Journal: Experimental and therapeutic medicine

Article Title: Scorpion venom component III inhibits cell proliferation by modulating NF-κB activation in human leukemia cells.

doi: 10.3892/etm.2012.548

Figure Lengend Snippet: Figure 5. THP-1 cells were incubated with BAY11-7082 (1 µM) alone or in combination with SVCIII (30 µg/ml) for 24 h. Protein from the total cell lysate was subjected to SDS-PAGE and western blot analysis using anti- IκBα, p-IκBα, cyclin D1 and GAPDH antibodies. Representative results are shown from three independent experiments. *P<0.05, SVCIII-treated groups compared to the control; #P<0.05, SVCIII in combination with BAY11‑7082 groups compared with SVCIII or BAY11‑7082 alone.

Article Snippet: Antibodies to cyclin D1, IκBα and p-IκBα were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Incubation, SDS Page, Western Blot, Control

Journal: British Journal of Pharmacology

Article Title: S timulation of autophagy prevents intestinal mucosal inflammation and ameliorates murine colitis

doi: 10.1111/bph.13860

Figure Lengend Snippet: Primary antibodies used in Western blots

Article Snippet: Results were normalized to β‐actin for total and cytoplasm proteins or nucleolin for nuclear proteins to control for unwanted sources of variation (Impellizzeri et al., 2015 ). table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Antibody Dilution Autophagy p‐mTOR (Ser 2448) (Santa Cruz Biotechnology) 1:1000 p62 (Santa Cruz Biotechnology) 1:1000 LC3 (Sigma‐Aldrich) 1:2000 Inflammation BCl‐10 (Santa Cruz Biotechnology) 1:1000 IκBα (Santa Cruz Biotechnology) 1:1000 p‐IκBα (Cell Signaling) 1:1000 NF‐κB (Invitrogen, Novex by Life Technologies) 1:250 β‐actin (Sigma‐Aldrich) 1:5000 Nucleolin (Sigma‐Aldrich) 1:1500 Open in a separate window Primary antibodies used in Western blots

Techniques: Western Blot

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: Biomolecules

Article Title: Redox-Regulated Pathway of Tyrosine Phosphorylation Underlies NF-κB Induction by an Atypical Pathway Independent of the 26S Proteasome

doi: 10.3390/biom5010095

Figure Lengend Snippet: Pervanadate (PV) stimulation induces tyrosine phosphorylation of IκBα but not its proteolytic degradation. ( A ) ILU-18 cells were either left untreated or treated with Pervanadate (100 μM) or TNF-α (20 ng/mL) for 20 min. At the end of treatment, cytosolic lysates were obtained and 30μg protein from each lysate was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Resolved proteins were detected by Western blotting using antibody to nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) and enhanced chemiluminescence (ECL). The blot was stripped and re-probed with antibody to β-actin to ensure equal protein loading. ( B ) ILU-18 cells were treated with PV (100 μM) for 20 min, with or without prior treatment with Aclacinomycin [Acla] (0.25 μM) for 2 h. At the end of incubation, cells were washed and cytosolic lysates prepared. As controls, cell lysates were made from ILU-18 cells either left untreated or treated for 140 min with 0.25 μM Aclacinomycin alone. Lysates, equalized for 30 μg protein, were resolved using SDS-PAGE, followed by Western blotting using a phospho-specific antibody recognizing IκBα (Tyr-42) or (Tyr-305). After stripping, the blot was re-probed with antibody to β-actin to demonstrate equal protein loading.

Article Snippet: Antibodies for phospho-specific IκBα (Tyr-42) and (Tyr-305) were purchased from ECM Biosciences (Versailles, KY, USA).

Techniques: Phospho-proteomics, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Incubation, Stripping Membranes

Journal: Biomolecules

Article Title: Redox-Regulated Pathway of Tyrosine Phosphorylation Underlies NF-κB Induction by an Atypical Pathway Independent of the 26S Proteasome

doi: 10.3390/biom5010095

Figure Lengend Snippet: Pretreatment with inhibitors of Src and mitogen-activated protein kinase kinase (MEK) but not p38, PI3K, c-Jun N-terminal kinase (JNK) and IkappaB kinase (IKK) complex interfere with PV-induced NF-κB activity. ( A ) ILU-18 cells were pretreated with 75 µM Piceatannol (Pic.) for 45 min or 20 µM PD98059 (PD.) for 30 min followed by activation with 100 µM pervanadate for 18 h. Cells treated with 100 µM pervanadate, 75 µM Piceatannol, or 20 µM PD98059 for 18 h, served as controls. At the end of 18 h, lysates were obtained and luciferase activity was determined employing the Promega luciferase assay kit. Fold induction represents a ratio of luciferase activity obtained in treatment-induced to that of untreated cells. Data represents mean ± standard error obtained from four independent experiments, following normalization. ** Denotes significant difference at p < 0.004, between treatment groups. ( B ) ILU-18 cells were treated with PV (100 μM) for 20 min, with or without prior treatment with Aclacinomycin (0.25 μM) for 2 h. At the end of incubation, cells were washed and cytosolic lysates prepared. As controls, cell lysates were made from ILU-18 cells left untreated or treated 140 min with 0.25 μM Aclacinomycin alone. Lysates, equalized for 30 μg protein, were resolved using SDS-PAGE, followed by Western blotting using antibodies for phospho-Syk (Tyr-352) and, as a control, Syk. ( C ) ILU-18 cells were treated with 100 µM pervanadate for 18 h, with or without pretreatment for 6 h with 2 µM Src Kinase Inhibitor I (SKI-1). As controls, cells were either left untreated or subjected to treatment with 2 µM Src Kinase Inhibitor I for 24 h . As described in (A), a luciferase assay was performed. Normalized data are presented as % activity with fold induction values from PV-treated cells set at 100%. Data represents mean ± S.E. from duplicates derived from two independent experiments. ( D ) ILU-18 cells were treated with 100 µM pervanadate for 20 min, with or without pretreatment for 4 h with 5 µM Src Kinase Inhibitor I (SKI-1). At the end of incubation, cells were washed and cytosolic lysates prepared. As controls, cell lysates were made from ILU-18 cells left untreated or treated 260 min with 5 µM Src Kinase Inhibitor I alone. Lysates, equalized for 30 μg protein, were resolved using SDS-PAGE, followed by Western blotting using a phospho-specific antibody recognizing IκBα (Tyr-42). The blot was stripped and re-probed with antibody to β-actin to ensure equal protein loading. ( E ) ILU-18 cells were treated with 100 µM pervanadate, 100nM SB23580 (SB.—p38 Kinase inhibitor), 10 µM Wortmannin (WRT—PI3K inhibitor), or 40 nM JNK Inhibitor for 18 h. ILU-18 cells were also pretreated for 1 h with 40 nM JNK Inhibitor or for 30 min with either 100nM SB23580 or 10 µM Wortmannin followed by activation with 100 µM pervanadate for 18 h. Fold induction was determined as in (A). Data represents mean ± S.E. from four independent experiments. ( F ) ILU-18 cells were either pretreated with 10 µM IKK-NBD for 1 h or left untreated. After 1 h, cells were either treated with 100 µM PV or 20 ng/mL TNFα for 18 h, yielding the following treatments: Untreated, PV, IKK-NBD+PV, TNFα, IKK-NBD+TNFα. Fold induction was determined as in (A). Data represents mean ± S.E. from duplicates derived from two independent experiments. ** Denotes significant difference at p < 0.001, between treatment groups.

Article Snippet: Antibodies for phospho-specific IκBα (Tyr-42) and (Tyr-305) were purchased from ECM Biosciences (Versailles, KY, USA).

Techniques: Activity Assay, Activation Assay, Luciferase, Incubation, SDS Page, Western Blot, Control, Derivative Assay

Journal: Biomolecules

Article Title: Redox-Regulated Pathway of Tyrosine Phosphorylation Underlies NF-κB Induction by an Atypical Pathway Independent of the 26S Proteasome

doi: 10.3390/biom5010095

Figure Lengend Snippet: PV-mediated activation of NF-κB involves oxidative stress. ( A ) ILU-18 cells were washed and briefly incubated in 1X Hank’s Balanced Salt Solution (HBSS), prior to incubation with 10 μM H 2 DCF-DA for 30 min at 37 °C in the dark. At the end of the incubation, cells were washed and resuspended in 1X HBSS. Intracellular reactive oxygen species (ROS) generation was detected following addition of PV (100 µM) or H 2 O 2 (200 µM), as described in the methods section. ( B ) ILU-18 cells were either untreated or treated with NAC (20 mM) for 2 h. After media replacement, cells were selectively treated with PV (100 μM) for 20 min. At the end of incubation, cells were washed and cytosolic lysates prepared. As controls, cell lysates were made from ILU-18 cells left untreated or treated with 20 mM NAC alone for 140 min. Lysates, equalized for 30 μg protein, were resolved using SDS-PAGE, followed by Western blotting using an antibody specific to Phospho-Tyrosine residues. Molecular weights derived from standards are indicated in kDa. ( C ) Cytosolic lysates (30 µg), obtained as in (B), were resolved using SDS-PAGE, followed by Western blotting using a phospho-specific antibody recognizing IκBα (Tyr-42). After stripping, the blot was re-probed with antibody to β-actin to demonstrate equal protein loading. ( D ) ILU-18 cells were either untreated or treated with NAC (20 mM) for 2 h. After media replacement, cells were selectively treated with PV (100 μM) for 18 h. Additionally, ILU-18 cells were treated with 100 μM H 2 O 2 for 18 h or subjected to treatment with 200 μM BSO for 24 h. Lysates obtained were evaluated for luciferase acitivity, as described previously. Data obtained from at least four independent experiments are presented; values represent data means ± standard error. ** Denotes significant difference at p < 0.001, between treatment groups.

Article Snippet: Antibodies for phospho-specific IκBα (Tyr-42) and (Tyr-305) were purchased from ECM Biosciences (Versailles, KY, USA).

Techniques: Activation Assay, Incubation, SDS Page, Western Blot, Derivative Assay, Stripping Membranes, Luciferase

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