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B. HEK293 cells were treated for 8 h with 100 or 200 μM H2O2. Nrf2 (left panel) and GSNOR (right panel) were evaluated by Western blot performed in nuclear and cytosol-enriched fractions. GAPDH and Lamin A/C were used as loading and purity controls of cytosol and nuclei, respectively. Densitometry of GSNOR immunoreactive bands is normalized to GADPH and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05.
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(E and F) W07G4.5::GFP is expressed in the intestine and a few aggregates are formed in a wild-type embryo at the fourfold stage. (E) DIC image of the embryo shown in F. (G and H) W07G4.5::GFP is expressed at dramatically elevated levels and accumulates into a large number of aggregates in atg-3 (G) and epg-7 (H) mutants.
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(A) HEK293A cells were transfected with either flag-tagged WT ATG16L1 or T300A ATG16L1. ULK1 was co-transfected in increasing amounts where indicated. Cleavage of ATG16L1 was analyzed by WB of whole cell lysates. Levels of ATG16L1 cleavage were quantified from 3 biological repeats (right panel). Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.
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Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green,1987) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. Identification of stem cells producing COLVII. Western blotting revealed that only clone 6 secreted COLVII in the culture supernatant, while clone 54 and surprisingly clone 22 did not (see A). RDEB cells were used as a negative control and healthy donor cells as a positive control. The secreted matrix metalloproteinase 2 (MMP2) was used as a loading control.
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0
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(C) SDS-PAGE and Western blot analysis with the indicated antibodies of input and elution (with FLAG peptide) from native anti-FLAG co-immunoprecipitations from cells expressing FLAG-Orm2-WT or FLAG-Orm2-K25,33R. Control cells expressed untagged Orm2 WT. The asterisks label unspecific cross-reactions of the respective antibodies.
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C. Cells were treated as in (A). Where indicated, lysosomal proteases inhibitors E64D and PepA were added. LC3-Parkin-mitochondria co-localization was calculated from 30 randomly selected cells per condition. Data represent mean ± SE of three independent experiments. Scale bar, 5μm
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Quantification of cell numbers 6.5 months after the induction of KO (n = 4 for Cont-8M, n = 3 for cKO-8M, n = 4 for Cont-15M,). (D) The density of RGL-ANSCs tends to be reduced in cKO. F (2, 8) = 3.98, P = 0.063, One-way ANOVA. (E) The density of ANPCs was reduced in cKO-8M and Cont-15M mice. F (2, 8) = 11.49, P = 0.0044, One-way ANOVA followed by Tukey's HSD test (** P < 0.01).
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(J) Time of anaphase I entry in oocytes that were treated with 15 µM IPZ after being cultured for 5h in control media. Oocyte numbers are indicated in brackets.
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(A) MEFs transiently co‐expressing Myc‐PAT4 and mStr‐Rab12 were immunostained with anti‐Myc antibody.
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Rab8a (T22N-3IS) mutant was not subject to polyubiquitin co-precipitation, while Rab8a (T22N) protein was.
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A. Schematic representation of the flow of the experiment. TCs were assembled on DNA-RNA scaffolds (DNA was biotinylated); RNA was labeled at 3' with radioactive UTPs (asterisks). The RNA length (including label) was 33 nt.
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(E) Downregulation of SLFN11 delays the decline of phosphorylated CHK1 and CHK2 in SF268 cells. SF268 cells were transfected twice with control siRNA or siRNAs specific for SLFN11. 48 hr after transfection, cells were treated with CPT (1 µM) for 1 hr. Cells were then washed, shifted to fresh medium (time 0), and harvested at the indicated time points for immunoblotting with the indicated antibodies.
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(D) Cytosolic aconitase activity in mouse quadriceps (n=4) measured following subcellular fractionation (n=4). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
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C) IRGB6 staining on PVM after infection of wild-type or Traf6 -/- MEFs for 3 h with Pru, PruΔgra15 and RH. On the right-hand side, a representative fluorescent image is shown of IRGB6 coating on the PVM of both wild-type and Traf6 -/- MEF. Scale bar is 10 µm. At least 100 different vacuoles were observed and analyzed for each experiment (n=3).
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(a) Top, FAK−/− cells were treated with 200 nM dasatinib for 24 h and then fixed and stained for anti-Src antibody (green). Solid arrows indicate Src at the cell periphery; dashed arrows indicate Src in autophagosomes. Scale bars, 20 μm. Bottom, quantification of dasatinib-treated cells with Src in puncta. Data are presented as mean±s.d. and significance is P0.001 (n=3). Middle, lysates from cells treated with dasatinib were immunoblotted with anti-Src and anti-pTyr-416-Src and lysates from cells treated with dasatinib and chloroquine were immunoblotted with anti-LC3B and anti-actin antibodies. Densitometry was carried out to calculate the increase in the amount of LC3B-II relative to actin for each condition after treatment with chloroquine and is presented as a percentage increase for the immunoblot shown.
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(E) The construct encoding Flag-Pdia4 or its mutants and that expressing Myc/Flag-tagged Ndufs3 (left) or p22phox (right) were co-transfected into 293T cells. Total lysates (TL) and anti-Myc immunoprecipitates (Myc IP) underwent immunoblotting analysis. N-terminal Flag tag, catalytic domains (a, a' and a''), non-catalytic domains (b and b'), and C-terminal KEEL are indicated. Pdia4* is a mutant of Pdia4 whose 3 CGHC motifs were changed into 3 SGHS motifs (a*, a'* and a''*).
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C. Histograms show the percentage of the different replication patterns observed in each experimental condition (average and SD of three assays). Circle dots in each column represent the values of individual replicates. Statistical analysis: two-way ANOVA followed by Bonferroni post-test comparing each condition to the WT. p-values of individual comparisons are indicated. Immunoblots showing the levels of V5-PrimPol proteins (WT and mutant derivatives). Ponceau-S staining is shown as loading control.
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A model for AR regulation of UPR in PCa cells: Liganded AR activates the IRE1α pathway and coordinately inhibits the PERK arm of the UPR. In addition, AR inhibits the proapoptotic JNK pathway that may be activated by IRE1α or other pathways. The end result of these AR effects is PCa cellproliferation and survival. An arrow with a solid line indicates direct promotion. An arrow with dashed line indicates indirect/unexplored interactions.
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A, B. Carfilzomib and proteasome inhibitor MG132 activate Il-1β luciferase in macrophages. (A) BMDMs were treated with IL-4 (20 ng/mL) for 24 hours and then stimulated by DMSO, Carfilzomib (1 μM), or MG132 (5 μM). (B) BMDMs were stimulated by DMSO, Carfilzomib (1 μM) or MG132 (5 μM)) for 1 hour, then induced with IL-4 (20 ng/mL). Luciferase assays were monitored 12 hours after stimulation. The data are means ± SD of three independent experiments. ***p < 0.001 (student's t-test).
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C,D Expression of pANJ::H2B-TdTomato in mature ovules. White dotted lines delineate the egg cell and synergid cells. Data information : Scale bars = 50 µm. M, micropyle. CC, central cell. EC, egg cell. SC, synergid cell.
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Mean corpuscular volume (MCV) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), *P = 0.0472, two-tailed unpaired Student's t-test) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times.
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C Steady state protein levels analyzed by SDS gel separation of indicated amounts of mitochondrial protein and subsequent Western- blotting with antibodies against SDHA and VDAC3 or visualizing of Flavin fluorescence after excitation with 488 nm. Lower panel: quantification of signal ratio SDHA/VDAC3 in shTAZ and control (set to 100%).
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(B) Quantification of engulfed vGluT2+ synapses in microglia. % Engulfment of vGluT2+ synapse = Volume of vGluT2+ synapse inside microglia / Volume of microglia. N = 5 for biological replicates.
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B Mice (n = 6-9 per group) were infected with each IAV as indicated. Body weight was recorded daily after infection for 2 weeks.
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HCT116 cells weretreated with AGK2 for 24 h (g), and the cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-Atg7
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WT B6 or B6 Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −3 and −1, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression.B, C Colitis scores based on histopathology and gross morphology of the colons were also used as measures of disease. Scale bar = 50 μm. n = 5 mice/group. Empty bars = WT; lined bars = KO; white bars = untreated; purple bars = DSS; red bars = DSS + NCK56; green bars = DSS + NCK2187; blue bars = DSS + SlpA.
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G Graph of the fold change in background-subtracted luminescence ± s.e.m. (relative to 0 hr measurement) of Cdc25A-CBRLuc spheroids (n = 3 independent experiments with 4 technical replicates). ****p<0.0001 for dmPGE2- and EP4i-treated spheroids compared to stem cells by repeated measures 2-way ANOVA (variable = treatment). p<0.001 at the 16 hr, 20 hr and 24 hr time points by Dunnett's post-test comparing dmPGE2-treated and EP4i-treated spheroids to the stem cell control.
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(A) Western Blot detection of SMCHD1, TRF2 and hnRNPA1 in SMCHD1 wild-type or SMCHD1 knockout HeLa inducible shTRF2 cells treated with or without doxycycline for the indicated number of days (d7, d8, d11).
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(G) Venn diagrams showing the numbers of differentially expressed Pol IV-dependent 24-nt siRNA clusters (P4-siRNAs) in arid2/3/4, epcr1/2, and hda6 relative to the wild type. All the overlaps are highly significant (P→0) as determined by hypergeometric test.
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(A) Principal component analysis of WT CD4+ naïve T cell (gray), WT Treg cells (blue), and Tet2/3 DKO Treg cells (red). The percent of variation explained by principle components 1 (PC1) and 2 (PC2) are shown in parenthesis.
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BT474 and BTRH cells were treated for the indicated days with EV20/MMAF (10 nM), lysed and the amount of different proteins analyzed by Western. Numbers below the blots show the quantitation of the signal of each band, referred to time 0.
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C Immunoprecipitation (IP) analysis of the interaction of NLRP3 with endogenous CCDC50 and ASC in THP-1 cells activated by LPS plus ATP. IgG was used as a negative control. Data information: actin was used as a loading control. All the experiments were repeated at least once.
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(D) Representative Western blotting bands and densitometric quantification of PYGL in livers of WT and AslNeo/Neo mice treated with either with TB-1 or vehicle (n=4 mice/group). *p<0.05 (One-way ANOVA). GAPDH was used as loading control. Data information: All values are shown as averages ± S.E.M.
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(D) CHD1-depleted cells show increased sensitivity to γ-radiation. For colony formation assay, both BHP1 and PC3 cells with shCont or shCHD1 were treated with the indicated doses of γ-radiation and surviving fractions were measured by counting colonies after 3 weeks, mean values are represented in the plot (n=3, ± SD). Data were normalized to the plating efficiency. p-values (0.0009 and 0.002, **p ≤ 0.01, ***p ≤ 0.001) were calculated using ANOVA. See also Figure EV2A-EV2B.
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Upper panel: expression of proteins associated with the acquisition of immortalisation process involved in senescence, in evasion of growth suppression and in apoptosis (Hanahan Weinberg, 2011). The level of P53, P21, RAS and P16 was similar in clone 6, untransduced cells and mass culture from four independent infections, significantly different from the squamous cell carcinoma cell line SCC-13. Lower panel: the phosphorylation state of the PRB restriction point was maintained in clone 6 and untransduced recessive dystrophic epidermolysis bullosa (RDEB) cells, whereas PRB was heavily phosphorylated in transformed cells (SCC-13). Appropriate loading controls were used for each cellular extract (GAPDH for cytoplasmic extracts, histone H3 for nuclear extracts and tubulin for whole-cell extracts. H3 for histone H3, ppRB for hyperphosphorylated pRB and pRB for hypophosphorylated PRB).
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(E) Human postmortem SN tissue of control donors exhibited PBX1 chromogenic staining (dark-blue/black, indicated by arrowheads) in the nuclei of neuromelanin+cells (NM+, cytosolic brown aggregates) of the SN. In contrast, PBX1 staining was severely reduced in PD patients. (F) Graphical representation of PBX1 intensity levels in the SN of NM+ neurons in all individual controls and PD patients analyzed. Each dot in the graph represents the level of PBX1 in the nuclei of one single NM+neuron.
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A Matrin3 crosslinking in Matrin3‐regulated pre‐mRNAs where position of crosslinked nucleotides was mapped onto the regulated exon and the 500 nucleotides upstream and downstream of its 3′ss and 5′ss, respectively, the upstream flanking exon and 500 nucleotides downstream of its 5′ss and the downstream flanking exon with 500 nucleotides upstream of its 5′ss. The iCLIP tags were mapped onto silenced ASE (blue, n = 421), enhanced ASE (red, n = 187) and to control ASE (grey, n = 16,242), and percentage of occupancy is plotted.
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Overall astrocytic and microglial coverage, as assessed by GFAP and Iba1 stainings, remained unchanged in APP/PS1-Stat3WT vs. APP/PS1-Stat3KO mice (n = 8 mice (4 females and 4 males) per group; age, 8 months; Mann-Whitney test for each comparison). Scale bars, 500 µm.
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The mRNA levels of EV71 virus and host factors in mock or EV71 infected mice were detected by RT-qPCR with GAPDH as a control. The expression of EV71 (D) were presented, and the levels of corresponding gene in mock infected mice were set as 100%
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(c) IL-8 enzyme-linked immunosorbent assay (ELISA) of conditioned media from the experiment described in b.
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C. GO was used to analyze the genes characterizing each cluster identified above. The average P-value was retrieved for each cluster taking the 3 best GO per cluster, then z-score ((P-value of each biological process-average of P-value of each biological process)/standard deviation) was calculated. Clusters 2 and 3 were undefined (un)
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A. T6SS secretion assay of A. tumefaciens wild type C58, ΔtssL, and various single, double, and triple toxin-immunity deletion strains.
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(B) In vitro, Bur1 directly phosphorylates the kinase domain of Sch9 as well as Bur1 itself. pRS425 BUR1-HA and pVT102-Ura BUR2, the essential cyclin for BUR1, were co-expressed in a bur1Δ mutant, immunoprecipitated using anti-HA antibody and protein A beads, and then incubated with [γ-32P]ATP and GST, GST-N-Sch9 (1-390) or GST-C-Sch9 (391-824). Proteins were eluted with SDS sample buffer, separated by SDS-PAGE, and transferred to nitrocellulose membranes. Membranes exposed to x-ray film (top) or stained with Ponceau S (bottom). Red arrowhead; phosphorylated GST-C-Sch9. Gray arrowhead; auto-phosphorylated Bur1.
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Two-photon representative images of wild-type and Nlrp3-/- mice (5 and 15 months old). Scale bar: 20µm. Quantification of morphological parameters for wild-type and Nlpr3-/- mice after LPS injection
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A MA-plot of RNA expression profiles from female Lin-Sca1+ progenitors 2 days after induction of differentiation with 100 nM Rosi, displaying the log2-ratio of Cited4-/- to Cited4+/+ intensities against the average log2-intensities for all microarray probe sets (n=3)
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A Nissl stained slice (left) and corresponding section from brain atlas (right). Left panel shows hippocampus and position of the stimulating (left) and recording (right) electrode. Scale bars: 500 µm (black bar).
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(A) Genome-wide screen and internalization assay workflow. Library-transduced Ramos Cas9 cells were incubated with fluorescent, biotinylated surrogate antigen, anti-IgM F(ab')2 (total antigen) and subsequently stained with fluorescent streptavidin (surface antigen). Flow cytometry plots show a control sample incubated on ice preventing anti-IgM F(ab')2 endocytosis and an experimental sample from which cells displaying high and low anti-IgM F(ab')2 internalization were flow sorted and analyzed for gRNA abundance.
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Model predictions for Aci2's monoculture growth for different resource supply ratios when started at a 104 dilution from carrying capacity. Each row of pixels is the calculation for a separate condition. The equivalent predictions for Pa's growth.
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D,E Images of HeLa cells transfected with CCDC50 (red) (D) or K63-Ub (red) (E) and Flag-NLRP3 (Purple) and GFP-LC3B for 24 h and then induced with LPS plus ATP; scale bars, 5 μM.
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(H) Flow-cytometric analysis of B220 and CD19 expression in B-ALL tumor cells from the lymph node of a Cd79a-Cre Ikzf1neo/+ Pax5LSL-Jak2/+ mouse (black; left). Pax5 expression in these B-ALL tumor cells (black line) and control Pax5+/+ lymph node B cells (grey filled) was determined by intracellular Pax5 staining (right).
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(B) SCOC is required for p62 degradation. p62 levels were determined by western blot (Supplementary Figure S5) after incubation in FM, ES for 2 or 4 h, or ES with BafilomycinA1 (ES+BAF) for 2 and 4 h. Quantification of averaged duplicates; error bars represent s.e.m. (n=3); RISCfree FM versus RISCfree ES 2 and 4 h, ***P>0.0001 and **0.0014, respectively.
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(G) ChIP assay using anti-Flag antibody following expression of Flag-HA tagged TSC22D1 in MDA-MB-231 cells. Quantitative PCRs were performed for indicated gene promoters and enrichments are plotted on the y-axis as ratio of precipitated DNA (bound) to total input DNA and then further divided by the same obtained in the empty vector transfected cells. SEM is derived from independent biological replicates.
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TNF-dependent induction of Caspase 3 activation in immortalized Hoip+/+ or HoipK778R/K778R MEFs measured by using DEVD-AFC. MEFs treated with hTNF (100ng/ml) and CHX (1µg/ml) for 4 hours subjected to the Caspase 3 activity assays.
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(D) qRT-PCR assay for Ezrin from RPE 2-month-old WT and miR-211-/- mice sacrificed 3h after light on at 10 AM. The graph shows the expression level of Ezrin normalized to Hprt. Bar graphs represent mean values ± s.e.m. Mann and Whitney test (miR-211-/- vs WT), ***p ≤ 0.005 (n=6 mice).
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(C) Examples of nearest genes that gained or lost SEs upon LPS treatment. Gene tracks for gained and lost loci (near Tnf and Csf1r) showing clusters of BRD4, Pol II and H3K27ac signals. Red bar represents SE length.
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Seeded HEK sensor cells stained with the amyloid dye (Amytracker-680). Scale bar=10 μm.
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(A, B) Knockdown of hnRNPA3 increases poly-GA expression while expressions of EGFP protein levels are not altered upon knockdown of hnRNPs. The control ("x0") vector lacks the G4C2 repeats but still contains the 5' flanking region and 3x TAG. ANOVA, Dunnett.
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C Top genes whose expressions are both up regulated or both down regulated in the NSG and uMT-/- co-transplantation groups. Shown are the genes whose expression passes the threshold p<0.05 and fold change <-2 or >2 Data information: Ingenuity Pathway Analysis (IPA) was used to perform gene ontology analysis on differentially expressed genes. Data were collected at month 7 post transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group. n = 3 mice for each group, except for the NSG control group where n=2 mice
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(A-E) Cbl expression in germaria. (E) Quantification of increased Cbl protein levels in cystoblasts using fluorescence intensity of immunostaining with 8C4 antibody. Fluorescence intensity was measured in arbitrary units using ImageJ, in germaria showing increased Cbl levels in cystoblasts. The number of scored cells (n) is indicated. Intensity in GSCs was set to 1. The error bar represents standard deviation. ***p-value <0.001 using the two-tailed Student's t test.
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Schematic illustration of mitochondrial dynamics and GAPDH-mediated micro-mitophagy in HD with expanded polyglutamine repeats.
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(H) Y245F STING is phosphorylated on S366 after stimulation of cells. Phosphorylation of S366 was detected in cells expressing the WT or the mutant STING after stimulation for the indicated time.
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B. Representative I-V curves (left) and a scatter plot and bar graph (right) showing that the PC2_AA is a GOF mutant and gave rise to a larger current than PC2_F604P. Scatter plot and bar graph shows the average current sizes at +60 mV. The cations included in the bath solution, 100 mM Na+ and 2 mM Ca2+ in this case, are indicated by the thick-lined boxes here and in all the following figures. Oocyte numbers for bar graph are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test).
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(A) Overview of VxrB-binding profiles across the V. cholerae genome after β-lactam exposure (PenG 100μg/ml [10x MIC] for 3 hours). Red peaks indicate VxrB binding sites above the significance cutoff (q-value<10-10; MACS).
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b, In-gel fluorescence assay showing myristoylated peptides of the indicated proteins in cells expressing IpaJ or catalytic mutant. The peptide-eGFP and IpaJ inputs are indicated. *Demyristoylation of GRASP65 and hVPS15 resulted in slower mobility of the resulting peptide, potentially owing to reduced mobility in SDS-polyacrylamide gel electrophoresis caused by proteolytic reaction.
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(B) Ethylene production was measured in the indicated genotypes 3.5 h after elicitation with mock or 500 nM flg22. Ethylene accumulation was normalized to the average of WT responses to flg22 set as 1. Shown is the mean of n=18 biological replicates from 3 pooled experiments -/+ SD (one-way ANOVA, Tukey post-hoc test, a-b p<0.001; a-c, p<0.001; b-c, p<0.001).
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(c) Yeast two-hybrid (Y2H) assay testing for the interaction of Atg11 with Atg19, the Atg19-Atg34 chimaera and Atg34, respectively.
|
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(F) Reactivity to HBHA was confirmed by Western Blot for a selected set of antibodies with HBHA ELISA reactivity.
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(A) HeLa cell lines with an intact (CTRL, carrying a non-coding gRNA) or inactive (Bax/Bak-double deficient) mitochondrial apoptosis apparatus were infected with Chlamydia trachomatis expressing GFP (MOI=0.2). Cells were analyzed by flow cytometry at the indicated time points. Cells were gated on the GFP-positive (infected) host cell population, and mean fluorescence intensity was recorded. Data are means/SEM of six experiments. (A) Growth of C. trachomatis was measured in HeLa cells as increase in GFP-fluorescence between 18 and 38 h. The data show means/SEM of six independent experiments (genotype effect (significance of the difference of GFP-expression between the cell lines overall, p = 0.08). Red line indicates the fold change calculation of the individual different time points and cell lines (significance tested (one sample t-test) for each time point for a difference between Bax/Bak vs CTRL).
|
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(C) Single-cell resolution trajectory of EGFR+ transitional glial cells (Cluster 14) was constructed from sample (GW11T-NC, GW14T-NC, GW18T-NC, GW20T-NC, GW21T-NC) during the transitional period using Monocle. Linear arrangement of the transition trajectory with developmental stages represents the transition process of a regulatory cell type. The arrow indicates the developmental trend.
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A. Immunoblotting analysis of Neuro2a (N2a) cells expressing Sig1R-FLAG variants.
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F LT-induced ubiquitination of NLRP1B. 293T cells expressing NLRP1B (1-983)-HA were treated with LT and/or MG132 for 6 h. Cells were then harvested for anti-HA immunoprecipitation and subjected to immunoblotting analyses using indicated antibodies.
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G Quantification of secreted soluble collagen proteins produced by TGFβ-treated HFL1 cells ± the IRE1α inhibitor 4μ8C. *P = 0,049.
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Dose response of A549 (black), NCI-H460 (yellow), NCI-H510A (blue), NCI-H82 (red) and DMS-53 (green) cell lines after treatment with (A) Carboplatin, (B) Etoposide, (C) Topotecan (D) Lurbinectedin Data representing the half maximal inhibitory concentration (IC50) expressed in molar concentrations. Cell survival is shown in percentage and 50% threshold is highlighted in dotted line. The data (presented as mean ± SEM) are the average of three independent experiments.
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Immunoblots show induction of HIF-1α and HIF-2α protein level, which is highest at 6 hours and falls by 48 hours
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(D) Quantification of Ki67 or pH3 positive cardiomyocytes in mice treated as described in (A); *p=0.0106 and **p=0.0052 between Con and CM-G4-KO after PBS treatment and *p=0.0439 or **p=0.0023 between CM-G4-KO mice with PBS or IL-13 treatment.
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Western blot analysis of collagen (Col) Ⅰ and Ⅲ expression in lung tissues from control and F-CRTH2 KO mice 14 days after bleomycin challenge.
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(E) Light microscopy pictures of MCF10A cells at passage 8 after infection with control or CIP2A-encoding lentivirus.
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ELISA of serum IL-1β from Abro1+/+ and Abro1−/− mice 3 h after intraperitoneal injection of LPS (15mg/kg) or vehicle (PBS). n = 6 for PBS groups and n = 12 for LPS groups.
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E: Immunofluorescence (IF) stainings of the 75 PDAC specimens were performed using α-p14ARF (red), α-CK8/18 (green, staining ductal cells in normal pancreas and neoplastic cells in PDAC) antibodies and DAPI (blue, nuclei/DNA). Representative IF images are shown for normal human pancreatic tissue, two PDAC specimens displaying no p14ARF expression and three PDAC specimens displaying strong p14ARF expression in tumor cells. Left and corresponding right images show the same tissue section. Asterisks indicate nucleolar p14ARF localization in neoplastic cells of PDAC. The last image pair shows a magnification of the image pair above (indicated by the rectangle). Scale bars: 35 μm.
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K. Quantifications of fluorescence intensity of internalized endogenous Syt (B, C) at single presynaptic boutons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector, SCRN1 shRNA alone or SCRN1 shRNA with GFP-SCRN1. N=2, n=201-300 boutons. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.
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Error bars are s.d.; n=3. (C-D) SLFN11 depletion in DU145 cells delays the decline of RPA foci. Wild-type or SLFN11-depleted DU145 cells were treated with CPT (1 µM) for 1 hr. Cells were then washed, shifted to fresh medium (time 0), and processed at the indicated time points for immunofluorescence by using anti-RPA2 antibody. Representative RPA2 foci were shown (C). Scale bar, 10 µm. Quantification of RPA2 foci formation using NIH Image J software (D).
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Computer prediction of the binding of miR-27a and miR-24 on the 3′ UTR or CDS of these pluripotency-associated genes.
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(c) Quantitative RT-PCR analysis of transcripts encoding spliced XBP-1 (sXBP-1), total XBP-1 (tXBP-1) and BiP in differentiating PCs at 0 d and 3 d after stimulation with LPS, presented relative to that of unstimulated Atg5f/f B cells at day 3.
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3D reconstruction showing the association of Prom1 (green) with AcTub-labeled (red) primary cilia in stem cell and transit amplifying cell regions. Note that the expression of Prom1 is not limited primary cilium but also to microvilli. A representative example of Prom1 association with one primary cilium at the stem cell to transit amplifying cell transition region. Green channel transparency was set up to 70%.
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(C) Bar plots illustrating the phospho-threonine enrichment of stress- and Igo1/Igo2 dependent S/T-P motifs. Phosphorylation sites quantified in both setup SR and setup SR igo1∆igo2∆ were analyzed. Grey bars show the percentage of phospho-threonine, white bars the percentage of phospho-serine. The enrichment was assessed using Fisher's Exact test.
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B Western blot analysis of kidney tissues from non-diabetic controls and diabetic nephropathy subjects. Expression levels of KDM6A, KLF10, nephrin and WT-1 in kidney tissues were determined by immunoblotting using the indicated antibodies. Relative protein levels in kidney tissues were normalized to actin. *P < 0.05 by Wilcoxon two-sample test (n = 6 for each group).
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I) Quantification graph of NSC diameter for genotypes in (G). n= n=127 NSCs for control; n=230 NSCs for msps RNAi; n=210 NSCs for UAS-E-cad7 + msps RNAi. ****p<0.0001.
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(a) A53T α-synuclein clearance in stable PC12 cells as in Figure 1a, treated with or without 100 nM PACAP, with or without 10 μM calpastatin or 500 μM 2′5′ddA for 24 h.
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a) Left, Experimental setup of surgically connected parabiotic mice. Acta1GFP/+ and Acta1+/+ mice underwent parabiosis for 2, 12 and 20 weeks before analysis. Right, quantification of GFP+Iba1+ microglia in the retina (rMG, squares, n.d. = not detectable), ciliary body (cbMΦ, triangles, Mann-Whitney ns p > 0.05) and cornea (cMΦ, circles, Kruskal-Wallis **p = 0.0024) of parabiotic mice. Blood chimerism of CD45+CD11b+Ly6ChiGFP+ cells in the analyzed wildtype mice was 37.7 ± 3.2 % (2 weeks), 27.5 ± 2.7 % (12 weeks) and 34.7 ± 3.7 % (20 weeks). Symbols represent mean ± s.e.m of three (2 weeks), four (12 weeks) or five (20 weeks) individual mice. Scale bars represents 50 µm.
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Cell death analysis by Celigo of PI-positive A375 treated with the indicated agents for 12 hrs. DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml). SM represents SM-164. Error bars represent SD. Displayed are representative results from n=3, and statistical analysis was performed with an unpaired t-test, **** P ≤ 0.0001.
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A) Representative confocal images of non-invasive or invasive human colorectal cancer explants collected from 8 patients with NOS adenocarcinoma The explants were fixed 4 days after recovery and stained for the lumen (Ezrin), F-actin (Phalloidin) and nuclei (DAPI). Boxed regions i, ii, iii and iv are displayed at high magnification. Arrowheads point to non-protruding cells, arrows point to protruding cells and white stars show off-centered nuclei. Scale bars: 20 μm.
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A AGO6 enrichment and depletion of 21-22nt siRNAs for the entire genome in 100‐bp tiles for both wt Col and ddm1. Tiles were categorized by annotation feature.
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D In the presence of PAI-1, plasminogen activation stimulates cell adhesion on VN. 293/uPARWT cells were seeded on VN-coated plates. Cells were treated with 10 nM sc-uPA and 10 nM PAI-1 either alone or in combination followed by the addition of 30 nM Plg and 100 nM a2AP or vehicle as control. A representative RTCA experiment is shown.
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(D) Representative FACS plots of digested skin from bone marrow (BM) transplanted αSMA-RFP mice 17 days post-wounding. (E) FACS quantification of αSMA-RFP+ cells in wounds of BM transplanted mice. αSMA-RFP with αSMA-RFP BM, N=2; WT with αSMA -RFP BM, N=3; αSMA-RFP with WT BM, N=4 biological replicates. One-way ANOVA with Tukey's multiple comparison test performed comparing WT with αSMA-RFP BM and αSMA-RFP with WT BM to αSMA-RFP with αSMA-RFP BM. (
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C) Explanation of the 2x2 linear model, where those cells labeled with 1 are compared to the cells labeled with 0. In the age comparison, mRNA expression in all 10-month-old (10M) mice is compared to all 4-month-old (4M) mice. In the genotype comparison, mRNA expression in all transgenic (TG) mice is compared to all wild-type (WT) mice. In the age*genotype comparison, we assess which transcripts are differentially expressed in the 10M TG mice compared to all other groups.
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(b) Autophagy detected by formation of mCherry-Atg8a punctate spots in midgut cells from wild-type animals at indicated stages. Representative images are shown.
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(A) Control or NRF2 siRNA transfected HeLa cells lysates were subjected to western blotting with indicated antibodies.
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Pooled clones (A-D) and single cell clones (E-H) of HEK293/sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ-secretase expression and APP processing.(A) PS1, PS2 and NCT were analyzed in cell lysates by immunoblotting using antibodies PS1N (PS1), BI-HF5C (PS2) and N1660 (NCT), respectively.(B) Full length APP and APP CTFs were analyzed by immunoblotting using antibody 6687. (C) Conditioned media were analyzed for secreted APPs by immunoblotting using antibody 22C11 and for total Aβ by combined immunoprecipitation/immunoblotting using antibodies 3552/2D8.
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Immunofluorescence was performed to study the expression and localization of E-cadherin and SMAD4 after TGFβ treatment (30 ng/ml for 24 hours) in control cell line and two Tgfbr2-mutant cell lines. Scale bars = 50 μm (fluorescent), scale bars = 100 μm (brightfield).
|
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Time lapses showing a TNT connecting two transfected cells. Top: GFP-vector transfected cells. Middle: GFP-βCaMKII WT transfected cells. Bottom: GFP-βCaMKII T287D transfected cells. Cells were stained with WGA (white). Yellow arrowheads point out a TNT in each condition. Scale bars represent 10 μm.
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