Journal: Experimental and Therapeutic Medicine

Article Title: Scutellaria barbata D. Don inhibits growth and induces apoptosis by suppressing IL-6-inducible STAT3 pathway activation in human colorectal cancer cells

doi: 10.3892/etm.2015.2692

Figure Lengend Snippet: Effect of EESB on the HT-29 cell cycle. Cells were pretreated with various concentration of EESB for 1 h, followed by stimulation with 10 ng/ml IL-6 for 24 h. (A) Cells were stained with propidium iodide and analyzed by fluorescence-activated cell sorting. The proportion of DNA in the S phase was calculated using ModFit LT version 3.0 software. (B) Quantification of fluorescence-activated cell sorting analysis. The data shown are averages with standard deviation from 3 independent experiments. #P<0.05 vs. cells treated with IL-6 but not EESB. EESB, ethanol extract of Scutellaria barbata D. Don; IL-6, interleukin-6.

Article Snippet: The HT-29 cell cycle progression was determined through flow cytometric analysis using a propidium iodide (PI) staining cell cycle assay kit (BD Biosciences, Franklin Lakes, NJ, USA).

Techniques: Concentration Assay, Staining, Fluorescence, FACS, Software, Standard Deviation

Journal: Experimental and Therapeutic Medicine

Article Title: Scutellaria barbata D. Don inhibits growth and induces apoptosis by suppressing IL-6-inducible STAT3 pathway activation in human colorectal cancer cells

doi: 10.3892/etm.2015.2692

Figure Lengend Snippet: Effect of EESB on HT-29 cell apoptosis. Cells were pretreated with various concentration of EESB for 1 h, followed by stimulation with 10 ng/ml IL-6 for 24 h. (A) Cells were collected and stained with Annexin V/PI, followed by fluorescence-activated cell sorting analysis. Double-negative stained cells indicate the live cell population; Annexin V-positive/PI-negative stained cells and Annexin V/PI double-positive stained cells represent early and late apoptosis, respectively; Annexin V-negative and PI-positive stained cells show dead cells. (B) Quantification of fluorescence-activated cell sorting analysis. The data shown are averages with standard deviation from 3 independent experiments. #P<0.05 vs. cells treated with IL-6 but not EESB. EESB, ethanol extract of Scutellaria barbata D. Don; IL-6, interleukin-6; UL, upper left; UR, upper right; LR, lower right; LL, lower left; PI, propidium iodide; FITC, fluorescein isothiocyanate.

Article Snippet: The HT-29 cell cycle progression was determined through flow cytometric analysis using a propidium iodide (PI) staining cell cycle assay kit (BD Biosciences, Franklin Lakes, NJ, USA).

Techniques: Concentration Assay, Staining, Fluorescence, FACS, Standard Deviation

Journal: Oncology Letters

Article Title: LIM and SH3 protein 1 knockdown suppresses proliferation and metastasis of colorectal carcinoma cells via inhibition of the mitogen-activated protein kinase signaling pathway

doi: 10.3892/ol.2018.8222

Figure Lengend Snippet: Effects of Lasp-1 in cellular proliferation, induction of apoptotic cell death and cell cycle progression in colorectal carcinoma cells. Cells were transfected with Lasp-1 siRNA or NC siRNA. (A) Cellular proliferation was determined using the Cell Counting Kit-8 assay (n=3; *P<0.05 vs. NC; # P<0.05 vs. siRNA). (B) Cellular apoptosis and (C) cell cycle progression were detected using flow cytometric analysis. Lasp-1, LIM and SH3 protein 1; NC, negative control; siRNA, small interfering RNA; PI, propidium iodide; FITC, fluorescein isothiocyanate.

Article Snippet: The propidium iodide/RNase staining kit (MultiSciences Biotech Co., Ltd., Hangzhou, China) was used according to the manufacturer's protocol.

Techniques: Transfection, Cell Counting, Negative Control, Small Interfering RNA

Journal: Molecules

Article Title: Anti-Tumor Activity and Mechanism of Silibinin Based on Network Pharmacology and Experimental Verification

doi: 10.3390/molecules29081901

Figure Lengend Snippet: ( A ) Cells stained with Hoechst 33342 and EdU dye after 24 h of incubation with different concentrations of silibinin. Scale bar = 100 μm. ( B ) Percentage of EdU-positive cells after treatment with different concentrations of silibinin. * p < 0.05, ** p < 0.01 vs. CTL, CTL: normal control group.

Article Snippet: Hoechst 33342/propidium iodide (PI) double staining kit was bought from Genview (Beijing, China), and a 5-ethynyl-20-deoxyuridine (EdU) assay kit was acquired from Ribobio (Guangzhou, China).

Techniques: Staining, Incubation

Journal: Molecules

Article Title: Anti-Tumor Activity and Mechanism of Silibinin Based on Network Pharmacology and Experimental Verification

doi: 10.3390/molecules29081901

Figure Lengend Snippet: ( A ) Cells stained with Hoechst 33342 and PI dye after 24 h of incubation with different concentrations of silibinin. Scale bar = 100 μm. ( B ) Percentage of PI-positive cells after treatment with different concentrations of silibinin. * p < 0.05, ** p < 0.01 vs. CTL, CTL: normal control group.

Article Snippet: Hoechst 33342/propidium iodide (PI) double staining kit was bought from Genview (Beijing, China), and a 5-ethynyl-20-deoxyuridine (EdU) assay kit was acquired from Ribobio (Guangzhou, China).

Techniques: Staining, Incubation

Journal:

Article Title: A selective sphingosine kinase 1 inhibitor integrates multiple molecular therapeutic targets in human leukemia

doi: 10.1182/blood-2008-02-138958

Figure Lengend Snippet: SK1-I and siSphK1 decrease cellular proliferation and viability. (A) U937 cells or (B) Jurkat cells (105 cells/mL) were cultured in medium containing 10% serum in the absence or presence of the indicated concentrations of SK1-I. Cell numbers were determined with a Coulter counter model Z1 (Beckman Coulter). (C) Knockdown of SphK1 by siRNA reduces cell growth. U937 cells were transiently transfected siRNA targeted to SphK1 (○) or siRNA control (●). Cells (105 cells/mL) were then cultured in medium containing 10% (top panel) or 2% (middle panel) serum for the indicated times and cell numbers determined with a Coulter counter. (Bottom panel) Equal amounts of cell lysate proteins (60 μg) from duplicate cultures were separated by SDS-PAGE and immunoblotted with anti-SphK1 antibody. Blots were stripped and probed with actin antibody to ensure equal loading and transfer. SphK1 mRNA levels were normalized to actin and the ratio relative to siControl from 3 independent experiments is shown. *P < .05. (D-G) SK1-I promotes apoptosis. U937 cells cultured in 10% serum were treated without or with 10, 15, or 20 μM SK1-I for the indicated times (D) or with 20 μM SK1-I for 24 hours (E) and stained with annexin V/PI, and apoptosis was determined by flow cytometry. Early apoptotic cells are annexin V positive, late apoptotic cells are both annexin V and PI positive, whereas necrotic cells are PI positive only. (F) Duplicate cultures were treated with the indicated concentration of SK1-I for 24 hours, and the fraction of TUNEL-positive cells was determined. (G) U937 cells were cultured in medium containing 2% serum in the absence or presence of 10 μM SK1-I for 24 hours and then stained with annexin V/PI, and apoptosis was determined by flow cytometry. The percentages in the lower right quadrant correspond to early apoptotic cells (annexin V positive), whereas percentages in the upper right quadrant correspond to late apoptotic cells (annexin V/PI-positive).

Article Snippet: FITC-labeled annexin V/propidium iodide staining kit for apoptosis was from BD Biosciences (San Jose, CA).

Techniques: Cell Culture, Transfection, SDS Page, Staining, Flow Cytometry, Concentration Assay, TUNEL Assay

Journal:

Article Title: A selective sphingosine kinase 1 inhibitor integrates multiple molecular therapeutic targets in human leukemia

doi: 10.1182/blood-2008-02-138958

Figure Lengend Snippet: Caspase inhibitors and overexpression of Bcl-2 protect against apoptosis induced by SK1-I. U937 cells were treated without or with 10 μM SK1-I (A) or 20 μM SK1-I (B) for the indicated times. Cell lysates were prepared and equal amounts of proteins (20 μg) analyzed by Western blotting with the indicated antibodies. Blots were stripped and reprobed with anti–Mcl-1 to ensure equivalent loading and transfer. (C) U937 cells were pretreated for 30 minutes without or with 20 μM Boc-D-FMK (BOC) or Z-VAD-FMK (ZVAD) and then treated with 20 μM SK1-I or 10 μM etoposide. After 4 hours, apoptosis was determined by flow cytometric analysis of annexin V/PI-stained cells. *P ≤ .01. (D) U937 cells stably expressing vector, Bcl-2, or Bcl-xL were treated with the indicated concentrations of SK1-I for 8 hours. Apoptosis was determined by flow cytometric analysis of annexin V/PI-stained cells. Insets show overexpression of Bcl-2 and Bcl-xL by immunoblotting.

Article Snippet: FITC-labeled annexin V/propidium iodide staining kit for apoptosis was from BD Biosciences (San Jose, CA).

Techniques: Over Expression, Western Blot, Staining, Stable Transfection, Expressing, Plasmid Preparation

Journal:

Article Title: A selective sphingosine kinase 1 inhibitor integrates multiple molecular therapeutic targets in human leukemia

doi: 10.1182/blood-2008-02-138958

Figure Lengend Snippet: SK1-I decreases S1P and increases ceramide levels and S1P protects against SK1-I–induced apoptosis. (A) U937 cells were treated without or with 20 μM SK1-I for 6 hours. Lipids were extracted and sphingosine (So), sphinganine (Sa), sphingosine-1-phosphate (SoP), sphinganine-1-phosphate (SaP), and ceramide species were determined by ESI-MS/MS. Data are means of triplicate determinations and are expressed as picomole of lipid per microgram of DNA. Numbers indicate fatty acid chain length followed by the number of double bonds. C16DH indicates C16-dihydroceramide. (B) U937 cells were treated without or with SK1-I (10, 15, 20 μM) in the absence and presence of S1P (1, 10 μM) for 4 hours. Cells were stained with annexin V/PI and apoptosis was determined by flow cytometry. *P ≤ .01.

Article Snippet: FITC-labeled annexin V/propidium iodide staining kit for apoptosis was from BD Biosciences (San Jose, CA).

Techniques: Tandem Mass Spectroscopy, Staining, Flow Cytometry

Journal:

Article Title: A selective sphingosine kinase 1 inhibitor integrates multiple molecular therapeutic targets in human leukemia

doi: 10.1182/blood-2008-02-138958

Figure Lengend Snippet: SK1-I alters survival signaling. (A-C) U937 cells were cultured in medium containing 2% serum in the presence of 10 μM SK1-I for the indicated times (A); in medium containing 10% serum in the presence of 20 μM SK1-I for the indicated times (B); or with the indicated concentrations of SK1-I for 5 minutes (C). Equal amounts of lysate proteins were resolved by SDS-PAGE and analyzed by Western blotting with antibodies against pAkt, Akt, p-ERK1/2, ERK1/2, p-p38, p-ATF-2, p-JNK, and p-cJun. Blots were stripped and reprobed with antitubulin (A), anti-Akt (B), or anti-ERK1/2 (C) to demonstrate equal loading. (D) Enforced expression of myristoylated Akt protects cells from SK1-I–mediated lethality. U937 cells stably expressing empty vector or constitutively active Akt (myristoylated Akt) were treated with the indicated concentrations of SK1-I for 24 hours. Cells were stained with annexin V/PI and apoptosis was determined by flow cytometry. (Inset) Immunoblot of lysate proteins from duplicate cultures with anti-Akt and antitubulin antibodies.

Article Snippet: FITC-labeled annexin V/propidium iodide staining kit for apoptosis was from BD Biosciences (San Jose, CA).

Techniques: Cell Culture, SDS Page, Western Blot, Expressing, Stable Transfection, Plasmid Preparation, Staining, Flow Cytometry

Journal:

Article Title: A selective sphingosine kinase 1 inhibitor integrates multiple molecular therapeutic targets in human leukemia

doi: 10.1182/blood-2008-02-138958

Figure Lengend Snippet: SK1-I induces apoptosis in primary AML blasts. (A) Primary blasts from 2 patients with AML (FAB classification F2; > 70% blasts) and peripheral blood mononuclear leukocytes from 2 healthy donors (B) were obtained as described in “Cells and cell culture,” cultured in medium containing 10% serum for 24 hours in the absence or presence of the indicated concentrations of SK1-I. Cells were stained with annexin V/PI and apoptosis determined by flow cytometry.

Article Snippet: FITC-labeled annexin V/propidium iodide staining kit for apoptosis was from BD Biosciences (San Jose, CA).

Techniques: Cell Culture, Staining, Flow Cytometry

Journal: American Journal of Translational Research

Article Title: Inhibitory effect of bufalin on retinoblastoma cells (HXO-RB44) via the independent mitochondrial and death receptor pathway

doi:

Figure Lengend Snippet: The effects of bufalin on HXO-RB44 cells. A. HXO-RB44 cells were treated with or without different concentrations of bufalin for 48 or 72 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were carried out as described. Data are presented as means ± standard deviation from three independent experiments. B. Flow cytometric analysis of HXO-RB44 apoptotic cells stained with Annexin V + propidium iodide (PI) after treatment with 0-10-1 μM bufalin, and Z-IETD-FMK or Z-LETD-FMK. All bufalin treated groups showed significant increases in apoptosis compared with the control groups. When the caspase inhibitor Z-LETD-FMK (5 mM) and (or) Z-LETD-FMK (5 mM) were added before exposure to 10-1 μM bufalin for 48 h, the apoptotic rates decreased (*P < 0.05; **P < 0.01).

Article Snippet: Materials used included the Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection Kit (Becton Dickinson, Franklin Lakes, NJ, USA), Hoechst-propidium iodide (PI) staining assay kit (Beyotime Institute of Biotechnology, Shanghai, China); anti-caspase-9, anti-caspase-3, anti-caspase-8, and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window caption a7 Chemical structure of Bufalin.

Techniques: Standard Deviation, Staining