Journal: EMBO reports

Article Title: eIF5A is required for autophagy by mediating ATG3 translation.

doi: 10.15252/embr.201846072

Figure Lengend Snippet: Figure 4. eIF5A regulates translation of ATG3.

Article Snippet: Primary antibodies used are as follows: LC3B (nanotools, 1:200) (LC3B CST, 1:1,000), GABARAP (Abgent 1:1,000), GATE-16 (MBL, 1:1,000), ATG3 (Sigma, 1:500), Vinculin (Sigma, 1:100,000), GAPDH (Santacruz, 1:20,000), eIF5A (Santacruz, 1:500), Lamin A1 (Santacruz, 1:1,000), histone H3 (Abcam 1:10,000), p62 (MBL, 1:5,000), RFP/ Cherry (Rockland, 1:6,000), p-mTOR (Ser 2448) (CST, 1:1,000), mTOR (Cell signaling, 1:1,000), RPL23A (Abcam, 1:50,000), RPS6 (CST, 1:1,000), RPL10A (Santacruz, 1:2,000), Hypusine (Merck Millipore 1:5,000).

Techniques:

Journal: EMBO reports

Article Title: eIF5A is required for autophagy by mediating ATG3 translation.

doi: 10.15252/embr.201846072

Figure Lengend Snippet: Figure 5. Regulation of autophagy by eIF5A is mediated via ATG3 translation.

Article Snippet: Primary antibodies used are as follows: LC3B (nanotools, 1:200) (LC3B CST, 1:1,000), GABARAP (Abgent 1:1,000), GATE-16 (MBL, 1:1,000), ATG3 (Sigma, 1:500), Vinculin (Sigma, 1:100,000), GAPDH (Santacruz, 1:20,000), eIF5A (Santacruz, 1:500), Lamin A1 (Santacruz, 1:1,000), histone H3 (Abcam 1:10,000), p62 (MBL, 1:5,000), RFP/ Cherry (Rockland, 1:6,000), p-mTOR (Ser 2448) (CST, 1:1,000), mTOR (Cell signaling, 1:1,000), RPL23A (Abcam, 1:50,000), RPS6 (CST, 1:1,000), RPL10A (Santacruz, 1:2,000), Hypusine (Merck Millipore 1:5,000).

Techniques:

Journal: Bioactive Carbohydrates and Dietary Fibre

Article Title: Resistant starch from a tuberous root from the Andes cordillera improves metabolic and immune parameters in broilers.

doi: 10.1016/j.bcdf.2024.100420

Figure Lengend Snippet: Fig. 3. CD3+, CD4+ and CD8+ intestinal T lymphocytes subpopulations in broilers on day 35 determined by flow cytometry (upper left image). The images A, B, C correspond to dot plot made in the FlowJo XV software based on readings made in the Facs Canto II flow cytometer (Becton Dickinson). The images show: A. Distribution by size (FSC-A) and complexity (SSC-A) of the events analyzed in broilers, B. Percentage of CD4+ T cells in broiler chickens fed with a control diet C. Percentage of CD4+ T cells in broiler chickens fed with a diet supplemented with RS2.

Article Snippet: The labeling mix 2 was prepared with 2 μL of each of the following anti-chicken monoclonal antibodies: anti-CD3 epsilon (PC3/188A), antiCD8 alpha (EP72), the anti-human antibody anti-MHC1 (F21-2) coupled to FITC (Novus biologicals, Colorado, USA), which was used as a positive control, and 94 μL of cytometer buffer.

Techniques: Flow Cytometry, Software, Control

Journal: PLoS ONE

Article Title: Cysteine (C)-X-C Receptor 4 Undergoes Transportin 1-Dependent Nuclear Localization and Remains Functional at the Nucleus of Metastatic Prostate Cancer Cells

doi: 10.1371/journal.pone.0057194

Figure Lengend Snippet: A , A human prostate tissue array, ranging from normal to high-grade prostate cancer, was evaluated by IHC for CXCR4 expression using standard methods. Samples were evaluated at magnification 40X, using a Q-Imaging camera of Olympus BX51 Microscope with Bioquant® Image Analysis Software (RtmBometrics). Normal prostate tissues demonstrated slightly weak or undetectable brown staining for CXCR4 (positive cells<5%), and no CXCR4 expression in the nucleus. Representative low grade prostate tissue (grade 2, stage II, T 2 N 0 M 0 , adenocarcinoma) demonstrated random/focal positive staining for CXCR4 in the nucleus (positive cells >11%, but less than 50%), indicating low expression of CXCR4. Representative high grade metastatic prostate tissue (grade 4, stage IV, T 4 N 1 M 1 , adenocarcinoma) demonstrated diffuse/intense staining (positive cells >50%), indicating high expression for CXCR4 in the nucleus. Scale bar represents 50 µm. B , CXCR4 IgG2B mouse monoclonal antibody was evaluated for specificity to CXCR4 protein by western blot analysis in PC3 (CXCR4 positive) or 293T (CXCR4 null) cell lines. C , CXCR4 antibody was evaluated for specificity to CXCR4 protein by immunoprecipitation for CXCR4 and western blot analysis for CXCR4. D , CXCR4 IgG2B antibody was evaluated for specificity to CXCR4 protein by immunoprecipitation with Fibronectin IgG2B mouse monoclonal antibody (unrelated isotype control) and western blot analysis for CXCR4; expression of Fibronectin protein was confirmed by western blot analysis. Beta-actin was used as a loading control.

Article Snippet: One milligram of PC3 whole cell lysates were immunoprecipitated for CXCR4 (Santa Cruz; 1 μg per 250 μg of protein) overnight at 4°C, followed by incubation with Protein A/G Plus-Agarose beads (Santa Cruz) for 2 hrs at 4°C.

Techniques: Expressing, Imaging, Microscopy, Software, Staining, Western Blot, Immunoprecipitation, Control

Journal: PLoS ONE

Article Title: Cysteine (C)-X-C Receptor 4 Undergoes Transportin 1-Dependent Nuclear Localization and Remains Functional at the Nucleus of Metastatic Prostate Cancer Cells

doi: 10.1371/journal.pone.0057194

Figure Lengend Snippet: A , Normal prostate epithelial (RWPE1) and PCa (PC3, DU145, 22RV1) cells were stimulated with SDF1α (100 ng/ µl) prior to subcellular fractionation into non-nuclear and nuclear fractions. Immunoblots were probed with anti-CXCR4. Anti-CD44 (non-nuclear) and anti-Topoisomerase1 (Topo 1, nuclear) were used as markers for fractionation purity and as loading controls. The bar graphs are quantitative results of the band density representing expression of CXCR4 in each fraction. Data were mean + S.E. from three independent experiments. *, P<0.05. B , Immunocytochemistry of PCa cell lines for CXCR4. PCa cells were stimulated with SDF1α (100 ng/ µl), fixed with methanol, blocked then incubated with an antibody mixture containing a mouse anti-CXCR4 monoclonal antibody and a rabbit polyclonal anti-Lamin A/C antibody, followed by secondary mixture containing a Cy3-conjugated anti-mouse antibody and FITC-conjugated anti-rabbit antibody. Imaging was with a Zeiss LSM-510 UV Confocal Microscope using the 63× Plan-Apochromat 63x/1.40 Oil DIC objective at excitation 488 nm for FITC and 543 nm for Cy3. Confocal images demonstrating the plasma membrane and cytosolic localization of CXCR4 (red), intact nuclear membrane (green), and nuclear-associated localization of CXCR4 (yellow/orange) are shown. Small arrows indicate co-localization of CXCR4 with the nucleus (yellow/orange). Scale bars represent 50 µm.

Article Snippet: One milligram of PC3 whole cell lysates were immunoprecipitated for CXCR4 (Santa Cruz; 1 μg per 250 μg of protein) overnight at 4°C, followed by incubation with Protein A/G Plus-Agarose beads (Santa Cruz) for 2 hrs at 4°C.

Techniques: Fractionation, Western Blot, Expressing, Immunocytochemistry, Incubation, Imaging, Microscopy, Clinical Proteomics, Membrane

Journal: PLoS ONE

Article Title: Cysteine (C)-X-C Receptor 4 Undergoes Transportin 1-Dependent Nuclear Localization and Remains Functional at the Nucleus of Metastatic Prostate Cancer Cells

doi: 10.1371/journal.pone.0057194

Figure Lengend Snippet: A , GFP-CXCR4 fusion protein localized similar to endogenous CXCR4. CXCR4-pEGFPN1 transfected PC3 cells were stimulated with SDF1α, fixed with methanol, blocked then incubated with a mouse anti-CXCR4 monoclonal antibody, followed by a Cy3-conjugated anti-mouse secondary antibody. Nuclei were stained with DAPI (blue). Images were taken at 40× maginification using Axiovision software 4.8.2 with a Zeiss Axio Imager.z1 fluorescence microscope at ex = 470 nm for FITC, ex = 358 nm for DAPI and ex = 551 nm for Cy3. Images demonstrate the co-localization (yellow) of endogenous CXCR4 (red) with GFP-tagged CXCR4 (green). B , Localization analysis of wild type CXCR4 (CXCR4-pEGFPN1), NLS-mutant of CXCR4 (pEGFPN1-CXCR4 R146A ,) and deleted NLS of CXCR4 (CXCR4 ΔNLS ) by immunocytochemistry in PC3 cells. Nuclei were stained with propidium iodide (red) and CXCR4 was detected as the fusion protein GFP-CXCR4 (green). Imaging was with a Zeiss LSM-510 UV Confocal Microscope using the 63× Plan-Apochromat 63x/1.40 Oil DIC objective at ex = 488 nm for FITC and ex = 543 nm for Cy3. Scale bars represent 50 µm. C , Transfected cells were stimulated with SDF1α prior to subcellular fractionation into non-nuclear and nuclear fractions. Immunoblots were probed with anti-GFP to detect the fusion protein GFP-CXCR4. Anti-CD44 (non-nuclear) and anti-Topoisomerase1 (Topo 1, nuclear) were used as markers for fractionation purity and as loading controls.

Article Snippet: One milligram of PC3 whole cell lysates were immunoprecipitated for CXCR4 (Santa Cruz; 1 μg per 250 μg of protein) overnight at 4°C, followed by incubation with Protein A/G Plus-Agarose beads (Santa Cruz) for 2 hrs at 4°C.

Techniques: Transfection, Incubation, Staining, Software, Fluorescence, Microscopy, Mutagenesis, Immunocytochemistry, Imaging, Fractionation, Western Blot

Journal: PLoS ONE

Article Title: Cysteine (C)-X-C Receptor 4 Undergoes Transportin 1-Dependent Nuclear Localization and Remains Functional at the Nucleus of Metastatic Prostate Cancer Cells

doi: 10.1371/journal.pone.0057194

Figure Lengend Snippet: A , Sixty micrograms of total protein were analyzed for TRN1 expression by western blot analysis using a TRN1 specific antibody. Alpha-tubulin served as a loading control. B , One milligram of PC3 whole cell lysate was immunoprecipitated with anti-CXCR4 and separated by SDS-PAGE. Immunocomplexes were probed with anti-TRN1 or anti-CXCR4 to ensure that CXCR4 interacted with TRN1 and was immunoprecipitated, respectively. Thirty micrograms of whole cell PC3 supernatant, post-immunoprecipitation, were separated by SDS-PAGE and harvested for western blot analysis to assess the efficiency of CXCR4 immunoprecipitation. C and D , Cells were transiently transfected with TRN1-specific siRNA to determine an effective concentration ( C ) , prior to harvesting for immunohistochemistry with anti-Lamin A/C and anti-CXCR4 ( D ) . Images were taken using Zeiss Axio Imager.z1 fluorescence microscope at 40× magnification at excitation 470 nm for FITC and 551 nm for Cy3. Small arrows indicate co-localization of CXCR4 with the nucleus (yellow/orange). Scale bar represents 50 µm.

Article Snippet: One milligram of PC3 whole cell lysates were immunoprecipitated for CXCR4 (Santa Cruz; 1 μg per 250 μg of protein) overnight at 4°C, followed by incubation with Protein A/G Plus-Agarose beads (Santa Cruz) for 2 hrs at 4°C.

Techniques: Expressing, Western Blot, Control, Immunoprecipitation, SDS Page, Transfection, Concentration Assay, Immunohistochemistry, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: Cysteine (C)-X-C Receptor 4 Undergoes Transportin 1-Dependent Nuclear Localization and Remains Functional at the Nucleus of Metastatic Prostate Cancer Cells

doi: 10.1371/journal.pone.0057194

Figure Lengend Snippet: A , Representative light images of whole cells and isolated nuclei confirmed the integrity of nuclear isolation at 20× magnification. B , Whole cells were treated with SDF1α prior to isolating and lysing intact nuclei. Nuclei lysates (1 mg) were immunoprecipitated with anti-CXCR4 and separated by SDS-PAGE. Immunocomplexes were probed for G αi (first row) or CXCR4 antibody (second row), respectively. Anti-CD44 (non-nuclear) and anti-Topoisomerase1 (Topo1, nuclear) were used as markers for fractionation purity and as loading controls. C , PC3 nuclei were isolated, incubated with FluoForte dye Ca 2+ probe, followed by incubation with AMD3100 or pertussis toxin (PTX) for 1 hr, then stimulated with SDF1α for 30 min. An increase in fluorescent-bound Ca 2+ was measured on a microplate reader at ex = 490 nm/em = 525 nm.

Article Snippet: One milligram of PC3 whole cell lysates were immunoprecipitated for CXCR4 (Santa Cruz; 1 μg per 250 μg of protein) overnight at 4°C, followed by incubation with Protein A/G Plus-Agarose beads (Santa Cruz) for 2 hrs at 4°C.

Techniques: Isolation, Immunoprecipitation, SDS Page, Fractionation, Incubation

Journal: Frontiers in immunology

Article Title: Investigating mammary glands of lactating goats for the presence of tertiary lymphoid organs.

doi: 10.3389/fimmu.2022.941333

Figure Lengend Snippet: FIGURE 1 Representative images of hematoxylin and eosin staining (HE) and immunohistochemistry against CD20 (B cells), CD3 (T cells), MECA79 (high endothelial venules; HEVs), CD40 (follicular dendritic cells), and BCL6 (germinal center) in a small-sized aggregation of lymphocytes in goat mammary gland tissues. Scale bar, 100 mm or 25 mm (clippings).

Article Snippet: The sections were then incubated overnight at 4°C with either rabbit polyclonal antibodies against goat-IgA (#A50-106A, Bethyl Laboratories), claudin-3 (#34-1700, Thermo Fisher Scientific), claudin-4 (#PA5-32354, Thermo Fisher Scientific), or mouse monoclonal antibodies against BCL6 (#sc7388, Santa Cruz Biotechnology), CD3 (#NBP2-53386, Novus Biologicals), CD20 (#NBP2-45454, Novus Biologicals), CD40 (#sc-13128, Santa Cruz Biotechnology), occludin (#sc-133256, Santa Cruz Biotechnology), or a rat monoclonal antibody against MECA79 (#sc-19602, Santa Cruz Biotechnology) diluted in PBS-T containing 2.5% bovine serum albumin.

Techniques: Staining, Immunohistochemistry

Journal: Frontiers in immunology

Article Title: Investigating mammary glands of lactating goats for the presence of tertiary lymphoid organs.

doi: 10.3389/fimmu.2022.941333

Figure Lengend Snippet: FIGURE 2 Representative images of hematoxylin and eosin staining (HE) staining and immunohistochemistry against CD20 (B cells), CD3 (T cells), MECA79 (high endothelial venules; HEVs), CD40 (follicular dendritic cells), and BCL6 (germinal center) in a large-sized aggregation of lymphocytes in goat mammary glands. Images of the same position are shown. Scale bar, 100 mm.

Article Snippet: The sections were then incubated overnight at 4°C with either rabbit polyclonal antibodies against goat-IgA (#A50-106A, Bethyl Laboratories), claudin-3 (#34-1700, Thermo Fisher Scientific), claudin-4 (#PA5-32354, Thermo Fisher Scientific), or mouse monoclonal antibodies against BCL6 (#sc7388, Santa Cruz Biotechnology), CD3 (#NBP2-53386, Novus Biologicals), CD20 (#NBP2-45454, Novus Biologicals), CD40 (#sc-13128, Santa Cruz Biotechnology), occludin (#sc-133256, Santa Cruz Biotechnology), or a rat monoclonal antibody against MECA79 (#sc-19602, Santa Cruz Biotechnology) diluted in PBS-T containing 2.5% bovine serum albumin.

Techniques: Staining, Immunohistochemistry

Journal: Frontiers in immunology

Article Title: Investigating mammary glands of lactating goats for the presence of tertiary lymphoid organs.

doi: 10.3389/fimmu.2022.941333

Figure Lengend Snippet: FIGURE 3 Representative images of immunohistochemistry against CD20 (B cells) and CD3 (T cells) in large aggregations of lymphocytes in goat mammary glands. Images of the same position are shown. Scale bar, 100 mm.

Article Snippet: The sections were then incubated overnight at 4°C with either rabbit polyclonal antibodies against goat-IgA (#A50-106A, Bethyl Laboratories), claudin-3 (#34-1700, Thermo Fisher Scientific), claudin-4 (#PA5-32354, Thermo Fisher Scientific), or mouse monoclonal antibodies against BCL6 (#sc7388, Santa Cruz Biotechnology), CD3 (#NBP2-53386, Novus Biologicals), CD20 (#NBP2-45454, Novus Biologicals), CD40 (#sc-13128, Santa Cruz Biotechnology), occludin (#sc-133256, Santa Cruz Biotechnology), or a rat monoclonal antibody against MECA79 (#sc-19602, Santa Cruz Biotechnology) diluted in PBS-T containing 2.5% bovine serum albumin.

Techniques: Immunohistochemistry

Journal: Oncotarget

Article Title: The antitumor potential of Interleukin-27 in prostate cancer.

doi: 10.18632/oncotarget.1425

Figure Lengend Snippet: Figure 1: Expression of IL-27R on hPCa cell lines and assessment of IL-27 effects on hPCa cells in vitro and in vivo. Panel A and B. Expression of WSX-1 (Panel A) and gp130 (panel B) was analysed in PC3, DU145, 22Rv1 and LNCaP cells by flow cytometry. Open profile: WSX-1 (panel A) and gp130 (panel B) staining. Dark profile: isotype matched mAb staining. Experiments were performed at least in triplicate. Panel C. Western blot analyses of WSX-1 in nuclear extracts (n.e.) and membrane extracts (m.e.) from 22Rv1 and DU145 cells. Nuclear extracts were used as negative controls. A specific 70 kDalton (Da) band, corresponding to WSX-1 protein, was observed in membrane but not nuclear extracts obtained from DU145 cells. Panel D. IL-27 inhibits PC3 cell proliferation in vitro, as assessed by CFSE staining. PC3 cells were cultured for 48 and 120 hours with medium alone (left panel) or in the presence of 100 ng/ml hrIL-27 (middle panel). Flow cytometry analyses showed that IL-27 inhibited PC3 cell proliferation after 120 hours of treatment (middle and right panel), as indicated by the higher CFSE intensity in hrIL-27 treated PC3 cells compared to untreated cells at this time point (right panel). Panel E. Flow cytometry analyses showed that IL-27 inhibited DU145 (left panel) cell proliferation after 120 hours of treatment, as indicated by the higher CFSE intensity in hrIL-27 treated cells (dark profile) compared to untreated cells at this time point (light profile). IL-27 did not affect 22Rv1 (middle panel) or LNCaP (right panel) cell proliferation at the same time point. Panel F. IL-27 did not induce apoptosis after 120 hours of treatment in PC3, DU145, 22Rv1 or LNCaP cells.

Article Snippet: Cell culture, antibodies, reagents, flow cytometry and western blot The human PC3, DU145, 22Rv1 and LNCaP PCa cell lines (LGC Standards, Teddington, UK) were cultured in RPMI 1640 with 10% FCS (Seromed-BiochromKG, Berlin, D). hrIL-27 (R&D System, Minneapolis, MN, USA) was used at 100 ng/ml following titration experiments.

Techniques: Expressing, In Vitro, In Vivo, Flow Cytometry, Staining, Western Blot, Membrane, Cell Culture

Journal: Oncotarget

Article Title: The antitumor potential of Interleukin-27 in prostate cancer.

doi: 10.18632/oncotarget.1425

Figure Lengend Snippet: Figure 2: Inhibition of human PC3 and DU145 cell growth in vivo by IL-27 treatment. Panel A. Tumor growth curve from day 14 to day 51 after PC3 s.c. cell injection into athymic nude mice was obtained by measuring tumor volume in situ using a caliper. Dark line represents growth curve (volume) of tumors formed in mice injected with PC3 cells and treated with PBS (controls). Grey light line represents growth curve of tumors formed in mice injected with PC3 cells and treated with hrIL-27. Asterisks mean significant differences (see results section). Panel B. Volume of tumor masses developed in mice injected with the DU145 cell line and subsequently treated with PBS (controls) or hrIL-27. Panel C and D. Morphological and immunohistochemical features of PC3 (C) and DU145 (D) tumor masses grown in hrIL-27 or PBS treated mice. Expression of WSX-1 was preserved in vivo by tumor developed after s.c. injection of PC3 or DU145 cells in both control (a) and hrIL-27 treated (b) mice. The proliferative activity of PC3 tumors grown in PBS treated animals (c) was considerably reduced in tumors from hrIL-27 treated animals (d). The histologic feature of poorly differentiated tumor with a solid growth pattern (e) and endowed with a well-developed vascularization (g) observed in PBS treated animals, was heavily compromised in tumors from hrIL-27 treated animals which showed multiple foci of ischemic necrosis (f) along with a deficient vascular supply (h). PC3 tumors revealed a strong expression of the anti-angiogenic chemokine IP-10/CXCL10 (j), which, on the contrary, was barely detected in tumors from PBS treated animals (i). (a-j: x400). Panel E. Expression profile of genes involved in angiogenic pathways in PC3 (white bars) and DU145 (black bars) cells treated with hrIL-27. Pooled results ± SD from two experiments performed in duplicate are shown. Histogram represents fold differences of individual mRNA between cells cultured in presence or absence of hrIL-27.

Article Snippet: Cell culture, antibodies, reagents, flow cytometry and western blot The human PC3, DU145, 22Rv1 and LNCaP PCa cell lines (LGC Standards, Teddington, UK) were cultured in RPMI 1640 with 10% FCS (Seromed-BiochromKG, Berlin, D). hrIL-27 (R&D System, Minneapolis, MN, USA) was used at 100 ng/ml following titration experiments.

Techniques: Inhibition, In Vivo, Injection, In Situ, Immunohistochemical staining, Expressing, Control, Activity Assay, Cell Culture