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:

Article Title: Inhibition of HDM2 and Activation of p53 by Ribosomal Protein L23

doi: 10.1128/MCB.24.17.7669-7680.2004

Figure Lengend Snippet: L23 inhibits HDM2-mediated p53 polyubiquitination and degradation. (A) Ectopic expression of L23 stabilizes HDM2 and p53. U2OS cells were transfected with the indicated plasmid DNA for 2 days, and cell extracts were resolved by SDS-PAGE, transferred onto a nitrocellulose membrane, and blotted with antibodies as indicated. Plasmid DNA expressing GFP was cotransfected as a control. +, present; −, absent; α-HDM2, anti-HDM2; α-p53, anti-p53; α-myc, anti-myc; α-GFP, anti-GFP. (B) Ectopic expression of L23 stabilizes HDM2 and p53 in normal human fibroblast cells. WI38 cells were infected with virus expressing HDM2 for 2 days, and cell extracts were resolved by SDS-PAGE, transferred onto a nitrocellulose membrane, and blotted with antibodies as indicated. Virus expressing GFP was coinfected as a control. α-actin, anti-actin. (C) L23 inhibits HDM2-mediated p53 polyubiquitination. U2OS cells were transfected with the indicated plasmid DNA for 2 days, and the cells were treated with MG132 (25 μM) for 5 h before lysing. Cell extracts were analyzed by Western blotting with antibodies to p53 (D01) and myc (9E10) as indicated. (D) L23 stabilizes HDM2 independent of p53. WI38-E6 cells were infected with viruses expressing GFP, HDM2, and myc-L23 as indicated. Cells were lysed 2 days after infection, and the cell lysates were blotted as described above. Endog, endogenous; α-L23, anti-L23.

Article Snippet: Mouse monoclonal antibody to HDM2 (Ab-1; Oncogene Research Products), goat polyclonal antibody to human p53 (FL393; Santa Cruz), and mouse monoclonal antibody to human p53 (DO-1; NeoMarkers) were purchased commercially.

Techniques: Expressing, Transfection, Plasmid Preparation, SDS Page, Infection, Western Blot

Journal:

Article Title: Inhibition of HDM2 and Activation of p53 by Ribosomal Protein L23

doi: 10.1128/MCB.24.17.7669-7680.2004

Figure Lengend Snippet: L23 induces a p53-dependent cell cycle arrest. (A and B) L23 overexpression stabilizes and activates p53. Normal human fibroblast WI38 cells and isogenic mutant WI38-E6 cells were infected with the indicated Ad for 2 days. Western blotting was performed as described above. α-myc, anti-myc; α-HDM2, anti-HDM2; α-p53, anti-p53; α-p21, anti-p21; α-actin, anti-actin. (C and D) L23 induces a p53-dependent cell cycle arrest. WI38 and WI38-E6 cells were infected with the indicated viruses. Cells were harvested 2 days after infection, fixed with 70% ethanol for 2 h, and stained with propidium iodide for 1 h, and the cell cycle distribution was determined by flow cytometry. Cell populations in the S phase are indicated as percentages of total cells. (E) L23 interacts with HDM2 in the nucleoplasm. U2OS cells were singly infected with Ad expressing myc-L23 for 2 days. Cells were then fixed with 3% paraformaldehyde for 10 min and immunostained with a rabbit anti-myc antibody (9E10) and a mouse anti-HDM2 antibody (N20). Nuclei were visualized by 4′,6′-diamidino-2-phenylindole (DAPI) staining. Fluorescence images were captured with a cooled charge-coupled device color digital camera (model 2.2.0; Diagnostic) on an Olympus IX70 inverted microscope equipped with the appropriate fluorescence filters.

Article Snippet: Mouse monoclonal antibody to HDM2 (Ab-1; Oncogene Research Products), goat polyclonal antibody to human p53 (FL393; Santa Cruz), and mouse monoclonal antibody to human p53 (DO-1; NeoMarkers) were purchased commercially.

Techniques: Over Expression, Mutagenesis, Infection, Western Blot, Staining, Flow Cytometry, Expressing, Fluorescence, Diagnostic Assay, Inverted Microscopy

Journal:

Article Title: Inhibition of HDM2 and Activation of p53 by Ribosomal Protein L23

doi: 10.1128/MCB.24.17.7669-7680.2004

Figure Lengend Snippet: Knocking down L23, but not L11, activates p53 and induces a cell cycle arrest. (A and B) U2OS cells were either untreated (Buffer) or transfected with a control scrambled RNA duplex (siScr), L23 siRNA (siL23), or L11 siRNA (siL11) for 2 days. Cell extracts were collected and analyzed by Western blotting with the indicated antibodies. α-HDM2, anti-HDM2; α-p53, anti-p53; α-p21, anti-p21; α-L23, anti-L23; α-actin, anti-actin. (C and D) U2OS cells were transfected with siRNA as described for panels A and B. Cells were harvested 2 days after transfection, fixed with ethanol, and stained with propidium iodide, and their cell cycle distribution was determined by flow cytometry. Percentages of cells in S phase are shown. The averages of the results from two independent experiments are shown as bar graphs.

Article Snippet: Mouse monoclonal antibody to HDM2 (Ab-1; Oncogene Research Products), goat polyclonal antibody to human p53 (FL393; Santa Cruz), and mouse monoclonal antibody to human p53 (DO-1; NeoMarkers) were purchased commercially.

Techniques: Transfection, Western Blot, Staining, Flow Cytometry

Journal:

Article Title: Inhibition of HDM2 and Activation of p53 by Ribosomal Protein L23

doi: 10.1128/MCB.24.17.7669-7680.2004

Figure Lengend Snippet: Down-regulation of L23-induced cell cycle arrest is dependent on the function of p53. (A and B) Normal human fibroblast WI38 cells and isogenic mutant WI38-E6 cells were transfected with either a control scrambled RNA duplex (siScr) or L23 siRNA (siL23) for 2 days, and cell extracts were analyzed by Western blotting with the indicated antibodies. α-HDM2, anti-HDM2; α-p53, anti-p53; α-p21, anti-p21; α-L23, anti-L23; α-actin, anti-actin. (C and D) WI38 and WI38-E6 cells were transfected siRNA as described for panels A and B. Cells were harvested 2 days after infection and stained with propidium iodide, and their cell cycle distribution was determined by flow cytometry. Percentages of cells in S phase are shown. (E) Down-regulation of L23 releases nucleolar B23. U2OS cells were transfected with the indicated siRNA for 2 days. The cells were then fixed and stained with a mouse anti-B23 (α-B23) antibody (Zymed) and an fluorescein isothiocyanate-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch). Fluorescence images were captured with a cooled charge-coupled device color digital camera (model 2.2.0; Diagnostic) on an Olympus IX70 inverted microscope equipped with the appropriate fluorescence filters.

Article Snippet: Mouse monoclonal antibody to HDM2 (Ab-1; Oncogene Research Products), goat polyclonal antibody to human p53 (FL393; Santa Cruz), and mouse monoclonal antibody to human p53 (DO-1; NeoMarkers) were purchased commercially.

Techniques: Mutagenesis, Transfection, Western Blot, Infection, Staining, Flow Cytometry, Fluorescence, Diagnostic Assay, Inverted Microscopy

Journal:

Article Title: Inhibition of HDM2 and Activation of p53 by Ribosomal Protein L23

doi: 10.1128/MCB.24.17.7669-7680.2004

Figure Lengend Snippet: Inhibition of ribosomal biogenesis decreases the protein level of L23. (A) Low concentrations of actinomycin D induce p53-dependent cell cycle arrest. U2OS cells were treated with the indicated concentrations of actinomycin D (Act D) for 24 h, and the cell lysates were analyzed by Western blotting as described above. α-HDM2, anti-HDM2; α-p53, anti-p53; α-L23, anti-L23; α-actin, anti-actin. (B) Time required for actinomycin D treatment to suppress L23. U2OS cells were treated with 5 nM actinomycin D for the indicated times, and the protein levels were analyzed as described above. (C) Inhibition of ribosomal biogenesis by 5 nM actinomycin D down-regulates L23 but not L11. U2OS cells were treated with 5 nM actinomycin D for 24 h before lysing, the cell lysates were resolved by SDS-PAGE, and Western blotting was performed as described above. α-L11, anti-L11. (D) Ectopic expression of L23 suppresses endogenous L23. U2OS cells were infected with the indicated viruses for 2 days, and cell extracts were harvested and resolved by SDS-PAGE. The proteins were transferred onto a nitrocellulose membrane and blotted with the indicated antibodies. α-myc, anti-myc. (E) Suppression of endogenous L23 by ectopically expressed myc-L23 was independent of HDM2 and p53. Normal human fibroblast WI38 cells were infected with the indicated viruses for 2 days. Cell extracts were harvested and resolved by SDS-PAGE, and the proteins were analyzed as described above. +, present; −, absent.

Article Snippet: Mouse monoclonal antibody to HDM2 (Ab-1; Oncogene Research Products), goat polyclonal antibody to human p53 (FL393; Santa Cruz), and mouse monoclonal antibody to human p53 (DO-1; NeoMarkers) were purchased commercially.

Techniques: Inhibition, Western Blot, SDS Page, Expressing, Infection

Journal: Molecular medicine reports

Article Title: Knockdown of biglycan expression by RNA interference inhibits the proliferation and invasion of, and induces apoptosis in, the HCT116 colon cancer cell line.

doi: 10.3892/mmr.2015.4383

Figure Lengend Snippet: Figure 1. Construction of the stable HCT116 cell line with biglycan down regulation. The (A) mRNA and (B) protein expression levels of biglycan in the shRNA‑biglycan/control‑transfected or non‑transfected cells was detected using reverse transcription‑quantitative polymerase chain reaction and western blotting, respectively. (C) The relative protein expression of big lycan was normalized against β‑actin. Each experiment was repeated three times and the data are expressed as the mean ± standard deviation (*P<0.05 or **P<0.01, compared with the shRNA‑control group). sh, short hairpin.

Article Snippet: The membrane was subsequently blocked with 5% non-fat dry milk at room temperature for 1 h, followed by incubation with the following diluted primary antibodies: Rabbit polyclonal anti-biglycan antibody (cat no. sc-33788; 1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit polyclonal anti-caspase-3 antibody (cat no. wl01992a; 1:1,000), rabbit polyclonal anti-p27 antibody (cat no. wl01769; 1:1,000), rabbit polyclonal anti-cyclin A antibody (cat no. wl01753; 1:200), rabbit polyclonal anti-p21 antibody (cat no. wl0362; 1:100), rabbit polyclonal anti-cyclin D1 antibody (cat no. wl01435a; 1:100) (all from Wanleibio, Shenyang, China), rabbit polyclonal anti-p38 antibody (cat no. sc-7149; 1:100) and anti-phosphorylated-p38 antibody (cat no. sc-101758; 1:100) (both from Abcam, Cambridge, MA, USA) at 4 ̊C overnight.

Techniques: Expressing, Polymerase Chain Reaction, Western Blot, Standard Deviation

Journal: Molecular medicine reports

Article Title: Knockdown of biglycan expression by RNA interference inhibits the proliferation and invasion of, and induces apoptosis in, the HCT116 colon cancer cell line.

doi: 10.3892/mmr.2015.4383

Figure Lengend Snippet: Figure 2. Downregulation of biglycan inhibits the proliferation of colon cancer cells and causes cell cycle arrest. (A) The effect of the downregulation of biglycan on HCT116 cell proliferation was measured using a cell counting kit‑8 assay, with each group containing six technical replicates. (B and C) The effect of the downregulation of biglycan on the HCT116 cell cycle distribution was analyzed by flow cytometry. (D) The protein expression levels of cyclin A, cyclin D1, p21 and p27 were detected by western blotting, and the results obtained from a representative experiment are shown. (E) The relative protein expres sion of these proteins were normalized against β‑actin. The data are expressed as the mean ± standard deviation (**P<0.01, compared with the shRNA‑control group). OD, optical density; sh, short hairpin.

Article Snippet: The membrane was subsequently blocked with 5% non-fat dry milk at room temperature for 1 h, followed by incubation with the following diluted primary antibodies: Rabbit polyclonal anti-biglycan antibody (cat no. sc-33788; 1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit polyclonal anti-caspase-3 antibody (cat no. wl01992a; 1:1,000), rabbit polyclonal anti-p27 antibody (cat no. wl01769; 1:1,000), rabbit polyclonal anti-cyclin A antibody (cat no. wl01753; 1:200), rabbit polyclonal anti-p21 antibody (cat no. wl0362; 1:100), rabbit polyclonal anti-cyclin D1 antibody (cat no. wl01435a; 1:100) (all from Wanleibio, Shenyang, China), rabbit polyclonal anti-p38 antibody (cat no. sc-7149; 1:100) and anti-phosphorylated-p38 antibody (cat no. sc-101758; 1:100) (both from Abcam, Cambridge, MA, USA) at 4 ̊C overnight.

Techniques: CCK-8 Assay, Flow Cytometry, Expressing, Western Blot, Standard Deviation

Journal: Molecular medicine reports

Article Title: Knockdown of biglycan expression by RNA interference inhibits the proliferation and invasion of, and induces apoptosis in, the HCT116 colon cancer cell line.

doi: 10.3892/mmr.2015.4383

Figure Lengend Snippet: Figure 3. Downregulation of biglycan suppresses the migratory and invasive properties of colon cancer cells (magnification, x200). (A) The motility of shRNA‑biglycan/control or non‑transfected cells was determined using a scratch wound assay over a 24 h time period. (B) The migration rate was determined by the distance traveled by the cells to the front of the denuded area. (C) The invasion ability of cells in each group was measured using a Transwell assay. (D) The number of invasive cells were counted under an inverted microscope and cell numbers were plotted. The results obtained from a representative experi ment are shown. The data are expressed as the mean ± standard deviation (**P<0.01, compared with the shRNA‑control group).. sh, short hairpin.

Article Snippet: The membrane was subsequently blocked with 5% non-fat dry milk at room temperature for 1 h, followed by incubation with the following diluted primary antibodies: Rabbit polyclonal anti-biglycan antibody (cat no. sc-33788; 1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit polyclonal anti-caspase-3 antibody (cat no. wl01992a; 1:1,000), rabbit polyclonal anti-p27 antibody (cat no. wl01769; 1:1,000), rabbit polyclonal anti-cyclin A antibody (cat no. wl01753; 1:200), rabbit polyclonal anti-p21 antibody (cat no. wl0362; 1:100), rabbit polyclonal anti-cyclin D1 antibody (cat no. wl01435a; 1:100) (all from Wanleibio, Shenyang, China), rabbit polyclonal anti-p38 antibody (cat no. sc-7149; 1:100) and anti-phosphorylated-p38 antibody (cat no. sc-101758; 1:100) (both from Abcam, Cambridge, MA, USA) at 4 ̊C overnight.

Techniques: Control, Scratch Wound Assay Assay, Migration, Transwell Assay, Inverted Microscopy, Standard Deviation

Journal: Molecular medicine reports

Article Title: Knockdown of biglycan expression by RNA interference inhibits the proliferation and invasion of, and induces apoptosis in, the HCT116 colon cancer cell line.

doi: 10.3892/mmr.2015.4383

Figure Lengend Snippet: Figure 4. Downregulation of biglycan activates the p38 signaling pathway and induces apoptosis in colon cancer cells. (A) The effect of biglycan downregula tion on the apoptosis of the HCT116 cells was detected using flow cytometry, and results are shown from a representative experiment. (B) The proportion of apoptotic cells in each group was determined. (C) The protein expression levels of caspase‑3 and p‑p38 were detected by western blotting, and the results obtained from a representative experiment are shown for each group. (D) The relative expression of these proteins was normalized against β‑actin. The data are expressed as the mean ± standard deviation (**P<0.01, compared with the shRNA‑control group; ##P<0.01, compared with the shRNA‑biglycan group). p‑, phosphorylated; sh, short hairpin.

Article Snippet: The membrane was subsequently blocked with 5% non-fat dry milk at room temperature for 1 h, followed by incubation with the following diluted primary antibodies: Rabbit polyclonal anti-biglycan antibody (cat no. sc-33788; 1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit polyclonal anti-caspase-3 antibody (cat no. wl01992a; 1:1,000), rabbit polyclonal anti-p27 antibody (cat no. wl01769; 1:1,000), rabbit polyclonal anti-cyclin A antibody (cat no. wl01753; 1:200), rabbit polyclonal anti-p21 antibody (cat no. wl0362; 1:100), rabbit polyclonal anti-cyclin D1 antibody (cat no. wl01435a; 1:100) (all from Wanleibio, Shenyang, China), rabbit polyclonal anti-p38 antibody (cat no. sc-7149; 1:100) and anti-phosphorylated-p38 antibody (cat no. sc-101758; 1:100) (both from Abcam, Cambridge, MA, USA) at 4 ̊C overnight.

Techniques: Flow Cytometry, Expressing, Western Blot, Standard Deviation

Journal: Journal of neurovirology

Article Title: Transgenic Mice Expressing HIV-1 Envelope Protein gp120 in the Brain as an Animal Model in NeuroAIDS Research

doi: 10.1007/s13365-017-0584-2

Figure Lengend Snippet: Sagittal brain sections of 6 months-old gp120-transgenic and wild-type (WT) littermate controls were immune-stained for neuronal MAP-2 (red) and astrocytic GFAP (green). DNA (blue) was labeled with H33342 and is shown to indicate nuclei. Fluorescence-labeled sections were analyzed using a Zeiss Axiovert 100 M inverted microscope and Slidebook software (Intelligent Imaging Innovations, Denver, CO) to record Z-stacks and perform deconvolution and 3D reconstruction. The upper six panels show 3D volume views, the bottom two panel are 3D surface views. Representative areas of mid-frontal cortex, layer 3, are shown. Note the difference in the density of MAP-2 immunoreactive neuropil and astrocyte morphology between WT and gp120tg samples, and the dimensions of the 10 μm grid.

Article Snippet: Fluorescence-labeled sections were analyzed using a Zeiss Axiovert 100 M inverted microscope and Slidebook software (Intelligent Imaging Innovations, Denver, CO) to record Z-stacks and perform deconvolution and 3D reconstruction.

Techniques: Transgenic Assay, Staining, Labeling, Fluorescence, Inverted Microscopy, Software, Imaging

Journal: PLoS ONE

Article Title: Rhabdastrellic Acid-A Induced Autophagy-Associated Cell Death through Blocking Akt Pathway in Human Cancer Cells

doi: 10.1371/journal.pone.0012176

Figure Lengend Snippet: A. Hep3B and A549 cells were transfected with an expression construct for LC3 fused to yellow fluorescent protein (YFP-LC3) for 24 h. Thereafter, cells were treated with or without 1 µg/mL or 4 µg/mL Rhabdastrellic acid-A for 36 h, and visualized under a confocal microscope. B. Electron micrograph showing autophagic vacuole of A549 cells following 4 µg/mL Rhabdastrellic acid-A treatment. C. Rhabdastrellic acid-A time-dependently induced the formation of LC3-II, a marker for autophagy. Hep3B and A549 cells were treated with 1 µg/mL Rhabdastrellic acid-A for the indicated times. Lysates were analyzed by immunoblotting with LC3 antibody.

Article Snippet: The following primary antibodies were used: caspase-3 antibody (Santa Cruz, sc-7272), PARP antibody (Santa Cruz, sc-7150), glyceraldehyde 3-phosphate dehydrogenase antibody (Santa Cruz, sc-47724), LC3 antibody (Novus Biologicals, NB100-2220), Atg5 antibody (Cell Signaling Technology, #2630), Phospho-Akt1/2/3(ser473) antibody (Santa Cruz, sc-7985-R), Akt1 antibody (Santa Cruz, sc-1618), Phospho-mTOR(ser2448) antibody(Cell Signaling Technology, #2971), mTOR antibody (Cell Signaling Technology, #2972), Phospho-FKHR(ser256) antibody (Cell Signaling Technology, #9461), FKHR antibody (Cell Signaling Technology, #9462), Phospho-STAT3(ser727) antibody (Santa Cruz, sc-8001-R), STAT3 antibody (Cell Signaling Technology, #9132).

Techniques: Transfection, Expressing, Construct, Microscopy, Marker, Western Blot

Journal: PLoS ONE

Article Title: Rhabdastrellic Acid-A Induced Autophagy-Associated Cell Death through Blocking Akt Pathway in Human Cancer Cells

doi: 10.1371/journal.pone.0012176

Figure Lengend Snippet: A. Hep3B cells were treated with 1 µg/mL Rhabdastrellic acid-A and/or 10 mM 3-MA for 36 h, and then examined by inverted microscope. *, p<0.05 vs. Rhabdastrellic acid-A−/3-MA-. **, p<0.05 vs. Rhabdastrellic acid-A+/3-MA-. B. Lysates from A549-Vector and A549-shAtg5 cells were analyzed by immunoblotting with Atg5 and LC3 antibodies. C. A549 vector cells and A549-shAtg5 cells were cultured at 6000 cells per well in a 96-well plate, exposed to different concentrations of Rhabdastrellic acid-A from 0.05 to 3.2 µg/mL for 72 h. The growth inhibition was detected using MTT assay. Reported values are mean ± SD of triplicate samples from a representative experiment. *P-vlaue<0.05 as compared with cells treated in the same way, but transfected without shRNA. D. A549 Cells were incubated with 4 µg/mL Rhabdastrellic acid-A and/or 10 mM 3-MA for 24 h. Cell death was quantified using flow cytometry as PI staining assay. The experiment was repeated 3 times.

Article Snippet: The following primary antibodies were used: caspase-3 antibody (Santa Cruz, sc-7272), PARP antibody (Santa Cruz, sc-7150), glyceraldehyde 3-phosphate dehydrogenase antibody (Santa Cruz, sc-47724), LC3 antibody (Novus Biologicals, NB100-2220), Atg5 antibody (Cell Signaling Technology, #2630), Phospho-Akt1/2/3(ser473) antibody (Santa Cruz, sc-7985-R), Akt1 antibody (Santa Cruz, sc-1618), Phospho-mTOR(ser2448) antibody(Cell Signaling Technology, #2971), mTOR antibody (Cell Signaling Technology, #2972), Phospho-FKHR(ser256) antibody (Cell Signaling Technology, #9461), FKHR antibody (Cell Signaling Technology, #9462), Phospho-STAT3(ser727) antibody (Santa Cruz, sc-8001-R), STAT3 antibody (Cell Signaling Technology, #9132).

Techniques: Inverted Microscopy, Plasmid Preparation, Western Blot, Cell Culture, Inhibition, MTT Assay, Transfection, shRNA, Incubation, Flow Cytometry, Staining

Journal: PLoS ONE

Article Title: Rhabdastrellic Acid-A Induced Autophagy-Associated Cell Death through Blocking Akt Pathway in Human Cancer Cells

doi: 10.1371/journal.pone.0012176

Figure Lengend Snippet: A. A. the positive clone stably expressed myr-Akt1 was analyzed by immunoblotting with Akt antibody. B. Hep3B cells were treated with 1 µg/mL Rhabdastrellic acid-A for 36 h in the absence or presence of constitutive active Akt ectopic expression. Then LC3 protein expression was analyzed. C. Cancer cells were treated with 1 µg/mL Rhabdastrellic acid-A and/or 10 µg/mL pepstatin A (pepA) for 36 h, Then LC3 protein expression was analyzed. D. After treatment, viable cells of two cell lines were measured. *, p<0.05 vs. Rhabdastrellic acid-A (vector).

Article Snippet: The following primary antibodies were used: caspase-3 antibody (Santa Cruz, sc-7272), PARP antibody (Santa Cruz, sc-7150), glyceraldehyde 3-phosphate dehydrogenase antibody (Santa Cruz, sc-47724), LC3 antibody (Novus Biologicals, NB100-2220), Atg5 antibody (Cell Signaling Technology, #2630), Phospho-Akt1/2/3(ser473) antibody (Santa Cruz, sc-7985-R), Akt1 antibody (Santa Cruz, sc-1618), Phospho-mTOR(ser2448) antibody(Cell Signaling Technology, #2971), mTOR antibody (Cell Signaling Technology, #2972), Phospho-FKHR(ser256) antibody (Cell Signaling Technology, #9461), FKHR antibody (Cell Signaling Technology, #9462), Phospho-STAT3(ser727) antibody (Santa Cruz, sc-8001-R), STAT3 antibody (Cell Signaling Technology, #9132).

Techniques: Stable Transfection, Western Blot, Expressing, Plasmid Preparation