Journal: International Journal of Molecular Sciences

Article Title: Resveratrol Modulates Chemosensitisation to 5-FU via β1-Integrin/HIF-1α Axis in CRC Tumor Microenvironment

doi: 10.3390/ijms24054988

Figure Lengend Snippet: Resveratrol’s reduction of inflammation, vascularisation as well as cancer stemness and elevation of apoptosis via β1-integrin receptors in HCT-116/HCT-116R cells shown by Western blot analysis. X -axis: HCT-116 ( A ) and HCT-116R ( B ) cells in alginate drops were left untreated alone (Co.) or in TME, where they were left untreated or were treated with 2 nM 5-FU, 5 µM resveratrol, 0.5 µM β1-SO, 0.5 µM β1-ASO or combinations thereof. Samples were immunoblotted with antibodies against NF-kB (unphosphorylated NF-kB), p-NF-kB (phosphorylated NF-kB), cleaved-caspase-3, HIF-1α, VEGF, CD44, CD133, ALDH1 and β-actin (loading control). Y -axis shows densitometric units. Relative to TME control, values were p < 0.05 (⋆) and p < 0.01 (⋆⋆).

Article Snippet: The monoclonal antibodies against phospho-p65-NF-kB (#MAB7226), p65-NF-kB (#MAB5078) as well as polyclonal anti-cleaved-caspase-3 (#AF835) were acquired from R&D Systems (Heidelberg, Germany), while monoclonal antibody against β1-integrin (#14-0299-82) was from Thermo Fisher Scientific (Langenselbold, Germany).

Techniques: Western Blot, Control

Journal: Journal of neuropathology and experimental neurology

Article Title: Linear Polyubiquitin Chain Modification of TDP-43-Positive Neuronal Cytoplasmic Inclusions in Amyotrophic Lateral Sclerosis.

doi: 10.1093/jnen/nlz135

Figure Lengend Snippet: FIGURE 4. Immunohistochemical localizations of linear polyubiquitin chain (L-Ub), phosphorylated NF-jB p65 (P-p65), and optineurin in the spinal motor neurons from patients with amyotrophic lateral sclerosis (ALS). Immunohistochemistry on the serial sections of the neuronal cytoplasmic inclusion (NCIs) of a spinal motor neuron from a patient with ALS, using antibodies for L-Ub (A; LUB6), P-p65 (B; No. 600-401-265), and optineurin (C; No. 100000). All the antibodies evidently detect the NCI formed in the same neuron. Scale bars: 20 lm. Double immunofluorescence analysis of spinal motor neurons from a patient with ALS, using antibodies against L-Ub (D; LUB6) and optineurin (E; No. 100000; F; merged). The parts of the NCI where immunoreactivity for L-Ub is robust also express optineurin reactivity (arrows). In contrast, the parts showing weak L-Ub signal lack optineurin immunoreactivity (arrowheads). Scale bars: 10 lm.

Article Snippet: We used the following primary antibodies: rabbit polyclonal antiubiquitin (U5379; 1:50; Sigma-Aldrich, St Louis, MO), rabbit monoclonal antiK48Ub (No. 8081, clone D9D5; 1:200; Cell signaling technology, Danvers, MA), mouse monoclonal antiK63-Ub (No. BMLPW0600-0100, clone HWA4C4; 1:200; Enzo Life Sciences, Farmingdale, NY), rabbit monoclonal antiK63-Ub (No. 5621, clone D7A11; 1:2000; Cell Signaling Technology), mouse monoclonal antiL-Ub (LUB6; 1:150; gift from JT Inc.) (21), rabbit polyclonal antiHOIL-1L interacting protein (HOIP) (ab187976; 1:200; Abcam, Cambridge, UK), rabbit polyclonal antiSHANK-associated RH domain-interacting protein (SHARPIN) (HPA044453; 1:50; Sigma-Aldrich), rabbit polyclonal antiphosphorylated NF-jB p65 (P-p65; No. 600-401- 265; 1:200; Rockland Antibodies & Assays, Limerick, PA), and rabbit polyclonal antioptineurin (No. 100000; 1:800; Cayman Chemicals, Ann Arbor, MI).

Techniques: Immunohistochemical staining, Immunohistochemistry, Immunofluorescence

Journal: Journal of neuropathology and experimental neurology

Article Title: Linear Polyubiquitin Chain Modification of TDP-43-Positive Neuronal Cytoplasmic Inclusions in Amyotrophic Lateral Sclerosis.

doi: 10.1093/jnen/nlz135

Figure Lengend Snippet: FIGURE 5. Description of ubiquitin modification and proposed involvement of ubiquitin modification in pathomechanism of sporadic ALS (sALS). (A) Schematic diagram of ubiquitin modification. Polyubiquitin chains are generated by isopeptide bond of a lysine (K) residue of a ubiquitin (Ub) molecule and the C-terminal (C-term) of another Ub. Linear polyubiquitin chain (L-Ub) is synthesized by peptide bond of the C-term of an Ub and the N-terminal (N-term) of another Ub. Physiological roles of each polyubiquitin chains are described in the square next to each chain. (B) Schema of our staining results and proposed involvement of ubiquitin modification in sALS pathomechanism. We showed that “wisp”, an immature form of TAR DNA- binding protein of 43 kDa-positive neuronal cytoplasmic inclusion (NCI), is attached K48-linked polyubiquitin chain (K48-Ub) first. As NCIs grow thicker, K63-linked polyubiquitin chain (K63-Ub) and L-Ub immunoreactivity were identified on NCIs. K48/ K63 branched chain or K63/Linear hybrid chain may exist in addition to the homotypic polyubiquitin chain. We found HOIP and SHARPIN, components of linear ubiquitin chain assembly complex (LUBAC), colocalize with L-Ub. Moreover, we found a part of L-Ub-positive NCI was immunopositive for optineurin, an autophagy receptor. Phosphorylated NF-jB p65 (P-p65) was detected to colocalize on L-Ub-positive NCIs. L-Ub is able to bind to IjB kinase (IKK) complex, and IKK complex phosphorylates and activates p65. P-p65 was detected on NCIs and does not apparently exist in the nucleus. The error of nuclear translocation of P- p65 is assumed to mediate tumor necrosis factor receptor (TNFR) complex II formation and finally lead to accelerate cell death. Polyubiquitin chains depicted in translucent color, K48/Linear-branched chain and K48/K63/Linear-branched chain, has not been yet established to exist in human cells.

Article Snippet: We used the following primary antibodies: rabbit polyclonal antiubiquitin (U5379; 1:50; Sigma-Aldrich, St Louis, MO), rabbit monoclonal antiK48Ub (No. 8081, clone D9D5; 1:200; Cell signaling technology, Danvers, MA), mouse monoclonal antiK63-Ub (No. BMLPW0600-0100, clone HWA4C4; 1:200; Enzo Life Sciences, Farmingdale, NY), rabbit monoclonal antiK63-Ub (No. 5621, clone D7A11; 1:2000; Cell Signaling Technology), mouse monoclonal antiL-Ub (LUB6; 1:150; gift from JT Inc.) (21), rabbit polyclonal antiHOIL-1L interacting protein (HOIP) (ab187976; 1:200; Abcam, Cambridge, UK), rabbit polyclonal antiSHANK-associated RH domain-interacting protein (SHARPIN) (HPA044453; 1:50; Sigma-Aldrich), rabbit polyclonal antiphosphorylated NF-jB p65 (P-p65; No. 600-401- 265; 1:200; Rockland Antibodies & Assays, Limerick, PA), and rabbit polyclonal antioptineurin (No. 100000; 1:800; Cayman Chemicals, Ann Arbor, MI).

Techniques: Ubiquitin Proteomics, Modification, Generated, Residue, Synthesized, Staining, Binding Assay, Translocation Assay

Journal: Molecular Oncology

Article Title: Oncogenic Ras mutant causes the hyperactivation of NF‐κB via acceleration of its transcriptional activation

doi: 10.1002/1878-0261.12580

Figure Lengend Snippet: Effect of H‐Ras (G12V) on TNFα‐induced signaling pathways. (A) Parental NIH‐3T3 cells were infected with control retroviruses or retroviruses harboring H‐Ras (G12V). After puromycin selection, cells were stimulated with 10 μg·mL −1 TNFα for the indicated periods. Then, the cells were lysed, and the degradation of IκBα and IκBβ was analyzed by immunoblotting analysis. (B) Cells were treated with 25 μg·mL −1 CHX for 30 min, and then, the cells were stimulated with 10 μg·mL −1 TNFα for the indicated periods. Cell lysates were prepared, and the degradation of IκBα was analyzed. (C) From the infected NIH‐3T3 cells shown in (A), nuclear extracts were prepared. The nuclear extracts were incubated with a biotin‐labeled DNA probe harboring κB‐responsive elements, and then, DNA·protein complexes were captured using streptavidin‐conjugated agarose. The captured proteins were eluted with sample buffer. Using these samples, the nuclear localization and DNA binding activity of NF‐κB were evaluated by immunoblotting analysis for p65/RelA.

Article Snippet: An anti‐phospho p65/RelA (S276) antibody was purchased from Rockland Immunochemicals (Limerick, PA, USA).

Techniques: Protein-Protein interactions, Infection, Control, Selection, Western Blot, Incubation, Labeling, Binding Assay, Activity Assay

Journal: Molecular Oncology

Article Title: Oncogenic Ras mutant causes the hyperactivation of NF‐κB via acceleration of its transcriptional activation

doi: 10.1002/1878-0261.12580

Figure Lengend Snippet: Oncogenic Ras mutants accelerate the transcriptional activation of p65. (A) Schemes of GAL4DBD‐p65/RelA and GAL4DBD‐p65/RelA (S276A) are shown. RHD, CBD, and TAD indicate the RHD, CBP/p300‐binding domain, and transcriptional activating domain, respectively. Numbers in parentheses indicate the original position of amino acids in p65/RelA. (B) NIH‐3T3 cells were transfected with pFR‐luciferase vector with the indicated combination of plasmids harboring GAL4DBD‐p65/RelA, GAL4DBD‐p65/RelA (S276A), H‐Ras (G12V). The cells were cultured in FBS‐free DMEM for 24 h, then stimulated with TNFα for 16 h. Cells were harvested, and their luciferase activity was measured. (C) Using the same experimental conditions with (B), the effects of dominant negative mutants of MKK6 and MKK7 and CDK inhibitor p21 Cip1 on H‐Ras (G12V)‐provoked transcriptional activation of p65/RelA were analyzed. In graphs, error bars indicate SD ( n = 3), and the results of calculations of independent t ‐tests are shown ( *P < 0.005).

Article Snippet: An anti‐phospho p65/RelA (S276) antibody was purchased from Rockland Immunochemicals (Limerick, PA, USA).

Techniques: Activation Assay, Binding Assay, Transfection, Luciferase, Plasmid Preparation, Cell Culture, Activity Assay, Dominant Negative Mutation

Journal: Molecular Oncology

Article Title: Oncogenic Ras mutant causes the hyperactivation of NF‐κB via acceleration of its transcriptional activation

doi: 10.1002/1878-0261.12580

Figure Lengend Snippet: MSK1/2 contributes to the oncogenic transcriptional activation of NF‐κB. (A) HEK293T cells were transfected with the indicated combinations of plasmids harboring FLAG‐MSK1 and H‐Ras (G12V). The cells were cultured in FBS‐free DMEM for 24 h, and then, the cells were lysed with NP‐40 lysis buffer. FLAG‐MSK1 was purified by immunoprecipitation with M2‐agarose, then incubated with 3 μg GST‐p65/RelA and 100 μ m ATP at 30 °C for 30 min. The phosphorylation of p65/RelA at Ser276 was detected by immunoblotting analysis using an antibody against phosphorylated p65/RelA (S276). (B) NIH‐3T3 cells infected with indicated retroviruses were stimulated with 10 ng·mL −1 TNFα for indicated periods, and then, their cell lysates were prepared as described in Materials and Methods. The phosphorylation of p65/RelA at Ser‐276 and Ser‐536 was detected by immunoblot analysis using indicated antibodies, respectively. The expressions of p65/RelA and H‐Ras were also detected by immunoblot analysis using indicated antibodies. (C) KF‐8 cells were infected with the indicated combinations of retroviruses harboring H‐Ras (G12V), shRNA, sh‐MSK1, or sh‐MSK2. Then, using these infected cells, luciferase assays were performed. In graph, error bars indicate SD ( n = 3), and the results of calculations of independent t ‐tests are shown ( *P < 0.005).

Article Snippet: An anti‐phospho p65/RelA (S276) antibody was purchased from Rockland Immunochemicals (Limerick, PA, USA).

Techniques: Activation Assay, Transfection, Cell Culture, Lysis, Purification, Immunoprecipitation, Incubation, Phospho-proteomics, Western Blot, Infection, shRNA, Luciferase

Journal: Molecular Oncology

Article Title: Oncogenic Ras mutant causes the hyperactivation of NF‐κB via acceleration of its transcriptional activation

doi: 10.1002/1878-0261.12580

Figure Lengend Snippet: Oncogenic K‐Ras signal is also required for NF‐κB activation in a human cell line. (A) Using retroviruses including shRNA against K‐Ras, the expression of K‐Ras mRNA was silenced in A549, a human lung cancer cell line. The amount of K‐Ras mRNA was evaluated using quantitative RT‐PCR (qPCR). (B) A549 cells were infected with retroviruses harboring sh‐luciferase (sh‐Luc) or K‐Ras. The cells were stimulated with 10 μg·mL −1 TNFα for 3 h, then harvested for the extraction of total RNA. Using the total RNA, qPCR was performed to evaluate the effect of knockdown of oncogenic K‐Ras on the TNFα‐induced expression of NF‐κB target genes such as COX‐2, ICAM1, and A20. In graphs, error bars indicate SD ( n = 3), and the results of calculations of independent t ‐tests are shown ( *P < 0.005). (C) Our observations summarize the mechanism how oncogenic Ras causes hyperactivation of NF‐κB. In the case of murine fibroblasts, Kinase for S276 is MSK1 and MSK2.

Article Snippet: An anti‐phospho p65/RelA (S276) antibody was purchased from Rockland Immunochemicals (Limerick, PA, USA).

Techniques: Activation Assay, shRNA, Expressing, Quantitative RT-PCR, Infection, Luciferase, Extraction, Knockdown

Journal: Molecular Oncology

Article Title: Oncogenic Ras mutant causes the hyperactivation of NF‐κB via acceleration of its transcriptional activation

doi: 10.1002/1878-0261.12580

Figure Lengend Snippet: Hyperactivation of NF‐κB by K‐Ras mutations in human cancer tissues. (A) Utilizing paired samples of tumor and normal tissues from K‐Ras (+) patients, immunohistochemical analyses were performed to detect the total p65/RelA and phosphorylated p65/RelA (Ser276). H&E staining is also shown. In the photograph, 50 μm scale bar was shown. (B) Extracts from tumor tissues and normal mucous membrane tissues were analyzed by immunoblotting analysis with anti‐phospho‐p65/RelA (S276), antitotal p65/RelA, and anti‐β‐actin antibodies. In the results, tumor and normal samples are shown labeled as T and N, respectively. (C) Using total RNA extracted from tumor tissues and normal mucous membrane tissues shown in (B), the mRNA expression levels of p65/RelA were analyzed by qPCR. In the graph, error bars = SD ( n = 3). (D) Using same cDNA with (C), quantitative RT‐PCR was performed to evaluate the mRNA expression of ICAM‐1 and A20 in tumor and normal tissues, respectively. In the graph, error bars = SD ( n = 3).

Article Snippet: An anti‐phospho p65/RelA (S276) antibody was purchased from Rockland Immunochemicals (Limerick, PA, USA).

Techniques: Immunohistochemical staining, Staining, Membrane, Western Blot, Labeling, Expressing, Quantitative RT-PCR

Journal: Frontiers in Pharmacology

Article Title: β1-Integrin plays a major role in resveratrol-mediated anti-invasion effects in the CRC microenvironment

doi: 10.3389/fphar.2022.978625

Figure Lengend Snippet: Impact of resveratrol or/and ASO against β1-integrin on TME-mediated activation and nuclear translocation of p65-NF-κB in CRC cells. HCT116 (A,B) and RKO (B) cells in TME on cover glasses without treatment or only treated with resveratrol (5 µM) or resveratrol-treated (5 µM) with addition of β1-SO (0.5 µM) or knocked down with β1-ASO (0.1, 0.2, 0.5 µM). Anti-phosho-NF-κB immunolabeled (red) and DAPI-stained (blue). White arrows = p65-NF-κB positive CRC cells. Microscope: Leica DM 2000. Magnification ×600; scale bar = 30 µm. Statistical evaluation: * p < 0.05 and ** p < 0.01, compared to TME control.

Article Snippet: Monoclonal antibodies to NF-κB (#MAB5078), and phospho-specific p65-NF-κB (#MAB7226), MMP-9 (#MAB911), polyclonal caspase-3 (#AF835) were purchased from R&D Systems (Heidelberg, Germany).

Techniques: Activation Assay, Translocation Assay, Immunolabeling, Staining, Microscopy, Control

Journal: Frontiers in Pharmacology

Article Title: β1-Integrin plays a major role in resveratrol-mediated anti-invasion effects in the CRC microenvironment

doi: 10.3389/fphar.2022.978625

Figure Lengend Snippet: Impact of resveratrol or/and ASO against β1-integrin or against NF-κB on TME-promoted CRC cell invasion in alginate cultures. Serum-starved HCT116 (A) and RKO (B) cells, cultured in 3D-alginate, emigrated under following treatments: untreated (Basal Co.), TME control, resveratrol (1, 5 µM), β1-SO/ASO (0.5 µM), NF-κB-SO/ASO (0.5 µM) or co-treatment of sense or antisense oligonucleotides with resveratrol (5 µM). CRC cells were stained with toluidine blue (T-Blue) after settling on the bottom of 12-well-plates. Statistical evaluation: Compared to TME control, * p < 0.05 and ** p < 0.01 for HCT116 (C) and RKO (D) .

Article Snippet: Monoclonal antibodies to NF-κB (#MAB5078), and phospho-specific p65-NF-κB (#MAB7226), MMP-9 (#MAB911), polyclonal caspase-3 (#AF835) were purchased from R&D Systems (Heidelberg, Germany).

Techniques: Cell Culture, Control, Staining

Journal: Frontiers in Pharmacology

Article Title: β1-Integrin plays a major role in resveratrol-mediated anti-invasion effects in the CRC microenvironment

doi: 10.3389/fphar.2022.978625

Figure Lengend Snippet: Impact of resveratrol or/and ASO against β1-integrin or against NF-κB on TME-promoted CRC cell metastasis and formation of colonospheres in alginate cultures. Serum-starved HCT116 (A,B) and RKO (D,E) cells from 3D-alginate, settled on square cover glasses as colonies under different treatment conditions: untreated (Basal Co.), TME control, resveratrol (1, 5 µM), β1-SO/ASO (0.5 µM), NF-κB-SO/ASO (0.5 µM) or combination of SO/ASO (0.5 µM) and resveratrol (5 µM). CRC cells were photographed using phase contrast (magnification ×100, Zeiss Axiovert 40 CFL microscope) and DAPI-staining (magnification ×50, Leica DM 2000 microscope). Statistical analysis for HCT116 (C) and RKO (F) : Relative to TME control, * p < 0.05 and ** p < 0.01.

Article Snippet: Monoclonal antibodies to NF-κB (#MAB5078), and phospho-specific p65-NF-κB (#MAB7226), MMP-9 (#MAB911), polyclonal caspase-3 (#AF835) were purchased from R&D Systems (Heidelberg, Germany).

Techniques: Control, Microscopy, Staining

Journal: Frontiers in Pharmacology

Article Title: β1-Integrin plays a major role in resveratrol-mediated anti-invasion effects in the CRC microenvironment

doi: 10.3389/fphar.2022.978625

Figure Lengend Snippet: Impact of resveratrol or/and ASO against β1-integrin on TME-promoted activation of metastasis and apoptosis parameters in CRC cells. HCT116 (A) and RKO (B) derived from 3D-alginate cultures were grown untreated or treated with 5 µM resveratrol alone or in combination with 0.5 µM β1-SO or 0.5 µM β1-ASO ( x -axis) and probed with antibodies against CXCR4, FAK, p-FAK, NF-κB, p65-NF-κB and cleaved caspase-3. Loading control: β-actin. Densitometric units complementing Western blot results ( y -axis). For densitometric analysis, data were compared to TME control: * p < 0.05 and ** p < 0.01.

Article Snippet: Monoclonal antibodies to NF-κB (#MAB5078), and phospho-specific p65-NF-κB (#MAB7226), MMP-9 (#MAB911), polyclonal caspase-3 (#AF835) were purchased from R&D Systems (Heidelberg, Germany).

Techniques: Activation Assay, Derivative Assay, Control, Western Blot

Journal: Frontiers in Pharmacology

Article Title: β1-Integrin plays a major role in resveratrol-mediated anti-invasion effects in the CRC microenvironment

doi: 10.3389/fphar.2022.978625

Figure Lengend Snippet: Western blot investigation on the efficacy of β1-integrin and NF-κB knockdown by antisense oligonucleotides in CRC cells. X -axis: HCT116 (A) and RKO (B) samples from 3D-alginate TME were untreated or treated with β1-SO/NF-κB-SO or β1-ASO/NF-κB-ASO (0.5 µM). Immunoblotting with anti-β1-integrin or with anti-p-65-NF-κB and β-actin as a loading control. Y -axis: Densitometric units ( y -axis) complementing Western blot results. Statistical analysis: * p < 0.05 and ** p < 0.01, comparison to TME control.

Article Snippet: Monoclonal antibodies to NF-κB (#MAB5078), and phospho-specific p65-NF-κB (#MAB7226), MMP-9 (#MAB911), polyclonal caspase-3 (#AF835) were purchased from R&D Systems (Heidelberg, Germany).

Techniques: Western Blot, Knockdown, Control, Comparison

Journal: Frontiers in Pharmacology

Article Title: β1-Integrin plays a major role in resveratrol-mediated anti-invasion effects in the CRC microenvironment

doi: 10.3389/fphar.2022.978625

Figure Lengend Snippet: Impact of resveratrol or/and ASO against β1-integrin or against NF-κB on TME-promoted activation of EMT-linked biomarkers in CRC cells. 3D-alginate RKO (A) and HCT116 (B) CRC cells were detected against E-cadherin, vimentin, slug and loading controlled with β-actin after 10–14 days of treatment. X -axis: untreated (Ba.Co.), TME control, resveratrol (1, 5 µM), β1-SO/ASO (0.5 µM), NF-κB-SO/ASO (0.5 µM) or combination of 0.5 µM SO/ASO and resveratrol (5 µM). Y -axis: Densitometric units ( y -axis) complementing Western blot results and for analysis, data were compared to TME control: * p < 0.05 and ** p < 0.01 were considered statistically significant.

Article Snippet: Monoclonal antibodies to NF-κB (#MAB5078), and phospho-specific p65-NF-κB (#MAB7226), MMP-9 (#MAB911), polyclonal caspase-3 (#AF835) were purchased from R&D Systems (Heidelberg, Germany).

Techniques: Activation Assay, Control, Western Blot

Journal: Frontiers in Pharmacology

Article Title: β1-Integrin plays a major role in resveratrol-mediated anti-invasion effects in the CRC microenvironment

doi: 10.3389/fphar.2022.978625

Figure Lengend Snippet: Impact of resveratrol or/and ASO against β1-integrin or against NF-κB on TME-promoted activation of metastasis- and apoptosis-linked biomarkers in CRC cells. Serum-starved RKO (A) and HCT116 (B) CRC cells, grown in 3D-alginate and differently treated for 10–14 days [untreated (Ba.Co.), TME control, resveratrol (1, 5 µM), β1-SO/ASO (0.5 µM), NF-κB-SO/ASO (0.5 µM) or combination of 0.5 µM SO/ASO and resveratrol (5 µM); x -axis] were investigated with antibodies against pan-NF-κB, p65-NF-κB, FAK, p-FAK, MMP-9, CXCR4 and cleaved caspase-3. β-actin served as loading control and statistical significance is shown by: * p < 0.05 and ** p < 0.01 compared with the TME control. Y -axis: Densitometric units ( y -axis) complementing Western blot results.

Article Snippet: Monoclonal antibodies to NF-κB (#MAB5078), and phospho-specific p65-NF-κB (#MAB7226), MMP-9 (#MAB911), polyclonal caspase-3 (#AF835) were purchased from R&D Systems (Heidelberg, Germany).

Techniques: Activation Assay, Control, Western Blot

Journal: Cancers

Article Title: Human Papillomavirus 16 E7 Promotes EGFR/PI3K/AKT1/NRF2 Signaling Pathway Contributing to PIR/NF-κB Activation in Oral Cancer Cells

doi: 10.3390/cancers12071904

Figure Lengend Snippet: HPV16 HPV E7 promotes nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) activation in oral cells. ( A ) pHAGE/NF-κB reporter vector map. ( B ) Luciferase activity normalized with GFP was evaluated in SCC143/E7 and SCC143/V cells transfected with the reporter vector pHAGE/NF-κB. ( C ) Luciferase activity normalized with GFP in SCC143/E7 cells co-transfected with pHAGE/NF-κB and siRNA for PIR or siRNA E7 knockdown and siRNA (SCR) as a control. ( D ) Protein array of NF-κB signaling pathway comparing the SCC143/V and SCC143/E7 cell extracts. Data were plotted in reference to the change that occurred in the presence of E7 compared to the empty vector control. ( E ) Western blot of nuclear and cytoplasmic protein fractions was performed to analyze the levels of Pirin, p65 S529 , p65, and C-Rel. β-actin or H3 were used as a load control in SCC143 cells transduced with empty (pLXSN) or E7 constructs. The graphs represent a densitometric analysis of three independent Western blots (WBs) for each protein normalized against β-actin. Data are presented as the mean ± SEM; average of three independent experiments, conducted in triplicate. * p < 0.05 (Mann–Whitney test).

Article Snippet: Membranes were incubated for 1 h at room temperature with blocking buffer (5% bovine serum albumin, Tris-buffered saline (TBS)–0.5% Tween 20, pH 7.6) and incubated overnight at 4 °C with primary antibody against Pirin (ab51360), pRb (ab24), EGFR (ab32562), EGFR Y1173 (ab32578), EGFR Y1068 (ab40815), p65 s536 (ab86299), β-actin (ab6276) (Abcam, Cambridge, UK), C-Rel (MAB2699), p65 s529 (MAB7624) (R&D System, Minneapolis, MN, USA), HPV16 E7 (SC6981), pAKT1-2-3 (SC514032), PHOSPHO AKT S473 (4060S), NRF2 (4060S) (Cell Signaling, Danver, MA, USA), ERK (SC514302), pERK (SC136521), AREG (SC5797), pMTORC (SC293133), MTORC (SC517464), Histone H3 (SC56616), GSK3 (SC7291), p65 (SC8008), (Santa Cruz Biotechnology, Dallas, TX, USA) diluted 1/1000 in Tris-buffered saline–Tween 20 (TBS–T20).

Techniques: Activation Assay, Plasmid Preparation, Luciferase, Activity Assay, Transfection, Knockdown, Control, Protein Array, Western Blot, Transduction, Construct, MANN-WHITNEY

Journal: Cancers

Article Title: Human Papillomavirus 16 E7 Promotes EGFR/PI3K/AKT1/NRF2 Signaling Pathway Contributing to PIR/NF-κB Activation in Oral Cancer Cells

doi: 10.3390/cancers12071904

Figure Lengend Snippet: HPV16 HPV E7 induces EGFR/PI3K/AKT1 signaling for PIR/NF-kB activation in oral cells. ( A ) Western blot was performed with protein extracts from SCC143/E7 cells previously exposed to Gefitinib (EGFR), U0126 (ERK) and LY294002 (PI3K) inhibitors for 24 h. The levels of total EGFR, pEGFR Y1173 , AKT1, pAKT1, ERK, pERK, Pirin and β-actin used as load control were analyzed. The graphs represent a densitometric analysis of three independent WBs for Pirin normalized against β-actin. ( B ) Time–response assay by exposure to Gefitinib for 1.5 to 24 h in SCC143/E7 cells. The levels of total EGFR, pEGFR Y1068 , Pirin and β-actin used as load control were analyzed. The graph represents a densitometric analysis of three independent experiments. ( C ) Time–response assay by exposure to 10 µM LY294002 for 3 to 24 h in SCC143/E7 cells. The levels of total AKT1, pAKT1, Pirin and β-actin used as load control were analyzed. The graph represents densitometric analysis of three independent experiments. ( D ) Western blot to evaluate AKT1, pAKT1, Pirin protein levels in organotypic raft cultures established from OKF6/TERT2 E7 oral cells treated with dimethyl sulfoxide (DMSO) or Gefitinib for 3 h (β-actin used as load control were analyzed). ( E ) Western blot to evaluate AKT1, pAKT1, Pirin protein levels in OKF6/TERT2 E7 oral organotypic raft culture cells treated with DMSO or 10 µM LY294002 for 12 or 24 h (β-actin used as load control were analyzed). ( F ) Western blot to evaluate c-Rel and p65 S529 protein levels in organotypic raft cultures established from OKF6/TERT2 E7 oral cells treated with DMSO or 10 µM LY294002 for 12 or 24 h (β-actin used as load control were analyzed) ( G ) Luciferase activity normalized against GFP was evaluated in SCC143/E7 cells transfected with the reporter vector pHAGE/NF-κB treated with DMSO or 10 µM LY294002 for 12 or 24 h. Data are presented as the mean ± SEM; average of three independent experiments, conducted in triplicate. * p < 0.05 and ** p < 0.01 (ANOVA test). LY294002 (LY).

Article Snippet: Membranes were incubated for 1 h at room temperature with blocking buffer (5% bovine serum albumin, Tris-buffered saline (TBS)–0.5% Tween 20, pH 7.6) and incubated overnight at 4 °C with primary antibody against Pirin (ab51360), pRb (ab24), EGFR (ab32562), EGFR Y1173 (ab32578), EGFR Y1068 (ab40815), p65 s536 (ab86299), β-actin (ab6276) (Abcam, Cambridge, UK), C-Rel (MAB2699), p65 s529 (MAB7624) (R&D System, Minneapolis, MN, USA), HPV16 E7 (SC6981), pAKT1-2-3 (SC514032), PHOSPHO AKT S473 (4060S), NRF2 (4060S) (Cell Signaling, Danver, MA, USA), ERK (SC514302), pERK (SC136521), AREG (SC5797), pMTORC (SC293133), MTORC (SC517464), Histone H3 (SC56616), GSK3 (SC7291), p65 (SC8008), (Santa Cruz Biotechnology, Dallas, TX, USA) diluted 1/1000 in Tris-buffered saline–Tween 20 (TBS–T20).

Techniques: Activation Assay, Western Blot, Control, Luciferase, Activity Assay, Transfection, Plasmid Preparation

Journal: Cancers

Article Title: Human Papillomavirus 16 E7 Promotes EGFR/PI3K/AKT1/NRF2 Signaling Pathway Contributing to PIR/NF-κB Activation in Oral Cancer Cells

doi: 10.3390/cancers12071904

Figure Lengend Snippet: HPV16 E7 expression and PIR induces migration of oral cells. ( A ) A migration assay in SCC143/E7 and SCC143/V cells was carried out for 7 h using fibronectin pretreated transwells, scale bar 25 µm. ( B ) A migration assay performed on SCC143/E7 cells previously transfected with control siRNA (SCR), siRNA PIR or HPV16 siRNA E7 was carried out for 7 h using fibronectin pretreated transwells, scale bar 25 µm. ( C ) A migration assay performed on SCC143/E7 cells previously transfected with control siRNA (SCR) and siRNA I-II p65 was carried out for 7 h using fibronectin pretreated transwells, scale bar 25 µm. ( D ) Western blot against eGFP performed for V corresponding to the empty vector (pcDNA 3.1–eGFP) and PIR corresponding to the vector containing the PIR sequence linked to eGFP (pcDNA 3.1–eGFP–PIR) transfected in SCC143 oral cell. ( E ) A migration assay in SCC143 V (pcDNA 3.1–eGFP) and SCC143 PIR (pcDNA 3.1–eGFP–PIR) cells was carried out for 7 h using fibronectin pretreated transwells. Scale bar: 40 µm. ( F ) Analysis of E-cadherin and N-cadherin protein levels, which were normalized with the expression of β-actin. The graphs represent a densitometric analysis of three independent assays. Data are presented as the mean ± SEM; average of three independent experiments, conducted in triplicate. * p < 0.05 and ** p < 0.01 (Mann–Whitney test).

Article Snippet: Membranes were incubated for 1 h at room temperature with blocking buffer (5% bovine serum albumin, Tris-buffered saline (TBS)–0.5% Tween 20, pH 7.6) and incubated overnight at 4 °C with primary antibody against Pirin (ab51360), pRb (ab24), EGFR (ab32562), EGFR Y1173 (ab32578), EGFR Y1068 (ab40815), p65 s536 (ab86299), β-actin (ab6276) (Abcam, Cambridge, UK), C-Rel (MAB2699), p65 s529 (MAB7624) (R&D System, Minneapolis, MN, USA), HPV16 E7 (SC6981), pAKT1-2-3 (SC514032), PHOSPHO AKT S473 (4060S), NRF2 (4060S) (Cell Signaling, Danver, MA, USA), ERK (SC514302), pERK (SC136521), AREG (SC5797), pMTORC (SC293133), MTORC (SC517464), Histone H3 (SC56616), GSK3 (SC7291), p65 (SC8008), (Santa Cruz Biotechnology, Dallas, TX, USA) diluted 1/1000 in Tris-buffered saline–Tween 20 (TBS–T20).

Techniques: Expressing, Migration, Transfection, Control, Western Blot, Plasmid Preparation, Sequencing, MANN-WHITNEY

Journal: bioRxiv

Article Title: CK2 inhibition suppresses glial inflammation in the brain

doi: 10.1101/2025.08.05.668554

Figure Lengend Snippet: A. Knockdown efficiency of experiments shown in (mean expression shown with each dot representing an experiment). B. Western blotting showing expression of CK2Α1 and GAPDH in individual THP-1 NF-κB-Lucia clones, quantified as a relative ratio. Heterozygotes and homozygotes confirmed by Sanger sequencing. Full blots in . C. Western blotting showing CK2Α2 expression in individual WT or KO THP-1 NF-κB-Lucia clones after immunoprecipitation of CK2Α2. Full blots in . D. Quantification of NF-κB reporter luciferase activity in CK2Α1 heterozygous (het) or homozygous (KO) knockout THP-1 NF-κB Lucia cell clones stimulated with 20 ng/mL LPS and CHR dilution series (mean ± sem, n = 3). E. Expression of CK2α1-HA and CK2α2-HA in GPCs confirmed by Western blotting. Full blots in . F. CK2 kinase-dead mutants block IL1β-induced IL6 upregulation in HCA (3 independent experiments shown with Poisson error; relative expression normalized to each WT). G. Quantification showing that pIκB/IκBα levels are reduced with CK2 inhibitors (6-hour treatment, mean ± SD, n = 2, pooled independent experiments; related to ). H. Immunoblot and quantification (n = 2, mean) of NF-κB IP showing NF-κB S529 phosphorylation is reduced with CHR (2-hour treatment). Full blots in .

Article Snippet: Primary Antibodies used: Chicken anti-GFP (1:300; Aves Labs: GFP-1020), Goat polyclonal anti-hSox9 (1:250; R&D Biosystems: AF3075), Rabbit monoclonal anti-NF-κB p65/RelA(1:400; CTS: 8242), Mouse monoclonal anti-Phospho-RelA/NFkB p65 (S529) (1:50; R&D Systems: MAB7624), Rabbit polyclonal anti-CSNK2A2 (1:100; Proteintech: 10606-1-AP); Chicken polyclonal anti-NeuN (1:250; Millipore: ABN91).

Techniques: Knockdown, Expressing, Western Blot, Clone Assay, Sequencing, Immunoprecipitation, Luciferase, Activity Assay, Knock-Out, Blocking Assay, Phospho-proteomics

Journal: bioRxiv

Article Title: CK2 inhibition suppresses glial inflammation in the brain

doi: 10.1101/2025.08.05.668554

Figure Lengend Snippet: A. CK2 levels increase with inflammation (time course by Western blot). Full blots in . B. Representative immunofluorescence images and quantification showing reduction of nuclear phospho-CK2α1 Y255/CK2α1 after 5 hours of CK2 inhibitor treatment (mean ± s.e.m., n = 4, one-way ANOVA with Dunnett’s post-hoc test). Scale bar = 100 µm. C. Representative immunoblots showing that pIκB/IκBα levels are reduced with CK2 inhibitors (6-hour treatment). Full blots in . D. Representative immunofluorescence images and quantification showing reduction of nuclear phospho-NF-κB S529/NF-κB after 5 hours of CK2 inhibitor treatment (mean ± s.e.m., n = 4, one-way ANOVA with Dunnett’s post-hoc test). Scale bar = 100 µm. E. Quantification of NF-κB reporter luciferase activity in a pool of phosphodeficient THP-1 NF-κB Lucia NF-κB-S529A-NeoR knockin cells or parental WT stimulated with various doses of LPS (mean of n = 3 biological replicates shown). F. X2K interaction network showing top enriched kinase modules, intermediate proteins (not labeled), and their downstream TF targets in IL1-β-stimulated astrocytes (gray nodes and edges unrelated to CK2, black nodes connected to CK2 via red edges). G. Immune signatures are significantly enriched in genes downregulated in inflamed astrocytes treated with API. H. CK2 inhibition reduces expression of “Astro-Inflammation” genes and increases expression of “Astro-Injury” genes. A-E: Representative of 3 independent experiments unless otherwise noted. ns = not significant; * P < 0.05; ** P < 0.01

Article Snippet: Primary Antibodies used: Chicken anti-GFP (1:300; Aves Labs: GFP-1020), Goat polyclonal anti-hSox9 (1:250; R&D Biosystems: AF3075), Rabbit monoclonal anti-NF-κB p65/RelA(1:400; CTS: 8242), Mouse monoclonal anti-Phospho-RelA/NFkB p65 (S529) (1:50; R&D Systems: MAB7624), Rabbit polyclonal anti-CSNK2A2 (1:100; Proteintech: 10606-1-AP); Chicken polyclonal anti-NeuN (1:250; Millipore: ABN91).

Techniques: Western Blot, Immunofluorescence, Luciferase, Activity Assay, Knock-In, Labeling, Inhibition, Expressing