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: IκB-α Double Nickase Plasmid (h) , Santa Cruz , Cat# sc-400034-NIC.

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

Journal: Oncotarget

Article Title: Transcriptional downregulation of microRNA-19a by ROS production and NF-κB deactivation governs resistance to oxidative stress-initiated apoptosis

doi: 10.18632/oncotarget.20235

Figure Lengend Snippet: (A) Western blotting analysis of CYLD protein levels in PC12 cells transfected with small interfering RNA (siRNA) targeting CYLD (CYLD.siRNA) in the absence or presence of BAY 11-7085 administration. (B) Western blotting analysis of CYLD protein levels in PC12 cells transfected with vector expressing Flag-tagged wild-type CYLD (Flag-CYLD) in the absence or presence of IκBα.siRNA transfection. (C) MTT assay measuring cell viability of CYLD.siRNA-expressed PC12 cells exposed to CoCl 2 (left panel) or H 2 O 2 (right panel)at the indicated concentrations with or without BAY 11-7085 administration. Experiments were performed three times and data are expressed as mean ± s.d. * p < 0.05, ** p < 0.01 versus Ctrl.siRNA; # p < 0.05 versus CYLD.siRNA, one-way ANOVA, post hoc comparisons, Tukey’s test. (D) MTT assay measuring cell viability of Flag-CYLD-expressed PC12 cells exposed to CoCl 2 (left panel) or H 2 O 2 (right panel) at the indicated concentrations with or without IκBα.siRNA transfection. Experiments were performed three times and data are expressed as mean ± s.d. * p < 0.05 versus vector; # p < 0.05 versus Flag-CYLD, one-way ANOVA, post hoc comparisons, Tukey’s test. (E and F) Representative histograms and quantification of flow cytometry with Annexin-V/PI staining in PC12 cells exposed to 0.6 mmol/L CoCl 2 (E) or 0.4 mmol/L H 2 O 2 (F) with CYLD.siRNA transfection in the absence or presence of BAY 11-7085 administration and with Flag-CYLD transfection in the absence or presence of IκBα.siRNA transfection, respectively. Experiments were performed three times and data are expressed as mean ± s.d. * p < 0.05, one-way ANOVA, post hoc comparisons, Tukey’s test.

Article Snippet: For siRNA and plasmid DNA transfection, cells were transfected with specific small interfering RNA (siRNA) duplex oligonucleotides targeting CYLD (GenePharma, Shanghai, China), RelA siRNA (Santa Cruz, CA), IκBα siRNA (Santa Cruz, CA) or pReceiver-M11 vector expressing CYLD (GeneCopoeia, Rockville, USA) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions as previously described [ ].

Techniques: Western Blot, Transfection, Small Interfering RNA, Plasmid Preparation, Expressing, MTT Assay, Flow Cytometry, Staining

Journal: Oncotarget

Article Title: Transcriptional downregulation of microRNA-19a by ROS production and NF-κB deactivation governs resistance to oxidative stress-initiated apoptosis

doi: 10.18632/oncotarget.20235

Figure Lengend Snippet: (A) Western-blotting analyses comparing the levels of IKKβ phosphorylation and total IκBα expression in PC12 cells treated with 0.6 mmol/L CoCl 2 in the presence of miR-19a mimics transfection and miR-19a mimics plus Flag-tagged wild-type CYLD cotransfection, respectively. (B) Coimmunoprecipitation assays examining the interaction between RelA and IκBα in PC12 cells treated with 0.6 mmol/L CoCl 2 in the presence of miR-19a mimics transfection and miR-19a mimics plus Flag-tagged wild-type CYLD cotransfection, respectively. IP, immunoprecipitation; WB, western-blotting. (C) Cellular ubiquitination assays comparing the poly-Ub levels of IκBα in PC12 cells treated with 0.6 mmol/L CoCl 2 in the presence of miR-19a mimics transfection and miR-19a mimics plus Flag-tagged wild-type CYLD cotransfection, respectively. PD, pull-down. (D) Western-blotting analyses detecting the levels of nuclear RelA accumulation in PC12 cells treated with 0.6 mmol/L CoCl 2 in the presence of miR-19a mimics transfection and miR-19a mimics plus Flag-tagged wild-type CYLD cotransfection, respectively. (E) ChIP analysis for RelA binding to VEGFA gene promoter in PC12 cells exposed to 0.6 mmol/L CoCl 2 and 0.4 mmol/L H 2 O 2 in the presence of miR-19a mimics transfection and miR-19a mimics plus Flag-tagged wild-type CYLD cotransfection, respectively. Enrichment of promoter region was normalized by input and data are expressed as mean ± s.d. of at least three experiments. * p < 0.05; ** p < 0.01, one-way ANOVA, post hoc comparisons, Tukey’s test. (F) ELISA assay for VEGF release from PC12 cell cultures exposed to 0.6 mmol/L CoCl 2 and 0.4 mmol/L H 2 O 2 in the presence of miR-19a mimics transfection and miR-19a mimics plus Flag-tagged wild-type CYLD cotransfection, respectively. Enrichment of promoter region was normalized by input and data are expressed as mean ± s.d. of at least three experiments. * p < 0.05 versus PBS; *** p < 0.001 versus CoCl 2 or H 2 O 2 plus miR-19a, one-way ANOVA, post hoc comparisons, Tukey’s test.

Article Snippet: For siRNA and plasmid DNA transfection, cells were transfected with specific small interfering RNA (siRNA) duplex oligonucleotides targeting CYLD (GenePharma, Shanghai, China), RelA siRNA (Santa Cruz, CA), IκBα siRNA (Santa Cruz, CA) or pReceiver-M11 vector expressing CYLD (GeneCopoeia, Rockville, USA) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions as previously described [ ].

Techniques: Western Blot, Phospho-proteomics, Expressing, Transfection, Cotransfection, Immunoprecipitation, Ubiquitin Proteomics, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: Transcriptional downregulation of microRNA-19a by ROS production and NF-κB deactivation governs resistance to oxidative stress-initiated apoptosis

doi: 10.18632/oncotarget.20235

Figure Lengend Snippet: (A) ChIP analysis for RelA binding to miR-19a promoter in PC12 cells transfected with control.siRNA (Ctrl.siRNA) and IκBα.siRNA, respectively. Enrichment of promoter region was normalized by input and data are expressed as mean ± s.d. of at least three experiments. ** p < 0.01. Two-sided Student’s t test was used to calculate the P value. (B) Western-blotting examining abundance of IκBα protein expression in PC12 cells transfected with control.siRNA (Ctrl.siRNA) and IκBα.siRNA, respectively. (C) RT-qPCR comparing levels of miR-19a mRNA expression in PC12 cells treated with CoCl 2 (left panel) or H 2 O 2 (right panel)for the indicated times in the presence of control.siRNA (Ctrl.siRNA) or IκBα.siRNA transfection. Experiments were performed five times, each with quantitative RT-PCR in technical duplicate and real-time values were normalized to RNU6b. Data are expressed as mean ± s.d. * p < 0.05, ** p < 0.01 versus IκBα.siRNA, one-way ANOVA, post hoc comparisons, Tukey’s test. (D) Luciferase assays of miR-19a promoter activity in PC12 cells treated with CoCl 2 (left panel) or H 2 O 2 (right panel)in the presence of control.siRNA (Ctrl.siRNA) or IκBα.siRNA transfection. Experiments were performed three times and data are expressed as mean ± s.d. * p < 0.05, ** p < 0.01 versus Ctrl.siRNA; # p < 0.05 versus Ctrl.siRNA plus CoCl 2 or H 2 O 2 , one-way ANOVA, post hoc comparisons, Tukey’s test. (E) RT-qPCR evaluating levels of miR-19a mRNA expression in CoCl 2 -treated PC12 cells with N-acetylcysteine (NAC) treatment in the presence or absence of BAY 11-7085 administration. Experiments were performed five times, each with quantitative RT-PCR in technical duplicate and real-time values were normalized to RNU6b. Data are expressed as mean ± s.d. (F) RT-qPCR comparing levels of miR-19a mRNA expression in CoCl 2 -treated PC12 cells with IκBα.siRNA transfection in the presence or absence of N-acetylcysteine (NAC) treatment. Experiments were performed five times, each with quantitative RT-PCR in technical duplicate and real-time values were normalized to RNU6b. Data are expressed as mean ± s.d.

Article Snippet: For siRNA and plasmid DNA transfection, cells were transfected with specific small interfering RNA (siRNA) duplex oligonucleotides targeting CYLD (GenePharma, Shanghai, China), RelA siRNA (Santa Cruz, CA), IκBα siRNA (Santa Cruz, CA) or pReceiver-M11 vector expressing CYLD (GeneCopoeia, Rockville, USA) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions as previously described [ ].

Techniques: Binding Assay, Transfection, Control, Western Blot, Expressing, Quantitative RT-PCR, Luciferase, Activity Assay

Journal: Oncotarget

Article Title: Transcriptional downregulation of microRNA-19a by ROS production and NF-κB deactivation governs resistance to oxidative stress-initiated apoptosis

doi: 10.18632/oncotarget.20235

Figure Lengend Snippet: (A) PC12 cells with miR-19a mimics transfection were treated with 0.6 mmol/L CoCl 2 (left panel) or 0.4 mmol/L H 2 O 2 (right panel) for the indicated times in the presence or absence of 100 ng/mL VEGF pretreatment and the cell viabilities were measured by MTT assay. Experiments were performed three times and data are expressed as mean ± s.d. ** p < 0.01 versus control; # p < 0.05 versus miR-19a, one-way ANOVA, post hoc comparisons, Tukey’s test. (B) Caspase-3 activity assays of miR-19a-expressed PC12 cells treated with CoCl 2 (left panel) or 0.4 mmol/L H 2 O 2 (right panel) for 24h in the presence or absence of 100 ng/mL VEGF pretreatment. Experiments were performed three times and data are expressed as mean ± s.d. * p < 0.05, one-way ANOVA, post hoc comparisons, Tukey’s test. (C) Representative pictures (top panel) and quantification (bottom panel) from Hoechst and PI double-staining assay of miR-19a-expressed PC12 cells treated with CoCl 2 (left panel) or 0.4 mmol/L H 2 O 2 (right panel) for 24h in the presence or absence of 100 ng/mL VEGF pretreatment. Data are expressed as mean ± s.d. * p < 0.05; ** p < 0.01; *** p < 0.001, one-way ANOVA, post hoc comparisons, Tukey’s test. (D) Cellular ubiquitination assays comparing the poly-Ub levels of IκBα in miR-19a-expressed PC12 cells treated with CoCl 2 (left panel) or 0.4 mmol/L H 2 O 2 (right panel) in the presence or absence of 100 ng/mL VEGF pretreatment. (E) Proposed schematic illustrating a pivotal role for miR-19a in promoting cell survival under OS by CYLD repression-mediated and NF-κB transactivation-dependent regulatory feedback loop.

Article Snippet: For siRNA and plasmid DNA transfection, cells were transfected with specific small interfering RNA (siRNA) duplex oligonucleotides targeting CYLD (GenePharma, Shanghai, China), RelA siRNA (Santa Cruz, CA), IκBα siRNA (Santa Cruz, CA) or pReceiver-M11 vector expressing CYLD (GeneCopoeia, Rockville, USA) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions as previously described [ ].

Techniques: Transfection, MTT Assay, Control, Activity Assay, Double Staining, Ubiquitin Proteomics

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