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(A) Confocal images of Proximity Ligation Assays performed on CD47 and CXCR4, CD47 and RAGE, CD47 and TLR4. Representative images from one experiment out of 3 performed are shown. MM cells were incubated overnight with 400 nM BoxA or PBS. Nuclei are in blue (DAPI), phalloidin is in green. Red dots represent physical contact of CD47 with CXCR4 or RAGE or TLR4. Scale bar, 20 μm. The intensity of red signal in PLA was quantified as described in Materials and Methods in individual cells (n as indicated). Insets are enlargements of the y axis. Mean and SD are indicated. Statistics: Kolmogorov-Smirnov test.
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(C) Fluorescence intensity based analysis of pFTAA-positive area covered in the cortex of APP23p40+/+ (n=10) and APP23p40-/- (n=8) mice (left) and representative images for each genotype (right), scale bar = 1 mm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, **p=0.0051.
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Representative immunofluorescence images of YAP distribution (right) and its nuclear quantification by Pearson's coefficient for correlation (left) of YAP and DAPI co-staining in H1299control and H1299RASSF1A plated on extracted ECM from H1299control or H1299RASSF1A cells with or without treatment with siP4HA2 or 4μM DPCA. (Correlation, Pearson's coefficient). (n=3, 300 cells per experiment) Scale bars 10µm. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test 2-(tailed) of n=3 experiments and error bars represent the mean ± S.E.M.
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U2OS-LacO cells transiently expressing Myc-LacI-Bub1 and EGFP-TOP2A (970-1531) were subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and GFP. The relative enrichment of EGFP-TOP2A at the LacO repeats was quantified in 130 cells (J). Data information: Means and error bars representing S.D. are shown unpaired t-test).
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A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of 5-AzadC or 5-AzaC. At 72 h post-treatment; and initiated (primers TAR) or elongated (primers tat) transcripts were quantified by RT-qPCR (D).
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Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis.Body weight change during seven weeks of HFD, expressed as average fold change in comparison with the starting weight. n = 16, WT; n = 6, Vegfd−/−; n = 16, VCiΔR26; n = 5, Vegfd−/−; VCiΔR26.Body weight comparisons at seven weeks of HFD. Significant differences were determined using one-way ANOVA and Bonferroni post hoc analysis compared to WT. *P = 0.004; **P = 0.003. n = 16, WT; n = 6, Vegfd−/−; n = 16, VCiΔR26; n = 5, Vegfd−/−; VCiΔR26.
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Measured log2(H/M) values at four time points as a function of their half-lives estimated here and elsewhere (Cohen et al, 2013). All log2(H/M) values shown here are averages of values obtained in 5 experiments (3 using lactacystin, 2 using epoxomicin). Proteins for which statistically significant differences at t=24 hours were observed (P<0.05, two-tailed Welch's t-test) are shown in red. Expected log2(H/M) values based on equation 4 are plotted as light blue lines. To minimize the masking of potential dependencies by the slight imperfections in the H/M normalization process (which introduces small offsets along the Y scale), average population log2(H/M) values of each time point (Fig. 6E) were subtracted from all measured Log2(H/M) values. Data for 1,416 proteins for which half-life estimates were available.
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(G) Model for the dual function of the Dsc complex in EGAD-dependent proteasomal as well as ESCRT-dependent lysosomal membrane protein degradation.
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H) Levels of ALDH1A3 differentiate between non-pro-invasive and pro-invasive tumors. High levels of ALDH1A3 in samples before treatment are associated to more invasive tumors after antiangiogenic treatment.
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(D) Cells were transfected and immunostained for IFT20 (red) and γ-tubulin (green). Scale bar, 10 μm.
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A-D. Example expression patterns for select genes identified by applying a general linear model (glm) including state, genotype, and interaction terms. Dots represent individual biological replicates (N = 3); lines highlight changes in mean values for replicates between each state (log2FC >1 and FDR < 0.05). A. State dependent expression is exemplified by Nanog which showed no difference between strains. B. Expression of Zfp277 exemplifies strain-dependence being consistently higher in B6. C & D. A significant genotype x state (GXS) interaction was identified for Nr5a2 (C, a marker for pluripotency) and Sox1 (D, a marker for neuronal differentiation).
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E. The efficiency of PGC differentiation was detected by IF, 4 d after induction, using C-KIT and CVH antibodies. Scale bar: 60 µm (n = 3 independent experiments).
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(c) Schematic representation of U2-pre-mRNA interaction. The U2 BSRR sequence and the pre-mRNA branch site sequence are shown. The arrows indicate several point mutations at the pre-mRNA branch site. Stem-loops I, IIa and IIb are also indicated.
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Western blotting showing endogenous PIF4 protein levels in the seedlings grown Total protein extracted from the seedlings was analyzed by immunoblotting with anti-PIF4 antibody. Ponceau S staining shows equal protein loading.
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CODIM super-resolution images (3 right side panels) of TNTs shown in the confocal expanded field images (left panels) of transfected cells showing expression of indicated GFP-tagged proteins (green). Cells were stained for F-actin with phalloidin (red). Scale bars in the expanded fields represent 5 µm and 1 µm in the insets.
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F) GO term enrichment analysis for biological processes of the 25 most significantly up-regulated genes upon SARS-CoV-2 infected in small airway cells. P-values were determined using PANTHER Overrepresentation Test.
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0,
0,
0,
0,
0
] |
Spleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. Blocking antibodies to IL-4 or isotype were added as indicated. IgG1+ and IgA+ class switched B cells were enumerated by flow cytometry after 4 days of coculture
|
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G) Presence of autophagic proteins (BNIP3, LC3 and Parkin) on mitochondrial enriched fractions obtained from control and Mfn2KD C2C12 myotubes untreated or treated with CCCP (n=3 independent experiments). Data were normalized by TIM44 as a loading control and expressed as a fold change compared to young control mice and represent mean ± SEM. #p<0.05 Mfn2KO vs. control mice or Mfn2KD vs. control cells, *p<0.05 old vs. young mice or treated vs. untreated cells.
|
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B UMAP visualization of all cells. Clusters are colored and labeled according to their inferred cell type identities.
|
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E) Quantification of the mean 45Ca2+-store content at the beginning of the measurement (t0) (n = 5).
|
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C. Scatchard analysis showing dissociation constant of 4D7 using I125-labelled and unlabelled 4D7 bound to full-length 30 amino acid peptide (n=3 biological replicates).
|
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BALF was collected on day 21 and centrifuged to isolate cell pellets. Cells (primarily macrophages) were analyzed by qPCR for CD86 (F) and IRAK4 (G) (n=5).
|
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(B) Immunoblot analysis of nuclear extracts isolated from Pax5+/+ (WT) pro-B cells and Pax5Jak2/+ lymph node tumor cells. The wild-type Pax5 and Pax5-Jak2 proteins were detected with an antibody raised again the N-terminal (N-term) paired domain of Pax5. The size of marker proteins is indicated in kilodaltons (kDa) to the left.
|
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E, F RT-PCR (E) and qPCR (F) of KPM cells and parental tumors show Trp53f/f allele deletion (Δ) and Bap1 and Cdkn2a overexpression compared with PMC.
|
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L Glucose uptake and lactate production were measured in Dgcr8−/−ESCs stably expressing Myc shRNAs and further infected with Mbd2 siRNAs.
|
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(B) Western blot of mitochondrial OXPHOS respiratory complexes in C2C12 myotubes treated for 48H with C26CM and ferric citrate. (n=3)
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g) Ubiquitylation at Lys 47 impairs RALB binding to EXO84. Flag-tagged RALB mutants were overexpressed in HEK293T cells and then immunoprecipitated with anti-Flag (M2) agarose followed by immunoblotting using anti-EXO84 antibody.
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(E) HEK293T cells were transfected with the indicated constructs. The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The lysate and immunoprecipitated products were subjected to western blotting. PY: PPEY motif; LY: LPTY motif. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.
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E) TE binning according to increasing 22G siRNA density in wild-type animals (grey, n=3). Mean normalized rpb-9 (mj261) mRNA reads (red, n=3) are overlaid with mean normalized wild-type mRNA reads (grey). (PolyA-selected RNA-seq libraries). The CEMUDR1 and Chapaev-2 transcripts are indicated with red (rpb-9 (mj261)) and grey (wild type) box plots. Central bands represent the median, boxes represent the 25th and 75th percentiles and whiskers represent the lowest and highest values, excluding outliers.
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Thiol tracker staining by flow cytometry 48h after seeding of the indicated untreated cells (n = 2) Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.
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Both wild-type (IWT) and R1285Q mutant (IR1285Q) FANCI efficiently associate with FANCD2. Fluorescence changes occurring when RED-tris-NTA-labelled FANCI (IWT or IR1285Q; both at 60 nM) is incubated at increasing concentrations of FANCD2 (ranging from 2.48 nM to 5.08 µM). Titrations were conducted twice (two technical replicates) and all data points for each protein-combination were used in fitting of a one-site binding model. Apparent Kd values (mean ± SEM) derived from fitting of a one-site binding model to the 24 data points of the two technical replicates are shown.
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Peak SARS-CoV-2 replication precedes innate immune activation. (A-I) (A,C) Representative images of NF-κB p65 (A) (red) and IRF3 (C) (red) nuclear localisation in mock or SARS-CoV-2 infected (MOI 0.4 TCID50VERO/cell) Calu-3 cells at 24 hpi. SARS-CoV-2 N protein (green. (E and G) Representative images of IL-6 mRNA (E) detected by FISH (red) and N protein (green) , or IFIT1 mRNA (G) (green) with N protein (red), both with nuclei (DAPI, blue) in mock or SARS-CoV-2 infected (MOI 0.4 TCID50VERO/cell) Calu-3 cells at 24 hpi. (B, D, F, H, I) Single cell analysis time course quantifying the Integrated Nuclear Intensity of NF-κB p65 (B), IRF3 (D) , or overall integrated intensity for IL-6 (F) or IFIT1 (H) mRNA over time in N protein positive cells and N protein negative cells (I). n=2. Kruskal-Wallis test with Dunn's multiple comparison. * (p<0.05), **** (p<0.0001). Scale bar represents 50µM.
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Automated microinjection of mouse neurons. Automated microinjection was performed on organotypic slices of mouse E16.5 dorsal telencephalon using Dx-A555 Phase contrast image of automated microinjection into mouse neurons. Scale bar is 100 µm.
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(E) Correlation statistics for the localization of endogenous MITOL and PMP70 in the presence of NMS-873. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines show the medians, box limits indicate the 25th and 75th percentiles as determined by the R software package, and whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and the X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.
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(G) The reads obtained by DNAse-seq (Song and Crawford, 2010) in T47D-MTVL cells around PRBs in the absence and in the presence of hormone were quantified and expressed as signal/bp (to account for peak length variation) for up and down-regulated genes. Up-genes: CORO2B, KCNH1, KLF15 and ATP10A; down-genes: KRT23, BCAS1, IGFBP5 and PGR. (*) P-value <0.05, ** P-value <0.01.
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(D) Tangential eye sections through MARCM clone mutants for ftfd that express either no transgene or an RNAi construct against yki (UAS-ykiIR) with Tub-Gal4 and aged 14 days at 29°C. Because the transgene UAS-ykiIR is on chromosomal arm 2 L, the same as ft, clones generated with this system carry two copies of UAS-ykiIR and therefore downregulate yki very effectively. No other cell outside the clones expresses the construct and is therefore wt for yki. Mask panel is presented on the right of each section. A quantification of photoreceptor numbers (far right) displays a very significant increase in the number of wt ommatidia (N=210 and 219 from four eyes). ***P0.001 in two‐tailed t‐test. If all classes of ommatidia are considered, χ2‐test=232.30; P0.001 for 3 degrees of freedom.
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Branching analysis of LPS-activated microglia by Monocle 2 leads to 9 distinct clusters in a two-dimensional state space inferred by generalized regression modelling (see Materials and Methods) showing the major difference of "subset LPS" (in yellow) compared to the other clusters corresponding to "main LPS" (in red). Monocle estimated a pseudotime for each cell along the inferred cell trajectory within the state space showing a delayed activation pattern of "subset LPS" compared to the other fractions.
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A. Electron micrograph of sections immunolabelled against RFP-SKIP (15nm gold) and GFP-Rab7 (10nm gold). Arrowheads and zoom inset (1.75x) highlight presence of RFP-SKIP and GFP-Rab7 on the same endosomal membrane, scale bar: 200nm.
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A. Representative FACS plots of spleens from leukemic recipient mice that had received LSK, GMP or DN2 cell grafts immediately after retroviral Myc/Bcl2 transduction. Numbers indicate frequencies (%) of CD45.2 donor cells within the indicated gates summarizing the mean±SEM of at least 8 recipients per group from a total of 10 independent experiments.
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(D) Maltoheptaose ubiquitylation was assayed for 60 s at 30°C in the presence of the indicated ubiquitin tetramers.
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(F) Heat-map analysis for GM3 species and clinical markers of six clusters. Sample sizes: sub-cluster-1 to -3 (total), n=22; cluster-4, n=7; cluster-5, n=9; cluster-6, n=12.
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(C) Cross-correlated motions of the Cα atoms of the WT and palmitoylated cGAS proteins.
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c, Immunoblot for ATG16L and clathrin in MEF homogenate (Hom), and the pellets from 1 h centrifugation at 100,000g (organelles (Org)), 300,000g (300k) and 500,000g (vesicles, 500k).
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(b) Parkin is recruited selectively to depolarized mitochondria and directs mitophagy. HeLa cells transfected with HA-Parkin were treated with CCCP for the indicated times. Mitochondria were stained by anti-TOM20 (pseudo coloured; blue) and a ΔΨm dependent MitoTracker (red). Parkin was stained with anti-HA (green). Without treatment, mitochondria are intact and stained by both mitochondrial markers, whereas Parkin is equally distributed in the cytoplasm. After 2 h of CCCP treatment, mitochondria are depolarized as shown by the loss of MitoTracker staining. Parkin completely translocates to mitochondria clustering at perinuclear regions. After 24 h of CCCP treatment, massive loss of mitochondria is observed as shown by the disappearance of the mitochondrial marker. Only Parkin-positive cells show mitochondrial clustering and clearance, in contrast to adjacent untransfected cells. Scale bars, 10 μm.
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C Slope of field excitatory postsynaptic potential (fEPSP) in response to theta-burst stimulation delivered to the Schaffer collateral pathway from WT mice (n = 10 recordings from 6 mice), Tau mice (n = 19 recordings from 6 mice) or Tau mice with TFEB injection (Tau + TFEB; n = 8 recordings from 6 mice). Insets: example fEPSP traces taken before (upper) or after (lower) stimulation from WT, Tau or Tau + TFEB mice. Calibration: 1 mV, 5 msec.
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(J) Intracellular K79 co-localization with EGFR and ezrin in control and S1P2 knockout HBMEC (as shown by arrows, cyan). Percentage of co-localization was calculated by counting numbers of all GBS and co-localized intracellular GBS from at least three representative fields, and expressing as numbers of co-localized GBS/all GBS x 100.
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C. MTT assay for NSCLC cell lines and Beas-2B cells with dasatinib or DMSO. Error bars represent mean ±S.E.M., n=9, *P<0.001 by two-tailed unpaired Student's t-test (H1915 P=1.09×10-5, H2110 P=0.009 and A549 P=6×10-6).
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(E) Selected stills from live imaging analysis of Rod-GFP streaming from KTs in neuroblasts expressing either PoloWT or PoloT182D. Asterisks indicate direction of chromosome segregation (putative spindle poles positions). Arrowheads highlight streaming of Rod-GFP from unaligned KTs. Representative kymographs are shown for Rod-GFP streaming from metaphase until anaphase onset (AO). Frames were acquired every 5sec. Data information: Scale bar: 5 μm.
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] |
PAR-binding motif of BRD7 is required for its ribosylation by PARP1. Recombinant Myc-BRD7-WT and Myc-BRD7-mutant were subjected to in vitro ribosylation assay and analysed by Western blot as indicated (n=3).
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5A) nt and PINK1 knockdown (kd) SH-SY5Y cells were either kept in RPMI medium with 5% FCS or starved with HBSS for 12 h. DEVD cleavage as parameter for caspase-3 activity was analyzed and normalized to the protein content. Cells with PINK1 knockdown demonstrated a significantly increased caspase-3 activity under both conditions; n = 4; RPMI+5% FCS: p<0.05; HBSS: p<0.01.
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(B) SUMO modification of endogenous PELP1 was monitored by anti‐PELP1 immunoblotting in HeLa cells transfected with siRNAs as indicated. Depletion of the respective proteins was verified by western blotting.
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A-B) Scatter plots showing ELF3 and FOXA1 levels (as determined by ChIP-seq) at bound genomic regions in wild type and KLF5-KO cells. Light blue dots indicate regions where ELF3 or FOXA1 signal is reduced in KLF5-deficient cells.
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E-S. Modeling hyperglycemia in HUVEC resulted in dysregulation of junctional assembly of CDH5 and CTNNB1 proteins and increased membrane localization of PRKCB protein. Complementation of VEAL2 and veal2 in hyperglycemic conditions reverted junctional disassembly of CDH5 and CTNNB1 and also kept PRKCB in cytoplasm to mitigate pathological conditions associated with hyperglycemia. (E-H,Q) CDH5 protein, (I-L,R) CTNNB1 protein, (M-P,S) PRKCB protein. (E-P) Magnification-60X, Scale bar-15μm. Arrowheads indicate representation of signals of proteins in HUVECs. (Q-S) Data from cells of different fields of 3 technical replicates of 1 biological replicate is presented as representation. Data is shown as individual values; the middle bar represents the mean and the error bar represents ± standard deviation.
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A Topology of GlpG, Rhom7, and the artificial substrate with a maltose-binding protein (MBP) domain, triple-FLAG tag (3xFLAG), the TMD of P. stuartii TatA, and a thioredoxin domain (Trx).
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F. Western blot of Gsnor was assessed in mouse adult fibroblasts (MAFs), obtained from wild type (WT) and Gsnor-null (KO) mice treated with 200 or 400 μM H2O2 for 24 h.
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(C) Acyl-RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h. HAM: hydroxylamine.
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One night after cell purification, CD8+-depleted PBMCs from 12 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added to the cultures. Six days after 5-AzadC treatment, WST-1 assay was performed. Mock value was arbitrary fixed at 100% for each individual.
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F Western blot for cleaved casp3, parp, and vinc in LIM1215 silenced for PHD1 and treated for 24 h with 200 μM 5-FU.
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A Diagram of the virally-mediated approach to induce Gadd45a-overexpression in hippocampal glutamatergic neurons of Nex-Cre(+/-) mice.
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The apoptosis (C) measured after FST knockdown in vitro. Scale bar = 10 μm. The apoptosis assays (C) were performed in triplicates. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).
|
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(G) 293T cells were transfected with shRNAs targeting CARS or NTC, then they were cultured with cystine-deficient medium for 8 h. Cell lysates were immunoprecipitated with anti-AMPKα and subjected to WB analysis with anti-AMPKα and anti-CaMKK2.
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Confocal microscopy of live unstained fibres from a GFP-LC3:WT mouse (WT) and from untreated (GAA−/−) or ERT-treated (GAA−/−; +ERT) GFP-LC3:GAA−/− mice. Images show typical centrally located autophagic build-up with multiple clusters of LC3-positive autophagosomes. Structures like these are never seen in WT muscle. Autophagic build-up is present virtually in all fibres derived from EDL or gastrocnemiusmuscles (not shown), but only in 60-70% of FDBfibres. Labelled recombinant human GAA (red) was administered i.v. into 3-4 month-old GFP-LC3:GAA−/− mice (n = 5) at a dose of 100 mg/kg twice with a 24 h interval. Mice were sacrificed the next day, and live fibres (shown for EDLmuscle) were analysed by confocal microscopy. The labelled recombinant GAA was detected almost exclusively in the autophagic vesicles. At least 500 fibres were analysed for each condition. Bar: 10 µm.
|
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F) Quantification of IHC shows percentage of cells with 2+ and 3+ PYCR1 staining intensity. Data is expressed as mean ± SD of three patient samples. Paired samples were compared by a Student's t-test. *p < 0.05 and *** p < 0.001.
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(A) The developmental phenotype of the arid2/3/4 mutant as compared to the wild type. Two-week-old seedlings grown on MS medium are shown.
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LDH-release from MDMs as treated in C. Bars represent the mean percentage of LDH release relative to the total cells lysed, ± S.D. n = 9 independent blood donors.
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Life imaging of wild-type (WT) and α1-tubulin(-) oocysts expressing ef1α:GFP (early and late oocysts) and csp:mCherry (late oocysts) to locate the oocyst cytoplasm. Microtubules were labeled with SiR-tubulin (green) and DNA with Hoechst (blue). Oocysts are shown in a chronological order from early (day 4) to late (day 12) development. (A, B) Early oocyst development with remaining subpellicular microtubules of the preceding ookinete stage (A) and subsequent DNA replication (B), where some but not all DNA is co-stained by microtubules in the mutant. (C) Nuclear alignments to the invaginated plasma membrane and budding of sporozoites. Note the strong SiR-tubulin signal only seen in sporozoites of WT oocysts and complete absence from the mutant. Sb: sporoblast. (D) Oocyst ready to burst with fully formed sporozoites in WT oocysts. Note that α1-tubulin(-) oocysts retained sporozoite nuclei predominantly within the sporoblast (Sb) during budding. Scale bars: 5 μm, magnification box in B: 1 µm,.
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Flow‐based, microfluidic device for temporal GF delivery. Computer‐controlled, pressure pump enables mixing of medium and GFs in the control part (left), and temporally defined GF delivery in the cell culture chamber (right and magnified inset).
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(B) Single confocal section of a population of S2 cells. Examples of interphase cells are boxed in blue. Mitotic cells positive for the anti-phosphoHistone3 (pH3) are boxed in red.
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(D) The IC50 value of the combination of CATR and cisplatin in CNE1-LMP1 cells was changed. Data are presented as means ± S.E.M. (n = 6, biological replicates per group).
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(F) The observed heterogeneity is reduced with increasing stress levels. Each line represents the cumulative fraction of cells (N=~50) with PsigV-YFP values that are higher than the half maximum of their final values (representing cells that have activated).
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K Quantification of the numbers of DMC1 foci in 6-month-old Usp26+/Y (n=50, Zygotene, n=31, Early-Pachytene) and Usp26-/Y (n=31, Zygotene, n=24, Early-Pachytene) spermatocytes. P=0.1893 for Zygotene. Red dots indicate Usp26+/Y mice and green dots indicate Usp26-/Y mice.
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B. Representative images of Parkin-mediated clearance of TOMM20 in HeLa/Parkin cells treated with PEX13 siRNA and transfected with indicated PEX13 siRNA-resistant plasmid and then treated with CCCP (10 μM, 16 h). Scale bars, 20 μm. See Fig EV3B for representative images of mitochondrial morphology (TOMM20 staining) in control cells treated with DMSO.
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Western-blot analysis of CYLD and BCL10 in total protein lysates from GSC#9 transfected with non-silencing RNA duplexes (sic) or BCL10 targeting duplexes (siBCL10, seq#1 and seq#3). GAPDH served as a loading control. Cell viability was measured using Cell TiterGlo luminescent assay in sic and seq#1 siBCL10-transfected cells. Data were normalized to their respective sic-treated controls and are presented as the mean + s.e.m of 3 independent experiments, in triplicate.
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B) Dendrogram representing the distance among the clusters. The tree was built on the correlation (Spearman) calculated on the regulon matrix from the NI dataset.
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C. Bar plots showing isoform specific RT-qPCR-based measurement of relative levels of PTC+ and PTC- isoforms of genes indicated below from wild-type (WT) or 3DKO#2 cells stably expressing the specified proteins. Relative levels from each replicate are shown by white circles. Error bars indicate standard errors of means. The asterisk (*) represents statistically significant difference (p<0.05, t-test) of indicated samples from EGFP-transfected control cells, which has a value of 1 (n=3 biological replicates).
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p53 has been show to response with a series of undamped pulse to ionizing irradiation leading to cell cycle arrest while intrinsic DNA damage during cell cycle does not induce regular pulsatile p53 and subsequent gene expression programs. Schematic representations of p53 dynamics in both cellular conditions are shown.
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G) WT and FAK−/− macrophages expressing HA-Akt were stained with anti-HA and anti-LAMP1 before analysis by confocal microscopy. White boxes show enlarged regions in lower panel. Arrows indicate HA+LAMP+ Salmonella evident in WT PEMs. (H) The percentage of ΔΣΠΙ1ΔΣΠΙ2 (first grouping) or ΔinvG (second grouping) Salmonella co-localizing with HA and LAMP-1 5 h post-infection. At least 100 bacteria were counted per condition. Values are means ± SEM, N = 3, *p<0.05.
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G: SP1 protein expression is reduced following mithramycin exposure. Western blot showing suppression of SP1 expression compared to loading control (GAPDH) after 100nM mithramycin exposure for 1h, 4h, 8h, 12h, 18h.
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A. Western blot (WB) analysis of specified proteins in small intestine of Pkd2wt/wt and Pkd2ki/ki male mice after one week of HFD. Quantification of the bands for each protein normalized to loading control and relative to Pkd2wt/wt. n=3.
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C) Downregulated genes following H2A.Z.1 depletion were analysed by STRING. The image shows the cluster of genes with gene onthology (GO) terms related to cell cycle. PPI=Protein-protein interaction.
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F. Representative pictures of intestines of Casp8WT/MLKLko and Casp8ECko/MLKLko mice at 30 days after tamoxifen treatment (n= 8 WT, 8 ECko).
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B) Blocking β1 integrins activity by using the specific anti-β1 Integrin monoclonal antibody prevents the PTX3-induced postsynaptic AMPARs recruitment (Ctr=1.000±0.074, PTX3=1.504±0.098, αCD29+PTX3=0.766±0.097, αCD29=0.810±0.108. Number of fields examined: 37, 33, 23, 24 respectively; one way ANOVA analysis of variance, p<0.0001 followed by post hoc Tukey test; 3 independent experiments, data are presented as normalized mean values±SEM)
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] |
E Immunoprecipitation assay showing the interaction of GFP-tagged NOT1 with HA-tagged DDX6 (wild-type or the indicated mutants) in HEK293T cells. Samples were analyzed as described in panels (A-D).
|
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G IFA of DHHC2 at the dhhc2::6HA zygotes treated with 100 μΜ 2-BP. Scale bar = 5 μm.
|
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(C) Confocal microscopy images of epidermal sheets from back skin of K5-R1/R2 and CTRL mice stained for IRF7 and K14, counterstained with DAPI. Arrows denote nuclear IRF7 (red) in basal and suprabasal keratinocytes of K5-R1/R2 mice. The strong red staining of the stratum corneum (sc) is unspecific background and is more pronounced in K5-R1/R2 mice due to the increased thickness of this layer.
|
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] |
SPRR5 induction during the course of keratinocyte (KC) differentiation is shown by northern blot (D) for four different primary keratinocyte isolates and compared to undifferentiated keratinocytes (n = 8 biological replicates). Data is presented as mean ± SD.
|
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(C) Patient (index case, IC PSMC3) fibroblasts transfected with either pcDNA3.1/empty vector (mock) or pcDNA3.1/PSMC3 were exposed to a 30-nM treatment of carfilzomib or left untreated (as a negative control). After 16 hours, cells were collected and subjected to RIPA-mediated protein extraction prior to SDS-PAGE and subsequent western-blotting using antibodies specific for ubiquitin, TCF11/Nrf1, Rpt1 (PSMC2), Rpt3 (PSMC4), Rpt5 (PSMC3), Rpt6 (PSMC5), β1, β2, β5, β5i, PA28-α and α-Tubulin (loading control), as indicated. For the TCF11/Nrf1 staining, two exposure times are shown.
|
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C-F; H-K; and N-Q. The tagged WT or mutant 7SK RNA was co-transfected with the indicated HA-tagged proteins into HeLa cells. Anti-HA immunoprecipitates (IP) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA.
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(C) Main functional categories encoded by the Vav1∆C-dependent transcriptome. For all of them, P ≤ 0.001 (Fisher's exact test).
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(F-G) Quantification of fluorescence intensity from stained embryos of FGFR::GFP (F) and of βPS-Integrin (G). Data are plotted as mean +/- SD; significance was assessed using one-way ANOVA with Dunnett's correction for multiple comparisons. **p=0.0022, ***p=0.0003, ****p<0.0001. Number of cells analyzed, for (F): Control n=8, 1hr at 34ºC n=6, Recovery n=8. (G). For (G): Control= 7, 1hr at 34ºC= 8, 2hrs at 34ºC= 8.
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NEDD4L was knocked out by the CRISPR Cas9 method in NHEKs. Western blot (WB) analysis of protein expression in NHEKs induced by IL-6 Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments
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(A) Primary screen anti-CD8 antibody uptake images for control cells (top) and GRINA KD cells (bottom).
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(F, G) HeLa cells were transfected with the indicated siRNAs. After 48 h, the cells were transfected with LAMP1-RFP (red), with or without HA-raptor for 24 h. The cells were stained with anti-mTOR (green) antibody and DAPI (blue), and then visualized using confocal microscopy. Regions within the dotted boxes are magnified in the insets. Scale bar, 5μm. The quantification of mTOR on lysosomes is shown in Figure 2G. Data from three independent experiments represented as means ± S.E.M., **, p<0.01, one-way ANOVA.
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I. STRING analysis of the proteins listed in figure 3H (string-db.org). PSMA2, PSMA6, PSMB1, PSMB3, PSMB4, PSMB6 are members of the gene ontology term (GO) ´"proteasome core complex" (GO:0005839).
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(O) Schematic diagram depicting a working model for Shh-regulation of epileptogenesis. Epileptic activity triggers sequential responses, including Shh release, inhibition of glutamate transporter activity, and increase in extracellular glutamate, leading to epilepsy development.
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E. Direct binding of HY5 to the FER promoter analyzed using ChIP-qPCR in 35S-HY5-3HA-overexpressing (HY5-OE) tomato plants. WT and HY5-OE plants were exposed to white light (200 µmol m-2s-1) grown under Fe-deficient (2 µM, -Fe) conditions for 7 d. Input chromatin was isolated from root samples at 7 d. The epitope-tagged HY5-chromatin complex was immunoprecipitated with an anti-HA antibody. A control reaction was processed side-by-side using mouse IgG. Input- and ChIP-DNA samples were quantified by RT-qPCR using primers specific for the promoter and exon fragment of the FER gene as indicated in (D). The ChIP results are presented as percentage of the input DNA.
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(D) Schematic representation of the creation of stable, untagged and siRNA resistant IRF2BP1 WT and K579R HeLa cells. Constructs expressing IRF2BP1 variants in an pIRES-hrGFP II ("pIRES") vector were transfected, selected with antibiotics and FACS sorted for low GFP expression.
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(G) iPSC-derived human neurons were transduced with shRNA targeting human TDP-43 (shTDP) or control (shCtrl) seven days after thawing for three days. VPS4B levels were analyzed by quantitative RT-PCR. Expression was normalized to the housekeeping gene YWHAZ and PGK1 (n = 3).
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(B-F) Basal views through living SCs, with dashed white circles approximating the outline of a single SC, and acidic compartments marked by the vital dye LysoTracker® Red (magenta). In merge images, a single non-acidic (C, D, F) and acidic (E) compartment containing intraluminal vesicles (ILVs) is boxed and magnified in the right panel (Zoom). ILVs appear as membrane-delineated vesicles, using super-resolution 3D-structured illumination (3D-SIM) microscopy for the brighter overexpressed GFP-tagged constructs (yellow; C and F). However, ILVs appear only as puncta, using lower resolution wide-field microscopy for the fainter endogenously expressed YFP-tagged Rab GTPases (yellow; D and E). (B) Wide-field fluorescence image, including differential interference contrast (DIC), of SC expressing a GFP-tagged version of human CD63 (CD63-GFP). CD63-GFP expression is apparent on the limiting membranes of non-acidic compartments and their ILVs, and also on the limiting membranes of the enlarged acidic compartments. Most large non-acidic compartments are Rab11-positive (D) and contain dense-core granules, which have a 'fried egg' appearance with DIC (arrowheads) (Corrigan et al., 2014; Redhai et al., 2016). (C) 3D-SIM image of CD63-GFP-expressing SC. Arrow highlights CD63-GFP-marked ILVs (Zoom). Many more ILVs are apparent in non-acidic compartments in a complete Z-stack of a non-acidic compartment (Movie EV1). (D) Wide-field fluorescence image of an SC expressing a YFP-Rab11 gene trap. YFP-Rab11 marks the limiting membranes of most non-acidic compartments and internal puncta (arrow in Zoom), but not the surface of acidic compartments (Appendix Fig S1B). (E) Wide-field fluorescence image of SC expressing a YFP-Rab7 gene trap. YFP-Rab7 marks the limiting membranes of acidic compartments and internal puncta (arrow in Zoom). Enlarged acidic compartments are also present in adjacent main cells. (F) 3D-SIM image of SC expressing a GFP-tagged version of Breathless (Btl-GFP). Btl-GFP marks the limiting membranes of non-acidic compartments and their ILVs (arrow in Zoom), but not the surface of acidic compartments (Appendix Fig S1C). Images from six-day-old male flies shifted to 29°C at eclosion. This induces GAL4/UAS-dependent SC transgene expression in (B), (C) and (F). The genotypes of flies carrying multiple transgenes are: w; P[w+, UAS-CD63-GFP] P[w+, tub-GAL80ts]/+; dsx-GAL4/+ (B, C); w; P[w+, tub-GAL80ts]/+; dsx-GAL4/P[w+, UAS-btl-GFP] (F).
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0,
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0,
0,
0,
0,
0
] |
G Correlation analysis of H3K4me1 (left) and H3K27ac (right) levels between WT and SclKOMES and ES cells and MES (Wamstad et al, ) around 4,393 Scl binding sites shows that establishment of these marks occurs independently of Scl.
|
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] |
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