Journal: Journal of Inflammation Research

Article Title: Lycopus lucidus Turcz Exerts Neuroprotective Effects Against H 2 O 2 -Induced Neuroinflammation by Inhibiting NLRP3 Inflammasome Activation in Cortical Neurons

doi: 10.2147/JIR.S305031

Figure Lengend Snippet: LLT protects cortical neurons against H 2 O 2 -induced neuronal damage. ( A ) Schematic depiction of experimental design for primary cortical neurons ( B ) Cell viability assay results of cortical neurons treated with various concentrations of LLT without H 2 O 2 induction at 24 h, n = 3. ( C ) CCK-8 results of H 2 O 2 -treated cortical neurons treated with various concentrations of LLT at 24 h, n = 3. ( D ) The total number of live and dead cells calculated from cellular fluorescence images of each group. n = 10. ( E ) The percentage of apoptotic cells analyzed by flow cytometry of Annexin-V-FITC/PI-PE double staining. n = 5. ( F ) Fluorescence images of live and dead (red) cells for analysis of the neuroprotective effect of LLT at different concentrations on H 2 O 2 -treated cortical neurons. Scale bar = 200 µm. ( G ) Representative flow cytometric plot using Annexin-V/PI to confirm the mode of cell death. Blank = non-treatment, H 2 O 2 = H 2 O 2 only, LLT = H 2 O 2 +LLT extract. Significant differences are indicated as follows and were analyzed via one-way ANOVA with Tukey’s post-hoc test: # p < 0.001 vs the blank group; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs the H 2 O 2 group.

Article Snippet: Apoptotic cell death was detected using an Annexin V-Fluorescein isothiocyanate (FITC) propidium iodide (PI)-phycoerythrin apoptosis detection kit (Abcam) as described previously.

Techniques: Viability Assay, CCK-8 Assay, Fluorescence, Flow Cytometry, Double Staining

Journal: International Journal of Oncology

Article Title: Eupalinolide A induces autophagy via the ROS/ERK signaling pathway in hepatocellular carcinoma cells in vitro and in vivo

doi: 10.3892/ijo.2022.5421

Figure Lengend Snippet: Effect of EA on apoptosis and necroptosis of human hepatocellular carcinoma cells. (A and B) Western blot analysis of the expression levels of apoptosis and necroptosis-related proteins at 48 h in MHCC97-L and HCCLM3 cells. β-actin was used as a control. (C and D) Effect of EA on apoptosis in MHCC97-L and HCCLM3 cells, as determined by flow cytometry. n=5. (E) Viability of MHCC97-L and HCCLM3 cells treated with or without apoptosis or necroptosis inhibitors was measured by Cell Counting Kit 8 assays. n=6. All data are shown as the mean ± SD. *** P<0.001. EA, eupalinolide A; C-, cleaved; p-, phosphorylated.

Article Snippet: The cells were then collected, washed with cold PBS and suspended in 200 μ l binding buffer (Annexin V-FITC/PI apoptosis assay kit; Neobioscience Technology Co., Ltd.) at a density of 5×10 5 /ml.

Techniques: Western Blot, Expressing, Control, Flow Cytometry, Cell Counting

Journal: Cellular signalling

Article Title: The canonical NF-?B pathway differentially protects normal and human tumor cells from ROS-induced DNA damage

doi: 10.1016/j.cellsig.2012.06.010

Figure Lengend Snippet: Suppression of canonical NF-κB signaling sensitizes A549 cells to H2O2-mediated apoptosis. (A) A549 vector and A549 IκBαSR cells were treated with increasing concentrations of H2O2 (0 - 1000 μM) and subjected to Annexin V/PI staining followed by flow cytometric analysis to quantify apoptosis. Values are the mean ± SEM of multiple independent experiments. Results were analyzed by paired two-way ANOVA, followed by Tukey post-hoc comparisons. Statistically significant differences in the viability of A549 vector and IκBαSR cells at each H2O2 dose in comparison to untreated controls (H2O2 = 0) are indicated (*p<0.05). Statistically significant increases or decreases in the viability profiles of A549 IκBαSR cells in comparison to A549 vector controls at the same H2O2 doses are also highlighted in the right side of Panel A (#p<0.05). Both early apoptotic (EA) and late apoptotic-necrotic cells (LA-N) were quantified. FACS gating of cell sub-populations is shown in Fig. S3. (B) Immunoblot analysis for the expression of caspase-3 and PARP-1 in total cell extracts as indicated. (C) Immunoblot analysis of PARP-1 in cytoplasmic and nuclear extracts prepared from untreated and H2O2-treated A549 Vector and A549 IκBαSR cells. β-actin served as a reference control. Immunoblots in (B) and (C) for PARP-1 and caspase-3 were overexposed to detect the cleaved forms of the corresponding proteins. Results are representative of several independent experiments.

Article Snippet: Simultaneous flow cytometric quantification of apoptotic and viable cells was performed with an annexin V/propidium iodide kit (Annexin V-FITC/PI) (Assay Designs, Ann Arbor, Michigan, USA).

Techniques: Plasmid Preparation, Staining, Comparison, Western Blot, Expressing, Control

Journal: Cellular signalling

Article Title: The canonical NF-?B pathway differentially protects normal and human tumor cells from ROS-induced DNA damage

doi: 10.1016/j.cellsig.2012.06.010

Figure Lengend Snippet: Generation and characterization of IKKβ-knockdown A549 cells. (A) A549 cells were stably transduced by a puromycin resistance retrovirsues harboring either a GL2 control or an IKKβ targeted shRNA (IKKβKD). Cells were selected in puromycin and total cell lysates extracted from 12 clones were randomly selected for analysis of IKKβ expression by immunoblotting. One specific A549 IKKβKD clone with >80% IKKβ ablation was chosen for subsequent experiments. (B) Immunoblotting for p53, caspase-3, PARP-1 and β-actin proteins in H2O2-treated A549 GL2 and A549 IKKβKD cells. (C) A549 GL2 and A549 IKKβKD cells were treated with increasing concentrations of H2O2 (0 - 1000 μM) and subjected to Annexin V/PI staining followed by flow cytometric analysis to quantify apoptosis. Values are the mean ± SEM of several independent experiments each performed in triplicate. Results were analyzed by paired two-way ANOVA, followed by Tukey post-hoc comparisons. Significant differences in percentages of live and apoptotic A549 vector and A549 IKKβKD cells induced by increasing H2O2 concentrations in comparison with untreated controls (H2O2 = 0) for each experimental series are indicated (*p<0.05). Significant increases or decreases in the percentages of A549 IKKβKD apoptotic or live cells respectively in comparison to control (GL2) values at the same H2O2 concentration are also shown (#p<0.05).

Article Snippet: Simultaneous flow cytometric quantification of apoptotic and viable cells was performed with an annexin V/propidium iodide kit (Annexin V-FITC/PI) (Assay Designs, Ann Arbor, Michigan, USA).

Techniques: Knockdown, Stable Transfection, Control, shRNA, Clone Assay, Expressing, Western Blot, Staining, Plasmid Preparation, Comparison, Concentration Assay

Journal: Oncology Letters

Article Title: lnc-NLC1-C inhibits migration, invasion and autophagy of glioma cells by targeting miR-383 and regulating PRDX-3 expression

doi: 10.3892/ol.2021.12901

Figure Lengend Snippet: Analysis of apoptosis, ROS level and cell cycle in U87MG cells. *P<0.05 and ***P<0.001. n=3. Lnc-NLC1-C, long non-coding RNA narcolepsy candidate region 1 gene C; ov, overexpression; sh, short hairpin RNA; NC, negative control; ROS, reactive oxygen species.

Article Snippet: After 200 µl binding buffer was added to the cells, 10 µl Annexin V-fluorescein isothiocyanate and 10 µl propidium iodide (part of the AnnexinV-FITC/PI Apoptosis Detection kit; cat. no. 556547; BD Biosciences) were added, and the cells were incubated at 4°C in the dark for 30 min and subjected to flow cytometry (FC500 MCL; Beckman Coulter, Inc.).

Techniques: Over Expression, shRNA, Negative Control