EMBO/sd-character-level-ner huggingface dataset:
Co‐IP between PAT1‐HA and LSM1‐GFP. Proteins were transiently co‐expressed in N. benthamiana and tissue harvested 3 days post‐infiltration. Immunoblots of GFP IPs (top panel) and inputs (20 μg each, bottom panel). Immunoblots were cut in half and probed with anti‐HA antibodies and anti‐GFP antibodies.
(C and D) Representative immunoblots (C) and densitometric quantification (D) of whole-cell acid extracts of wild-type and Δach1 cells combined with or without knockdown of ACS2 (tet-ACS2). Cells were chronologically aged to day 3 in the presence of 1 ng/ml doxycycline. Blots were probed with antibodies against total Histone H3 (loading control) or H3 acetylated lysines (K9Ac, K14Ac, K18Ac). Data represent means ± SEM (n = 8).
Model of the relationship between nucleocytoplasmic channels, PA metabolism, and NPC maturation.
C Venn diagram presenting the number of common genes from RNA-seq results as compared with dinaciclib-target genes (GSE8882226[]) and the PolII ChIP-seq results (GSE7202328[]).
D Infarct size relative to left ventricular area (LV) and plasmatroponin I levels 24 hours after ZT5 or ZT13 MI. Student`s t-test; For infarct size n = 3 mice in both groups for ZT5 and n = 4 mice for isotype and n = 3 mice for Ly6G at ZT13; Isotype vs. Ly6G: *P = 0.0158 (ZT13). For troponin levels n = 8 mice for isotype and n = 6 mice for Ly6G with ZT13 MI; *P = 0.0012.
Figure 8: A working model. In wild type, ddaC neurons establish minus-end-out MT orientation which is likely required for dendrite pruning. In msps or tacc mutant neurons, minus-end-out MT orientation in dendrites is disrupted, which likely results in impaired dendrite pruning. MT depolymerization, which is induced by both MDA treatment and katanin overexpression, also causes mixed MT orientation in dendrites as well as dendrite pruning defects. This study supports a link between minus-end-out MT orientation and dendrite pruning. Figure EV legends
Fig. 3 Network derived from large-scale fractionation predicts 48 protein communities. Integration of experimental elution data, known functional associations and predicted interaction interfaces from homologous proteins allow the creation of a high-quality network with interconnected protein complexes (Appendix Figs. S8-S10). Here, known protein complexes are shown in blue and other physically associated proteins in grey, predicted interactions of complexes as grey lines and cross-links as red lines, cross-links between different subunits of a heteromultimeric complex are represented with red loops (see insert). Communities containing multiple complexes are highlighted with yellow; numbering and naming of complexes and communities is described in the legend of Appendix Fig. S9.
C. Representative RPA fluorescence staining for labile mitochondrial iron in H9c2 cells with indicated iron chelator treatment. Scale bar = 100μm.D. Labile mitochondrial iron measured by RPA fluorescence in H9c2 cells with indicated iron chelator treatment. ANOVA followed by post-hoc Tukey test was performed.* P=0.016 PBS vs. BPD. * P=0.04 DFO vs. BPD. N=8 independent samples for each group.
H The ATP level of mice after knocking down CD39 in the hippocampal (n = 5 mice/group. Treatment F2, 12 = 6.04, P = 0.0153; CSDS - LV-GFP vs Ctrl - LV-GFP, P = 0.0444; CSDS - LV-CD39 vs CSDS - LV-GFP, P = 0.0051. One-way ANOVA with Fisher's LSD test).
(B) Pattern correlation used to find proteins that accumulate upon ABA/NAA treatment and are removed in WT but not in atg2 after swapping to flg22/6-BA.
D, E Western blot analysis (D) and quantification (E) of NB-3 expression in the sensorimotor cortex after SCI. NB-3 expression levels were normalized by b-tubulin. *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni's post-test. Data were analyzed from 3 independent experiments each including 3 mice per group.
Back skin sections of K15CrePR+/T; Wls∆/∆ and Control K15CrePR+/+; Wlsflox/flox mice treated with mifepristone from P7 during 12 weeks (P90), immunostained for LYVE1 (red), and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 100 μm. epi, epidermis; der, dermis.
(C) Heatmap of differentially mRNA-seq expressed genes in stiff, soft or ALDH+ cells (CSC) MCF-7 cells. The unit for the color scale was z-score of log2 expression data shown.
(A) Immuno-EM of GFP-p62. S-GFP-p62 cells were labeled with rabbit anti-GFP (Abcam) followed by protein A-gold (15 nm). We observed labeling in membrane-free cytosolic structures and sequestosomes (arrows) as well as in endosomes (Endo).
(b) A549 cells stably expressing GFP-LC3 were subjected to Rubicon knockdown (KD) and then transfected with mStrawberry, mStrawberry-Rubicon or mStrawberry-Rubicon mutant lacking the Beclin 1 binding region (Δ393849). Cells with strong expression of mStrawberry-Rubicon showed an overexpression-dependent dominant-negative phenotype; therefore, we selected cells with modest expression. The number of GFP-LC3 puncta was counted and mean ± s.d. are shown. mStrawberry, n = 12 (12 cells); mStrawberry-Mm-Rubicon, n = 14 (14 cells); mStrawberry-Mm-RubiconΔ393-849, n = 13 (13 cells).
(a) Immunostaining of TCF20, NESTIN, and merged with DAPI in E13 and E16 embryonic brain sections. TCF20 is widely expressed in the brain. Data information: Scale bar in (A) e13, 50 μm, e16 100 μm
(E) HIV- or HIV∆Vpu-infected CD4 T cells (strain NLAD8) were incubated with NHS and either 10-1074 (left panel) or 3BNC117 (right panel). Both WT or E430G-mutated antibodies were used. After 24h, C3 surface levels on infected cells were analyzed by flow cytometry. Results are expressed as MFI of staining on infected (Gag+) cells. The MFI of C3 staining on Gag- cells was included as a control. Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells.
(A) Rescue of growth phenotype. Wild-type (WT) or mdm10Δ cells were transformed with the empty plasmid pYX142 (). In addition, mdm10Δ cells were transformed with pYX142 encoding MDM10 or MCP3. Cells were grown to an OD600 of 1.0 and spotted on YPD or YPG plates in a 1:5 dilution series. Plates were incubated for growth at the indicated temperatures.
RT-PCR analysis of pre‐mRNA splicing efficiencies of rap1+ and poz1+ pre‐mRNAs in cay1+ and cay1Δ cells expressing Cay1‐YFP or Rap1‐YFP. Amplification products corresponding to unspliced and spliced mRNAs are indicated by asterisks and gene names, respectively. rap1T indicates amplification products obtained with oligonucleotides spanning rap1 exon 3 and thereby amplifying both spliced and unspliced mRNA. Intronless act1+ was used as a loading control. Numbers at the bottom of each panel are ratios between spliced and unspliced forms (s/u) and spliced and act1+ (s/act1). Values are expressed as fold increase over wt.
Color-coded time-traces of fluorescence (left panel) averaged for 50 individual cells per strain (mean ± SD) treated with MMC (0.5 μg/ml) for 2 hours (gray shading). Fluorescence induction during 6 hours from MMC exposure (right panel). Asterisks denote significance by Kruskal-Wallis and Dunn's multiple comparison test: ns, not significant; ****p < 0.0001. The data shown are from 2 independent experiments.
D. Gene Set Enrichment Analysis (GSEA) was performed to compare transcriptomes of genotype comparisons (wt/Act vs wt/wt; HPV8/Act vs HPV8/wt; HPV8/Act vs wt/wt, [all]/Act vs [all]/wt) to 3 published datasets of Tumor-Associated Macrophages (TAM) (Franklin et al. 2014; Ojalvo et al, 2009; Galletti et al, 2016). Gene sets are composed of top genes upregulated (TAM UP) or downregulated (TAM DOWN) in TAM vs normal tissue macrophages. Positive Normalized Enrichment Score (NES) corresponds to enrichment of gene set in activin-upregulated genes; negative NES corresponds to enrichment in activin-downregulated genes. Data are presented as pseudo-heatmap with magnitudes of NES color-coded as indicated in the legend; all non-grey comparisons are FDR<0.10.
F, G RT-PCR analysis using primers specific to the ST7 exon 11 (F) and ABI2 exon 8 (G) splicing reporters, respectively, of samples where Matrin3 or PTB expression vectors were co‐transfected, in control siRNA and PTB/nPTB siRNA‐transfected cells. Quantification of at least three replicates for each condition is shown as a histogram of the percentage of exon inclusion. *P 0.01 when compared with control sample in each condition (‐).
(C) GFP, siRNA-resistant GFP-WIPI2b, GFP-WIPI2b RERE, GFP-WIPI2b FTTG, or GFP-WIPI2b FTTG RERE was expressed in HEK293A cells treated for 72 hr with WIPI2 siRNA. Cells were left in full medium (F) or starved for 2 hr with EBSS (S) or EBSS with BafA (B) before immunoblot analysis.(D) Statistical analysis of (C) was performed by one-way ANOVA with Tukey's posttest. The SEM for LC3 (n = 2) and p62 (n = 4) are shown. ∗p < 0.05.
A. Plot of the percentage of nup116Δ cells incubated for 3 h at 37°C with nuclear rim-like accumulations of Chm7-GFP. Error bars are SD from the mean from 3 independent replicates of >100 cells. p values from unpaired student's T-test where *** is p ≤ 0.001.B. Deconvolved inverted fluorescence micrographs of Chm7-GFP in nup116Δ cells at either 23°C or grown for 3 h at 37°C. Percentages ± SD of the proportion of cells with Chm7-GFP NE foci are shown under each panel (see Figure EV5F).
K. Active histone marks increase in the B compartment upon BAZ2A knockdown. Boxplots indicating the log2 fold-changes of normalized H3K27ac and H3K4me3 read counts in ESC+2i treated with siRNA-Baz2a and siRNA-control at boundaries and centers of A and B compartments. ). Statistical significance (P-values) for the experiments was calculated using the unpaired two-tailed t-test (*** < 0.001, * < 0.05). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.
(C) Cells were incubated with 1.0 × 10−7 M Tf(59Fe)2 for 24 h followed by incubation for 6 h in the absence or presence of 10 μM Ponasterone A. Cells were then treated with or without 10 μM MG132 in the presence of Ponasterone A for 10 h and harvested and the amount of 59Fe in immunoprecipitatedferritin was determined.
8-week-old female C57Bl/6 mice were infected intranasally with 150 pfu of influenza A/Scotland/20/74 (H3N2) virus (IAV) and treated or not simultaneously with 4 mg of succinate (Suc; by the intranasal route). (i, j) In separate experiments, body weight loss (n=8) (i) and animal survival (n=10) (j) were monitored daily. Data are represented as the mean ± SEM. Data information: Statistical analysis was performed using the Mann-Whitney test , i), the log rank test (j) (*P < 0.05).
C. U2OS cells transfected with indicated siRNAs and then treated as in (B) were collected in late S phase or mitosis (M), subjected to stringent pre-extraction and immunostained with DNMT1 antibody. DNMT1 foci formation was quantified by QIBC analysis (red bars, mean; >7400 cells analyzed per condition). Data are representative of three independent experiments.
(H) Number of iPS colonies generated from GOF18 EpiSCs transfected transiently with a piggyBac vector containing GFP or Klf4 and treated with LIF for 48hours and rotenone as indicated. Mean and s.e.m. of two technical replicates of a representative experiment is shown. Unpaired t-test: ** P<0.01 *** P<0.001. See also Appendix Figure S10H.
(C) The screening efficiency of four different types of chip was detected in 4T1 cells. n =100. Data information: Kruskal-Wallis test (C) The data represent mean ± SD of three independent experiments.
(D) atg1Δ and atg1Δatg13Δ cells expressing HA‐tagged Atg1 and wild‐type (wt) cells containing an empty plasmid were grown to mid log phase (rich), and then treated with rapamycin (rapa) or starved for 4 h in SD‐N medium (starv). Atg1 was immunoprecipitated and its association with Atg13 analysed by immunoblotting. Note that phosphorylated Atg13 isolated from cells grown in rich medium migrates slightly slower on SDS-PAGE, and the extent corresponds to the shift typically observed under our gel conditions.
After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. C, Inflammation score
Immunoblots of p-YAP (ser127), YAP, p-LATS (Thr1079), LATS, p-MST (Thr1083), MST, Kibra, PTPN14 and GADPH for Ctrl, Blebb and Blebb + AP conditions.
C. Western blot and densitometry analysis shows that levels of phosphorylated Foxo3a at S315 and T32 are increased in sgk1tg.
(G) ePAT assay of CblL mRNA. Ovaries from 1-day-old (germarium to stage 8) wild-type, aub and twin mutant females, and from 4- to 7-day-old bam mutant females were used.
J Superoxide (a), apoptosis (b), and Wnt signalling (c, d) were over-activated in ATRA-treated mice neural tubes and restored by AHT or NAC treatment. (a) Whole mount DHE staining of E10.5 mice embryos. Scale bar: 500 μm. Neural tube defect (white arrows) and heart (green arrow) regions show especially higher fluorescence intensities. (b) TUNEL (green) staining detected apoptotic cell death in E8.5 neural tube (E) sections. MARS (red). Scale bar: 300 μm. (c, d) IHC staining for β-catenin (green) in E8.5 (c) and E10 (d) mice neural tube sections. Scale bar: 300 μm (c), 150 μm (d). DAPI (blue).
B Chromogenic assay performed on plasma samples to detect F8 activity in AAV-treated mice compared to controls groups. Data are presented as mean ±SEM. The same animals were analysed at the various time-points. Each dot within the same group and within different groups of treatment corresponds to a single animal. The baseline includes all animals (n=20) before the treatment: CodopV3: n=8; N6 intein: n=5; CodopN6 intein: n=5. The statistical difference between groups has been assessed at 4weeks post-injection (wpi) with the One-way Anova p value = 0.056; at 8 wpi with the Kruskal-Wallis test p value = 0.68; at 12wpi with the Kruskal-Wallis test p value = 0.45; at 16wpi with the Kruskal-Wallis test p value = 0.58.
K - Average whole-body oxygen consumption rate (OCR) of ChATfl/fl (n = 10) and ChATfl/fl;LysM-Cre (n = 14) mice housed in metabolic chambers at RT or CE for 6 h (from 9 a.m. to 3 p.m.).
G. The transcription of pericentric satellites is normal after LincGET depletion. Embryos injected with LNA were collected at phCG 50 hours at the late 2-cell stage for TM-qPCR analysis. The error bars represent s.e.m. About 50 embryos were used for each group and three experimental replicates were performed. n.s., p > 0.05.
C Immunofluorescence for SOX9 (red) co-stained with CK19, CK7, α1AT, and αSMA (green). Arrowheads show elongated nuclei in αSMA-positive HSCs.
B Relative mRNA expression of NFATc1‐ and EMT‐related marker genes on administration of CsA (1 μM) in KPNC cells. Data represents means ± SD from at least three independent experiments. Asterisks show significance (*P 0.05).
(f) Flow cytometry of untreated macrophages (control) or of LPS-primed macrophages left unstained or stained with MitoSOX, then stimulated for 15 min with ATP.
(D) SYPRO-stained (left) and Western blot (right) time course SUMO conjugation reaction using and ΔHinge/Smc5-Nse2 truncation construct in the presence of ssDNA. T7-tagged Smc5 and E1 were immunodetected by an anti-T7 antibody. Reactions were run at 30 °C and stopped at indicated times by adding SDS-loading buffer
(b) HEK293 cells were cotransfected with MEF-hVps34 and GFP or GFP-Atg14LCAAX and stained with anti-Flag antibody. Bars, 10 µm.
Radiolabeled [ [35S]TIMM8A (G) precursors were imported into mitochondria that were isolated from HEK293 MIA40 WT/WT and WT/Del53-60 cells. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.
Fluorescent and histological analysis of liver morphology in WT and yap-/- mutant larvae on a lf:CFP background at 5 dpf. The blue colour overlay represents the fluorescent liver tissue. Scale bar: 200μm (bright field, BF) and 50μm (histology, H+E) Quantitative analysis of fluorescent liver area in WT and lf:Yap larvae at 5 dpf. n>30, ****p<0.0001, two-sided Student's t-test; values represent the mean ± standard error of the mean (s.e.m.)
A Biochemical fractionation into RIPA-soluble (S) and RIPA-insoluble (I) fractions to analyze solubility of the different myc-TDP-43 variants (Wt, 12D and 12A) expressed in HeLa cells for 48 h. TDP-43 was detected by TDP-43 Western blot (upper blot, rabbit anti-TDP-43 C-term, Proteintech) and Myc Western blot (lower blot, mouse anti-Myc 9E10). B Quantification of myc-TDP-43 variants (Wt, 12D and 12A) in (S) versus (I) fractions extracted from TDP-43 Western blots of 4 independent replicates ± SD, plotted as S/(S+I). *p < 0.0332 by one way ANOVA with Dunnett´s multiple comparison test to Wt.
A PBL27 phosphorylates MAPKKK5-C in vitro. The in vitro phosphorylation reaction was carried out using [32P]γ-ATP, and the phosphorylated proteins were detected by autoradiography. CBB, Coomassie brilliant blue.
(c) Overexpression of Fbxo7 also suppresses dopaminergic (DA) neurodegeneration parkin mutants.
(C) Distribution of the translational efficiency, in mESCs, of mRNAs (n=7156, red), micropeptide encoding transcripts (n=43, pink) and bona fide cytosolic (n=298, blue) and nuclear (n=1857, grey) lncRNAs. Two-tailed Mann-Whitney U test: *-p-value<0.05, **-p-value<0.01 and ***-p-value<0.001. Central band of boxplot represents median, box depicts 25-75 quantiles of distribution and whiskers represent the 5th and 95th quantiles of the distribution.
(C) Representative images from immunofluorescence staining of LC3B and CYCS in BAT from mice at the normal temperature (30 ℃) or 4℃ for 72 h.
Sequence alignment of TDP-43 segments containing the identified O-GlcNAcylation sites in different species. The amino acids with red color display the conserved O-GlcNAcylated sites.
A) Left panels: representative images of ORO staining on ACM and HC C-MSC in AM supplemented or not with 150µg/ml oxLDL. Middle panel: image quantification (n=11 biological replicates; Two-Way Anova). Right panels: representative images of Western Blot of CD36, PPARγ and GAPDH protein expression of ACM and HC C-MSC protein extracts in AM supplemented or not with 150µg/ml oxLDL (n=5 biological replicates) and d.a. normalized on the house-keeping GAPDH (Two-Way Anova). Data information: mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.
Scheme of possible mechanisms for SOCS1- and USP18-induced receptor degradation. Models with different structures concerning SOCS1- and USP18-catalyzed degradation of active receptor complexes (aRecIFN) were tested. Vertical lines denote separate enzymatic reactions, multiplication sign denotes cooperative enzymatic reactions. For each of the four different model structures, the resulting model trajectories of the best fit of pSTAT1 in the nucleus are exemplarily shown as dashed lines. Data is displayed as filled circles with errors representing 1σ confidence intervals estimated from biological replicates (N=1 to N=38).
A. Schematic representation of cGAMP- and Empty-VLP production. HEK293T cells were transfected with plasmids encoding HIV-1 Gag-GFP and VSV-G envelope to enable VLP production. Overexpression of cGAS WT in the same cells generated cGAMP that was then incorporated into nascent VLPs (cGAMP-VLPs). As control, Empty-VLPs were produced in cells where a catalytically inactive cGAS (cGAS AA) was overexpressed.
Diagram illustrating co-occurrence of endocytic morphology phenotypes associated with protein complex perturbation. Shown are significant protein complexes from (A) with biological processes and linked phenotype pairs. # denotes a phenotype pair without significant enrichment. Phenotype names are color-coded by endocytic marker, using the colour key described in Figure 1 and indicated on the yeast cell diagram.
B. Quantification of TACC3 positive centrosomes per metaphase I spermatocyte. Immunolabeling was performed on three biological replicates, with ≥20 spermatocytes quantified per replicate. The total number of cells quantified for control, Aurka cKO, Plk1 cHet, and Plk1 cKO mice were 97, 103, 96, and 105, respectively.
p47phox (E) colocalization with M.tb were analyzed. Data shown are representative of three independent infections. The results are the mean ± SD of three independent experiments. n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).
Montages of representative DAPI-stained nuclei are shown for gene knockdowns that led to reduced nuclear size. Cell-to-cell variability in the amount of ELYS knockdown likely accounts for apparent nuclear size variability. Ten nuclei per column, organized by maximum nuclear size z-score. Scale bar, 25 µm. Median nuclear size z-scores are plotted for gene knockdowns that led to reduced nuclear size. Data are based on three different siRNA oligo sequences for each gene and two biological replicates. Error bars represent the SEM for biological replicates.
(f) Pulldown experiment using the GST-prApe1 propeptide (propep.) to pull down Atg8 through the indicated Atg19 proteins. The graph is based on three experiments (N = 3), one of which is shown below the graph. Shown are the averages and s.d. See Supplementary Fig. 2 for input gel.
By means of its affinity for cholesterol, uncleaved C99 at the ER induces the uptake and retrograde transport of cholesterol from the PM to the ER, resulting in the formation of a lipid raft domain, or MAM. These C99-dependent lipid rafts passively segregate and organize lipid-binding proteins, thereby facilitating their interaction and the regulation of specific signaling pathways. Failure to cleave C99 completely would result in a futile cycle of continuous uptake of extracellular cholesterol and its mobilization from the PM to the ER, resulting in the upregulation of MAM formation and activation, which in turn would cause the upregulation of SMases, ACAT activity, and LD deposition. Closing the cycle, this accumulation of cholesterol in membranes also induces APP internalization and its interaction and cleavage by BACE1, and the downregulation of α-secretase activity.
Growth curves of the MCF7 (left) and T47D (right) cells cultured in the media collected from the siRNA-transfected cells. Each sample has 3 replicates. Error bars represent mean ± SD.
qPCR analysis of M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophage polarization markers in mouse bone marrow-derived macrophages (BMDMs) incubated for 24 h in conditioned media (CM) collected from control (siCTRL#1), Vps4a- and/or Vps4b-depleted CT-26 cells. For double Vps4a+b depletion various combinations of siVps4a#2 or #5 and siVps4B#3 or #4 duplexes were used. Data were normalized and are presented as the fold change of expression of a given M1 or M2 marker in BMDMs treated with CM from Vps4-depleted cells compared to its expression in BMDMs treated with CM from siCTRL#1-transfected cells (set as 1). Data are means of 4 independent experiments ±SEM. One-sample t-test; ns - non-significant (p≥0.05), *p<0.05, **p<0.01.
Representative flow cytometric analysis and comparison of MHCII+ F4/80+ macrophage gated on DAPI- CD45+ cells from jejunum and ileum of WT and DTR mice (n = 5 mice/group).
(D-E) RT-qPCR analysis o MuRF1 (E) mRNA expression from the tibialis anterior of three mice per cohort (n = 3). Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from saline controls; †, p < 0.05; ††, p < 0.01 from C26 controls
K. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, lncRNA-MIF harboring nonsense mutation or miR-586 binding defective lncRNA-MIF. Forty-eight hours after transfection, cell lysates were analyzed by Western blotting.