Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

Article Title: The Pro-Tumoral Activity of Heparan Sulfate 3- O -Sulfotransferase 3B (HS3ST3B) in Breast Cancer MDA-MB-231 Cells Is Dependent on the Expression of Neuropilin-1

doi: 10.3390/molecules23102718

Figure Lengend Snippet: Stable expression of HS3ST3B in MDA-MB-231 cells. Cells were transfected with the expression vector encoding HS3ST3B and then cultured in complete DMEM medium in the presence of 400 µg/mL G418. After 14 days of culture, individual colonies were isolated by limit dilution and amplified in medium supplemented with G418. In parallel, cells were transfected with the empty vector to obtain the control parental cells (pEmpty). ( A ) Following RNA extraction, the mRNA level of HS3ST3B was quantified by real-time RT-PCR in each clone. Relative abundance of the transcripts was normalized to endogenous HPRT mRNA. Data are means ± SD of triplicates. ( B ) HS3ST3B expression in the clones C and D was analyzed by confocal microscopy. To this end, cells were seeded on glass coverslips, permeabilized and then incubated in the presence of anti-HS3ST3B antibodies. After wash, they were immunostained with secondary antibodies conjugated to Alexa-568, in order to highlight the enzyme in red fluorescence. For the detection of 3- O -sulfated motifs, recombinant HSV-1 gD (10 μg/mL) was incubated with primary anti-gD antibody for 30 min at 4 °C, and the immune complex was added to cells for an additional 30 min-incubation. After washing, cells were fixed and incubated for 1 h with Alexa 488-conjugated secondary antibody (green fluorescence). In all the microscopy experiments, nuclei were stained in blue with DAPI, in order to visualize cell nuclei ( N = 3 separate experiments; n = 30 cells). Scale bar = 10 µm.

Article Snippet: Transfection was performed with Lipofectamine ® 2000, according to the manufacturer’s instructions (Thermo Fisher), after which cells were cultured in culture medium in the presence of 400 μg/mL G418 (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing, Transfection, Plasmid Preparation, Cell Culture, Isolation, Amplification, Control, RNA Extraction, Quantitative RT-PCR, Clone Assay, Confocal Microscopy, Incubation, Fluorescence, Recombinant, Microscopy, Staining

Journal: Gut Microbes

Article Title: ADP-heptose attenuates Helicobacter pylori -induced dendritic cell activation

doi: 10.1080/19490976.2024.2402543

Figure Lengend Snippet: TLR2 mediated Helicobacter pylori uptake is essential for potent dendritic cell activation. (a) TLR2 mRNA expression upon stimulation with H. pylori wt or mutant (Δ) (MOI 5) at indicated time points. Mean±SD of three individual donors is shown. (b) TLR2 surface expression was monitored by flow cytometry 16 h post-infection (MOI 5) ( n = 4). (c,d) H. pylori strains were stained with eFluor670 proliferation dye prior to infection. One hour post infection with H. pylori wt or the ADP-heptose-deficient mutant (Δ) (MOI 5), DCs were subjected to immunofluorescence and stained for CD45, DAPI and TLR2. Internalization of bacteria as well as TLR2 localization was analyzed by confocal fluorescence microscopy. Orthogonal views of confocal z-stacks of one out of three representative donors are shown. Scale bar: 5 µm. (e) DCs were treated with a TLR2 neutralizing antibody 20 min prior to infection. After 1 h of infection with eFluor670 stained H. pylori (MOI 5), DCs were subjected to immunofluorescence and stained for CD45 and DAPI. Maximum intensity projections of confocal z-stacks of one representative out of three donors are shown. Scale bar: 5 µm. (f,g) DCs were treated with a TLR2 neutralizing antibody 20 min prior to infection with H. pylori (wt) or the ADP-heptose deficient mutant (Δ) at an MOI of 5. After 16 h CD40 expression (f) and IL-12p70 secretion (g) was monitored by flow cytometry and multiplex assay ( n = 6). For statistical analysis one-way ANOVA with a šídák’s post-hoc test was performed.

Article Snippet: Following primary antibodies were used: mouse anti-human CD45 (ab30470 Abcam, 1:200), rabbit anti-human TLR2 (NB100–56720 Novus Biologicals, 1:400).

Techniques: Activation Assay, Expressing, Mutagenesis, Flow Cytometry, Infection, Staining, Immunofluorescence, Bacteria, Fluorescence, Microscopy, Multiplex Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: The natural product vioprolide A exerts anti-inflammatory actions through inhibition of its cellular target NOP14 and downregulation of importin-dependent NF-ĸB p65 nuclear translocation.

doi: 10.1016/j.biopha.2021.112255

Figure Lengend Snippet: Fig. 7. Vioprolide A reduces importin subunit alpha-1 total protein level and influences importin subunit alpha-1/beta-1 nuclear localization. a-c Confluent HUVECs were treated with vioprolide A (10 nM) as indicated. Total protein levels of importin subunit alpha-1 (a), importin subunit alpha-3 (b) and importin subunit beta-1 (c) were determined by western blot analysis. One representative blot out of 3 is shown. d-f Confluent HUVECs were treated with vioprolide A (10 nM) for 16 h followed by activation with TNF (10 ng/ml) as indicated. The nuclear fractions were separated and total nuclear protein expression of importin subunit alpha-1 (d), importin subunit beta-1 (e) and importin subunit alpha-3 (f) was determined by western blot analysis. One representative blot out of 3 is shown. VioA, vioprolide A; Imp, importin; Topo1, topoisomerase 1. Data are expressed as mean ± SEM. n = 3. *P ≤0.05 vs. negative control (a-c) or TNF control (d-f). #P ≤0.05 vs. negative control (d-f).

Article Snippet: Unspecific binding sites were blocked with 0.2% BSA (MilliporeSigma) for 30 min and cells were treated with primary antibody for 2 h. The following primary antibodies were used: rabbit anti-human NF-ĸB p65 (1:400, sc8008; Santa Cruz Biotechnology) and rabbit anti-human importin subunit alpha-1 (1:400, #14372; Cell Signaling/New England Biolabs).

Techniques: Western Blot, Activation Assay, Expressing, Negative Control, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: The natural product vioprolide A exerts anti-inflammatory actions through inhibition of its cellular target NOP14 and downregulation of importin-dependent NF-ĸB p65 nuclear translocation.

doi: 10.1016/j.biopha.2021.112255

Figure Lengend Snippet: Fig. 8. NOP14 knockdown influences TNF- activated inflammatory processes in HUVECs. a Confluent HUVECs were treated with 300 nM vioprolide A for 1 h or left untreated. Cells were exposed to increasing temperatures between 42.3 and 57.8 ◦C and lysed by repeated freeze- thaw cycles in liquid nitrogen. Thermal aggre gation curves of NOP14 were analyzed by western blot. One representative blot out of 4 is shown. b-e Subconfluent cells were transfected with siRNA against NOP14 (siNOP14, 60 nM) or non-targeting siRNA (nt siRNA, 60 nM) as control using GeneTrans II Transfection Re agent. 30 h after transfection, HUVECs were treated with 10 nM vioprolide A for 16 h. b HUVECs were activated with TNF (10 ng/ml) for 6 h and fluorescence-labeled THP-1 cells were allowed to adhere to the HUVEC mono layer for 5 min. The amount of adhered THP-1 was detected by fluorescence measurement. c HUVECs were activated with TNF (10 ng/ml) for 4 h. The mRNA expression of ICAM1 was analyzed by quantitative PCR. d HUVECs were activated with TNF (10 ng/ml) for 30 min p65 was visualized by immunocytochemistry and fluorescence microscopy. One representative experiment out of 3 is shown. Scale bar, 200 µm. e Importin subunit alpha-1 was visu alized by immunocytochemistry and fluores cence microscopy. One representative experiment out of 3 is shown. Scale bar, 50 µm. Tagg, aggregation temperature; VioA, vioprolide A. Data are expressed as mean ± SEM. n = 4 (a), n = 5 (b), n = 3 (c-e). *P ≤0.05 vs. TNF control.

Article Snippet: Unspecific binding sites were blocked with 0.2% BSA (MilliporeSigma) for 30 min and cells were treated with primary antibody for 2 h. The following primary antibodies were used: rabbit anti-human NF-ĸB p65 (1:400, sc8008; Santa Cruz Biotechnology) and rabbit anti-human importin subunit alpha-1 (1:400, #14372; Cell Signaling/New England Biolabs).

Techniques: Knockdown, Western Blot, Transfection, Control, Fluorescence, Labeling, Expressing, Real-time Polymerase Chain Reaction, Immunocytochemistry, Microscopy

Journal: Scientific Reports

Article Title: Nexilin/ NEXN controls actin polymerization in smooth muscle and is regulated by myocardin family coactivators and YAP

doi: 10.1038/s41598-018-31328-2

Figure Lengend Snippet: Nexilin/ NEXN is expressed in striated and smooth muscle cells (SMCs) and correlates at the mRNA level with the smooth muscle myosin. Panel A shows RNA-Seq data (GTExPortal.org) for the top-twelve NEXN -expressing human tissues (N = 6–430, Esophagus-M: muscular layer, -GEJ: Gastroesophageal Junction). Panel B shows immunohistochemical staining for NEXN in human heart (top left), skeletal muscle (top right), esophagus (bottom left) and gall bladder (bottom right), all from the Human Protein Atlas (proteinatlas.org). Panel C shows correlation between smooth muscle myosin ( MYH11 ) and NEXN in human coronary artery (N = 133, data from the GTExPortal). Panel D shows our own double staining for Nexilin (green, ab213628) and CAV1 (red) in cross-sectioned human urinary bladder SMCs (white scale bar represents 10 µm, N = 2) imaged in a conventional fluorescence microscope.

Article Snippet: Sections were blocked with 3% BSA for 60 min at room temperature before incubation with the following primary antibodies overnight: Nexilin (1:200 in 3% BSA, Abcam, ab213628 or HPA011185, Sigma), CAV1 (1:400 in 3% BSA, Cell Signaling Technology, #3267) and YAP1 (1:200 in 3% BSA, Cell Signaling Technology, #4912).

Techniques: RNA Sequencing, Expressing, Immunohistochemical staining, Staining, Double Staining, Fluorescence, Microscopy

Journal: Scientific Reports

Article Title: Nexilin/ NEXN controls actin polymerization in smooth muscle and is regulated by myocardin family coactivators and YAP

doi: 10.1038/s41598-018-31328-2

Figure Lengend Snippet: Nexilin localizes to puncta in the cytosol and at the membrane and is excluded from caveolae domains at the membrane. Nexilin ( NEXN ) and CAV1 immunofluorescence was imaged by confocal microscopy and using two different Nexilin antibodies (ab213628 in ( A) and HPA011185 in ( B ). Both show punctate/granular distribution of Nexilin (green) in the cytoplasm and at the membrane in human bladder SMCs that overlaps poorly with CAV1 (red) staining. Correlation of the two labels (CAV1 and NEXN ) indicated poor co-localization (panel C, R = 0.073 and 0.18, for the respective NEXN antibodies). The fluorescence intensity ratio for the two labels was also plotted for three membrane profiles ( D ). Panel E shows immuno-electron microscopy for Nexilin. The gold particle on the secondary antibody appears as a black sphere with a diameter of 10 nm, and examples of dense bodies (dbo) and dense bands (dba) are shown. White arrowheads highlight gold particles.

Article Snippet: Sections were blocked with 3% BSA for 60 min at room temperature before incubation with the following primary antibodies overnight: Nexilin (1:200 in 3% BSA, Abcam, ab213628 or HPA011185, Sigma), CAV1 (1:400 in 3% BSA, Cell Signaling Technology, #3267) and YAP1 (1:200 in 3% BSA, Cell Signaling Technology, #4912).

Techniques: Membrane, Immunofluorescence, Confocal Microscopy, Staining, Fluorescence, Immuno-Electron Microscopy

Journal: Scientific Reports

Article Title: Nexilin/ NEXN controls actin polymerization in smooth muscle and is regulated by myocardin family coactivators and YAP

doi: 10.1038/s41598-018-31328-2

Figure Lengend Snippet: NEXN correlates with gene products that control and respond to changes in actin polymerization and Nexilin is reduced by depolymerization of actin. Correlations of NEXN versus all other RNAs in the top-ten NEXN expressing tissues were examined (data from GTExPortal). The sum of correlation coefficients for individual RNAs across tissues was calculated (R sum ) and the positive extreme of this distribution was plotted ( A ). Actin controlling and responding gene products represented in the extreme are highlighted in blue colors. Examples of NEXN correlations in the human coronary artery (N = 133) are shown in panels B through ( D ). P-values and Spearman Rho values are given in the respective panels. Panels E and F show mRNA data for NEXN in cultured human bladder (HBSMCs, N = 8) and coronary artery (HCASMCs, N = 9) SMCs after treatment with Latrunculin B (LatB). Panels H and I show protein data for Nexilin/ NEXN in the presence and absence of LatB (HBSMCs, 300 nM, N = 12; HCASMCs, 100 nM, N = 10). The top micrographs in panel J shows confocal imaging of YAP (red) on the left, and YAP (green) and CAV1 (red) on the right. The bottom row shows YAP (red) and Nexilin (green). The high magnification overlay at the bottom right shows partial colocalization of YAP and Nexilin at the cell membrane in yellow. All micrographs are from cross-sectioned HBSMCs and white scale bars represent 5 μm throughout.

Article Snippet: Sections were blocked with 3% BSA for 60 min at room temperature before incubation with the following primary antibodies overnight: Nexilin (1:200 in 3% BSA, Abcam, ab213628 or HPA011185, Sigma), CAV1 (1:400 in 3% BSA, Cell Signaling Technology, #3267) and YAP1 (1:200 in 3% BSA, Cell Signaling Technology, #4912).

Techniques: Control, Expressing, Cell Culture, Imaging, Membrane

Journal: Archives of Toxicology

Article Title: Building predictive in vitro pulmonary toxicity assays using high-throughput imaging and artificial intelligence

doi: 10.1007/s00204-018-2213-0

Figure Lengend Snippet: Pulmonotoxic chemicals may induce DDR pathway activations dependent or independent of DNA strand breaks. a Fluorescence microscopy images showing the staining patterns of Hoechst and anti-γH2AX in single BEAS-2B cells treated with the indicated chemicals for 16 h. Cells with raw F 2 values (indicated below the cells) close to the average raw F 2 values across all the cells under the same treatment conditions are shown (scale bar = 30 µm). To allow visual comparisons, all the shown images have been scaled to the same intensity ranges with respect to the solvent controls. b Fluorescence microscopy images showing the DNA spots obtained from the Comet assays of BEAS-2B cells treated with the indicated chemicals for 4 h. DNA spots with % tail DNA values (indicated below the patterns) close to the average % tail DNA values across all the spots obtained under the same treatment conditions are shown (scale bar = 20 µm). To allow visual comparisons, all the shown images have been scaled to the same intensity ranges with respect to the solvent controls. c Changes in the median % tail DNA values obtained from the Comet assays of BEAS-2B cells treated with the indicated chemicals or solvent controls for 4 or 16 h (bars = mean of the log 2 -ratio values obtained from at least three independent biological replicates, error bars = 95%-tile confidence intervals; X marks = ≤ 5% cells left). d Fluorescence microscopy images showing the staining patterns of Hoechst and anti-phospho-p53 (Ser15) in BEAS-2B cells treated with the indicated chemicals for 16 h. e Changes in the median nuclear phospho-p53 intensity levels of these cells quantified from the images with respect to the solvent controls (dots = mean of the median values, error bars = 95%-tile confidence intervals.) All P values shown in this figure are obtained from two-sided t tests and adjusted for false discovery rates (*** P -adjusted ≤ 0.01, ** P -adjusted ≤ 0.05, * P -adjusted ≤ 0.10)

Article Snippet: For the p53 imaging assay, similar cell staining procedures were performed, except that we stained the cells with 1:400 mouse anti-phospho-p53 (Ser15) primary antibody (#9286, Cell Signaling Technology, Danvers, USA) and 1:500 goat anti-mouse secondary antibody conjugated to Alexa 488 (#A21121; Invitrogen, Waltham, USA).

Techniques: Fluorescence, Microscopy, Staining, Solvent

Journal: The Journal of Experimental Medicine

Article Title: Promyelocytic Leukemia Protein (Pml) and Daxx Participate in a Novel Nuclear Pathway for Apoptosis

doi:

Figure Lengend Snippet: Con A activation of splenocytes induces Daxx expression. (A) Daxx localization in NBs is induced upon Con A activation. PML +/+ splenocytes, before and after Con A activation, were immunostained with anti-PML and anti-Daxx antibodies. Both Daxx and PML display a strong staining upon Con A activation. Single staining ( a , b , a ′, and b ′) and superimposed images ( c and c ′) are shown. The nuclei were visualized by DAPI staining ( d and d ′). Representative confocal pictures are shown. Bar, 10 μM. (B) Upregulation of Daxx mRNA expression upon Con A treatment. Northern blot analysis was performed on total RNA from PML +/+ primary splenocytes, before and after 72 h of Con A activation using a Daxx cDNA probe, which detected a single 2.6-kb band. Normalization with 28S RNA is also shown. (C) Analysis of Daxx protein levels in cytosolic (C) and nuclear NP-40 (N)–soluble (SOL) and insoluble fractions (INSOL). Levels of heat shock protein 90 (Hsp90) were measured as a control of equal loading.

Article Snippet: Either a mouse anti-PML mAb (1:50–1:100 dilution, sc-966; Santa Cruz Biotechnology), a mouse anti-Daxx mAb (1:400 dilution, sc-8043; Santa Cruz Biotechnology), or a rabbit polyclonal anti-Daxx antibody (1:400 dilution, sc-7152; Santa Cruz Biotechnology) was added to 50 μl of lysate, and the solution was incubated at 4°C overnight.

Techniques: Activation Assay, Expressing, Staining, Northern Blot, Control

Journal: The Journal of Experimental Medicine

Article Title: Promyelocytic Leukemia Protein (Pml) and Daxx Participate in a Novel Nuclear Pathway for Apoptosis

doi:

Figure Lengend Snippet: Daxx and PML colocalize in NBs and physically interact. (A) Colocalization of transfected Daxx and PML. Cos-1 cells cotransfected with GFP-Daxx and BFP-PML were analyzed by confocal microscopy. Green dots (GFP) are Daxx specific, blue dots (BFP) are PML specific, and turquoise dots (GFP plus BFP) represent colocalizing proteins. Orange and white arrows indicate cells transfected with Daxx only and with PML only, respectively. Representative pictures are shown. Bar, 10 μM. (B) Coimmunoprecipitation of transfected Daxx and PML in Cos-1 cells. The left panel shows that PML is specifically detected in the Daxx immunoprecipitant, and the right panel shows that Daxx is coimmunoprecipitated with PML. The whole cell lysates (inputs) used for the immunoprecipitation are also included to display the migration of specific immunoreactive species. The anti–human PML antibody used for the coimmunoprecipitation analysis does not cross-react with the endogenous monkey PML. However, Cos-1 cells do express PML as revealed by Northern blot analysis. (C) Coimmunoprecipitation of endogenous Daxx and PML in Con A–activated PML +/+ and PML −/ − splenocytes. The left panel shows that Daxx is specifically detected in the PML immunoprecipitant, and the right panel shows that PML is coimmunoprecipitated with Daxx. The migration of specific immunoreactive species detected in the whole cell lysates is also shown. As expected, the endogenous Daxx was coimmunoprecipitated along with PML in PML +/+ but not in PML −/ − cells. (D) Yeast two-hybrid assay between the full-length Daxx and PML. Yeast strain Y187 was transformed with pAS2-1-Daxx and/or pACT-2-PML. Three to four colonies per transformation were grown in liquid culture, and the β-galactosidase expression (β-Gal) was determined using ONPG as substrate. The expression level of β-galactosidase measured by quantitative liquid assays in the cotransformed colonies was ∼50-fold higher than that in the single-transformed colonies.

Article Snippet: Either a mouse anti-PML mAb (1:50–1:100 dilution, sc-966; Santa Cruz Biotechnology), a mouse anti-Daxx mAb (1:400 dilution, sc-8043; Santa Cruz Biotechnology), or a rabbit polyclonal anti-Daxx antibody (1:400 dilution, sc-7152; Santa Cruz Biotechnology) was added to 50 μl of lysate, and the solution was incubated at 4°C overnight.

Techniques: Transfection, Confocal Microscopy, Immunoprecipitation, Migration, Northern Blot, Y2H Assay, Transformation Assay, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Promyelocytic Leukemia Protein (Pml) and Daxx Participate in a Novel Nuclear Pathway for Apoptosis

doi:

Figure Lengend Snippet: PML is required for the NB localization of Daxx. (A) Immunofluorescence analysis of Daxx in PML −/ − splenocytes. Splenocytes, before and after Con A activation, were immunostained with anti-PML and anti-Daxx antibodies. Single staining ( a , b , a ′, and b ′) and superimposed images ( c and c ′) are shown. The nuclei were visualized by DAPI staining ( d and d ′). Representative confocal pictures are shown. Bar, 10 μM. (B) Western blot analysis of nuclear extract from PML +/+ and PML −/ − splenocytes, before and after Con A activation. Hsp90 protein levels are shown as a control of equal loading.

Article Snippet: Either a mouse anti-PML mAb (1:50–1:100 dilution, sc-966; Santa Cruz Biotechnology), a mouse anti-Daxx mAb (1:400 dilution, sc-8043; Santa Cruz Biotechnology), or a rabbit polyclonal anti-Daxx antibody (1:400 dilution, sc-7152; Santa Cruz Biotechnology) was added to 50 μl of lysate, and the solution was incubated at 4°C overnight.

Techniques: Immunofluorescence, Activation Assay, Staining, Western Blot, Control

Journal: The Journal of Experimental Medicine

Article Title: Promyelocytic Leukemia Protein (Pml) and Daxx Participate in a Novel Nuclear Pathway for Apoptosis

doi:

Figure Lengend Snippet: Daxx is selectively induced in B lymphocytes. PML +/+ and PML −/ − splenocytes were treated with Con A and at 72 h B and T subpopulations were obtained (Materials and Methods). Daxx expression levels at 0 and 72 h were measured in B and T cells by Western blot with the polyclonal anti-Daxx antibody. The differential expression of Daxx is also visible in extracts from untreated B and T cells upon longer exposure (not shown). Hsp90 was used as a normalization marker for protein input.

Article Snippet: Either a mouse anti-PML mAb (1:50–1:100 dilution, sc-966; Santa Cruz Biotechnology), a mouse anti-Daxx mAb (1:400 dilution, sc-8043; Santa Cruz Biotechnology), or a rabbit polyclonal anti-Daxx antibody (1:400 dilution, sc-7152; Santa Cruz Biotechnology) was added to 50 μl of lysate, and the solution was incubated at 4°C overnight.

Techniques: Expressing, Western Blot, Quantitative Proteomics, Marker

Journal: The Journal of Experimental Medicine

Article Title: Promyelocytic Leukemia Protein (Pml) and Daxx Participate in a Novel Nuclear Pathway for Apoptosis

doi:

Figure Lengend Snippet: Daxx induction correlates with B cell apoptosis. PML +/+ and PML −/ − splenocytes were treated with Con A. At 0, 24, 48, and 72 h after stimulation, B lymphocytes were isolated (see Materials and Methods) and scored for apoptosis by in situ TUNEL assay (A) or by cytometric analysis of subdiploid DNA content (B).

Article Snippet: Either a mouse anti-PML mAb (1:50–1:100 dilution, sc-966; Santa Cruz Biotechnology), a mouse anti-Daxx mAb (1:400 dilution, sc-8043; Santa Cruz Biotechnology), or a rabbit polyclonal anti-Daxx antibody (1:400 dilution, sc-7152; Santa Cruz Biotechnology) was added to 50 μl of lysate, and the solution was incubated at 4°C overnight.

Techniques: Isolation, In Situ, TUNEL Assay

Journal: The Journal of Experimental Medicine

Article Title: Promyelocytic Leukemia Protein (Pml) and Daxx Participate in a Novel Nuclear Pathway for Apoptosis

doi:

Figure Lengend Snippet: PML is required for the ability of Daxx to induce apoptosis. (A) Time course of apoptosis in PML +/+ and PML −/ − keratinocytes transfected with His(6×)-Daxx or, as control, His(6×)-LacZ. Apoptotic cells were scored by TUNEL as described in B. The values are expressed as mean percentage of apoptosis ± SD ( n = 3). (B) TUNEL staining of PML +/+ (top) and PML −/ − (bottom) keratinocytes 24 h after transfection. Transfected cells were recognized by immunofluorescence staining with an anti-His mAb. His(6×)-positive, red; TUNEL-positive, green; DAPI, blue. White arrows point to His(6×)-positive nonapoptotic (TUNEL-negative) cells; red arrowheads point to His(6×)-positive apoptotic cells (yellow). The pictures were taken with an Olympus fluorescence microscope.

Article Snippet: Either a mouse anti-PML mAb (1:50–1:100 dilution, sc-966; Santa Cruz Biotechnology), a mouse anti-Daxx mAb (1:400 dilution, sc-8043; Santa Cruz Biotechnology), or a rabbit polyclonal anti-Daxx antibody (1:400 dilution, sc-7152; Santa Cruz Biotechnology) was added to 50 μl of lysate, and the solution was incubated at 4°C overnight.

Techniques: Transfection, Control, TUNEL Assay, Staining, Immunofluorescence, Fluorescence, Microscopy

Journal: The Journal of Experimental Medicine

Article Title: Promyelocytic Leukemia Protein (Pml) and Daxx Participate in a Novel Nuclear Pathway for Apoptosis

doi:

Figure Lengend Snippet: Daxx is delocalized in the NB4 cells and relocalized to the NB upon RA treatment. NB4 cells were grown with or without 1 μM RA for 24 h before harvest. Cells were immunostained with a polyclonal anti-Daxx antibody and an anti-PML mAb. Single staining ( a , b , a ′, and b ′), superimposed images ( c and c ′), and DAPI staining ( d and d ′) are shown. Representative confocal pictures are shown. Bar, 10 μM.

Article Snippet: Either a mouse anti-PML mAb (1:50–1:100 dilution, sc-966; Santa Cruz Biotechnology), a mouse anti-Daxx mAb (1:400 dilution, sc-8043; Santa Cruz Biotechnology), or a rabbit polyclonal anti-Daxx antibody (1:400 dilution, sc-7152; Santa Cruz Biotechnology) was added to 50 μl of lysate, and the solution was incubated at 4°C overnight.

Techniques: Staining

Journal: eFood

Article Title: Docosahexaenoic Acid and Nervonic Acid Synergically Enhance Cognitive Memory in Normal Mice via Brain Fatty Acids Remodeling

doi: 10.1002/efd2.70059

Figure Lengend Snippet: FIGURE 3 | DHA and NA enhanced c‐Fos expression in the hippocampus of normal mice. (A) Representative c‐Fos immunopanned photos in the hippocampus. (B) The number of c‐Fos+ neurons in the hippocampal DG area. (C) The expression of PSD95 in the hippocampus. (D) The expression of synaptophysin (SYP) in the hippocampus. *p < 0.05, **p < 0.01 vs control group. One‐way ANOVA post‐hoc Dunnett's test (mean ± SEM, n = 4).

Article Snippet: Immunostaining was performed using monoclonal antibodies against c‐Fos (1:1000; Cell Signaling Technology, cat#2250S) and synaptophysin (SYP, 1:400; Santa Cruz, sc‐17750), polyclonal antibody against PSD95 (1:200; Invitrogen, cat#51‐6900), followed by incubation at 4°C for 24 h. Goat anti‐ mouse (Alexa Fluor 594, 1:500, ab150116) and goat anti‐rabbit (Alexa Fluor 488, 1:500, ab150077) IgG were used as secondary antibodies and incubated in the dark at room temperature for 2 h. Fluorescent images were captured using an ECHO Revlove fluorescence microscope (ECHO, San Diego, California, USA) and quantitatively analyzed using ImageJ software (NIH, Bethesda, Maryland, USA).

Techniques: Expressing, Control