Journal: Oncology Letters

Article Title: Hedyotis diffusa willd extract suppresses colorectal cancer growth through multiple cellular pathways

doi: 10.3892/ol.2017.7244

Figure Lengend Snippet: EEHDW inhibits cellular proliferation and induces apoptosis in xenograft tumors. (A) Staining for Ki-67 and TUNEL were performed to examine the in vivo effect of EEHDW on proliferation and apoptosis. The photographs are representative images captured at a magnification of ×400. The percentage of positively stained cells was also quantified. To rule out any non-specific staining, PBS was used to replace the primary antibody as a negative control. Data are expressed as the mean ± standard deviation from 10 individual mice in each group. *P<0.05 vs. control mice. (B) A total of three tumors were randomly selected from each group, and the level of cytochrome C, caspase-3, −9 and PARP in tumor tissues was determined by western blotting to examine the in vivo effect of EEHDW on apoptosis. β-actin was used as the internal control. For each tumor sample, western blotting was performed in triplicate. In densitometric analysis, the expression of the target proteins was normalized to the mean protein expression of control. *P<0.05, vs. control mice. EEHDW, ethanol extract of Hedyotis Diffusa Willd. TUNEL, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling; PARP, poly(ADP-ribose)polymerase 1.

Article Snippet: TumorTACS in situ Apoptosis kit (catalog no. 4815–30-K) was purchased from R&D Systems Inc. (Minneapolis, MN, USA).

Techniques: Staining, TUNEL Assay, In Vivo, Negative Control, Standard Deviation, Control, Western Blot, Expressing, End Labeling

Journal: Gene therapy

Article Title: Tumor cells expressing a fusion protein of MULT1 and Fas are rejected in vivo by apoptosis and NK cell activation.

doi: 10.1038/gt.2008.77

Figure Lengend Snippet: Figure 3 Mouse UL16-binding protein-like transcript 1 E (MULT1E)/FasTI induces apoptosis. A total of 1 106 cells of TC-1 and clones L-5, L-7 and L-10 were treated with 1 mg/ml NKG2D/Fc for 16 h. The cells were then analyzed for apoptosis and necrosis using Annexin V assay (a–c) or caspase-3 assay (d) according to the manufacturers’ protocols. (a) An example of the fluorescence-activated cell sorting (FACS) data. (b, c) Summaries of data from three separate experiments. The statistical analyses were conducted between the controls (open bars) and NKG2D/Fc-treated cells (solid bars) using two-way analysis of variance (ANOVA). The difference between NKG2D/Fc-treated L-5 cells and NKG2D/Fc-treated L-10 cells was also compared using Student’s t-test. *Po0.05; **Po0.01 and ***Po0.001.

Article Snippet: Induction of apoptosis in cells expressing the fusion protein To determine if cells expressing the fusion protein can be induced to undergo apoptosis, one million cells of TC-1 and clones L-5, L-7 and L-10 were treated with 1 mg/ml of NKG2D/Fc for 16 h. Apoptosis of the cells was measured using two systems: a TACS Annexin V-FITC Apoptosis Kit (R&D Systems) and a caspase-3 fluorometric assay (R&D Systems).

Techniques: Binding Assay, Clone Assay, Annexin V Assay, Caspase-3 Assay, FACS

Journal: Cancer Discovery

Article Title: A Ubiquitination Cascade Regulating the Integrated Stress Response and Survival in Carcinomas

doi: 10.1158/2159-8290.CD-22-1230

Figure Lengend Snippet: Validation of BIRC6 dependency in vitro and in vivo. A, Consequences of CRISPR-mediated BIRC6 knockout on cell viability. Five putatively dependent cells and six putatively nondependent cells [as defined by Chronos score (see Methods)], all of which constitutively express Cas9, were analyzed using an ATP-based assay 7 days after transducing an sgRNA against BIRC6 (three different sgRNA sequences were tested). Viability scores relative to the average viability of cells transduced with cutting control sgRNAs and the average viability of cells with knockout (KO) of common essential genes are shown. Values = means ± SD ( n = 9). ****, P < 0.0001 (dependent vs. nondependent; for each guide). B, Consequences of CRISPR interference (CRISPRi)–mediated BIRC6 knockdown on long-term cell fitness. Clonogenic growth of the cells was evaluated 14 days after the transduction of an all-in-one CRISPRi construct targeting the indicated gene. Two sgRNA sequences against BIRC6 were tested. Presented are the representative images of cells with crystal violet staining (left) and the mean staining intensities per sample ( n = 3, right). *, P < 0.05; ****, P < 0.0001 (sgCiCh2-2 vs. sgCiBIRC6). C and D, Cell cycle ( C ) and cell death ( D ) analysis following BIRC6 knockout. Cas9-expressing derivatives of indicated cells were transduced with a cutting control sgRNA (sgCh2-2) or an sgRNA targeting BIRC6 (sgBIRC6-1, sgBIRC6-4). Cells were harvested 4 ( C ) or 7 ( D ) days later, stained, and analyzed by flow cytometry. In C , the proportion of cells in the S-phase was reduced upon BIRC6 knockout in the three dependent models, but not in the three nondependent models. In D , the proportion of dead cells (late apoptosis + nonapoptotic death + early apoptosis) was increased following the knockout of BIRC6 in all of the three dependent cell lines, but only one of the three nondependent cell lines. ns, P ≥ 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 ( n = 3). E–G, In vivo validation of the BIRC6 dependency. In E , ZR751 breast cancer cells expressing a doxycycline (DOX)-inducible shRNA against BIRC6 (shBIRC6–2) were implanted into the mammary fat pads of NRG mice. Following tumor formation, some of these mice were treated with DOX, while others were left untreated. In F and G , KYSE450 esophagus cancer cells ( F ) and HCC95 lung cancer cells ( G ), both of which were engineered to express an sgRNA against BIRC6 in a tamoxifen (TAM)-inducible fashion, were implanted subcutaneously via intraperitoneal injection (IP) into the NSG (NOD- scid Il2rg −/− ) mice. Following tumor formation, some mice were injected with TAM, while others were treated with vehicle control. In both cases, the tumor growth is plotted to compare the two different groups of mice. Data are represented as means ± SEM [ n = 8 (Keep w/o TAM group, G ), 9 (Keep w/o TAM and TAM(-) groups, F ; TAM hereafter group, G ), 10 (Keep w/o DOX and DOX(-) groups, E ; TAM hereafter and TAM (+) groups, F ; TAM(-) and TAM(+) groups, G ), 12 (DOX hereafter and DOX (+) groups, E )]. ns, P ≥ 0.05; ****, P < 0.0001 (for each of the last five time points for the tumor growth curves). All the experiments were performed twice except for A , which was conducted three times, and E – G , which were conducted once.

Article Snippet: Subsequently, 7 days after the lentiviral transduction, cells were collected and labeled withFITC-tagged Annexin V and propidium iodide (PI) using the TACS Annexin V-FITC Apoptosis Detection Kit (R&D Systems, catalog no. 4830–250-K).

Techniques: Biomarker Discovery, In Vitro, In Vivo, CRISPR, Knock-Out, ATP Assay, Transduction, Control, Knockdown, Construct, Staining, Expressing, Flow Cytometry, shRNA, Injection