Journal: PLoS ONE

Article Title: Sinomenine Sensitizes Multidrug-Resistant Colon Cancer Cells (Caco-2) to Doxorubicin by Downregulation of MDR-1 Expression

doi: 10.1371/journal.pone.0098560

Figure Lengend Snippet: Then MDR-Caco-2 cells were treated with or without sinomenine (500 µM) and celecoxib (25 µM) for 48 hours. ( A ) immunocytochemistry targeting P65 (green). ( B ) Western blot analysis of sinomenine and celecoxib mediated effect on nuclear translocation of P65 in Caco-2 cells and MDR-Caco-2 cells. Antibody to beta-actin was used to ensure equal loading of protein in each lane. ( C ) The relative band density values in the nuclear expression P65 lanes are described in the bar chat. ( D ) The relative band density values in the cytoplasmic p-IκB-α lanes are described in the bar chat. All the results above are expressed as the means ± SE (n = 3) of three independent experiments. *, P<0.05 and **, P<0.01 compared with MDR-Caco-2 group.

Article Snippet: P-glycoprotein (P-gp) mouse anti-human monoclonal antibody, p-IκB-α (Ser 32/36) and IκB-α rabbit anti-human polyclonal antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.).

Techniques: Immunocytochemistry, Western Blot, Translocation Assay, Expressing

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: 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