EMBO/sd-character-level-ner huggingface dataset:
(A) Axonal spheroids stain diffusely positive for LC3B. IHCLC3B, scale bar 100 μm. (B,C) The granular cores of the spheroids are positive for (B) ubiquitin (arrow) and (C) p62. IHCubiquitin and p62, scale bars 20 and 100 μm, respectively. (D) Smooth axonal swellings in the cerebellar cortex contain ATG4D. Inset: control. IHCATG4D, scale bar 100 μm. (E) Affected neurons show increased perinuclear granular LC3B positivity. Inset: control. IHCLC3B, scale bar 100 μm. (F) Neuronalvacuoles are partially LAMP2 positive (arrow) and partially negative (arrow head). Inset: control. IHCLAMP2, scale bar 20 μm.
MTT assays comparing the effects of docetaxel and mocetinostat in the prostate cancer cell line DU-145 and the docetaxel-resistant subclone DU-145 DR (left). Mocetinostat treatment of DU-145 DR downregulates ZEB1, upregulates E-cadherin, miR-200, and miR-203 expression. For relative miRNA expression, the expression levels in original DU-145 were set to 1 (middle panels, the immunoblot panel derives from the same experiment shown in Fig1A). Mocetinostat sensitizes DU-145 DR to docetaxel (right). For calculation of the CI and synergy between the drugs, see Table2. n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are *P = 0.01-0.05; for exact P-values, see Supplementary Table S4).
B. Heatmap showing the codon optimality code underlying the genetic code in zebrafish and (C) Xenopus. Based on the consistent identification by two methods (rCSI) and (CSC) we defined: Optimal codons in red and non-optimal codons in blue. The intensity of the color represents the strength (light, weak; dark, strong) based on different CSC values, which corresponds to different cumulative effects in mRNA half life (Fig 1F).
Immunoblot analysis of the indicated proteins in HFSC colonies from Sirt7+/+, Sirt7-/- and Sirt7-TG mice (D)
(I-J) IHC analyses of Nfatc1 expression in hair follicles of Sirt7f/f, Sirt7f/f ;K15-Cre and Sirt7-TG mice at the indicted time points. The black dashed line indicates the bulge (Bu) of hair follicle.
(B) Representative kymographs of dense-core vesicle motility in proximal dendrites of transfected neurons. DIV 10-14 hippocampal neurons were co-transfected for 4 days with NPY-GFP and pSUPER (control) or pSUPER encoding DCLK1 shRNA in the absence or presence of DCLK1 truncation constructs and dense core vesicle motility in dendrites was recorded during live-cell imaging. Scale bar = 5 µm.
B Oxygen consumption rate of female mice shown in (A) adjusted for body weight by ANCOVA. 3-day averages of VO2 were calculated for each mouse (n=9/10). *P=0.025 in ANCOVA with Bonferroni posttest (Cited4-/- vs. Cited4+/+)
D. Oxr1 causes disassembly of holo V-ATPase. In this model, Oxr1 binding destabilizes the interaction between EG2, Cfoot and the distal domain of aNT, which leads to release of V1 from the membrane.
F. Quantification of the nociceptive responses in TRPA1-/- mice within 5 min after intraplantar injection of control saline or 1,000 ppm iodine, following intraperitoneal injection of saline or vehicle.
Western blot of individual E9.5 wildtype (n=4) and Slc7a5-null (n=4) embryo lysates immunoblotted using an antibody against phospho-GCN2 (Thr899) and total GCN2 (loading control), ***p = 0.0001 ; band intensities measured with FIJI and analysed with unpaired t-test Data information: All error bars indicate SEM.
WT PINK1 was expressed in HEK293T cells with or without PGAM5 or a GFP control plasmid after (g) DMSO or (h) CCCP treatment. Cells were permeabilized with digitonin or Triton X-100 separately, followed by treatment with the indicated amounts of trypsin on ice for 30 min. Cell lysates were immunoblotted for PINK1, PGAM5, Tomm20 or HSP60 as indicated. Full-length (*63 kDa) and cleaved (#54 kDa) PINK1 are shown.
(A) Representative traces of [Ca2+]i transients in Indo-1 AM-loaded hiPSC-CMs paced at 1 Hz. (B) Time to reach 50% of [Ca2+]i transient amplitude, t1/2; [Ca2+]i concentrations during systole and diastole (C) Amplitude and tau decay of the [Ca2+]i transient.
C. RNA immunoprecipitation of veal2 was performed by pull-down of Prkcbb and Epbhb3, followed by qRT-PCR. IgG pull-down was performed as control. Gel image shows the amplification of veal2 in different RNA immunoprecipitation samples, along with 10% input control, as marked by arrowheads.
MSN cultures were stimulated with HiK (55 mM) for 1h in the presence of various pharmacologicals to dissect the synapse-to-nucleus cascades mediating transcription of Npas4, Fosb and Arc. Values were normalized to the induced condition to compare for differences in induction between treatments. Mean + SEM and one-way ANOVA with Bonferroni post-hoc correction were used in all experiments. p* < 0.05, p** < 0.01, p***<0.001, p****<0.0001. n-number indicated in each panel. a, co-incubation with the NMDA receptor blocker APV (100 mM, applied 5min before) reveals that all IEG inductions through HiK in MSNs depend on NDMA receptor activity. Mean + SEM; n = 4 independent cultures as biological replicates. b, blockade of voltage-dependent calcium channels through verapamil (30 µM, 5min before) and TTX (1 µM, 5min, to prevent verapamil-induced bursting) leads to significant reduction in gene-inductions for Npas4 and Fosb. Mean + SEM; n =4 independent cultures as biological replicates. c, inhibition of ERK1/2 through UO126 (10 µM, 20min before) leads to significant blockade of mRNA induction for Arc and Fosb, but not Npas4. Mean + SEM; n = 3 independent cultures as biological replicates. d, inhibition of p38alpha and p38beta through SB203580 or SB202190 respectively (both 10 µM and 20min before) leads to significant reductions in mRNA induction for Arc and Fosb, but not Npas4. Mean + SEM; n = 3 independent cultures as biological replicates. e, blockade of PKA activity through H-89 (10 µM, 20min before) leads to reduced induction of Arc, unaffected induction of Fosb and a superinduction of Npas4. Mean + SEM; n = 3 independent cultures as biological replicates. f, inhibition of calcineurin through co-application of Cyclosporin and FK506 (1 µM each, 20min before) leads to unaffected induction of mRNAs for Arc and Fosb, but significantly reduced levels of Npas4. Mean + SEM; n = 3 independent cultures as biological replicates.
LSM images of VEGFR3/β1 integrin PLA dots in human LECs transfected with control or ILK siRNA. Scale bars: 10 µm Quantification of VEGFR3/β1 integrin PLA dots per human LEC after transfection with control siRNA or ILK siRNAs (n = 5 independent transfections per siRNA), P = 0.234 (control versus ILK-1), *P = 0.024 (control versus ILK-2), *P = 0.001 (control versus ILK-3)
(B) Venn diagram showing the various counts and overlaps between the microarray and network analysis. Also shown are the Ub-associated Nrf2 targets featured in the autophagy network mapped to mouse orthologues. Probes detecting transcripts exhibiting differential expression (Benjamini-Hochberg [BH]-adjusted P < 0.05) in at least one of the four comparisons are shown. Heat map represents Log2 (fold changes) with respect to either wild type (WT) or Atg7 KO (as indicated). Labels show the symbol, RefSeq (accession numbers from the NCBI Nucleotide database), gene probe name, and the gene row and gene column (i.e., array coordinates).
(D) Human neural progenitor cells (NPCs) and neurons differentiated from NPCs for 1-6 weeks express SLC6A20 proteins, as revealed by immunoblot analysis using the SLC6A20 antibody directed against a region of mouse SLC6A20A (aa 301-369; KATFNYENCLKKVSLLLTNTFDLEDGFLTASNLEQVKGYLASAYPSKYSEMFPQIKNCSLESELDTAVQ; Sigma) that is 83% identical to the human SLC6A20 sequence. Two different splice variants of human SLC6A20 (v1 and v2) expressed in HEK293T cells were used as positive controls. PSD-95, an abundant excitatory postsynaptic scaffold protein, was used as a maker for neuronal maturation, and β-actin was used as a loading control. Note that the band intensity of 'unboiled' SLC6A20 at 2-week time point is stronger relative to the boiled SLC6A20 band at 2 weeks, in line with that SLC6A20 is a transmembrane protein with twelve transmembrane domains that easily forms larger protein aggregates upon boiling and get stuck at the start location of western gels, decreasing the protein band intensity of SLC6A20. We did not try the unboiling of other samples at different time points because one sample was enough for proof of concept. U, untransfected HEK293T cells; V1/V2, HEK293T cells expressing human SLC6A20-v1/v2.
(B) DOR overexpression enhances autophagosomal formation. LC3‐GFP‐positive vacuoles were counted in 100 transfected HeLa cells (with GFP‐LC3 and TRα1, or GFP‐LC3 and DOR). *Significant effects of starvation, P0.001; τsignificant effects of DOR overexpression, P0.001.
TCHP knock-down or control cells were stained for anti-PCM1 antibody. Panel below: quantification (n=50 cells; unpaired t-test; **p=0.004 vs control). Scale bars, 50 µm and 5 µm in the inset.
A Structure of USP2 (red) bound to Ub (cyan) [pdb‐id 2hd5 (Renatus et al, )], with a close‐up on Ub Ser65 in the distal Ub. A negative charge at the USP2 Asp345 position is conserved in most USP enzymes (Ye et al, ).
F ChIP analysis of Ccq1HA and FLAG-TetR-Clr4* (see scheme). Expression and tethering of FLAG-TetR-Clr4* are controlled by thiamine/AHT addition (red dots indicate induced/tethered FLAG-TetR-Clr4*; empty circles indicate non-induced/non-bound controls) (n = 3 independent experiments) Data information data are normalized to input and represented as mean ± SEM
F. Quantification of soft agar colony formation assay of LNCaP and DU145 prostatic cancer cell lines treated with increasing concentrations of siRNA targeting GOLPH3 or LCS (data are means ± SEM of at least 3 independent experiments; ***p <0.001 [Student's t-test]).
(a) Confocal time-lapse images of an MCF10A cell-in-cell structure expressing GFP-LC3 and H2B-mCherry. The arrow marks recruitment of LC3 to the entotic vacuole (see Supplementary Movie S1). Scale bar, 15 μm.
A-C The kinetic block of SV endocytosis at excitatory synapses depleted of PI(3) P depends on prior neuronal activity and Ca2+. VPS34IN1-induced defects in SV endocytosis are reverted by parallel treatment with TTX (0.2 µM) in A and EGTA (2 mM) in B and AP5 (20 µM) in C, in Synaptophysin-pHluorin-expressing neurons N = 3 independent experiments (≥ 10 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM ; n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Acute application of CTα16 and recAPPsα elevates LTP in cDKO slices to the same extent. O, P After 1 hour pre-incubation with either 10 nM CTα16 or 10 nM recAPPsα fEPSPs were recorded. The TBS induced LTP curve was closely overlapping for both conditions and resulted in similar, statistically not significantly different LTP values Q CTα16 and recAPPsα modulate presynaptic function of cDKO mice in the same manner. Age: 5-6 months. n=number of recorded slices. N=number of animals. Data represent mean ± SEM. Data were analyzed by unpaired two-tailed Students t-test.
(A-B) Coronal brain section, bregma: - 2.88 from The Mouse Brain Atlas shows the site of injection in the CA1 cell body layer of the right hippocampus (A). 8 h post-injection, majority of the injected Fib-Tau-1N3R-ATTO550 (red) is taken up by the corpus callosum of the injected side (B). Images are shown at low (left, 10X) and high (right, 63X) magnification. Data information: Scale bar, 100µm in the left panel in (B), 10µm everywhere else.
ATG3 and LC3B gene expression pattern in neutrophils after stimulation with PMA or rapamycin. ATG3 mRNA expression analysis (fold) after PMA (2.44±0.78) and rapamycin (RAP: 1.81±0.41) stimulation. LC3B gene alterations (PMA: 4.98±0.59) and rapamycin (RAP: 1.7±0.55). Effect of 3‐MA pre‐treatment, for both stimuli, was also assessed (PMA+3‐MA: ATG3: 1.36±0.64, LC3B: 1.5±0.49 and rapamycin+3‐MA: ATG3: 1.28±0.46, LC3B: 1.24±0.57). No other significant alteration in ATG genes mRNA levels was detected. Representative data of ATG7 mRNA expression analysis after treatment with PMA (1.21±0.42) and rapamycin (0.94±0.64). Data are representative of six independent experiments and presented as mean relative expression±SD. Wilcoxon matched‐pairs test, *p<0.05 and n.s.=not significant compared with control conditions (dot line) as indicated in the figure. Relative expression (folds) of each gene is derived from substitution of DCT values (normalized by GAPDH DCT value) in 2DDCT equation 36.
(C) Pie charts showing the effect of treatment on the preventing infection of Vero-E6 cells. The number of cells above and below the cut-offs for being positive for Spike were counted and the percent cells in each category were determined. All analyses were done in Tibco Spotfire.
C. Percentage of TF-1 TpoR CALRdel61 human CD33+ cells measured in peripheral blood at 3 weeks post-tail vein engraftment (n=5 mice per treatment). Data information: a student's unpaired t-test was used to determine statistical significance.
Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Concentrations used: 200ng/mL Sema3A; Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). , isolated axons (C) were used for FRAP analysis.
E. ST2 cells were pretreated with TBK1/IKKε inhibitor MRT67307 (2 µM) or IKKα/IKKβ inhibitor TPCA1 (10 µM), stimulated with IL-17 (500 ng/ml) as indicated and analyzed by immunoblotting.
Catalytic activity of cathepsin D, B, and L. Total brain lysates from 4.5-month-old mice were incubated with quenched fluorogenic substrate for Cat D, Cat B and Cat L. The linear increase of fluorescence signal was measured and then normalized to wildtype. Data represent the mean normalized activity ± SD. (n = 3 biological replicates per genotype).
(A-B) SPHK2 inhibition using ABC294640 and SLM6031434 prevents DHS-induced DNA damage as shown here by staining for γH2A.X (green) in hCMs.
The possible interaction of macrophages with kugeln was studied in the transgenic Tg(kdrl:HRAS-mCherry)s916, Tg(fms:GAL4.VP16)i186, Tg(UAS-E1b:nfsB.mCherry)il149, visualizing macrophages and the vasculature simultaneously. Coloured depth-coding indicates the different anatomical depths of kugeln and macrophages (ventral purple, dorsal white).
Representative images of recombined IAHCs following IgG or αDll4 conditions. Kit is shown in white in the image, in black in the label. Multistack reconstruction of confocal images. Scale bar: 5μm.
D-F. MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIVmac virus‐like particles containing Vpx/Vpr (SIV VLP). DNA was isolated 18 h post‐infection for qPCR quantification of F. Late RT products; G. 2LTR circles; H integrated viral DNA.Integrated copies of viral DNA, Alu-Gag qPCR. (n = 3, mean ± s.e.m.; (ns) non‐significant; **P‐value ≤ 0.01, paired t‐test).
(F) Atg16L1-Δ/Δ MEFs stably expressing the indicated constructs were cultured in growth medium (Fed) or EBSS (Starved) for 1 h and then subjected to immunocytochemistry using an anti-LC3 antibody. The number of LC3 puncta in each cell was counted for more than 50 cells. The average ± SD is shown for three independent experiments.
Polyubiquitin chains were co-immunoprecipitated with FLAG-PSMD4, an ubiquitin receptor for the 26S proteasome, from cells expressing either USP14 wild-type, C114A, or C114R. One representative experiment of two is shown.
C, Quantification of the mitochondrial phenotypes observed in b. Errors bars show the standard error of the mean (SEM). (15-20 images each with an average of 20-25 cells from triplicate independent experiments were analyzed).
F Mouse body weight was measured at the indicated day. Data shown are mean ± SEM (n = 5 to 10 mice).
(A) Interaction between HsAtg4B and the ubiquitin core of LC3. HsAtg4B is coloured salmon red, and LC3 is coloured green. The side chains of the residues involved in the HsAtg4B-LC3 interaction are shown as stick models. Possible hydrophilic interactions are shown as broken lines.
WT and SNX10 KO Caco-2 cells were treated with OMVs (100 μg/mL) for the indicated time. Protein expression of E-cadherin and SNX10 was quantified by ImageJ software (E) (n = 3 independent experiments). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001.
Western blots of FLAG, HA and Dpm1 from cell lysates or anti-FLAG immunoprecipitates of WT or ∆nem1 cells containing Spo7-FLAG, or Ice2-HA as indicated (SSY122, 2421, 3183, 3184, 3195, 3196, 3197).
c) Biochemical protease protection assay of NRK cells expressing YFP-CD3δ, CD3δ-CFP and GFP. Transfected cells were subjected to SDS-PAGE and Western blot analysis before (lane 1) and after separation of the cytosolic (lane 2) and membrane (lane 3) fractions by ultracentrifugation. Equal amounts of the membrane fraction were treated with trypsin in the absence (lane 4) or presence (lane 5) of Triton X-100. Protein extracts were analyzed by immunoblotting with monoclonal anti-GFP antibody JL-8.
We treated 30-day-old Mecp2 KO mice (n=10) and Mecp2 KO/HTTSA mice (n=10) with 5mg/kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests. (C) Body weight of FK506-treated KO, FK506-treated KO HTTSA and vehicle-treated KO mice at P35 and P55 (Mann-Whitney test). Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Data are means ± SEM.
(E) Transcriptional activity driven by ATF6. WT and Mfn2 KO cells were co‐transfected with the 5xATF6‐GL3 and TK‐Renilla plasmids and treated with 1 μM Tg for 24 h after transfection. Data are mean±s.e.m. (n=4). *P0.05 versus WT group.Source data for this figure is available on the online supplementary information page.
(d) Similar to c. WT and KO MEFs were treated with CCCP for 6 and 12 h and immunoblotted for anti-COXIV, anti-Tomm20 or anti-actin as indicated. For c and d, the numbers below each lane indicate the fractional band density compared with the 0 h time point, which has been set at 1.0;
Mice were pre-treated with LicoB or MCC950 for 1 h, then i.p. injected with LPS (20 mg/kg) and treated for 3 h. The levels of IL-1β in the serum and peritoneal lavage fluid were measured using ELISA.
J. Percent of IFN-γ+ TNF+ resident CD8+ T cells following stimulation with increasing amount of NP 366-374 peptide (0.01 - 10 ng/mL). A are pooled data from 2 experiments. G-J are representatives of 3 experiments. * p<0.05 Student's two-tailed t-test with unequal variance.
(d) HeLa cells with a transient knockdown using siCont. and siBeclin1-3′-untranslated region for 30 h were further left untreated or treated with 10 μM Y27632. Cells were then incubated in control or HBSS media for 8 h. Whole cell extracts were prepared, resolved by SDS-PAGE and were analysed for Beclin1, LC3 and β-actin.
(C, D) Length distribution of 3'UTRs (C) and proportions of putative PTC-containing isoforms (D) in 10,136 mRNA isoforms detected in N2 and/or smg-2 (All), the 219 isoforms depleted from and the 420 isoforms enriched in the smg-2 mutant.
D. Overview of organoid fusion co-culture method at Day 20 and 40. All images taken on a widefield cell-culture microscope. Scale bars, 500μm.
(A) Scheme of mouse Atg16L1 and deletion mutants showing the N-terminal Atg5 interacting domain (white box), a coiled-coil domain (black box), and a WD propeller-repeat domain (striped box).
Expression of mutant proteins from pBAD plasmid on the WT background does not result in filamentation For the boxplots in panels the central band represents the median of the distribution, the box ends represent the 25th and 75th percentile, the whiskers represent the 10th and 90th percentile, while the dots represent the 5th and 95th percentile. Data was usually obtained from 2-3 biological replicates. The number of cells used to derive the boxplot distributions in the different panels range usually between 200 to 450 (please refer to Figure 1 source data for exact number of cells for each dataset). See related Figure EV1B.
Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC-KO hCOs. Sections were stained with TUJ1 and PAX6. Scale bar: 200 μm. Quantification of the ratio of TUJ1 area versus PAX6 area. (H9, n=15; #6, n=6; #23, n=9 from 2 independent experiment). Each plot represented an individual organoid. Three to five rosettes were analyzed for each organoid. Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC-KO hCOs. Sections were stained with TBR1 and PAX6. Scale bar: 200 μm. Quantification of the ratio of TBR1-positive area around PAX6-positive area versus PAX6-positive area in each rosette. (H9, n=10; #6, n=10; #23, n=7 from 2 independent experiment). Each plot represented an individual organoid. Three to five rosettes were analyzed for each organoid. Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC-KO hCOs. Sections were stained with CTIP2 and PAX6. Scale bar: 200 μm. Quantification of the ratio of CTIP2 area versus PAX6 area. (H9, n=8; #6, n=8; #23, n=8 from 2 independent experiment). Each plot represented an individual organoid. Three to five rosettes were analyzed for each organoid.
A and B, Efficiency of AAV9-mediated gene delivery. AA9 viruses expressing the reporter GFP gene were stereotaxically injected into the SN of mice, and GFP expression in astrocytes, microglia, and neuronal cells was quantified. Data are represented as mean ± SEM. n=3 mice (>5 randomly selected microscopic fields per animal were counted). Significantly different at p=0.006*, 5.1E-09#, 5.36E-09+ in graph B.
(E-F) HCT116 cells were transiently transfected with the indicated shRNAs for 48 hr before treatment with 1 μM doxorubicin/ 0.4 μM TSA for 6 hr. Cell lysates were collected and blotted for the indicated proteins (E). Quantitation (n=3 biological replicates) of normalized cleaved PARP levels is shown (F).
d IFN-γ production of cells treated as in c was assessed by intracellular staining after 24 h of stimulation.
C-E Immunofluorescence microscopy analysis with the indicated antibodies reveals that mutation of tyrosines 65, 85 and 120 to phenylalanine abrogates the ability of GFP‐KSHV‐TK to induce loss of phosphotyrosine epitopes, phospho‐paxillin and phospho‐FAK from focal adhesions.
Transcriptome of female gametocyte by RNA-seq between DFsc7 (WT) and DFsc7;Δap2-o3 (KO) strains. The genes were ranked by transcripts level (FPKM average of three sample replicates) in the heatmap. In spite of global upregulation of vast genes with low or medium transcript levels (left panel), the highly expressed genes (FPKM>1500, right panel) are downregulated in KO over WT. The FPKM values in each row (each gene) were normalized by Z-score normalization.
(E) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h. Cells were harvested and subjected to measurement of caspase-3/7 activation. Caspase-3/7 activity is reported as relative light units (RLU) of infected samples minus uninfected samples. *P<0.05 (one-way ANOVA).
B. BMDM cells were transfected with control or TBK1 siRNA. 72 hours after transfection, cells were analyzed by immunoblotting with antibody for phospho-RKIP or TBK1.
G. RK and RF mutations disrupt SIRT7 methylation. Flag-tagged wildtype SIRT7 and its RK/RF mutants were immunoprecipitated and blotted with α-meSIRT7(R388) antibody
(A) Anti-CMS dot blot assay assessing the global amount of 5hmC levels in CD4+ naïve T cells and WT Treg cells. Data are representative of three biological replicates.
A. Representative intracellular Ca2+ signals in hTRPA1-expressing HEK293 cells in response to different concentrations of iodine. 2-APB, a TRPA1 agonist, was subsequently applied to fully activate TRPA1. RFU: relative fluorescence unit.