Journal: International journal of molecular medicine

Article Title: Ginsenoside Rg1 exerts a protective effect against Aβ₂₅₋₃₅-induced toxicity in primary cultured rat cortical neurons through the NF-κB/NO pathway.

doi: 10.3892/ijmm.2016.2485

Figure Lengend Snippet: Figure 2. Ginsenoside Rg1 (Rg1) attenuates amyloid β-peptide 25-35 (Aβ25-35)-induced nuclear factor κ-light-chain‑enhancer of activated B cells (NF-κB) activation. Primary cultured neurons were preincubated with 20 µM Rg1 for 24 h, and subsequently treated with 10 µM Aβ25-35 for another 24 h. (A) The fluo rescence images indicated NF-κB translocation to the nucleus after Aβ25-35 treatment (as shown by arrows). Scale bar, 50 µM. (B and C) After stimulation with Aβ25-35 and 150 µM hydrogen peroxide (H2O2) solution for 24 h, the nucleoprotein was extracted using a nuclear and cytoplasmic protein extraction kit. NF-κB expression was detected by western blot analysis. Lamin B was used as a housekeeping protein for the nuclear protein and GAPDH was used for cytoplasmic protein. (D) Phosphorylation of IκB-α was detected by western blot analysis. Data are represented as the means ± standard deviation of three independent experiments. The statistical significance was defined using Student's t-test as *P<0.05, **P<0.01, ***P<0.001 vs. control group, #P<0.05, ##P<0.01 vs. model group. N, nuclear fractions; C, cytoplasmic fractions.

Article Snippet: Primary antibodies against NF-κB (p65) (sc-372), IκB-α (sc-371), phosphorylated (p-)IκB-α (sc-8404), lamin B (sc-6217), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (sc-47724), inducible nitric oxide synthase (iNOS) (sc-651), Bcl-2 (sc-7382), Bax (sc-7480) were all purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Activation Assay, Cell Culture, Translocation Assay, Protein Extraction, Expressing, Western Blot, Phospho-proteomics, Standard Deviation, Control

Journal: Cell Reports Medicine

Article Title: Haploinsufficiency of NFKBIA reshapes the epigenome antipodal to the IDH mutation and imparts disease fate in diffuse gliomas

doi: 10.1016/j.xcrm.2023.101082

Figure Lengend Snippet:

Article Snippet: For near-complete knockdown of NFKBIA , G418-resistant primary human astrocytes transduced to stably express wildtype IDH1 or mutant IDH1- ( R132H ) were transfected with Accell human NFKBIA small interfering (si)RNA or non-targeting control siRNA (Dharmacon), at 20nM concentration using lipofectamine 2000 reagent (Invitrogen) at 1:1 ratio for 48 h. For complete clustered regularly interspaced short palindromic repeats (CRISPR) knockout of NFKBIA , astrocytes were transfected with IκBα Double Nickase Plasmid (h) (sc-400034-NIC, Santa Cruz)—consisting of a pair of plasmids each encoding a D10A mutated Cas9 nuclease and a target-specific 20-nucleotide (nt) guide RNA (gRNA)—using plasmid transfection medium (sc-108062) and Ultra-Cruz transfection reagent (sc-395739) at 1:1.5 ratio and were incubated for 72 h. After incubation, cells were screened for GFP-positivity to select for successfully transfected cells.

Techniques: Plasmid Preparation, Recombinant, Transfection, Activation Assay, Methylation, Marker, DNA Methylation Assay, Sequencing, Empire Assay, Software

Journal: Breast Cancer Research : BCR

Article Title: Hsp27 participates in the maintenance of breast cancer stem cells through regulation of epithelial-mesenchymal transition and nuclear factor-κB

doi: 10.1186/bcr3042

Figure Lengend Snippet: Knockdown of Hsp27 decreased the activity of NF-κB in BCSCs . ( A ) AS-B145 or ALDH+ AS-B244 cells were transfected with negative control siRNA (Ctrl) or Hsp27 siRNA (KD) for 24 h or 48 h and total proteins were harvested to detect the expression of IκBα, phosphor-IκBα (p-IκBα), Hsp27 and β-actin by Western blot. Inserted values indicated relative proteins expression in comparison with ctrl siRNA. ( B ) AS-B145 or ALDH+ AS-B244 cells were transfected with negative control siRNA (Ctrl) or Hsp27 siRNA (KD) for 48 h and nuclear (Nuc) and cytosolic (Cyto) proteins were isolated to detect NF-κB, histone H1, Hsp27 and β-actin by Western blot. Inserted values indicated relative proteins expression in comparison with ctrl siRNA. ( C ) AS-B145 or ALDH+ AS-B244 cells were co-transfected with NF-κB reporter vector, Renilla luciferase vector and negative control siRNA (ctrl-si)/or Hsp27 siRNA (si-Hsp27) for 48 h. Luciferase activity was determined by dual luciferase assay. Data were presented as relative luciferase activity of ctrl siRNA. *, P < 0.05. ( D ) AS-B145 or AS-B244 cells were treated with JSH-23 (20, 10 or 5 μM) or 0.1% DMSO for 48 h and ALDH+ population were determined by ALDEFLUOR assay. Data were presented as relative percentage of DMSO. *, P < 0.05; #, P < 0.01. ALDH, aldehyde dehydrogenase; BCSCs, breast cancer stem cells; NF-κB, nuclear factor kappa B

Article Snippet: The specific siRNA oligos of Hsp27 (sc-29350) or IκBα (sc-29360), or negative control siRNA oligos was purchased from Santa Cruz Biotechnologies, Inc.

Techniques: Knockdown, Activity Assay, Transfection, Negative Control, Expressing, Western Blot, Comparison, Isolation, Plasmid Preparation, Luciferase

Journal: Breast Cancer Research : BCR

Article Title: Hsp27 participates in the maintenance of breast cancer stem cells through regulation of epithelial-mesenchymal transition and nuclear factor-κB

doi: 10.1186/bcr3042

Figure Lengend Snippet: Restoring NF-κB activity attenuates the suppressive effect of Hsp27 knockdown in ALDH+ breast cancer cells . AS-B145 or AS-B244 cells were transfected with negative control siRNA (Ctrl-siRNA, 100 nM), ctrl-siRNA (50 nM)+si-Hsp27 (50 nM) or si-Hsp27 (50 nM) +si-IκBα (50 nM) for 48 h. ( A ) The expression of Hsp27 and IκBα was determined by Western blot. Inserted values indicated relative expression in comparison with ctrl-siRNA. ( B ) The NF-κB activity was determined by co-transfected with NF-κB-Luc reporter vector (0.5 μg) and RLuc (0.05 μg) vector. The results were presented as relative luciferase activity to ctrl-siRNA. ( C, D ) The percentage of ALDH+ population among groups was determined by ALDEFLUOR assay. DEAB was used to set the cutoff line for ALDH+ cells. The quantitative results were presented as relative percentage of negative control siRNA (ctrl-siRNA) group and were shown in bar graph (D). *, P < 0.05; #, P < 0.01. ALDH, aldehyde dehydrogenase; NF-κB, nuclear factor kappa B.

Article Snippet: The specific siRNA oligos of Hsp27 (sc-29350) or IκBα (sc-29360), or negative control siRNA oligos was purchased from Santa Cruz Biotechnologies, Inc.

Techniques: Activity Assay, Knockdown, Transfection, Negative Control, Expressing, Western Blot, Comparison, Plasmid Preparation, Luciferase

Journal: Oncology reports

Article Title: Diallyl disulfide inhibits the metastasis of type Ⅱ esophageal‑gastric junction adenocarcinoma cells via NF-κB and PI3K/AKT signaling pathways in vitro.

doi: 10.3892/or.2017.6113

Figure Lengend Snippet: Figure 10. Inhibitory effects of DADS on protein levels of the NF-κB pathway in OE19 cells. OE19 cells were treated with various concentrations of DADS (0, 2.5, 5 and 10 µg/ml) for 24 h. (A) Representative western blot of protein levels of nuclear NF-κB p65, cytoplasmic NF-κB p65, IκBα and p-IκBα is shown. (B) Densitometric analysis of the protein expression of nuclear NF-κB p65, cytoplasmic NF-κB p65, IκBα and p-IκBα in OE19 cells treated with DADS is shown. *P<0.05, **P<0.01 compared with the control group. DADS, diallyl disulfide.

Article Snippet: Primary antibodies against u-PA mouse antibody (sc-59729), NF-κB (p65) mouse antibody (sc-71675), IκBα mouse antibody (sc-52900), p-IκBα mouse antibody (sc-52943) and β-actin mouse antibody (sc-47778), as well as HRP-conjugated goat anti-mouse and anti-rabbit secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, uSA).

Techniques: Western Blot, Expressing, Control