EMBO/sd-character-level-ner huggingface dataset:
Figure 5Accumulation of endogenous PLN in the testis of SPPL2c-/- mice. PLN was immuno-precipitated (IP) from equal protein amounts of total lysates of wild type or SPPL2c-/- testis with the mouse monoclonal antibody. Western blot analysis of the IPs was performed with the rabbit monoclonal PLN antibody. SPPL2c and actin were detected from total lysates directly. Quantification of normalised PLN/Actin ratios from n=3 biological replicates per genotype. Expression of PLN in murine testis from wild type (wt) SPPL2c-/- (2c-/-) was quantified by qPCR. Immunohistochemical detection of PLN in paraformaldehyde (PFA)-fixed testis from wild type and SPPL2c-/- mice. Cryosections were stained using a rabbit monoclonal PLN antibody or normal rabbit IgG as negative control and diaminobenzidine as peroxidase substrate. Testis suspensions were stained with 2.5 µM DRAQ5 to determine cellular DNA amount. 1C-co = elongated spermatids, 1C = other haploid cells including round spermatids, 2C = spermatogonia, secondary spermatocytes, Sertoli cells, other somatic cells, 4C = primary spermatocytes, G2 spermatogonia. Intracellular Ca2+ concentrations were analysed in testis suspensions from either wild type or SPPL2c-deficient mice using the Ca2+-sensitive probe Fluo4-AM. Individual germ cell populations were identified Bars indicate Median Fluo4-AM fluorescence ± SD of 8 individual samples from 3 mice per genotype normalised to those of wild type samples. Median forward scatter (FSC) was controlled in the 1C-co population from the datasets Median FSC ± SD of 8 individual samples from 3 mice per genotype. Expression of SPPL2c in human testis. (H) Western blot analysis of lysates from HEK293 cells expressing human (h) SPPL2c, human and murine testis using antisera against a C-terminal epitope of murine SPPL2c or human SPPL2c as indicated. Immunohistochemical detection of SPPL2c with the monoclonal 1A1 antibody in paraffin-sections from Bouin's-fixed human testis. In addition, sections were incubated with control immunoglobulins. Arrows indicate elongated spermatids.
Inhibition of GRK2 activity decreases Smoothened accumulation in cilia and GLI3 processing.Figure 5Defective SMO cilia accumulation and inhibited Hh signaling in chondrocytes treated with GRK2 inhibitors. Control human R00-082 chondrocytes were serum starved in the presence of a GRK2 inhibitor, either CMPD101 (C) or paroxetine (P), for 24 hours before they were treated with SAG for additional 12 (A, B) (A,B) Cilia were stained by ARL13B and SMO antibodies, and the intensity of ciliary SMO was analyzed and plotted. Note the impaired SMO accumulation in cilia caused by GRK2 inhibition (black arrow). Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cilia are indicated. Scale bar, 1 µm. Defective SMO cilia accumulation and inhibited Hh signaling in chondrocytes treated with GRK2 inhibitors. Control human R00-082 chondrocytes were serum starved in the presence of a GRK2 inhibitor, either CMPD101 (C) or paroxetine (P), for 24 hours before they were treated with SAG for additional 24 hours (C D). (C) Cell lysates were immunoblotted for GLI3 processing and GLI1 upregulation; actin levels served as a loading control. GLI3FL and GLI3R, full-length and repressor GLI3 variants, respectively. (D) Quantification of GLI3 and GLI1 levels by densitometry. Note the impaired GLI3 processing and no GLI1 upregulation in cells treated with CMPD101 or paroxetine. Mean ± SEM. Mann-Whitney U test (GLI3 ratio) and Welch´s t-test (GLI1 levels); number of biological experiments is indicated. Defective SMO cilia accumulation in chondrocytes with downregulated GRK2. (E) Doxycycline (DOX)-inducible GRK2 downregulation in control human R00-082 chondrocytes, tested by western blot, normalized to actin and plotted below. Mean ± SEM. Welch´s t-test; number of biological experiments is indicated. shScr, scramble shRNA. (F) Cells pre-treated with DOX for three days were serum starved, SAG treated, stained and analyzed as in (A,B), and the intensity of ciliary SMO was analyzed and plotted. Note the impaired SMO translocation to cilia caused by GRK2 loss. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cilia are indicated. Scale bar, 1 µm.
Fig.Figure 6(A) FKRP-NSCs treated with 4BPPNit showed increased IIH6 reactivity, compared with untreated control cells. Note that the augmented IIH6 reactivity by 4BPPNit is much weaker than the that in CRISPR-corrected NSCs. Under the same exposure time, the signal intensity of IIH6 reactivity is saturated in corrected-NSCs. Data information: Intensities of glycosylation are normalized to GAPDH protein expression. (B) Representative immunoblots with the IIH6 antibody using FKRP- and corrected-NSCs treated with 4BPPNit for 24, 48 and 72 h, and DMSO only controls. Data information: Intensities of glycosylation are normalized to GAPDH protein expression. Note that the left and right panels of the immunoblots are not equivalent exposure time. (C) Quantification of α-dystroglycan glycosylation (IIH6), compared with DMSO only controls. Data information: Intensities of glycosylation are normalized to GAPDH protein expression. Values indicate mean ± s.d. (n= 3 or 4 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01). (D) Representative immunoblots of laminin-binding analysis using FKRP- and corrected-NSCs treated with 4BPPNit and DMSO only controls. (E) Quantification of laminin-binding activities, compared with DMSO only controls. Data information: Intensities of laminin-binding activity are normalized to GAPDH protein expression. Note that the left and right panels of the immunoblots are not equivalent exposure time. Values indicate mean ± s.d. (n= 3 or 4 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01). (F) Representative immunoblots with the AF6868 antibody detecting both α-dystroglycan core protein and β-dystroglycan in FKRP- and corrected-NSCs treated with 4BPPNit and DMSO only controls. (G) Quantification of α-dystroglycan core protein and β-dystroglycan expression, compared with DMSO only controls.Data information: Intensities of protein expression are normalized to GAPDH protein expression. Values indicate mean ± s.d. (n= 3 or 4 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).
Figure 1A Heat-map of significantly differentially expressed genes in CCDC50-knockdown A375 cells versus the control cells. B KEGG pathway enrichment analysis of upregulated pathways in CCDC50- deficient A375 cells. The dot size represents the size of pathway, and the color represents Benjamini & Hochberg (BH) adjusted hypergeometric test p-values. C Heat-map of selected genes related to inflammatory cytokines and chemokines. D GSEA analysis of NLRP3 inflammasome complex assembly genes. E,F qRT-PCR analysis of IL-1β, Tnfα and Il6 mRNA levels in BMDMs transfected with siCCDC50 or control siRNA for 48 h and then stimulated with LPS for 3 h in combination with ATP (E) for another 1 h or Nigericin (F) for another 0.5 h (n = 3 biological replicates). G,H BMDCs transfected and treated as in BMDMs were collected and subjected to qRT-PCR analysis (n = 3 biological replicates). Data information: mRNA results are normalized to GAPDH and relative to untreated non-targeting control cells. Data are representative of three biological replicates and shown as mean with SEM (E-H); **P < 0.01, ***P < 0.001; two-tailed unpaired Student's t-test.
Loss of CCN1 increased quiescent NSCs in the V-SVZ A Numbers of GFAP+BrdU+ label-retaining NSCs in the V-SVZ.Figure 5Numbers of GFAP+BrdU+ label-retaining NSCs in the V-SVZ. Arrowheads point to label-retaining NSCs. n=4, *p= 0.039. V-SVZ coronal sections stained for GFAP (cyan) and BrdU (red). Scale bar: 10 μm. Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts 24h after TMZ treatment. Arrowheads point to dividing NSCs under regenerative condition. n=6, *p= 0.0173. Scale bar: 10 μm. Densities (cells/mm2) of DCX+ neuroblasts in V-SVZ whole-mounts 3 days after TMZ treatment. n=3, *p= 0.0179. V-SVZ whole-mounts stained for DCX. Scale bar: 500 μm. Densities (cells/mm2) of BrdU+ cells in the OB GCL labeled 24h after TMZ treatment. n=5 (Ctrl) and 6 (Ccn1cKO), *p= 0.0484. OB sections stained for BrdU (yellow) and nuclei (blue). Scale bar: 50 μm. Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts of aged mice 24h after TMZ treatment. Arrowheads point to dividing NSCs under regenerative condition. n=3, *p= 0.0321. Scale bar: 10 μm. Densities (cells/mm2) of DCX+ neuroblasts in V-SVZ whole-mounts of aged mice 3 days after TMZ treatment. n=3, n.s. not significant. Scale bar: 500 μm. Densities (cells/mm2) of BrdU+ cells in the OB GCL of aged mice labeled 3 days after TMZ treatment. n=4 (Ctrl) and 3 (Ccn1cKO), n.s. not significant. Scale bar: 50 μm.
Unsupervised analysis of proteomics of neoadjuvant treatment A) Clinical parameters of 35 patients.Figure 1A) Clinical parameters of 35 patients. Subtype at diagnosis (HP : ER+ and/or PR+ , TP : ER+ and/or PR+, HER2+, TN : ER-, PR-, HER2- and Her2 overexpressing : ER-, PR-, and HER2+, H: Healthy Breast ducts, N: Tumor Adjacent normal ); Tumor Grade at diagnosis (1,2 or 3); Miller & Payne score( 1:no reduction in tumor cellularity, 2:minimum reduction in cellularity,3:partial reduction in cellularity); Relapse (yes/no); Death (yes/no); Ki67 intensity measured by immunohistochemistry at diagnosis (1-3); Tumor size at diagnosis ( 1: < 2 cm, 2:2-5 cm, 3:> 5 cm, 4 indicates that tumor has grown into chest wall or skin); Age (years); Co-clustering of matched pre and post-treatment samples after hierarchical clustering (yes/no, p<0.05); Relapse free survival time (years); Overall survival time (years). B) Association between patient pathological response and tumor relapse in our cohort. Significance was determined using Chi square test. C) Kaplan Meier survival curve for Miller and Payne pathological response score. Global Log Rank p value and corrected p value for pairwise comparison is indicated. D) Dendrogram colors indicate co-clustering of matched pre and post-treatment tumor samples coming from the same patient. E) Association between co-clustering of matched pre - and post-treatment tumor samples with relapse. Significance was determined using Chi square test. F) Association between co-clustering of matched pre - and post-treatment tumor samples with M&P score. Significance was determined using Chi square test.
NeutrobodyPlex enables a differentiated analysis on neutralizing IgGs compared to total SARS-CoV-2 binding IgGs in individuals.Figure 7A ,B Total IgGs derived from spike-binding IgGs were plotted against normalized MFI values from IgGs binding to RBD (MFI RBD (%control)) in the presence of both concentrations of NM1267 (1 µM; 1 nM,). C, D Total IgGs derived from N-binding IgGs were plotted against normalized MFI values from IgGs binding to RBD (MFI RBD (%control)) in the presence of both concentrations of NM1267 (1 µM; 1 nM,). F
NeoR accesses an early endosomal compartment.Figure 4(A) 293T cells transfected with pINCO‐NeoR were harvested 6 and 96 h post transfection and NeoR localization within various cell fractions determined by Western blot. In addition, supernatant of NeoR‐pINCO transfected 293T cells (96 h post transfection) was included to test for release of NeoR into the culture supernatant. At 6 h post transfection, NeoR protein becomes detectable in the cytoplasm, while at 96 h post transfection a substantial amount of NeoR protein is also localized in the endosomal/lysosomal fraction. For further characterization of the various fractions, blots were stripped and re‐probed with antibodies specific for the cytoplasmic protein LDH, the nuclear protein c‐myc, early endosomes (Rab‐5), late endosomes and lysosomes (Lamp‐1) and mitochondria (cytochrome c), which co‐purify with endosomes/lysosomes. (B) Endosomal/lysosomal fractions were prepared from pINCO‐NeoR‐transfected cells 96 h post transfection as in (A). To prevent lysosomal degradation of NeoR, half of the cells were treated with leupeptin for the last 48 h. These fractions were separated on a 27% Percoll gradient and eight different fractions collected. Successful separation into fractions with increasing density was verified by Western blot analysis with antibodies directed against Lamp‐1, Rab‐5 and cytochrome c. Inhibition of NeoR degradation by leupeptin causes accumulation of NeoR protein in the late endosomal/lysosomal fractions 7 and 8. (C) To determine where NeoR gains access to the vacuolar system, 293T cells were transfected with pINCO‐NeoR, treated with leupeptin to prevent NeoR degradation, and the endosomal/lysosomal fractions prepared at various time points post transfection and separated by Percoll gradient centrifugation. Starting at 16 h post transfection, NeoR becomes detectable in the Rab‐5‐positive fraction 5, and then spreads into late endosomal/lysosomal fractions. At 26 h post transfection an almost identical NeoR distribution pattern is observed in cells treated with or without BfA.
Figure 4A) Fluorescence analysis of RPE-1 cells expressing endogenously tagged γ-tubulin-EGFP. Cells were synchronized in G2, using RO-3306, to allow for a clear distinction of the two centrosomes present at this cell cycle stage. Panels illustrate sum projections of the stacks from an original picture (left), a whole cell mask (middle) and a centrosome mask (right); both masks are based on intensity thresholding. Bar: 10 μm. B) EGFP-tagged viral like particles (GFP-VLP2/6) are used as a reference for quantification of γ-tubulin-EGFP. The histogram shows the distribution of the total fluorescence intensity associated with single GFP-VLP2/6; the 2 dashed lines delimit the population used for assigning an average fluorescence intensity value to represent the 120 GFP molecules associated with each VLP2/6 particle (Charpilienne et al, 2001). The inset shows a sum projection of EGFP-VLPs fluorescence image. Bar: 10 μm. C) Scatter plots show the numbers of γ-tubulin molecules per centrosome determined by calibrations using either SRM measurements (n=46 cells) or EGFP-VLP2/6 fluorescence (n=40 cells). The black lines represent mean values; numbers ± SEM are indicated on top of each scatter plot.
Figure 2Western blot analysis of MsrB2 and LC3 I/II in Meg-01 cell after shMsrB2 transfection (72 hrs). Cells were then treated with H2O2 (1 mM for 1 hr) alone or with NAC (100 μM for 30 min). GAPDH was used as the loading control. Quantification and analysis of individual groups. GAPDH served as the loading control. (shMsrB2 vs. shCon; *p=0.024, shMsrB2/H2O2 vs. shCon/H2O2; *p=0.0295, shMsrB2/H2O2/NAC vs. shCon/H2O2/NAC **p=0.0100, n=3) Western blot analysis of MsrB2, pp53(S15) and LC3 I/II in Meg-01 cell after 25 mM HG treatment for 24 hrs. GAPDH was used as the loading control. Quantification analysis on individual groups. GAPDH served as the loading control. (MsrB2: shCon/HG vs. shCon; **p=0.0017, shMsrB2/HG vs. shCon/HG; *p=0.0189, pp53: shMsrB2/HG vs. shCon/HG; *p=0.0317, LC3II: shCon/HG vs. shCon; *p=0.0215, shMsrB2/HG vs. shCon/HG; *p=0.0158, n=3) The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.
a) mCherry-GFP-LC3b MSCV retroviral constructs (presented as in Fig.figf3(a) mCherry-GFP-LC3b MSCV retroviral constructs (presented as in Fig. 2a) used for transduction of P14 cells (Supplementary Fig. 2a). (b) Flow cytometry of splenocytes from recipient mice (n = 6) given adoptive transfer of P14 cells transduced with retrovirus as in a, assessed on days 5 and 8 after infection of recipients with LCMV (left), and quantification of P14 cells per spleen (right). Numbers adjacent to outlined areas (left) indicate percent P14 (CD45.1+) cells. *P ≤ 0.0005 (unpaired t-test). (c) Flow cytometry of the size of P14 cells transduced with wild-type mCherry-GFP-LC3b retrovirus (assessed as in Fig. 1b) in mice treated as in b, assessed at days 5 and 8 after infection. (d) Efficiency of retroviral transduction (left) and expression of GFP and mCherry (right) in retrovirus-transduced P14 cells, assessed by flow cytometry gated on CD45.1+ P14 cells (left) or CD45.1+Thy-1.1+ P14 cells (right). Numbers in quadrants indicate percent CD45.1+Thy-1.1− (untransduced) P14 cells (top left) or CD45.1+Thy-1.1+ (transduced) P14 cells (top right; magenta) (left) or percent cells in each (right). (e) Ratio of the geometric mean fluorescence intensity (gMFI) of GFP to that of mCherry (GFP/mCherry) for the cells in d; each symbol represents an individual mouse; numbers above lines connecting groups indicate difference in ratio. Data are representative of two independent experiments with three or more mice per group (error bars (b), s.e.m.).
Figure 1(A) Wild type (left and middle panels) and p53-/- (right panel) HCT116 cells were transfected with control (mixture of control DS scrambledNeg, siLuci, and siGFP) or GRWD1-targeting (mixture of siGRWD1-3 and 4) siRNAs for 36 h, treated with 5 nM actinomycin D, and harvested at the indicated times. Whole cell extracts were analyzed by immunoblotting with the indicated antibodies. Coomassie Brilliant Blue (CBB) staining serves as a loading control. (B) Control U2OS cells infected with control retroviruses with the empty vector and U2OS cells overexpressing the siRNA-resistant HA-GRWD1 were treated and analyzed as above. (C) HCT116 cells were transfected with control (mixture of control DS scrambledNeg, siLuci, and siGFP) or GRWD1-targeting (mixture of siGRWD1-3 and 4) siRNAs for 24 h, treated with 50 µg/ml cycloheximide, and harvested at the indicated times. Whole cell extracts were analyzed by immunoblotting with the indicated antibodies. (D) HFF2/T cells stably overexpressing HA-GRWD1 were established by retroviral infection. Cells were cultured in the presence or absence of actinomycin D (5 nM) for 12h and then treated with or without MG132 (20uM) for 6 h as indicated. Whole cell extracts were analyzed as above. (E) Control and HA-GRWD1-overexpressing HFF2/T cells were first treated with actinomycin D (5nM) for 12 h, then further treated with 50 ug/ml cycloheximide, and harvested at the indicated times. Whole cell extracts were analyzed by immunoblotting with the indicated antibodies. The means and SDs from two independent experiments are shown.
Figure 5Sequences of Plasmodium falciparum Glu-tRNAGAA (A The arrows indicate T1 digestion sites LC-MS/MS analysis was performed on T1-digested tRNA and deconvolution yielded precise masses of the unmodified (3055.425 Da) and modified (3143.43 Da) oligos for Glu-tRNAGAA (B Collision-induced dissociation of the parent molecular ions for m/z 1046.477-3 from Glu-tRNAGA produced expected -c, -w and -y ions for the sequences CU[mcm5s2U]UCACCC Sequences of Plasmodium falciparu Gln-tRNACAA (D) The arrows indicate T1 digestion sites LC-MS/MS analysis was performed on T1-digested tRNA and deconvolution yielded precise masses of th unmodified (1835.20 Da) and modified (1923.262 Da) oligos for Gln-tRNACAA (E) Collision-induced dissociation of the parent molecular ions fo m/z 1923.262 from Gln-tRNACAA produced expected -c, -w and -y ions for the sequence CC[mcm5s2U]CCAA Ratios of modified oligonucleotides from the anticodon stem-loop o (H)GluGA isoacceptors expressed as percentages of their sum total Ratios of modified oligonucleotides from the anticodon stem-loop o (G)GlnCA Ratios of modified oligonucleotides from the anticodon stem-loop o (I)GlyGGA isoacceptors expressed as percentages of their sum total
Figure 1Decreasing ∆ψm from ∆III to ρ0 cells. WT and mutant yeast cells were cultured to log phase and stained with TMRM for FACS analysis. Decreasing proliferation rates from ∆III to ρ0 cells. Cells were cultured from 0.05 OD600 in SCD. OD600 was measured every 2 hours. Transcriptome analysis of OXPHOS mutants. WT and mutant yeast cells were cultured to log phase. RNAseq was performed with three biological replicates per genotype (Dataset EV1). All the transcripts and four categories of transcripts (gene lists in Table EV1B) are shown. Significantly changed genes (P value < 0.05; fold change > 1.5 or < 0.67) are highlighted with dashed lines; numbers of significantly changed genes are indicated. qRT-PCR analysis of selected transcripts in ρ0 cells.
Figure 3(A) Immunofluorescence microscopy. Primary hepatocytes prepared from p62f/f mice were infected with or without adenovirus Cre recombinase for 48 hrs and then additionally infected with adenovirus LacZ or NBR1. 24 hrs after the infection, the hepatocytes were immunostained with anti-Keap1, anti-p62 and anti-NBR1 antibodies. Each inset is a magnified image. Bars: 10 µm. (B) Immunoprecipitation analysis. Crude extracts were prepared from primary hepatocytes as described in (A) and then immunoprecipitated with anti-FLAG (as a negative control) or anti-NBR1 antibody. The resulting samples were analyzed by immunoblot analysis. Data shown are representative of three separate experiments. (C) Primary hepatocytes prepared from p62f/f; Alb-Cre mice were co-infected with adenovirus GFP or NBR1 in combination with wild-type p62 or the mutants for 48 hrs. Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin. Data are shown as means ± s.e. *P < 0.05, **P < 0.01, and ***P < 0.001 as determined by Welch's t-test. (D) RT qPCR analysis. Total RNAs were prepared from hepatocytes described in (C). Values were normalized against the amount of mRNA in the hepatocytes expressing GFP. The experiments were performed three times. Data are shown as means ± s.e. *P < 0.05, and **P < 0.01 as determined by Welch's t-test.
Figure 2A, B. Mice were infected with PR8 and treated with IFNα (circles, 1.45μg/50μl), IFNλ (triangles, 2.6μg/50μl) or Veh Ctrl (squares) as previously stated. Concentrations of stated proinflammatory cytokines in BAL fluid was measured by multiplex cytokine assay (A) (data shown is representative of two independent experiments, n=2-6). A, B. Mice were infected with PR8 and treated with IFNα (circles, 1.45μg/50μl), IFNλ (triangles, 2.6μg/50μl) or Veh Ctrl (squares) as previously stated. Concentrations of stated proinflammatory cytokines in BAL fluid was measured by flow cytometry quantification of pDCs and Inflammatory Monocytes in the lung was performed (B) (data shown is representative of two independent experiments, n=2-6). C, D. Lung sections from control and infected mice treated as indicated, were stained by TUNEL for apoptotic cells at 6dpi. Quantification of TUNEL+ cells in whole lung slides by Icy Spot Detector (ICY-R3M2Y2) (C) (data shown is pooled from three independent experiments, n=3-8). Red arrowheads indicate TUNEL signal (D). Scale bar, 200 μM (data shown is representative of two independent experiments, n=3-4).
Figure 7(A) A scheme showing peptide and DSS administration regime. (B) Weight loss of DSS challenged mice on day 6. (C) Real-time PCR analysis showing mRNA expression levels of IL-6, IL-12p35 and IL-23p19 of FACS isolated monocytes collected from mice with the indicated treatments. Data are obtained from two to three independent experiments (n=6-7 mice pooled per group).