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

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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: International Journal of Molecular Medicine

Article Title: MicroRNA-199a-5p functions as a tumor suppressor in oral squamous cell carcinoma via targeting the IKKβ/NF-κB signaling pathway

doi: 10.3892/ijmm.2019.4083

Figure Lengend Snippet: IKKβ is a direct target of miR-199a-5p. (A) miR-199a-5p sequence is shown to be highly conserved among species. (B) Putative binding sites of miR-199a-5p and IKKβ. (C) Luciferase assay of 293T cells co-transfected with firefly luciferase constructs containing the IKKβ wt or mut 3′-UTRs and miR-199a-5p mimics, mimics NC, miR-199a-5p inhibitor or inhibitor NC, as indicated (n=3). Data are presented as the mean ± standard deviation of three independent experiments. ** P<0.01 vs. mimics NC, ## P<0.01 vs. inhibitor NC. (D) Protein expression of IKKβ following transfection with miR-199a-5p mimics or miR-199a-5p inhibitor was measured by western blot analysis. ** P<0.01 vs. mimics NC, ## P<0.01 vs. inhibitor NC. (E) Expression of IKKβ was assessed by immunohistochemistry in OSCC tissues and matched tumor-adjacent tissues (magnification, x200). (F) Expression was of IKKβ examined in four OSCC cell lines (SCC-25, CAL-27, Tca8113 and SCC-4) and HOK cells, used as a control, by RT-qPCR analysis. Data are presented as the mean ± standard deviation of three independent experiments. ** P<0.01 vs. HOK cells. (G) Expression of IKKβ was measured by RT-qPCR analysis in OSCC tissues and matched tumor-adjacent tissues (n=60). ** P<0.01 vs. Normal group. (H) Spearman's rank correlation analysis revealed a negative correlation between the expression of IKKβ and miR-199a-5p (r=-0.6512, P<0.01). miR, microRNA; 3′-UTR, 3′-untranslated region; wt, wild-type; mut, mutant; NC, negative control; IKKβ, inhibitor of nuclear factor-κB kinase β; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; HOK, Human Oral Keratinocyte.

Article Snippet: The membranes were blocked for 1 h with 5% non-fat milk at room temperature and then incubated with primary antibodies against IKKβ (cat no. 8943; 1:1,000 dilution; Cell Signaling Technology, Inc., Danvers, MA, USA), total p65 (cat. no. 8242; 1:1,000 dilution; Cell Signaling Technology, Inc.), nuclear phosphorylated (p-)p65 (cat. no. 3033; 1:1,000 dilution; Cell Signaling Technology, Inc.), inhibitor of NF-κB (IκB)-α (cat. no. sc-52900; 1:1,000 dilution; Santa Cruz Biotechnology, Inc.), p-IκB-α (cat. no. 2859; 1:1,000 dilution; Cell Signaling Technology, Inc.), Histone H3 (cat. no. 9728; 1:1,000 dilution; Cell Signaling Technology, Inc.), β-actin (cat. no. 4970; 1:2,000 dilution; Cell Signaling Technology, Inc.) and α-tubulin (cat. no. ab7291; 1:2,000; Abcam) at 4°C overnight.

Techniques: Sequencing, Binding Assay, Luciferase, Transfection, Construct, Standard Deviation, Expressing, Western Blot, Immunohistochemistry, Control, Quantitative RT-PCR, Mutagenesis, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Medicine

Article Title: MicroRNA-199a-5p functions as a tumor suppressor in oral squamous cell carcinoma via targeting the IKKβ/NF-κB signaling pathway

doi: 10.3892/ijmm.2019.4083

Figure Lengend Snippet: Overexpression of IKKβ abrogates the inhibitory effects of miR-199a-5p mimics on cell proliferation and apoptosis. Tca8113 and SCC-4 cells were co-transfected with miR-199a-5p mimics, mimics NC, pcDNA-IKKβ and pcDNA-vector for 48 h, and the cells were used for further analysis. (A) Protein levels of IKKβ were detected by western blot analysis. (B) Protein bands were analyzed semi-quantitatively using ImageJ software, normalized to β-actin density. (C) Cell viability was measured using a Cell Counting Kit-8 assay. (D) Apoptosis was detected by flow cytometry. Data are presented as the mean ± standard deviation of three independent experiments. * P<0.05 and ** P<0.01 vs. mimics NC group, ## P<0.01 vs. miR-199a-5p + pcDNA-vector group. miR, microRNA; NC, negative control; IKKβ, inhibitor of nuclear factor-κB kinase β.

Article Snippet: The membranes were blocked for 1 h with 5% non-fat milk at room temperature and then incubated with primary antibodies against IKKβ (cat no. 8943; 1:1,000 dilution; Cell Signaling Technology, Inc., Danvers, MA, USA), total p65 (cat. no. 8242; 1:1,000 dilution; Cell Signaling Technology, Inc.), nuclear phosphorylated (p-)p65 (cat. no. 3033; 1:1,000 dilution; Cell Signaling Technology, Inc.), inhibitor of NF-κB (IκB)-α (cat. no. sc-52900; 1:1,000 dilution; Santa Cruz Biotechnology, Inc.), p-IκB-α (cat. no. 2859; 1:1,000 dilution; Cell Signaling Technology, Inc.), Histone H3 (cat. no. 9728; 1:1,000 dilution; Cell Signaling Technology, Inc.), β-actin (cat. no. 4970; 1:2,000 dilution; Cell Signaling Technology, Inc.) and α-tubulin (cat. no. ab7291; 1:2,000; Abcam) at 4°C overnight.

Techniques: Over Expression, Transfection, Plasmid Preparation, Western Blot, Software, Cell Counting, Flow Cytometry, Standard Deviation, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: MicroRNA-199a-5p functions as a tumor suppressor in oral squamous cell carcinoma via targeting the IKKβ/NF-κB signaling pathway

doi: 10.3892/ijmm.2019.4083

Figure Lengend Snippet: miR-199a-5p inhibits the IKKβ-mediated activation of the NF-κB pathway. Tca8113 and SCC-4 cells were transfected with the miR-199a-5p mimics or mimics-NC for 48 h, and were used for western blot and NF-κB activity assays. (A) Levels of nuclear p-p65, cytoplasm-p-p65, total p65, p-IκB-α and IκB-α were measured by western blot analysis in the whole cell lysate (upper), cytoplasm (middle) and nuclei (lower). β-actin protein was used as the inner control of total proteins; α-tubulin and Histone H3 protein was used as the inner control of the cytoplasmic and nuclear proteins, respectively. (B) Phosphorylation levels of IκB-α were quantified as (p-IκB-α/control)/(total IκB-α/control). Expression levels of p-p65 in the (C) cytoplasm and (D) nucleus were quantified. α-tubulin protein was used as the inner control of the cytoplasmic proteins; Histone H3 protein was used as the inner control of the nuclear proteins. (E) NF-κB activity was quantified using a Promega luciferase assay kit. Data are presented as the mean ± standard deviation of three independent experiments. * P<0.05 and ** P<0.01 vs. mimics NC group; ## P< 0.01 vs. miR-199a-5p mimics group. miR, microRNA; NC, negative control; NF-κB, nuclear factor-κB; IκB-α, inhibitor of NF-κB-α; IKKβ, inhibitor of NF-κB kinase β; p-, phosphorylated.

Article Snippet: The membranes were blocked for 1 h with 5% non-fat milk at room temperature and then incubated with primary antibodies against IKKβ (cat no. 8943; 1:1,000 dilution; Cell Signaling Technology, Inc., Danvers, MA, USA), total p65 (cat. no. 8242; 1:1,000 dilution; Cell Signaling Technology, Inc.), nuclear phosphorylated (p-)p65 (cat. no. 3033; 1:1,000 dilution; Cell Signaling Technology, Inc.), inhibitor of NF-κB (IκB)-α (cat. no. sc-52900; 1:1,000 dilution; Santa Cruz Biotechnology, Inc.), p-IκB-α (cat. no. 2859; 1:1,000 dilution; Cell Signaling Technology, Inc.), Histone H3 (cat. no. 9728; 1:1,000 dilution; Cell Signaling Technology, Inc.), β-actin (cat. no. 4970; 1:2,000 dilution; Cell Signaling Technology, Inc.) and α-tubulin (cat. no. ab7291; 1:2,000; Abcam) at 4°C overnight.

Techniques: Activation Assay, Transfection, Western Blot, Activity Assay, Control, Phospho-proteomics, Expressing, Luciferase, Standard Deviation, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: MicroRNA-199a-5p functions as a tumor suppressor in oral squamous cell carcinoma via targeting the IKKβ/NF-κB signaling pathway

doi: 10.3892/ijmm.2019.4083

Figure Lengend Snippet: Schematic diagrams showing that miR-199a-5p is downregulated in OSCC tissues and cell lines, and miR-199a-5p acts as tumor suppressor that inhibits NF-κB signaling pathways by targeting IKKβ, thereby inhibiting the progression of OSCC. miR, microRNA; OSCC, oral squamous cell carcinoma; NF-κB, nuclear factor-κB; IKKβ, inhibitor of NF-κB kinase β.

Article Snippet: The membranes were blocked for 1 h with 5% non-fat milk at room temperature and then incubated with primary antibodies against IKKβ (cat no. 8943; 1:1,000 dilution; Cell Signaling Technology, Inc., Danvers, MA, USA), total p65 (cat. no. 8242; 1:1,000 dilution; Cell Signaling Technology, Inc.), nuclear phosphorylated (p-)p65 (cat. no. 3033; 1:1,000 dilution; Cell Signaling Technology, Inc.), inhibitor of NF-κB (IκB)-α (cat. no. sc-52900; 1:1,000 dilution; Santa Cruz Biotechnology, Inc.), p-IκB-α (cat. no. 2859; 1:1,000 dilution; Cell Signaling Technology, Inc.), Histone H3 (cat. no. 9728; 1:1,000 dilution; Cell Signaling Technology, Inc.), β-actin (cat. no. 4970; 1:2,000 dilution; Cell Signaling Technology, Inc.) and α-tubulin (cat. no. ab7291; 1:2,000; Abcam) at 4°C overnight.

Techniques: Protein-Protein interactions