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disease.Eight adult rhesus macaques were inoculated with SARS-CoV-2 isolate nCoV-WA1-2020. After inoculation, animals were observed for disease signs and scored according to a pre-established clinical scoring sheet (a).
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(H)-(J) mRNA expression of (H) U2AF1, (I) SMN2-FL, or (J) SMN2∆7 in SMA patient-derived fibroblasts (red bars) 72 hours post-transfection with SMN2 SSO, scrambled siRNA, or U2AF1 siRNA at the indicated dose. Unaffected, SMA carrier fibroblasts (blue bars) treated with scrambled siRNA. Data information: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; empirical p-values are determined by a simulation and sampling method The error bars show the Standard Error of Mean (SEM). Data represent 3 biological replicates. Three technical replicates were performed during qPCR for each biological replicate and averaged. Scrambled siRNA condition for each experiment was set to 1.
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Representative images showing hematoxylin-eosin (HE) staining and ANG IHC staining in mild and severe IBD samples.
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F PBL27 interacts with MAPKKK5-C6xA in yeast two-hybrid experiments. The growth of yeast colonies on plates (-ULWH) lacking uracil (U), leucine (L), tryptophan (W), and histidine (H) with 10 mM 3-AT indicated a positive interaction.
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(D) Representative images of hypoxia marker pimonidazole in A375 xenografts treated as indicated. Bar graphs show the % of tumor hypoxic area in A375 and COLO205 xenografts (n=5 tumors), **P < 0.01, ***P < 0.001 versus vehicle (A375: PLX4720 P = 6.94E-12; COMBO P = 3.34E-12) (COLO205: PLX4720 P = 0.0015; bevacizumab P = 0.0004; COMBO P = 0.0003).
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First panel: In WT condition the lncRNA DUXAP8 is not expressed and the chromatin of Celf2a regulatory region is characterized by a permissive signature (H3K27ac and H3K4me3) that allows the expression of this factor. In the presence of Celf2a protein the ex45 is normally included in the mature form of DMD mRNA. Second panel: In GS∆44 cells the lncRNA DUXAP8 is aberrantly expressed. The chromatin of Celf2a regulatory region is marked by the repressive H3K27me3, Celf2a is not expressed and the ex45 is not included in the mature form of DMD mRNA allowing the production of an in-frame DMD transcript (∆44-∆45). Third panel: in ∆44 condition DUXAP8 is absent, Celf2a is expressed and ex45 is included in the mature form of DMD mRNA producing an out of frame transcript (∆44). Fourth panel: In ∆44 cells the depletion of Celf2a isoform is paralleled by a recovery of dystrophin protein (5%) obtained by the exclusion of ex45 from the mature DMD transcript (∆44-∆45).
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(H) Experimental overview of a therapeutic B-ALL model. Replicate experiments are shown in Figure EV3 A,B.
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(a) Localization using confocal microscopy of endogenous DICER (monoclonal antibody 13D6) and NDP52 in HeLa cells treated with RAP or control. The arrows highlight some examples of co-localization. The lower left insets show higher magnifications of the areas outlined in the main panel. Scale bars, 5 μm. (b) Quantification of DICER co-localization with NDP52. Total fluorescence intensity of DICER co-localized with punctae (>0.2 μm3) of NDP52 was quantified with Volocity software in RAP-treated cells/control-treated cells over several Z-stacked microscope fields per experiment (n = 4, error bars s.e.m.).
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Representative curves show the lifespan of wt and gas-1(fc21) nematodes compared to (B) age-1 and age-1; gas-1 mutants animals. Average median lifespans ± SEM from all replicates indicated beneath representative curves.
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(i) Quantitative real-time PCR analysis of P4-P5 retinas from Pald1+/+ and Pald1-/- pups. Ccnd1 transcript levels, normalized to Gapdh. Mean±SEM, unpaired t-test. n=10 Pald1+/+ and 14 Pald1-/- pups. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.
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A Mitochondrial membranes, isolated from cardiac tissue, were solubilized in 1% digitonin and analyzed by BN-PAGE and Western- blotting with the indicated antibodies.
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(C) Left: γ-secretase inhibitors do not induce the formation of TLN accumulations. Wild-type hippocampal neurons were treated with γ-secretase inhibitors (125 nM, daily from d 7-14 post-plating), fixed, and immunostained for TLN and the percentage of neurons with TLN accumulations was scored. Culturing neurons for 25 d increases the size and number (insets), but not the frequency of TLN accumulations (mean ± SEM, n = 3, 200-700 neurons per time point). Right: Western blot showing that only APP-CTF accumulates after chronic treatment with γ-secretase inhibitors.
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F. Confocal microscopic images displaying the coexpression of MAG or MBP with Phalloidin-594 (a-b and c-d, respectively) in rat primary OPC cultured in absence or in presence of 100 μM D-Asp for 4 days. (e), Single representative MBP++ cell. Scale bars: 50 μm in a-d; 10 μm in e. (f-g); Quantitative analysis of Alexa-594-phalloidin-positive oligodendrocytes (f) or MAG+ and MBP++ oligodendrocytes (g) scored in control and D-Asp exposed cells. Alexa-594-phalloidin-positive oligodendrocytes were scored in 4 categories, according to their morphological complexity. In each category, data were normalized on the total number of oligodendrocytes Data information: The values represent the means ± S.E.M. Level of significance was determined by usin F, for each category two-tailed Student's t test, *P<0.05 versus control (n=3)
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(E) 2.0 x 10ˆ5 cells were seeded and challenged with 10 ng of HIV-1NL4-3 in the presence of 2 μg/mL DEAE-Dextran. Productive infection was scored by immunostaining with KC57 and flow cytometry at 48 hours post-infection. The 'Colobus native' sequence represents the full IFITM3 sequence identified in the colobus monkey. (F) As in (E), except cells were challenged with Influenza A virus H1N1 PR/8/34, (Charles River Laboratories), at a dose that resulted in ˜50% infection of control (Empty) cells (equivalent to 103 TCID50 / 0.2 mL). Infection was scored by immunostaining with an anti-IAV NP antibody and flow cytometry at 18 hours post-infection. Results are presented in a logarithmic scale. Means + SD of 3-5 experiments are shown. Unpaired t-test, *p<0.05, **p<0.005.
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D, BiFC between cYFP-TSN2 and nYFP-TSN-interacting proteins in N. benthamiana leaf cells or protoplasts after HS (39ºC for 40 min). BiFC analysis of cYFP-TSN2 and nYFP-TSN-interacting proteins (TIPs) with empty vectors (EV) encoding nYFP and cYFP, respectively was used as a negative control. Only one representative example of BiFC between cYFP and nYFP-TIP is shown. Scale bars = 5 μm.
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Adipose tissue weight normalized to body weight in male mice following 9 weeks of HFD feeding (n=4 per group).
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(E) Model fit to the target concentration curve (mean interpolation of plasma L-carnitine over 24 hours) with standard deviation indicated in gray.
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C) WT VPS15 or VPS15 with 6SA, 2SA or individual phosphoacceptors mutated to alanine were incubated in vitro with Ulk1 1-427 and analysed as in B. The Coomassie-stained gel shows VPS15 levels in assay. Mean +/-SEM, n=3.
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I MOAP-1 deficiency does not alter total protein levels of p62 in the LO2 cells. Western blotting analysis of p62 protein levels in the WT, MOAP-1 KO and p62 KO LO2 cells lysed in 1x Laemmli sample buffer containing 2% SDS. Actin as loading control.
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D Treatment response by therapy tier. 14 of 29 patients with molecular-based therapeutic options received the treatment.
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(D) Bar plot showing the S/EGFR MFI ratio determined by flow cytometry analysis with Jurkat-S of serum samples from 30 sanitary personnel repeatedly tested as PCR negative for SARS-CoV-2 at the Ramón y Cajal Hospital of Madrid. A negative result is considered for a ratio lower than 0.5. Two clear cases of sera positive for anti-S IgG1 are indicated (RyC52 and RyC65). A borderline sample just above the threshold line (RyC58) is also indicated. Data show the mean±s.e.m. All datapoints are shown.
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] |
(F) Mitochondria were isolated from immortalized patient-derived skin fibroblasts treated with bortezomib (10 nM) for 12 h and recovered for another 6 h. Mitochondria were solubilized in digitonin buffer and analysed by 4-13 % gel BN-PAGE and Western blot. SC, supercomplexes.
|
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D Internalization of CXCR4 with 10 nM CXCL12 alone and in the presence of 10 nM Gal-3 CRD was assessed by incubation with an APC-conjugated anti-CXCR4 antibody (baseline signal = 100%, n = 8, two technical replicates).
|
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B, C Anti-IL-6 ELISA assay (mean ± SD) using CM or protein extracts from SOM230-treated or not CAF (from left to right: §§§P = 0.0002, §§§P = 0.0002) (B), or CM from siCTR- or si4E-BP1-transfected CAFs treated or not with SOM230 (from left to right: ***P = 0.0003, $$P = 0.004) (C) (n = 3).
|
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C. pbl34/35/36 triple mutant displayed decreased callose depositions in response to 3-OH-C10:0 treatment. Two-week-old seedlings were sprayed with 1 μM 3-OH-C10:0. The leaves were sampled for callose deposition assays 12 hrs later. The stained callose was counted for 1 mm2 of the leaves. Mock is solvent control. Scale bar=100μm.Values are means ± SD (n=6 leaves). (Student's t-test, **P< 0.01).
|
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] |
the mRNA expression of HSL and ATGL n the gWAT of male OXGR1KO mice after 14-day resistance exercise (n = 4 per group).
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G. Verification of the efficiency of knockout of exon 3 of NDIME in mESCs via qRT-PCR.
|
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C Representative live-cell STED super-resolution images (t=0 s), showing WT Hela cells expressing MIC13-SNAP, from a time-series of images acquired at a time interval of 1.3 s stained with silicone-rhodamine. Box marks selection shown as a zoom in panel (D). Scale bar 500 nm. D Time-lapse image series of a mitochondrion expressing MIC13-SNAP imaged at a time interval of 1.3 s/ frame. Green and magenta asterisks show cycles of cristae merging and splitting marked by MIC13-SNAP. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Scale bar 500 nm.
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The bar graphs show the relative percentages of neurons showing PS exposure only in the soma (left) and overall PS exposure (right) (n = 30-40 cells for each group) in control and Cdc50a cKO mice.
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H&amp;E staining of adult head sections of (A) repo>GFP.nls and (B) repo>Shits1 at 29°C for 3 days. Lamina degeneration was identified as vacuoles in (B). Cryosection of adult (C) repo>H2B-RFP exhibited the expression of the nuclear red fluorescent protein (RFP) in the glia (epithelialglialnucleus: arrowhead) and (D) repo>Shits1 at 28°C for 12 days. Vacuoles were identified in the lamina neuropile (D). Epithelial (arrowhead) (E), marginal (arrow) and distal satellite (arrowhead) glianuclei (F) are labeled by HisCl-GAL4 and NP2109-GAL4, respectively. Note that HisCl-GAL4 is not expressed in all epithelialglia. (G, H) Weak lamina vacuolization was identified in HisCl>Shits1 (G, 0.92%) and NP2109>Shits1 (H, 1.46%) at 29°C for 14 days. (I, J) A single MARCMglia clone (GFP, green) expressed Shits1 at 21°C (I) and 29°C (J). (J) A vacuole occurred within a glial clone. DAPI (white) stains the nuclei (C, D, and G-J). (K) The percentage of the vacuole area in the lamina of the repo>Shits1flies at 28°C progressively increased. n indicated in each column. P-values were calculated using one-way ANOVA with Bonferroni's post-test. (L) When repo>Shits1flies were shifted to 28°C for 12 days and then shifted to 17°C for 9 additional days, the vacuolization was not alleviated. P-values were calculated using one-way ANOVA with Tukey's post-test. n indicated in each column.
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(A-C) Numerical solution of the HPA model after a prolonged pulse of input (u=4) lasting 3 months, followed by recovery to baseline input u=1 (A). During the pulse, gland masses (B) increase over weeks, leading to exact adaptation of ACTH and CRH levels after a few weeks (C) despite the increased input level. After stress ends, gland masses adjust back over weeks. During this adjustment period, the HPA axis is dysregulated.
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(G) Electron microscopic quantification of autophagy vacuole in H1299 cells transfected with the indicated shRNA in normal medium or after 3 hr HBSS starvation or treated with H2O2 (500μM). Data are presented as mean±s.e.m. from 3 independent experiments; *P < 0.05; **P < 0.01 (Student's t-test).
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A Lysates of 293T cells transfected with plasmid for Flag-Beclin-1 and HA-K11-linked-Ub, together with empty vector or expression vector for Myc-USP5 or Myc-USP19 were immunoprecipitated with anti-Flag and immunoblotted with anti-HA.
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Evaluation of CD69 (G) and CD25 (H) proteins, expressed as Mean Fluorescence Intensity (MFI), was determined by flow cytometry on the surface of responders CD8+ T cells (n=4).
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F) Immunohistochemical stainings of cross-sectioned seminiferous tubules of SPPL2c+/+ and SPPL2c-/- mice using anti-Cab45 antibody. Stages VII/VIII are depicted. Higher magnifications are indicated with dashed lines and black arrows indicate pre-acrosomal structures. Scale bar 50 µm.
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MtDNA heteroplasmy analyzed by PCR/RFLP, 24 hours post mitoTev-TALE and mitoTALEN transfection. The RFLP analysis shows increased %WT mtDNA in sorted cells when compared to the the untransfected. mitoTALEN monomers positive for both eGFP and mCherry were isolated as "Yellow". The "Black" cells represent the mitoTALEN sorted population of cells negative for eGFP and mCherry and the "GFP-+" population represents mitoTev-TALE sorted cells with low levels of eGFP fluorescence. GFP++ were cells positive for GFP after mitoTev-TALEs transfections
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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.
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Tfh cell development in the spleen (SPL), mesenteric lymph nodes (MLN) and Peyer's patches (PP) of WT:Pou2af1-/- mixed bone marrow chimeras seven days after immunization with SRBC. (B) Frequencies of Pou2af1+/+ and Pou2af1-/- B220+FAS+GL7+ GC B cells.
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A. Yeast strains (200 cells) containing chromosomal Flag-tagged rbd2, rbd2-G244R, rbd2-G246R or rbd2-S130A in rbd2Δ background and sre1Δ yeast were grown on rich medium in the absence or presence of cobalt chloride (CoCl2).
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Phylogenetic analysis of nucleotide sequences of coronavirus. (A) Whole genome; (B) Spike gene; (C) RdRp gene.
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B Expression levels of RBM15 and AS-RBM15 in different blood lineages by meta-analysis of RNA-seq data [38]: HSC (hematopoietic stem cells), MPP (multipotent progenitor cells), CMP (common myeloid progenitor cells), CLP (common lymphoid progenitor cells), GMP (Granulocyte-macrophage progenitor cells), MEP (megakaryocyte- erythrocyte progenitor cells), MK (megakaryocyte), and EB (erythroid body).
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(E-F) Immunostaining of YFP and Tunel assay (E) and immunohistochemistry of YAP and TAZ (F) in Lgr5CREER/KRasG12D/P53 KO/Rosa-YFP mice and Lgr5CREER/KRasG12D/TP53/YAP TAZ triple KO/Rosa-YFP mice 3.5days after tamoxifen administration. Scale bars: 20 μm
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Representative super-resolution images of TOM-20 immune-labelled mitochondria in ESCs following 48h culture in 2i/Lif/KSR media, and EpiLC-inducing conditions in the presence of 4mM dm-αKG and DMSO, respectively, are displayed. Scale bar, 3um.
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C. Predicted concentrations of TET in the cytoplasm as a function of TET concentration in the media, κ, and γ. The κ and γ values held constant in the plots are those for wild-type TetB.
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Primary adult microglia were cultivated in the presence of TGF-β (50 μg/ml) and M-CSF (10 ng/ml), while neonatal cells were stimulated for 24 hours with TGF-β (50 μg/ml) followed by 6 hours stimulation with LPS (1 ng/mL) or left untreated. Expression levels of microglia homeostatic (Olfml3, Tmem119, Gpr34) and inflammatory (Il1β, Tnf, Ccl2) genes were analysed by qPCR. Bars represent mean of relative expression (Gapdh as housekeeping gene) ± SEM (**p < 0.01 by two-tailed Student's test; n=3).
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(I) Hematoxylin and eosin (HE)‐stained epididymal white adipose tissue (eWAT) from 6‐mo‐old RD‐fed Con and KO mice. Quantification for cell size is shown (n=3). Scale bar, 50 μm.
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C) Changes in AGR2 dimer and monomer ratio in TMED2 overexpressing cells. DSP-stabilized AGR2 was analyzed under non-reducing (top blot) or reducing conditions (bottom blot). D=dimeric, M=monomeric AGR2.
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(g) The disease-causing mutation T22M interferes with Fbxo7's interaction with Parkin. Coimmunoprecipitation was performed as in b using lysates from U2OS cells expressing Flag-Parkin and WT or T22M Fbxo7-HA.
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Western blot showing expression of TRF1-FOK1-mCherry fusion protein following doxycycline induction, both WT and D450A (nuclease dead). Cells were treated with 40 ng/mL doxycycline for 16 hrs and then 1µM Shield-1 and 1µM 4-OHT for 4 hrs
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B The estimated fractions of cells containing an insertion for all somatic TE insertions identified in neoplastic samples. The onset of neoplasia is represented with a green horizontal line. Three samples (P45B, P51 and P21), where timing of the neoplasia onset could not be unambiguously estimated, were excluded from this analysis.
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(D) Localization of netrin-1 protein in the adult rat striatum. (Scale bar, 600µm). Left panel, confocal image of the rat striatum. Netrin-1 (red), Dopamine transporter (DAT) (green). Right panels, higher magnification.
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C Western blot for representatives of all five respiratory complexes in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose. GAPDH is from the same blot as panel A. Quantification based on two biological replicates for RNASEH1 and ISCU mutant fibroblasts Significant changes in protein levels in RNASEH1 and ISCU mutant fibroblasts compared to controls have been boxed in red.
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(K) Representative coronal brain section (left panel) and example of c-Fos immunoreactivity in the MPOA (right panel) of Shank2+/+ and Shank2-/- pup exposed females (scale bar: 200 µm). The dotted line outlines the third ventricle (3V). Arrowheads indicate c-Fos positive cells.
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G, H (G) Change in fat mass (%) (P<0.0001) and (H) lean mass (%) (P<0.0001) from baseline to 3-weeks in CTRL and BAM15 treated animals (N=8 per group).
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B.-C. Cataracts with opacification of the lens (B., black arrow) and extruded nuclei (C., white arrow) are observed with progressive age in VEGF-Ahyper mice (here a representative 30-months old lens with a cataract is shown).
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A. pICLpt was replicated in egg extracts immunodepleted with indicated antibodies. Replication samples were amplified by PCR and analyzed by next-generation sequencing for deletions and insertions. Results from two independent experiments are shown.
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J: Flow cytometric measurement of specific TFAM level (total TFAM/TOMM20) in iPSC-derived DA neurons from Detroit 551 control, WS5A and CP2A POLG patients (n=8, technical replicates per clone for control; n=5, technical replicates per clone for WS5A; n=4, technical replicates per clone for CP2A).
|
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F Immunoblot analysis of lysates from HEK293 cells cotransfected with Flag- AIM2, -NLRP3, -ASC and HA-CCDC50 or empty control vector. The expression levels of Flag-tagged proteins and Actin were quantitated using ImageJ software. Data information: actin was used as a loading control. All the experiments were repeated at least once.
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Immunoblot (left) and ELISA (right, n = 3) analyses of CCL6 in IMC-CM from primary Npc2+/+ IMCs cultured with or without 50 μg/ml bNPC2 for 48 h (immunoblots) or 72 h (ELISA). The data in the bar graph (ELISA) represent mean + SD.
|
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Genes with major PAS located in 3' UTR have on average higher major PAS usage (B) and gene expression level (C) compared to genes with upstream major PASs (Mann-Whitney U test, ***P < 0.001).
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(n, o). Label-free relative quantitative mass spectrum analysis of Drp1S616 phosphorylation. Representative spectra for the Drp1 phospho-peptide (S616) SKPIPMPA(p) SPQK and non-phospho-peptide SKPIPMPA(p) SPQK. Spectra obtained for peptides from PINK1WT HEK293 cells are shown (n). The relative ratios of the phospho-peptides containing phosphor-Ser616 to peptides containing Ser616 from PINK1WT and PINK1KO HEK293 samples were calculated and plotted (o).
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] |
Samples were taken at the indicated times and used to isolate TAP-SLD5 by immunoprecipitation on IgG beads. The indicated proteins were monitored by immunoblotting. For reasons of space, samples A, S, M and 1-6 were resolved in a separate gel to samples 7-8.
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(d-f) Representative immuno-TEM images of Uba1IRclone cells at puparium formation. Uba1IR-expressing cells possess gold particles; control cells lack gold particles (d). Control cells possess numerous autolysosomes in the cytoplasm (arrowheads) and few mitochondria (asterisks); Uba1IR-expressing cells possess numerous mitochondria and few autophagic structures. Scale bars, 20 μm (a-c) and 1 μm (d-f).
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(D) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 (WT or K134R) and HA-K11-Ub, together with empty vector or Myc-USP8. Lower panel shows the intensity analysis of the bands from the three independent experiments.
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F) As above, in a proliferation assay, H3122 and CrizR1 cells were treated in parallel with DMSO or 1uM crizotinib as drug control. H3122 vs DMSO P <0.0001, CrizR1 vs DMSO P=0.1 (n=4).
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C t-SNE plot of the integrated scRNA-seq profiles of fibroblasts from pre-and post-menopausal tissue Cell colors correspond to tissue specimens. D Same t-SNE plot as in (C) showing 5 distinct clusters (clusters 3 and 5 were specific to one patient).
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A. The gene-specific RNA to protein correlation factors are shown for all the 55 genes with a box-plot showing the average correlation factor for each gene and the variation observed in the nine cell lines and eleven tissues. All the values for each of the cell lines and tissues are found in Table EV7.
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D) ERGIC53 (green) to mark ERGIC compartment. (n=3 independent experiments, n≥14 fields analysed). Analysis of ULK1 localization with each compartment is reported in each graph. Co-localization analyses were performed by ImageJ plugin Jacop. Values of Mander's coefficient for ULK1 are expressed in percentage as mean ± SEM. Statistical analyses were performed by two-way ANOVA followed by Tukey's multiple comparison test. **, P<0,01; ***, P<0,001; ****, P<0,0001; ns, not significant. Scale bars 5 μm. White arrowheads point at ULK1 puncta associated to the analysed markers; N, nucleus.
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A-C. Scanning electron microscopy (SEM) images of (A) nanowires (VNw; scale - 5 µm) and (B) crude nanosheets (VSh; scale - 200 nm), (C) ultrathin-nanosheets (Vs), scale - 20 µm.
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(C) Sashimi plot representing coverage on the Dppa4 gene of an RNA sequencing analysis of murine zygotes, early 2C, mid 2C and late 2C Arcs depict splicing events, and numbers their relative frequency (i.e. reads across junctions).
|
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a, Northern blot analysis of lamp-2 expression. Total RNA was hybridized using a lamp-2 cDNA2 probe and a murine glyceraldehyde-3-phosphate dehydrogenase probe.
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A Transfected GFP−Atg8 homologs were immunoprecipitated with μMACS™ from total U2OS cell extracts followed by immunoblot analysis with anti−GFP and anti−ALFY antibodies. Data are representative of two independent experiments.
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Relative amount of viral DNA at time of reactivation (8dpi) in SCG neurons infected with HSV-1 in the presence or absence of IFNα (600 IU/ml). n=9 biological replicates.
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B UCSC genome browser capture of wild-type (WT, N=3) and R6/1 (N=3) mice hippocampus ATAC-seq data, hippocampal H3K9ac and unchanged, closed in R6/1 and open in R6/1 accessible detected regions (left) in Tuba1α, Gabra2α and Dlx2 gene locus. Arrows in blue (closed in R6/1) and red (open in R6/1) indicate differential accessible regions. Bar graphs of significant (Benjamini's adjusted P-value < 0.05) Biological Processes terms from DAVID for genes associated with decreased (right top) or increased (right bottom) chromatin accessibility regions. Gene-term enrichment westimated by DAVID using a modified Fisher's exact test and Benjamini multiple correction test. Bars represents the -log10 (Benjamini's adjusted P-value). Exact P values are reported in Appendix Table S3.
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E Representative images of spheroid formation from sg-CTRL and sg-HMGCL (#2, #3 and #4 clones) PANC-1 cells after 4 or 15 days of culture. Photos are representative of n=3 independent experiments.
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IHC analysis of USP28 and ∆Np63 protein abundance in lung cancer and non-transformed human samples (n=300). The staining intensity was quantified in arbitrary units from 0 up to 3 by three independent pathologists. In box plots, the centre line reflects the median, the cross represents the mean and the upper and lower box limits indicates the first and third quartile. Whiskers extend 1.5x the IQR and outliers are marked as dots. p-values were calculated using two-tailed t-test.
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F The combination of net1-mAb and DAC reduces humanbreasttumor growth in immuno-compromised mice. After anesthesia, mice were engrafted in the interscapular area with a ≈ 60 mm3 patient-derived tumor. When the tumors reached 120-150 mm3, mice were injected subcutaneously with decitabine (0.4 mg/kg) or PBS and/or intraperitoneally with net1-mAb (10 mg/kg) or with a humanIgG1 control isotype antibody (Ctrl IgG1, 10 mg/kg) from day 1 to day 21. Tumor volumes were measured twice a week; n = 7 mice per group. The statistical significance of the differences obtained between the DAC + Ctrl IgG1-group and the DAC + net1-mAb-group was determined by two-way ANOVA2 and a post-hoc Tukey-test, **** P<0.0001. Error bars = s.e.m.
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Overviews of hNCAM+/TH+ cell grafts at 8 weeks post-transplantation. Insets, TH+ DA neuron morphology in grafts shown with high magnification.
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B. Characterization of the various B cell progenitor fractions as described by [38] in the bone marrow of 4 wildtype and 4 Shld2−/− littermate controls.
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c, d. Incidence of papilloma formation (ns: non-significant; Gehan-Breslow-Wilcoxon test) (c) and number of tumours per mouse (d) in wild-type (WT, n=17) and PRSS35-/- (n=22) mice treated with DMBA-TPA (*p=0.0171; Wilcoxon matched-paired rank test). Data represent means ± S.E.M.
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B: Cell lysates from control and ELDR depleted Cal27 or JHU029 cells using two different siRNAs to ELDR were subjected to Western blot analysis for the EGFR using specific antibodies. The membrane was reprobed with an antibody against Actin as an internal control. The same actin blot was used in Fig. 2D (for PCNA). The bottom panel shows a quantitative representation of Western blot band intensities. n = 3 biological replicates. Data are represented as the mean ± SD,
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VEGFR2 expression is regulated by TFEB through a miR-dependent mechanism. qPCR of VEGFR2 in human scr-shRNA and sh-TFEB ECs and in control treated with a specific miR-control, miR-15a-5p and miR-16-5p mimics Data are expressed as relative fold-change compared with the expression in control cells after normalization to the housekeeping gene TBP
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F CRISPR guides enriched (positive hits) in IMT1-treated RKO cells plotted as log2 fold change (LFC) versus log10 of adjusted p-value. mTORC1 and VHL pathway-related genes are reported in green and blue, respectively.
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MPSIIIA GAG primes a TLR4 mediated innate inflammatory response, resulting in production of intracellular pro-IL-1β (blue arrows). This step can potentially also be achieved by delivery of cholesterol crystals, amyloid beta deposition or high levels of cellular ATP. Cholesterol, amyloid beta and ATP are each able to activate the formation of the NLRP3 inflammasome, usually via lysosomal destabilisation, inducing cleavage of pro- I-1β by caspase 1 and secretion of IL-1β (red arrows), but only if cells are first primed. Extracellular IL-1β is able to bind IL-1R1 expressed on astrocytes and neurons, leading to astrocyte activation and changes in neuronal activity. Inhibition of IL-1 signalling reduces potentiation of microglial and astrocytic responses, presumably by reducing astrocyte activation, which leads to prevention of behavioural and cognitive changes in MPSIIIA
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LINC00665 ORF-His plasmid was transfected into MDA-MB-231 cells. Whole-cell lysates were subjected to co-immunoprecipitation using anti-His antibody and immunoblot analysis with CIP2A antibody to verify that CIP2A bound to CIP2A-BP.
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(C) Detection of endogenous TAB1 ubiquitination at 1-cell or 2-cell stages. Endogenous TAB1 proteins were immunoprecipated by anti-TAB1 antibody at either 1-cell or 2-cell stages, the immunoprecipates were detected by either anti-TAB1 antibody or anti-ubiquitin antibody. The results show that more Ub chains on TAB1 were detected in the two cell stage embryos than in the zygotes, when the TAB1 proteins were at comparable level.
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C-D Cleavage of VN in the RGD-motif is responsible for the negative feedback of plasminogen activation on cell adhesion to VN. 293/uPARR83/89A cells were seeded on different VN variants and treated with 10 nM sc-uPA, 10 nM tc-uPA (C) or with a combination of 10 nM sc-uPA, 30 nM Plg and 100 nM α2AP (D). To merge independent experiments, the cell index measured after 6 h of treatment is shown in % of the one measured for cells seeded on the same substrate, but treated with sc-uPA, as depicted in panel B. Dots are data from independent experiments and means ± SD are shown. The significance levels were evaluated using Student's t-test (two-tailed).
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H. Kaplan-Meier survival curves of the indicated groups of mice. Tie2-GFP (n=3); Tie2-IFNα (n=5); p=0.007 by log-rank/Mantel-Cox test.
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Graph showing body weight of WT, KO and KO+AAV-hWWOX at different days after birth. n=3 mice per genotype.
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In the presence of glucose or glucose 6-phosphate (left panel), the expression of RsaI is inhibited, in contrast to RsaG. In the absence of glucose or when glucose is metabolized (right panel), repression of RsaI is alleviated to regulate its RNA targets. In blue, are the transcriptional protein regulators, and in red the regulatory sRNAs. Grey arrows represent functional links, black arrows are for activation, bars for repression. Red lines corresponded to post-transcriptional regulation and black lines to transcriptional regulation. Dotted lines represented the regulatory events for which the regulation is not yet demonstrated. A schematic view of the post-transcriptional icaR mRNA regulation is represented in the insert. The 3'UTR of icaR contains an anti-Shine and Dalgarno (anti-SD) sequence able to bind the SD sequence in the 5'UTR The CU-rich unpaired sequence of RsaI (in red) binds to the 3'UTR of icaR mRNA downstream of the anti-SD, and represses the translation indirectly either by stabilizing the interaction between the two UTRs or by preventing the action of trans-acting activators (protein or RNA).
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(B) Correlation of rhythmic Srsf10 expression (orange, left y-axis) and exon 3 inclusion (black, right y-axis) in mouse liver samples from the indicated ZTs (n=4, mean +/- SEM). Quantifications were obtained by RNA-seq analysis of datasets from (Atger et al., 2015).
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B. Distribution of iMet positions giving rise to 323 N-terminal proteoforms with assigned turnover times (to avoid redundancy a single iMet processed proteoform was considered).
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C ChIP analysis of histone H3 levels at centromeric (dg) repeat sequences was determined by qPCR. Data are the mean of three independent ChIP experiments and error bars are ±SEM. P-values were calculated using a two-tailed unpaired t-test.
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(C) EdU labeling assays of multiple HKC strains plus/minus HSD17B7 silencing as in the previous panels. Cells were plated in triplicate dishes 5 days after lentiviral vector infection and selection and pulse labeled with EdU for 3 hrs. Shown are % of EdU positive cells +/- SD. n(dishes per condition)= 3. *p<0.05, **p<0.01; ***p<0.0005; p****<0.0001, 2-tailed unpaired t-test.
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Analysis of shScramble (gray, n = 2,400 cells)‐, shIP3R(1,3) A (blue, n = 1,611 cells)‐, and shIP3R(1,3) C (green, n = 1,454 cells)‐expressing DCs migrating in the device described in Supplementary Fig S3I, stained with Hoechst and imaged every 2 min during 24 h at 10× magnification. Values were obtained from three independent experiments. Cell trajectories were obtained by tracking their nucleus.C Representation of individual DC trajectories.
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Immunoblot detection of NCOA4 and ACTIN levels from normoxic lysates of shCntl and shBNIP3 cells expressing either Luc or HIF-1α-AA, collected and treated for 24h in the absence or presence of 5nM BafA (n=3). Densitometric quantifications relative to ACTIN levels are shown below each corresponding band and they were analyzed with a one-sample T-test against shCntl Luc.
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Distribution table of PSAT1 expression in the cohort of 41 human breast carcinomas according to the staining grade.
|
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E Normalized distribution of E-box motifs that occur within Myf6 peaks compared to their occurrences in the genome normalized per kilo base of DNA (two-tailed t-test).
|
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D Sequence logo for an experimentally-validated NF-κB binding motif (Wong et al, 2011). The genomic sequence around rs6927172 is aligned below.
|
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