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