Journal: The Journal of cell biology

Article Title: ARFRP1 functions upstream of ARL1 and ARL5 to coordinate recruitment of distinct tethering factors to the trans-Golgi network.

doi: 10.1083/jcb.201905097

Figure Lengend Snippet: Figure 2. Both ARL5 and ARFRP1 are required for localization of GARP to the TGN. (A) KO of TGN-localized small GTPases in HeLa cells confirmed by immunoblot analysis with antibodies to the proteins indicated on the right. In this figure and subsequent figures, ARL5 KO represents KO of both ARL5A and ARL5B, and RAB6 KO represents KO of both RAB6A and RAB6B. α-Tubulin was used as a loading control. The positions of molecular mass markers are indicated on the left. (B) Immunofluorescence microscopy of WT and KO HeLa cells transfected with a plasmid encoding VPS54-13Myc and stained for the Myc epitope (red), giantin (green), and nuclei (DAPI; blue). Scale bars: 10 μm. Insets are magnified views of the boxed areas. Inset scale bars: 5 μm. (C) Quantification of the percentage of cells exhibiting VPS54-13Myc staining at the TGN. Values are the mean ± SEM from three independent experiments. More than 100 cells per sample were counted in each experiment. The statistical significance of the differences relative to WT cells was determined using Dunnett’s test. **, P < 0.01; ***, P < 0.001.

Article Snippet: The following antibodies were used for immunoblotting and/or immunofluorescence microscopy: rabbit anti-VPS51 (HPA039650; Atlas Antibodies), rabbit anti-VPS52 made in our laboratory (Pérez-Victoria et al., 2008), rabbit anti-VPS53 (HPA024446; Atlas Antibodies), mouse anti-VPS50 (FLJ20097, monoclonal antibody M01, 2D11; Abnova), mouse anti-Myc epitope (9E10; Santa Cruz Biotechnology), sheep anti-TGN46 (AHP500G; Bio-Rad), mouse anti–β-actin (G043; Applied Biological Materials), rabbit antigiantin (ab80864; Abcam), mouse HRP-conjugated anti-α-tubulin (DM1A; Santa Cruz Biotechnology), rabbit anti-ARL1 (16012-1-AP; Proteintech), rabbit anti-ARFRP1 (PA5-50606; Thermo Fisher Scientific), mouse anti-ARL5A (sc-514680; Santa Cruz Biotechnology), rabbit anti-RAB6A (GTX110646; GeneTex), rabbit anti-GCC88 (HPA021323; Sigma-Aldrich), rabbit anti-GCC185 (HPA035849; Sigma-Aldrich), mouse anti-Golgin-245 (611281; BD Biosciences), mouse anti-GM130 (610822; BD Biosciences), mouse anti–Golgin-97 (A-21270; Thermo Fisher Scientific), rabbit anti-TMF1 (HPA008729, Sigma-Aldrich), monoclonal HRP-conjugated anti-GFP (Miltenyi Biotec Inc.), rabbit anti-GFP (A-11122; Thermo Fisher Scientific), HRP-conjugated goat anti-rabbit and donkey anti-mouse antibodies (Jackson ImmunoResearch), HRP-conjugated donkey antisheep (R&D Systems), and Alexa Fluor–conjugated secondary antibodies for immunostaining (Thermo Fisher Scientific).

Techniques: Western Blot, Control, Immunofluorescence, Microscopy, Transfection, Plasmid Preparation, Staining

Journal: The Journal of cell biology

Article Title: ARFRP1 functions upstream of ARL1 and ARL5 to coordinate recruitment of distinct tethering factors to the trans-Golgi network.

doi: 10.1083/jcb.201905097

Figure Lengend Snippet: Figure 5. Small GTPases required for localization of golgins to the TGN. (A) Immunofluorescence microscopy of WT, ARL1-KO, ARFRP1-KO, ARL5-KO, and RAB6-KO cells immunostained for endogenous Golgin-245, Golgin-97, GCC88, GCC185, or TMF1 and counterstained with DAPI (blue). Scale bars: 10 μm. Insets are magnified views of the boxed areas. Inset scale bars: 5 μm. Notice that ARL1 KO or ARFRP1 KO caused complete disappearance of Golgin-245 and GCC88, and a partial decrease in the intensity of Golgin-97, at the TGN; quantification in 10 cells per sample in three independent experiment showed that Golgin-97 decrease was 75.6% ± 2.4% in ARL1-KO cells and 55.0% ± 1.9% in ARFRP1-KO cells. (B) SDS-PAGE and immunoblot analysis of endogenous golgins and α-tubulin (loading control) in WT and KO cells. The positions of molecular mass markers are indicated on the left. (C) Immunofluorescence microscopy of RAB6- KO cells transfected with a plasmid encoding GFP-tagged mouse Rab6A (green), immunostained for endogenous GCC185 and TMF1 (red), and counterstained with DAPI (blue). Cells were examined for GFP fluorescence by confocal microscopy. Scale bars: 10 μm.

Article Snippet: The following antibodies were used for immunoblotting and/or immunofluorescence microscopy: rabbit anti-VPS51 (HPA039650; Atlas Antibodies), rabbit anti-VPS52 made in our laboratory (Pérez-Victoria et al., 2008), rabbit anti-VPS53 (HPA024446; Atlas Antibodies), mouse anti-VPS50 (FLJ20097, monoclonal antibody M01, 2D11; Abnova), mouse anti-Myc epitope (9E10; Santa Cruz Biotechnology), sheep anti-TGN46 (AHP500G; Bio-Rad), mouse anti–β-actin (G043; Applied Biological Materials), rabbit antigiantin (ab80864; Abcam), mouse HRP-conjugated anti-α-tubulin (DM1A; Santa Cruz Biotechnology), rabbit anti-ARL1 (16012-1-AP; Proteintech), rabbit anti-ARFRP1 (PA5-50606; Thermo Fisher Scientific), mouse anti-ARL5A (sc-514680; Santa Cruz Biotechnology), rabbit anti-RAB6A (GTX110646; GeneTex), rabbit anti-GCC88 (HPA021323; Sigma-Aldrich), rabbit anti-GCC185 (HPA035849; Sigma-Aldrich), mouse anti-Golgin-245 (611281; BD Biosciences), mouse anti-GM130 (610822; BD Biosciences), mouse anti–Golgin-97 (A-21270; Thermo Fisher Scientific), rabbit anti-TMF1 (HPA008729, Sigma-Aldrich), monoclonal HRP-conjugated anti-GFP (Miltenyi Biotec Inc.), rabbit anti-GFP (A-11122; Thermo Fisher Scientific), HRP-conjugated goat anti-rabbit and donkey anti-mouse antibodies (Jackson ImmunoResearch), HRP-conjugated donkey antisheep (R&D Systems), and Alexa Fluor–conjugated secondary antibodies for immunostaining (Thermo Fisher Scientific).

Techniques: Immunofluorescence, Microscopy, SDS Page, Western Blot, Control, Transfection, Plasmid Preparation, Fluorescence, Confocal Microscopy

Journal: Science signaling

Article Title: Neuropilin-1 mediates vascular permeability independently of vascular endothelial growth factor receptor-2 activation.

doi: 10.1126/scisignal.aad3812

Figure Lengend Snippet: Fig. 1. VEGF-A165, a ligand-blocking anti-NRP1, and a tetrameric CendR peptide induce NRP1 accumula- tion at endothelial cell–cell contacts. (A to D) Epifluorescence microscope images of permeabilized HUVEC monolayers. (A) Cells were stimulated with VEGF-A165 and stained with an antibody against NRP1 (red). (B) After incubation with the ligand-blocking anti-NRP1 antibody (anti-NRP1) (lower panel) or its cor- responding control sheep IgG (upper panel), cells were stimulated with VEGF-A165. HUVECs were stained with an antibody specific for NRP1 (red) and secondary anti-sheep antibody (green). Accumulation of NRP1 at cell-cell contacts was observed in the absence of VEGF-A165 (white arrows). (C) Cells were stimulated with anti-NRP1 and stained with secondary anti-sheep antibody (green). (D) Cells were stimulated with NA-RPARPAR peptide and stained with an antibody specific for NRP1 (red). Nuclei were stained with Hoechst (blue). Images in (A) to (D) are representative of n > 3 independent experiments. Scale bars, 20 mm.

Article Snippet: The ligand-blocking sheep anti-human NRP1 and goat anti-rat NRP1 antibodies were purchased from R&D Systems.

Techniques: Blocking Assay, Microscopy, Staining, Incubation, Control

Journal: Science signaling

Article Title: Neuropilin-1 mediates vascular permeability independently of vascular endothelial growth factor receptor-2 activation.

doi: 10.1126/scisignal.aad3812

Figure Lengend Snippet: Fig. 2. VEGF-A165, NA-RPARPAR, and anti-NRP1 induce NRP1 junctional localization and endothelial leakage in vitro and in vivo. (A) Confocal microscopy images of permeabilized HUVEC monolayers stimu- lated with VEGF-A165, NA-RPARPAR, and anti-NRP1 and stained with an antibody against VE-cadherin (green). NRP1 (red) was stained with an antibody against NRP1 (upper and middle panels) or directly with the secondary anti-sheep antibody (lower panel). Nuclei were stained with Hoechst (blue). White arrows indicate colocalization between NRP1 and VE-cadherin. Images are representative of four independent experiments. Scale bar, 20 mm. (B) HUVEC monolayers were seeded on top of Transwell filters and stimulated with PBS, VEGF-A165 (VEGF), NA-RPARPAR (NA-R), and anti-NRP1 (Ab). Leakage of FITC- dextran 70 kD from the upper to the lower well was measured by comparing fluorescence values at 520 nm (mean ± SEM; n = 9 independent experiments; the median from three to six replicates per independent experiment was used for statistical analyses; Friedman test followed by Dunn’s multiple com- parison post hoc test; *P < 0.05 and **P < 0.01). (C) Wild-type mice were systemically injected with Evans Blue and then with PBS, VEGF-A165, NA-RPARPAR, anti-NRP1 antibody, and their respective controls. The extravasated dye concentration was measured at 620 nm, and results were expressed as a ratio between the tested substance and its control (mean; n = 10 to 15 mice per treatment; Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test; **P < 0.01 and ***P < 0.001).

Article Snippet: The ligand-blocking sheep anti-human NRP1 and goat anti-rat NRP1 antibodies were purchased from R&D Systems.

Techniques: In Vitro, In Vivo, Confocal Microscopy, Staining, Fluorescence, Injection, Concentration Assay, Control, Comparison

Journal: Science signaling

Article Title: Neuropilin-1 mediates vascular permeability independently of vascular endothelial growth factor receptor-2 activation.

doi: 10.1126/scisignal.aad3812

Figure Lengend Snippet: Fig. 3. Unlike VEGF-A165, NA-RPARPAR and anti-NRP1 do not activate VEGFR-2, Akt, p38, ERK, or FAK. (A to C) HUVEC monolayers were stimu- lated with VEGF-A165, NA-RPARPAR, or anti-NRP1 antibody. (A) Cell ly- sates were immunoprecipitated with an antibody against VEGFR-2 and blotted with anti–VEGFR-2 and anti-phosphotyrosine (pY) antibodies. The corresponding total lysates were blotted for VEGFR-2 (n = 4 independent experiments). (B and C) Blotting was performed on total lysates with anti- bodies against pAkt, pERK1/2, p-p38 (n = 3 independent experiments) (B),

Article Snippet: The ligand-blocking sheep anti-human NRP1 and goat anti-rat NRP1 antibodies were purchased from R&D Systems.

Techniques: Immunoprecipitation

Journal: Science signaling

Article Title: Neuropilin-1 mediates vascular permeability independently of vascular endothelial growth factor receptor-2 activation.

doi: 10.1126/scisignal.aad3812

Figure Lengend Snippet: Fig. 4. VEGF-A165, NA-RPARPAR, and anti-NRP1 induce NRP1 relocalization and vascular leakage inde- pendently of VEGFR-2 activation. (A and B) HUVEC monolayers were treated with PTK/ZK or dimethyl sulfoxide (DMSO) before stimulation. ct, control. (A) Cell lysates were immunoprecipitated (IP) with VEGFR-2 antibody. Immunoprecipitates were blotted for VEGFR-2 and phosphotyrosine, and corresponding total lysates for VEGFR-2 (R2). Representative scans of five experiments. (B) HUVEC mono- layers were stained for NRP1 (red), nuclei were stained with Hoechst (blue), and cells were imaged with an epifluorescence microscope. Representative images from three experiments. Scale bar, 25 mm. (C and D) HUVECs were transfected with noncoding (NC) small interfering RNA (siRNA) or siRNAs coding for VEGFR-2 (KDR) (siRNA 1 and siRNA 2). (C) KDR mRNA relative expression was quantified by quantita- tive real-time polymerase chain reaction (qRT-PCR) (mean ± SEM; n = 3 independent experiments). (D) Epifluorescence images of transfected HUVECs in (C), stimulated with VEGF-A165, NA-RPARPAR, or anti- NRP1. Cells were stained for NRP1 (red), and nuclei were stained with Hoechst (blue). Representative images of three experiments. Scale bar, 20 mm. (E to H) Mice were injected intraperitoneally with DMSO or PTK/ZK before systemic treatment with Evans Blue. Leakage was induced with VEGF-A165 (E), NA-RPARPAR (F), anti-NRP1 (G), or VEGF-A121 (H), and results were expressed as a ratio between the tested substance and its respective control (mean; n = 10 to 20 mice per condition; Mann-Whitney test).

Article Snippet: The ligand-blocking sheep anti-human NRP1 and goat anti-rat NRP1 antibodies were purchased from R&D Systems.

Techniques: Activation Assay, Control, Immunoprecipitation, Staining, Microscopy, Transfection, Small Interfering RNA, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Injection, MANN-WHITNEY

Journal: Science signaling

Article Title: Neuropilin-1 mediates vascular permeability independently of vascular endothelial growth factor receptor-2 activation.

doi: 10.1126/scisignal.aad3812

Figure Lengend Snippet: Fig. 5. The NRP1 cytoplasmic domain mediates vascular leakage. (A and B) HUVECs overexpressing GFP, full-length NRP1, or NRP1 deprived from the cytoplasmic domain (NRP1DC) were stimulated with VEGF-A165. (A) Cell lysates were immunoprecipitated with VEGFR-2 antibody. Immunopre- cipitates were blotted for VEGFR-2 and phosphotyrosine, and total lysates were blotted for NRP1 and actin (n = 3 independent experiments). (B) HUVECs overexpressing GFP, NRP1, or NRP1DC were stained with anti- NRP1 (red). Nuclei were stained with Hoechst (blue). Representative images of three independent experiments. Scale bar, 20 mm. (C to E) Wild-type mice (NRP1cyto+/+) and mice expressing cytoplasmatically trun-

Article Snippet: The ligand-blocking sheep anti-human NRP1 and goat anti-rat NRP1 antibodies were purchased from R&D Systems.

Techniques: Immunoprecipitation, Staining, Expressing

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Tadalafil Reduces Myeloid-Derived Suppressor Cells and Regulatory T Cells and Promotes Tumor Immunity in Patients with Head and Neck Squamous Cell Carcinoma

doi: 10.1158/1078-0432.CCR-14-1711

Figure Lengend Snippet: MDSCs identification. A, the indicated myeloid cell subsets were tested for suppressive activity against CFSE-labeled autologous T cells stimulated with beads coated with anti-CD3/anti-CD28 antibodies. Data normalized on the control (no MDSC) are cumulative of five independent experiments using PBMCs from 5 patients. P value for the ANOVA test (Pa) and the Tukey post hoc test are reported. B, example of multicolor FACS analysis for MDSC phenotype CD33+IL4Rα+ cells are highlighted in blue. C, intratumoral CD33+ IL4Rα+ cells were retrospectively evaluated in the tumor specimen of recurrent or nonrecurrent OSCC patients by immunofluorescence microscopy. P value for t test is reported.

Article Snippet: FACS sorting For the suppressive assay, cryoconserved PBMCs were thawed and stained with Percp-Cy5.5–conjugated anti-human HLADR, FITC-conjugated anti-human CD33 (BD) and PE-conjugated anti-human IL4Rα (R&D Systems).

Techniques: Activity Assay, Labeling, Control, Immunofluorescence, Microscopy

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Tadalafil Reduces Myeloid-Derived Suppressor Cells and Regulatory T Cells and Promotes Tumor Immunity in Patients with Head and Neck Squamous Cell Carcinoma

doi: 10.1158/1078-0432.CCR-14-1711

Figure Lengend Snippet: An intermediate tadalafil dose modulates most effectively tumor immunity. The ratio between the MDSCs (A) or the log2-ratio of the CD8 proliferation (B) after (t2) and before (t1) pharmacologic treatment was plotted against the weight-normalized tadalafil dose. Best-fitting quadratic curve and confidence interval (gray area) are reported. cGMP (C) and cAMP (D) were measured by ELISA in the following FACS-sorted cell population from patients (n = 3) treated with intermediate or high dosage of tadalafil: CD33+IL4Rα+ (MDSCs), HLADRhigh (APC), or CD3+ (T cells). Pt, paired t test; BDL, below detection limit.

Article Snippet: FACS sorting For the suppressive assay, cryoconserved PBMCs were thawed and stained with Percp-Cy5.5–conjugated anti-human HLADR, FITC-conjugated anti-human CD33 (BD) and PE-conjugated anti-human IL4Rα (R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Tadalafil Reduces Myeloid-Derived Suppressor Cells and Regulatory T Cells and Promotes Tumor Immunity in Patients with Head and Neck Squamous Cell Carcinoma

doi: 10.1158/1078-0432.CCR-14-1711

Figure Lengend Snippet: Tadalafil modulates tumor microenvironment. CD33/IL4Rα (A), CD4/FoxP3 (B), or CD8/CD69 (C) intratumoral concentration was evaluated by immune-fluorescence microscopy. Pa, P ANOVA test.

Article Snippet: FACS sorting For the suppressive assay, cryoconserved PBMCs were thawed and stained with Percp-Cy5.5–conjugated anti-human HLADR, FITC-conjugated anti-human CD33 (BD) and PE-conjugated anti-human IL4Rα (R&D Systems).

Techniques: Concentration Assay, Fluorescence, Microscopy

Journal: Developmental biology

Article Title: Developmental Guidance of Embryonic Corneal Innervation: Roles of Semaphorin3A and Slit2

doi: 10.1016/j.ydbio.2010.04.032

Figure Lengend Snippet: Anterior eyes from embryonic ages E4-E7 as brightfield images – overlayed by fluorescent images showing nerves (arrows) labeled red with the TuJ-1 antibody and growing toward the cornea. In E4 the cornea is designated with a “C”. At E7 arrowheads designate small nerve branches growing towards the cornea, but not into it. The scale in all figures is the same, as shown by the bar with the E4 image, which is 1mm.

Article Snippet: Then, to visualize axons, the cultures were fixed in 4% paraformaldehyde and immunolabeled with the TuJ-1 antibody followed by donkey anti-mouse Northern lights Red fluorescent secondary antibody (R&D Systems) Images were captured using a fluorescent stereo dissecting microscope (SMZ 1500, Nikon Instruments, Melville, NY) equipped with a SPOT Flex real time CCD camera (Diagnostic Instruments, Inc., Sterling Heights, MI) and neuron numbers and lengths were analyzed using ImageJ software.

Techniques: Labeling

Journal: Developmental biology

Article Title: Developmental Guidance of Embryonic Corneal Innervation: Roles of Semaphorin3A and Slit2

doi: 10.1016/j.ydbio.2010.04.032

Figure Lengend Snippet: (A and B) Fluorescent images of E7 TuJ-1 labeled OTG co-cultured with an E7 lens “L” in the presence of (A) 10μg/ml rabbit IgG antibody or (B) 10μg/ml Slit2 antibody. (C) Neurite numbers and (D) neurite lengths from OTG co-cultured with lens or cornea and treated with 10μg/ml of either Slit2 antibody or control rabbit IgG. Neurite numbers (E) and neurite lengths (F) from co-cultures of E7 OTG with E7 cornea treated with 10μg/ml rabbit IgG antibody, 10μg/ml Slit2 antibody, 3μg/ml recombinant mouse Slit2, or 10μg/ml Slit2 antibody and 3μg/ml recombinant mouse Slit2. Error bars in C - F show the SEM. Scale bar in B = 0.3mm.

Article Snippet: Then, to visualize axons, the cultures were fixed in 4% paraformaldehyde and immunolabeled with the TuJ-1 antibody followed by donkey anti-mouse Northern lights Red fluorescent secondary antibody (R&D Systems) Images were captured using a fluorescent stereo dissecting microscope (SMZ 1500, Nikon Instruments, Melville, NY) equipped with a SPOT Flex real time CCD camera (Diagnostic Instruments, Inc., Sterling Heights, MI) and neuron numbers and lengths were analyzed using ImageJ software.

Techniques: Labeling, Cell Culture, Control, Recombinant

Journal: Oncology letters

Article Title: Luteolin induces apoptosis by impairing mitochondrial function and targeting the intrinsic apoptosis pathway in gastric cancer cells.

doi: 10.3892/ol.2023.13913

Figure Lengend Snippet: Figure 1. Viability of HGC‑27 and MFC cells is affected by luteolin. HGC‑27 and MFC cells were treated with different doses of luteolin for 24 h. Human gastric cancer HGC‑27 cells viability was inhibited by luteolin. (A) Morphological changes of HGC‑27 cells were observed under a light microscope (magni‑ fication, x100). (B) HGC‑27 cell viability was detected by the Cell Counting Kit‑8 assay. Mouse forestomach carcinoma MFC cells viability was inhibited by luteolin. (C) Morphological changes of MFC cells were observed under a light microscope (magnification, x200). (D) MFC cell viability was detected using the Cell Counting Kit‑8 assay. The decrease in the viability rate was analyzed using the GraphPad Prism 6.0 software. Experiments were repeated at least three times. **P<0.01 vs. DMSO group. HGC‑27, human gastric cancer HGC‑27 cell line; MFC, mouse forestomach carcinoma cell line.

Article Snippet: Luteolin was dissolved in dimethyl sulfoxide (DMSO) (D8371, Solarbio, Beijing, China) and diluted with complete medium to the required concentration.

Techniques: Light Microscopy, CCK-8 Assay, Software

Journal: Oncology letters

Article Title: Luteolin induces apoptosis by impairing mitochondrial function and targeting the intrinsic apoptosis pathway in gastric cancer cells.

doi: 10.3892/ol.2023.13913

Figure Lengend Snippet: Figure 2. HGC‑27 and MFC cells apoptosis are induced by luteolin. Morphological changes indicative of (A) HGC‑27 cells and (B) MFC cells apoptosis were observed using Hoechst 33258 staining method under an inverted fluorescence microscope (magnification, x100). Red arrows indicated the apparent apoptotic morphological features, such as karyopyknosis, nucleosome and chromosome condensation. Following double‑staining with Annexin‑V FITC and PI, the flow cytometry was used to test the apoptosis in (C) HGC‑27 cells and (E) MFC cells, and the quantitative determination of apoptosis of (D) HGC‑27 cells and (F) MFC cells was showed on the histogram. Experiments were repeated at least three times. *P<0.05 and **P<0.01 vs. DMSO group. HGC‑27, human gastric cancer HGC‑27 cell line; MFC, mouse forestomach carcinoma cell line.

Article Snippet: Luteolin was dissolved in dimethyl sulfoxide (DMSO) (D8371, Solarbio, Beijing, China) and diluted with complete medium to the required concentration.

Techniques: Staining, Fluorescence, Microscopy, Flow Cytometry

Journal: Oncology letters

Article Title: Luteolin induces apoptosis by impairing mitochondrial function and targeting the intrinsic apoptosis pathway in gastric cancer cells.

doi: 10.3892/ol.2023.13913

Figure Lengend Snippet: Figure 3. ROS accumulation in HGC‑27 and MFC cells is induced by luteolin. The fluorescence intensity of (A) HGC‑27 cells and (B) MFC cells were visualized under a fluorescence inverted microscope (magnification, x100). Luteolin‑induced ROS levels were detected using DCFH‑DA staining and flow cytometry in (C) HGC‑27 cells and (E) MFC cells. The quantitative analysis of ROS levels in (D) HGC‑27 cells and (F) MFC cells was showed on the histo‑ gram. (G) SOD activity was assessed in HGC‑27 and MFC cells using a microplate reader at an absorbance of 560 nm. Experiments were repeated at least in triplicate. Data were presented as mean ± SD. **P<0.01 vs. DMSO group. ROS, reactive oxygen species; SOD, superoxide dismutase; HGC‑27, human gastric cancer HGC‑27 cell line; MFC, mouse forestomach carcinoma cell line.

Article Snippet: Luteolin was dissolved in dimethyl sulfoxide (DMSO) (D8371, Solarbio, Beijing, China) and diluted with complete medium to the required concentration.

Techniques: Fluorescence, Inverted Microscopy, Staining, Flow Cytometry, Activity Assay

Journal: Oncology letters

Article Title: Luteolin induces apoptosis by impairing mitochondrial function and targeting the intrinsic apoptosis pathway in gastric cancer cells.

doi: 10.3892/ol.2023.13913

Figure Lengend Snippet: Figure 4. Mitochondrial membrane potential, ATP levels and some enzyme activities in HGC‑27 and MFC cells induced by luteolin. The luteolin‑treated (A) HGC‑27 cells and (C) MFC cells were stained with JC‑1, and analyzed using flow cytometry. Quantitative statistics of the mitochondrial membrane potential was based on flow cytometry in (B) HGC‑27 cells and (D) MFC cells. The (E) ATP levels, (F) Na+/K+‑ATPase activities and (G) Ca2+/Mg2+‑ATPase activities were showed based on the microplate system at the absorbance values of 660 nm. The data were presented as mean ± SD. The experiments were repeated in triplicate. *P<0.05, **P<0.01 vs. DMSO group. HGC‑27, human gastric cancer HGC‑27 cell line; MFC, mouse forestomach carcinoma cell line.

Article Snippet: Luteolin was dissolved in dimethyl sulfoxide (DMSO) (D8371, Solarbio, Beijing, China) and diluted with complete medium to the required concentration.

Techniques: Membrane, Staining, Flow Cytometry

Journal: Oncology letters

Article Title: Luteolin induces apoptosis by impairing mitochondrial function and targeting the intrinsic apoptosis pathway in gastric cancer cells.

doi: 10.3892/ol.2023.13913

Figure Lengend Snippet: Figure 5. Luteolin induces the enzyme activities of the METC complexes in HGC‑27 and MFC cells. Complexes (A) Ⅰ, (B) Ⅲ and (C) Ⅴ were assessed by testing kits. The data were obtained using a microplate reader at 340, 550 and 660 nm, respectively. The data were presented as mean ± SD. The experiments were performed at least in triplicate. *P<0.05 and **P<0.01 vs. DMSO group; HGC‑27, human gastric cancer HGC‑27 cell line; MFC, mouse forestomach carcinoma cell line.

Article Snippet: Luteolin was dissolved in dimethyl sulfoxide (DMSO) (D8371, Solarbio, Beijing, China) and diluted with complete medium to the required concentration.

Techniques:

Journal: Oncology letters

Article Title: Luteolin induces apoptosis by impairing mitochondrial function and targeting the intrinsic apoptosis pathway in gastric cancer cells.

doi: 10.3892/ol.2023.13913

Figure Lengend Snippet: Figure 6. Luteolin unbalanced Bcl‑2 and Bax protein expression in HGC‑27 and MFC cells. Bcl‑2 and Bax protein levels in (A) HGC‑27 cells and (B) MFC cells were examined using western blot. (C) Ratio between Bcl‑2 and Bax protein expression levels is showed in the histogram. The data were presented as mean ± SD. Experiments were repeated at least in triplicate. *P<0.05 and **P<0.01 vs. DMSO group. Bcl‑2, B cell lymphoma‑2; Bax, Bcl‑2‑associated X; HGC‑27, human gastric cancer HGC‑27 cell line; MFC, mouse forestomach carcinoma cell line.

Article Snippet: Luteolin was dissolved in dimethyl sulfoxide (DMSO) (D8371, Solarbio, Beijing, China) and diluted with complete medium to the required concentration.

Techniques: Expressing, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Topogenesis of Peroxisomal Membrane Protein Requires a Short, Positively Charged Intervening-loop Sequence and Flanking Hydrophobic Segments

doi: 10.1074/jbc.m003304200

Figure Lengend Snippet: FIG. 2. Functional and topogenic regions of PMP34. C-terminally HA- tagged or GFP-fused PMP34 and its vari- ants were verified for intracellular local- ization in CHO-K1. A, constructs of deletion mutants of PMP34. DN30HA, PMP34-HA with deletion of N-terminal residues from 1 to 30; 204HA, HA-tagged PMP34 with residues 1–204; 204GFP, PMP34 comprising residues 1–204 fused with GFP. Others likewise representing respective constructs were indicated. Numbers in box represent the positions of transmembrane segments; L1–L5 desig- nate the intervening-loop region between two flanking TMs. Peroxisomal targeting activity of each variant verified (see be- low) was shown: 1, active; 1/2, partially active; 2, inactive. B, PMP34 variants represented in A were expressed in CHO- K1. a and b, DN30HA; c and d, DN125HA; e and f, DN186HA; g and h, DN125GFP; i and j, DN186GFP; k, DN204HA; l, 186HA; m and n, 204HA; o and p, 204GFP. C- terminally HA-tagged PMP34 variants were verified for peroxisomal localization by immunostaining using mouse (a, c, e, and m) and rabbit (k and l) anti-HA anti- body and FITC-labeled second antibody, where peroxisomes were assessed by anti- Pex14p antibody and Texas Red-labeled second antibody (b, d, f, h, j, n, and p). PMP34 truncation mutants fused with GFP were verified by GFP fluorescence (g, i, and o). Arrowheads indicate PMP34- positive particles, positive in expressed PMP34-variants, that were absent from Pex14p. Original magnification, 3630; bar, 20 mm. C, transmembrane topology of GFP fusion proteins, DN125GFP and 204GFP, was determined. CHO-K1 cells expressing DN125GFP (a and b) and 204GFP (c and d) were fixed, then treated with 25 mg/ml digitonin. Localization and membrane orientation were verified by GFP fluorescence (a and c) and immuno- fluorescence staining of GFP with anti- GFP antibody and Texas Red-labeled sec- ond antibody (b and d). Bar, 20 mm.

Article Snippet: Antigen-antibody complexes were detected under a Carl Zeiss Axioskop FL microscope, using fluorescein isothiocyanate (FITC)-labeled sheep anti-mouse antibody (Amersham Pharmacia Biotech, Tokyo, Japan), FITC-labeled sheep anti-rabbit immunoglobulin (Ig) G antibody (Cappel), or Texas Red-labeled goat antibodies to guinea pig IgG (Vector Laboratories) and rabbit IgG (Leinco Technologies).

Techniques: Functional Assay, Construct, Activity Assay, Variant Assay, Immunostaining, Labeling, Fluorescence, Expressing, Membrane, Staining

Journal: Journal of Biological Chemistry

Article Title: Topogenesis of Peroxisomal Membrane Protein Requires a Short, Positively Charged Intervening-loop Sequence and Flanking Hydrophobic Segments

doi: 10.1074/jbc.m003304200

Figure Lengend Snippet: FIG. 5. Coordinated function of the membrane targeting se- quence and transmembrane segments. A, constructs of the loop region and transmembrane segments (loop plus TM) fused with GFP. B, intracellular localization of the (loop plus TM)-GFP fusion protein. a and b, 86/204GFP; c and d, 30/204GFP; e, 125/273GFP; f, 86/273GFP. Each construct was expressed in CHO-K1 cells and detected by GFP fluorescence (a, c, e, and f). Cells expressing 86/204GFP were also stained using anti-malate dehydrogenase antibody and Texas Red- labeled second antibody (b); peroxisomes in 30/204GFP-expressing cells were assessed by anti-Pex14p antibody (d). Bar, 20 mm.

Article Snippet: Antigen-antibody complexes were detected under a Carl Zeiss Axioskop FL microscope, using fluorescein isothiocyanate (FITC)-labeled sheep anti-mouse antibody (Amersham Pharmacia Biotech, Tokyo, Japan), FITC-labeled sheep anti-rabbit immunoglobulin (Ig) G antibody (Cappel), or Texas Red-labeled goat antibodies to guinea pig IgG (Vector Laboratories) and rabbit IgG (Leinco Technologies).

Techniques: Membrane, Construct, Fluorescence, Expressing, Staining, Labeling