skeletal muscle 24 h after iron injections. Representative western blot images and quantification of LC3-B and p62 to tubulin in skeletal muscles 24 h after iron injections.
D hydrogels with different pore sizes.Figure 2(A) Infected salivary glands were sandwiched between a soft PA hydrogel and a glass coverslip inducing sporozoites to move into the hydrogel. (B) Overlay of sporozoite tracks (black) and DIC image showing sporozoites inside a soft hydrogel at the end of a recorded image sequence. Scale bar, 20 µm. (C) Speed of WT sporozoites moving in the skin in vivo (Douglas et al, 2018b) and in soft hydrogels with small or large pores. (D, E) Speed of indicated mutant and control (Ctrl) parasites in hydrogels with small (D) and large (E) pores. (F, G) Speed of drug-treated and control parasites in hydrogels with small (F) and large (G) pores. Data information: Note the faster speed in gels with larger pores. Data from at least two independent experiments. Numbers above bars indicate the number of sporozoites analyzed. Significance determined by Mann-Whitney test. Box-and-whisker plots (C-G) depict the 25% quantile, median, 75% quantile and nearest observations within 1.5 times the interquartile range (whiskers). Outliers beyond this range are shown as black dots.
Figure 4Effects of the ion selectivity-perturbing R371Q mutation in Ttyh1 E14.5 neural progenitor cells were transduced with retroviral vectors expressing each indicated gene, and 2 days posttransduction, cells were harvested for qPCR analyses of Notch target mRNAs (n = 3). Effects of the ion selectivity-perturbing R371Q mutation in other Ttyh family members on Notch-dependent transcription. E14.5 neural progenitor cells were transduced with retroviral vectors expressing each indicated gene, and 2 days posttransduction, cells were harvested for qPCR analyses of Notch target mRNAs (n = 3). Immunolabeling of E15.5 brain sections electroporated in utero with indicated plasmids at E13.5 using anti-GFP and Alexa Fluor 488-conjugated secondary antibodies. GFP immunofluorescence (green) was merged with DAPI-counterstained images (blue). (D) Quantification of GFP+ cell localization in (C) (n = 3). VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm. Schematic representation of a series of Ttyh1 mutants with deletions in the C-terminal cytoplasmic tail region (Ttyh1 gene structure was not drawn in proportion to the actual domain size), series of Ttyh1 mutants with deletions in the C-terminal cytoplasmic tail region , and (F) their effects on cell distribution assessed by intraventricular in utero electroporation at E13.5 followed by immunolabeling at E15.5 using anti-GFP antibody. Scale bar: 100 μm.
Figure 8(A) CD68 coverage in the old APPPS1 tissue (14 DIV) treated with GM-CSF and vehicle Ctr. CD68 coverage is increased upon treatment with GM-CSF. The values are normalized to CD68 coverage of the Ctr and represent mean ± SEM from 3 independent experiments, each experiment including 2 independent slice culture dishes (*P < 0.05). (B) Quantitative analysis of core-only plaques in the old APPPS1 tissue (14 DIV) treated with GM-CSF and Ctr reveals an increased number of core-only plaques upon treatment with GM-CSF. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments, each experiment including 3 independent slice culture dishes (***P < 0.001). (C) CD68 coverage in the old APPPS1 tissue (14 DIV) incubated with conditioned media collected from young WT slices (young CM) or with non-conditioned slice culture media (Ctr). CD68 coverage is increased upon incubation of the APPPS1 tissue with the young CM. The values are normalized to CD68 coverage of the Ctr and represent mean ± SEM from 3 independent experiments, each experiment including 2 independent slice culture dishes (**P < 0.01). (D) Immunofluorescence analysis of the old APPPS1 slice cultured alone (old alone) or in co-culture with young GM-CSF-/- brain slices (old co-culture) at 14 DIV and immunostained with CD68 (red) and M3.2 (green) reveals an increased number of core-only plaques upon co-culturing with young GM-CSF-/- brain slices. Scale bar: 50 µm.(E) Quantitative analysis of core-only plaques in the old APPPS1 slice cultured alone or in co-culture with young GM-CSF-/- brain slices (14 DIV) reveals an increased number of core-only plaques upon co-culturing of old APPPS1 brain slices together with young GM-CSF-/- brain slices. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques. The values represent mean ± SEM from 3 independent experiments, each experiment including 3 independent slice culture dishes (***P < 0.001).(F) CD68 coverage in the old APPPS1 tissue (14 DIV) cultured alone or in co-culture with young GM-CSF-/- brain slices. CD68 coverage is increased upon co-culturing of old APPPS1 brain slices together with young GM-CSF-/- brain slices. The values are normalized to CD68 coverage of the old slice cultured alone and represent mean ± SEM from 3 independent experiments, each experiment including 2 independent slice culture dishes (*P < 0.05).
Figure 6A549 cells expressing control shRNA, p53 shRNA, or p53 shRNA plus Flag-WDR63 were subjected to wound-healing (A) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar in (A): 200 μm. A549 cells expressing control shRNA, p53 shRNA, or p53 shRNA plus Flag-WDR63 Lysates from these cells were also analyzed by western blot (B). A549 cells expressing control shRNA, p53 shRNA, or p53 shRNA plus Flag-WDR63 were subjected to transwell migration (C) The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bars 100 μm. A549 cells expressing control shRNA, p53 shRNA, or p53 shRNA plus Flag-WDR63 were subjected to transwell invasion (D) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bars 100 μm. A549 cells expressing either control shRNA or p53 shRNA were infected with lentiviruses expressing Flag-tagged full-length or truncated WDR63 proteins as indicated. Cells were then subjected to transwell migration (E) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; ***, P ˂ 0.001; One-way ANOVA. Scale bar: 100 μm. A549 cells expressing either control shRNA or p53 shRNA were infected with lentiviruses expressing Flag-tagged full-length or truncated WDR63 proteins as indicated. Cells were then subjected to transwell invasion (F) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; ***, P ˂ 0.001; One-way ANOVA. Scale bar: 100 μm. (G) A549 cells expressing control shRNA, p53 shRNA, or p53 shRNA plus Flag-WDR63 (each also expressing luciferase) were injected via tail vein into nude mice (n=6 for each group). Three weeks after injection, tumor formation was monitored by bioluminescence imaging. Data shown are mean ± SD; *, P ˂ 0.05; One-way ANOVA.
Figure 4(A) Upper row: DiI tracing of the visual cortex axons at P9 (before pruning), sagittal sections. Branching point of the tract is shown. Lower row: visual cortex axons in adult mice after pruning period. Note significantly reduced number of axons continuing into pyramidal tract in WT (dashed arrow). (arrows - 2 branches of corticospinal visual axons). Maximum projections are shown. Scale bars: 200 μm. (B) Schematic drawing of DiI injection and axon tracing. Axons that initially enter pyramidal tract fail to prune in crmp2-/- mice (red line). (C) Quantification of VP (visual pruning) index (fluorescence intensity of pyramidal axons after vs. before the branch point). In WT animals after the pruning period, only a minor part of axons descends towards the pyramidal tract (WT: P9 0.65±0.09, adults 0.17±0.04, p=0.006). However, in crmp2-/- mice, corticospinal axons are still largely present, and their VP indexes are not significantly different between adult and P9 stages (crmp2-/-: P9 0.54±0.18, adults 0.62±0.27, p=0.24). Mean ± SD, **p<0.01, 2-way ANOVA with Bonferroni's multiple comparisons test. P9: n=3 pups/genotype, adults: n=5 mice/genotype)
Figure 2Schematic experimental workflow: Patient-derived tumour samples were lysed according to the FASP protocol; the resulting proteins were mixed in equimolar amounts with a Super-SILAC spike-in standard and digested with trypsin. The MS analysis of each sample was performed in duplicate and data analysis was performed with the software packages MaxQuant, Perseus and R. Representative histogram showing the SILAC ratio distribution between the Super-SILAC standard and SQCLC (left) and HNSCC (right) samples. Boxplot showing the numbers of quantified proteins, derived from 44 SQCLC and 30 HNSCC tissue samples. The central line in the boxes represents the median number of proteins over all samples, upper and lower borders of boxes correspond to 25% and 75% quantiles. Whiskers indicate minimum and maximum. Distribution of MS-based protein signal intensities in all 74 tissue samples of the training cohort. Principal-component analysis of SQCLC (n=44; blue) and HNSCC (n=30; red) protein expression. Shown are the first two principal components, accounting for 8.51% and 8.03% of the total variance in the data, respectively.
Figure 4A. Analysis of CD4T cells isolated from the dLN and restimulated with PMA/Ionomycin for 4 h. Data are representative FACS plots, gated on VD-CD4+cells with mean frequencies per group.Cells (A-D) were isolated at day 9 after MOG/CFA/PTx immunization. Data consist of n=5 mice of each genotype. *p < 0.05, **p < 0.01, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed twice with similar results. B. Analysis of cytokine expression by CD4T cells isolated from the spleen and restimulated with MOG for 6 h. Data are representative FACS plots, gated on VD-TCRβ+CD4+CD44+cells (upper row) with mean frequencies among CD4T cells per group ± SEM, and (lower row) with mean frequencies per group.Cells (A-D) were isolated at day 9 after MOG/CFA/PTx immunization. Data consist of n=5 mice of each genotype. *p < 0.05, **p < 0.01, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed twice with similar results. C. Total cell numbers (mean +SEM) of MOG-specific Th17 cells isolated from the spleen, shown in (B, upper row).Cells (A-D) were isolated at day 9 after MOG/CFA/PTx immunization. Data consist of n=5 mice of each genotype. *p < 0.05, **p < 0.01, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed twice with similar results. D. Frequencies and total numbers (mean +SEM) of MOG-specific GM-CSF+IFNγ+/- Th17 cells isolated from the spleen, shown in (B, lower row).Cells (A-D) were isolated at day 9 after MOG/CFA/PTx immunization. Data consist of n=5 mice of each genotype. *p < 0.05, **p < 0.01, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed twice with similar results.
Figure 6(A) α-Synuclein, CSPα, VAMP2 and SNAP-25 were immunoblotted in total brain homogenates and/or synaptosomal fractions of 1.5 month-old WT (CSPα+/+/α-syn+/+), CSPα-/-, CSPα+/-, α-syn-/-, α-syn+/- and CSPα+/-/α-syn+/-. Synapsin-1 was also blotted as a control. Quantitation of WB in synaptosomal samples was shown. (B) SDS-resistant complex levels were evaluated in brain homogenates derived from mice with indicated genotypes by immunoblotting of non-boiled samples with VAMP2 or Syntaxin-1 antibodies. Quantitation of WB was shown. (C) EM analysis of cortical synapses derived from mice with indicated genotypes. The number of synaptic vesicles per synapse was quantified from 40 different images (taken from 5 different mice for each genotype), normalized by the length of synaptic cleft and expressed as % of WT. The size of synaptic vesicles was quantified from 400-500 vesicles (taken from 5 different mice for each genotype) and expressed as the average of vesicle diameter (nm) Arrows indicate the synaptic cleft. (D) Extracellular recordings (fEPSPs) in hippocampal brain slices from 1.5 months-old WT (CSPα+/+/α-syn+/+) and CSPα+/-/α-syn+/- mice. The left panel shows representative fEPSP traces. Summary graphs show the fEPSPs slope as a function of either the applied stimulus intensity or the prevolley amplitude. Data are the average of the values from 5 slices (from 4 mice) in the WT group and 5 slices (from 4 mice) in the CSPα+/-/α-syn+/-. *p<0.05, repeated measure ANOVA.
Beclin‐1 depletion prolongs prometaphase by delaying chromosome congression.Figure 2Beclin‐1 depletion prolongs prometaphase by delaying chromosome congression. (A-H) Time‐lapse films of HeLa cells stably expressing YFP‐α‐tubulin and H2B‐Cherry and treated with the indicated siRNA. The pictures are acquired every 10 min. Time lapse starts at nuclear envelope breakdown. (A) Mitosis in control cells. (B) Western blot of whole‐cell lysates from Control, Beclin‐1 and Beclin‐1+Mad2 siRNA‐treated cells. (C-E) Three different classes of mitotic progression were observed upon Beclin‐1 depletion. Higher magnification of lagging chromatids in the anaphase of Beclin‐1‐depleted cells is shown on the right of (D). (F) The prometaphase delay is dependent on MAD2 and the spindle checkpoint. Cells treated simultaneously with siRNA targeting MAD2 and Beclin‐1 rapidly exit mitosis. (G) Average duration of mitosis in different classes of Beclin‐1‐depleted cells, compared to control and MAD2 depletions. n=number of counted cells in three independent experiments. Error bars on graphs represent s.d. (H) The frequency of the different Beclin‐1 phenotypes depended on the efficiency of Beclin‐1 knockdown. Mitotic progression in cells treated with increasing concentrations of Beclin‐1 siRNA was followed by video‐microscopy. Left panel: western blot of Beclin‐1 upon RNAi treatment; right panel: quantification of the appearance of the three different mitotic progression patterns, that is, prolonged mitosis with normal anaphase (as C), with lagging chromatids (as D) and mitotic exit without clear anaphase (as E) upon increased Beclin‐1 knockdown experiments. NEB, nuclear envelop breakdown; RNAi, RNA interference; siRNA, small interfering RNA; YFP, yellow fluorescent protein.
Figure 4(A_B) Vero E6-TMPRSS2 (A) or Vero E6 (B) cells were inoculated with a total of 126 clinical NP swab samples representing a range of viral RNA levels, both total E RNA (Clinical Sample E CT detected by WHO-E primer/probe set) and subgenomic E RNA (CT detected by the Mills-SgE primer set developed in this study). Culture supernatants were harvested when cells displayed CPE or at day 7 if no CPE was observed earlier, clarified, and presence (blue square) or absence (yellow circle) of SARS-CoV-2 determined by immuno-focus assay.
Mucinous tumors from KLN model are resistant to phenformin treatment.Figure 5(A) Statistic analysis of ADC numbers of vehicle (Veh) or phenformin (Phen) treatment group in KL mice (n=4 in Veh group, n=3 in Phen group). Results were shown as mean ± SD. (B) IHC staining of Ki67- and CC3-positive cells in lung ADC of vehicle and phenformin (Phen) groups. Scale bar, 50 μm. (C Statistical analysis of Ki67 activity in ADC lung sections of vehicle (Veh) and phenformin (Phen) groups. The numbers of tumors analyzed over vehicle and phenfortmin groups were 25, 22 for Ki67 Results were shown as mean ± SEM. Significance was calculated by two-tailed unpaired Student's t-test with Welch's correction. ****p<0.0001 (C) D) Statistical analysis of Ki67 CC3 (D) activity in ADC lung sections of vehicle (Veh) and phenformin (Phen) groups. The numbers of tumors analyzed over vehicle and phenfortmin groups were 32, 23 for CC3. Results were shown as mean ± SEM. Significance was calculated by two-tailed unpaired Student's t-test with Welch's correction. p=0.0162 (D). (E) Statistic analysis of ADC numbers of vehicle (Veh) or phenformin (Phen) treatment group in KLN mice (n=7 per group). Results were shown as mean ± SD. Significance was calculated by two-tailed unpaired Student's t-test with Welch's correction. *p<0.05. (F) Representative photos of H&E, AB and PAS staining in IMA treated with phenformin (Phen). Scale bar, 150 μm. Inset: Representative enlarged photos of lung lobe of KLN mice treated with phenformin. Scale bar, 50 μm. (G) Statistic analysis of IMA numbers of vehicle or phenformin (Phen) treatment group in KLN mice (n=11 per group). Results were shown as mean. (H) IHC staining of Ki67- and CC3-positive cells in murine lung ADC and IMA of vehicle and phenformin (Phen) groups. Scale bar, 50 μm. (I, J) Statistical analysis of Ki67 (I) and CC3 (J) activity in ADC and IMA lung sections of vehicle and phenformin (Phen) groups. The numbers of tumors analyzed over vehicle group and phenformin groups for ADC and IMA were 17, 8, 9 for Ki67, 10, 10, 7 for CC3. Results were shown as mean ± SEM. Significance was calculated by one-way ANOVA with Dunnett's multiple comparisons test. **p=0.0010 (I), ***p=0.0009 (I), *p=0.0427(J).
Figure 7A. Cell viability of T3359 GSCs treated with indicated doses of C646 or the vehicle control (n=5). B. Tumorsphere images of T3359 GSCs treated with indicated doses of C646 or the vehicle control for 6 days. Scale bar: 100 μm. C. qPCR analysis of SATB2, FOXM1 and FOXM1 downstream targets in T3359 GSCs treated with indicated doses of C646 or the vehicle control for 24 hours (n=3). D. Schematic diagram showing the treatment of mice bearing the GSC-derived xenografts with C646. After GSC transplantation for 7 days, mice were treated with C646 or the vehicle control daily. Mice were monitored by IVIS bioluminescent imaging and maintained until neurological signs occur. E. Bioluminescent imaging of tumor growth in mice bearing xenografts derived from the luciferase-labeled T3359 GSCs treated with C646 or the vehicle control at indicated days after GSC transplantation (n=5 mice per group). F. Quantification of tumor growth from (E) (n=5 mice per group). G. Kaplan-Meier survival curves of mice bearing T3359 GSC-derived xenografts treated with C646 or the vehicle control (Vehicle control: n=9 mice; C646: n= 8 mice). Median survival: Vehicle control, 27 days; C646, 33.5 days. H. Immunofluorescence of Ki67 (Green) in T3359 GSC-derived xenografts from mice treated with with C646 or the vehicle control (n=6 tumors per group). Scale bar: 40 μm.
UBE2SHTH substitutions partially rescue the defects in Ub chain elongation created by disrupting the UBE2S-APC2 interaction.Figure 7A Structural view of UBE2SHTH (green) interacting with APC2 for diUb synthesis (Brown et al., 2016). Residues of interest at the UBE2SHTH-APC2 interface are indicated as red spheres. B Representative fluorescent scan of an SDS-PAGE gel (left) and its quantification (right) where the charged-swapped variants of the UBE2SHTH enhance substrate ubiquitination of APC/Cs harboring APC2 substitutions. Statistical significance was assessed by two-way ANOVA (* p ≤ 0.05, *** p ≤ 0.001, n=3 independent, technical replicates). Error bars: SEM. Data normalized to the negative control substrate band from reactions lacking an E2. C APC/C-UBE2S-dependent diUb synthesis was monitored by fluorescent scanning (left), quantitated (right), and assessed for statistical significance by two-way ANOVA (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, n=3 independent, technical replicates), demonstrating that the UBE2SHTH substitutions rescue the defects in diUb synthesis created by mutating APC2. Error bars: SEM. Data normalized to WT APC/C-UBE2S diUb reactions. D APC/C Platform-dependent activation of UBE2S-mediated diUb synthesis was monitored by fluorescent scanning (left), quantitated (right), and assessed for statistical significance by two-way ANOVA (* p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001, n=3 independent, technical replicates). Error bars: SEM. Data normalized to WT APC/C Platform-UBE2S diUb reactions. E Similar to B, the role of the "closed" conformation in UBE2S-dependent Ub chain elongation of the APC/C substrate Ub-CycB*, monitored by the incorporation of C118A-substituted UBE2S variants (top). Quantification of ubiquitinated Ub-CycB* was assessed (bottom) and statistical significance by one-way ANOVA between C118A and WT, C118A/E139R, and C118A/E143R (** p ≤ 0.01, **** p ≤ 0.0001, n=3 independent, technical replicates). Error bars: SEM. Data normalized to WT UBE2S 4+ Ub additions.