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: Cancer Research

Article Title: Nucleolar Targeting of RelA(p65) Is Regulated by COMMD1-Dependent Ubiquitination

doi: 10.1158/0008-5472.can-09-1397

Figure Lengend Snippet: Figure 1. Nucleolar translocation of RelA and proteasome activity. A, SW480 colon cancer cells were treated with aspirin (0–5 mmol/L, 16 h). 20S proteasome activity was measured in whole-cell lysates using fluorometric assay. Columns, percentage of basal (0 mmol/L) activity (n = 4); bars, SE. B, SW480 cells were treated with 10 mmol/L aspirin for the times specified and then 20S proteasome activity was measured as above. Columns, percentage of basal (0 h) activity (n = 4); bars, SE. C, SW480 cells were transfected with HA-tagged ubiquitin 24 h before treatment with aspirin (10 mmol/L) for the specified times. Anti-HA Western blot analysis was done on whole-cell extracts. Actin is used as a protein loading control. D, SW480 cells treated with aspirin (0 or 5 mmol/L, 16 h) were fractionated using sucrose gradients. 20S proteasome activity was measured in lysates from the nuclear and nucleolar fractions as above. Columns, percentage of nontreated (NT) nuclear proteasome activity (n = 3); bars, SE. Inset, anti-C23, fibrillarin (nucleolar markers), and lamin B (nonnucleolar protein) Western blot analysis done on lysates from the nuclear and nucleolar fractions.

Article Snippet: The following primary antibodies were used: p65(C-20), p65(F-6), nucleolin (C23), fibrillarin, glutathione S-transferase (GST), and green fluorescent protein (GFP; all from Santa Cruz); His [generated in-house (purified form)]; ubiquitin (rabbit antibody; Dako Cytomation); ubiquitin (mouse monoclonal; Stressgen); filamin A (Bethyl Laboratories); phospho-p65 (Ser468) and phospho-p65 (Ser536) (Cell Signaling Technology).

Techniques: Translocation Assay, Activity Assay, Transfection, Ubiquitin Proteomics, Western Blot, Control

Journal: Cancer Research

Article Title: Nucleolar Targeting of RelA(p65) Is Regulated by COMMD1-Dependent Ubiquitination

doi: 10.1158/0008-5472.can-09-1397

Figure Lengend Snippet: Figure 2. Ubiquitination of RelA precedes nucleolar translocation of the protein. A, SW480 cells were treated with aspirin (0–5 mmol/L, 16 h). Top, the levels of high molecular weight [indicative of ubiquitination (Ub.)] and native RelA were determined by Western blot analysis of whole-cell lysates. Bottom, RelA was immunoprecipitated (IP) from lysates, and recovered protein analyzed using anti-ubiquitin Western blot analysis (WB Ub.). Immunoprecipitation with isotyped IgG controlled for specificity. Stripped gels were reprobed for RelA to monitor the amount of RelA immunoprecipitated (WB. RelA). B, SW480 cells were transfected with WT 6His-ubiquitin (ub) or the R7 mutant that cannot form polyubiquitin chains. Twenty-four hours after transfection, cells were either left untreated (NT) or treated with aspirin (5 mmol/L) or MG132 (25 μmol/L) for a further 16 h. His-tagged proteins were precipitated from whole-cell lysates using Ni-agarose beads and then subjected to anti-RelA and anti-histidine (his) Western blot analysis (WB). NS, nonspecific band. C, SW480 cells were treated with aspirin (10 mmol/L) for the times specified. Top, the levels of native and high molecular weight forms of RelA were determined using Western blot analysis. Bottom, the nuclear distribution of RelA was assessed by immunocytochemical staining (magnification, ×63). D, SW480 cells were exposed to UV-C radiation (40 J/m2) or TNF (10 ng/mL) for the times specified. Top, anti-RelA Western blot analysis done on whole-cell lysates. Bottom, immunomicrographs showing the cellular distribution of RelA (magnification, ×63).

Article Snippet: The following primary antibodies were used: p65(C-20), p65(F-6), nucleolin (C23), fibrillarin, glutathione S-transferase (GST), and green fluorescent protein (GFP; all from Santa Cruz); His [generated in-house (purified form)]; ubiquitin (rabbit antibody; Dako Cytomation); ubiquitin (mouse monoclonal; Stressgen); filamin A (Bethyl Laboratories); phospho-p65 (Ser468) and phospho-p65 (Ser536) (Cell Signaling Technology).

Techniques: Ubiquitin Proteomics, Translocation Assay, High Molecular Weight, Western Blot, Immunoprecipitation, Transfection, Mutagenesis, Staining

Journal: Cancer Research

Article Title: Nucleolar Targeting of RelA(p65) Is Regulated by COMMD1-Dependent Ubiquitination

doi: 10.1158/0008-5472.can-09-1397

Figure Lengend Snippet: Figure 3. Proteasome inhibition induces ubiquitination and nucleolar translocation of RelA. A, SW480 cells were treated with carrier (DMSO), MG132 (25 μmol/L), or lactacystin (Lact.; 35 μmol/L) for 16 h. Immunomicrographs (×63) show the cellular localization of RelA. C23 staining identifies nucleoli. DNA is stained by DAPI. The percentage of cells showing nucleolar RelA (as indicated by colocalization with C23) was determined in at least 200 cells from at least five randomly selected fields of view (n = 3). B and D, SW480 cells were treated with MG132 (25 μmol/L) or lactacystin (50 μmol/L) for the times specified. Top, anti-RelA Western blot analysis showing native and high molecular weight forms of the protein, indicative of the addition of multiple ubiquitin chains (Ub. RelA). Middle, the cellular distribution of RelA was determined by immunocytochemical staining (magnification, ×63). Bottom, for each time point, the percentage of cells showing nucleolar localization of RelA (as depicted by areas devoid of DAPI staining) was determined as in A (n = 3). B, bottom, gray line, SW480 cells were transiently transfected with the NF-κB–dependent 3×κB ConA-luc and the control pCMVβ reporter constructs. Twenty-four hours after transfection, cells were treated with MG132 (25 μmol/L) for the times specified. Results were normalized using β-galactosidase activity and are presented as the percentage of relative luciferase activity compared with basal (carrier treated) controls (n = 2); bars, SD. C, SW480 cells were transfected with GFP-RelA 24 h before treatment with MG132 (25 μmol/L, 0 or 2 h). Immunoprecipitation (IP) of ubiquitinated proteins followed by anti-RelA Western blot analysis (WB) of recovered complexes confirmed an increase in ubiquitinated forms of RelA 2 h after MG132 treatment. Control immunoprecipitations were done with preimmune (IgG) serum. RelA in input samples is shown.

Article Snippet: The following primary antibodies were used: p65(C-20), p65(F-6), nucleolin (C23), fibrillarin, glutathione S-transferase (GST), and green fluorescent protein (GFP; all from Santa Cruz); His [generated in-house (purified form)]; ubiquitin (rabbit antibody; Dako Cytomation); ubiquitin (mouse monoclonal; Stressgen); filamin A (Bethyl Laboratories); phospho-p65 (Ser468) and phospho-p65 (Ser536) (Cell Signaling Technology).

Techniques: Inhibition, Ubiquitin Proteomics, Translocation Assay, Staining, Western Blot, High Molecular Weight, Transfection, Control, Construct, Activity Assay, Luciferase, Immunoprecipitation

Journal: Cancer Research

Article Title: Nucleolar Targeting of RelA(p65) Is Regulated by COMMD1-Dependent Ubiquitination

doi: 10.1158/0008-5472.can-09-1397

Figure Lengend Snippet: Figure 4. Amino acids 27–30 are required for ubiquitination and nucleolar translocation of RelA. A, SW480 cells were transfected with the specified plasmids and treated with MG132 (25 μmol/L) or lactacystin (35 μmol/L) for 16 h as above; then the cellular localization of GFP-tagged RelA was determined in live cells using an Axiovert 100 inverted fluorescent microscope (×40; left). Nucleoli are indicated by arrows in phase-contrast images. Right images, fixed cells counterstained with DAPI (DNA stain). Nucleoli are depicted by areas devoid of DAPI staining (magnification, ×63). B, SW480 cells were transfected with the specified GFP plasmids, then either left untreated (Asp. −) or treated with aspirin (10 mol/L), carrier (MG132 −), or MG132 (25 μmol/L) for 2 h. The levels of native and high molecular weight (Ub) RelA and total protein ubiquitination were determined by Western blot analysis. C, SW480 cells were transfected and treated with MG132 (25 μmol/L) or aspirin (10 mmol/L) as above. The levels of ubiquitinated RelA were determined by anti-ubiquitin immunoprecipitation (IP) followed by anti-RelA Western blot analysis (WB). Membranes were stripped and reprobed to show the effects of the agents on total protein ubiquitination. Right, SW480 cells constitutively expressing GFP-RelA WT or Δ27–30 were transfected with 6His-ubiquitin. Cells were then treated for 2 h with aspirin (10 mmol/L), and ubiquitinated proteins precipitated using Ni-agarose beads. Precipitated proteins were subjected to anti-RelA and anti-His Western blot analysis. D, SW480 cells were transfected with the specified vectors and then treated with MG132 (25 μmol/L) or lactacystin (0–75 μmol/L) for 16 h. Annexin V-biotin staining, with a Texas red-streptavidin conjugate, was used to identify apoptotic cells. The percentage of cells expressing GFP-tagged RelA undergoing apoptosis was determined by fluorescent microscopy in at least 250 transfected cells for each sample. Columns, mean of at least three independent experiments; bars, SE.

Article Snippet: The following primary antibodies were used: p65(C-20), p65(F-6), nucleolin (C23), fibrillarin, glutathione S-transferase (GST), and green fluorescent protein (GFP; all from Santa Cruz); His [generated in-house (purified form)]; ubiquitin (rabbit antibody; Dako Cytomation); ubiquitin (mouse monoclonal; Stressgen); filamin A (Bethyl Laboratories); phospho-p65 (Ser468) and phospho-p65 (Ser536) (Cell Signaling Technology).

Techniques: Ubiquitin Proteomics, Translocation Assay, Transfection, Microscopy, Staining, High Molecular Weight, Western Blot, Immunoprecipitation, Expressing

Journal: Cancer Research

Article Title: Nucleolar Targeting of RelA(p65) Is Regulated by COMMD1-Dependent Ubiquitination

doi: 10.1158/0008-5472.can-09-1397

Figure Lengend Snippet: Figure 5. COMMD1 modulates the nuclear distribution of RelA. A, immunomicrographs (magnification, ×63) showing aspirin (5 mmol/L, 16 h)-mediated nucleolar translocation of RelA in SW480 cells transfected with GST-COMMD1 or control (GST) vector. NT, nontreated. The percentage of cells in the total cell population showing nucleolar RelA (as indicated by colocalization with the nucleolar marker fibrillarin) was determined in at least 200 cells. Columns, mean (n = 4); bars, SE. Inset, anti-COMMD1 immunoblot showing levels of GST-COMMD1 in transfected SW480 cells. B and C, SW480 cells were transfected with control or COMMD1 siRNA (C1/1 and C1/2) and then treated with aspirin (10 mmol/L) for 0 to 8 h. B, anti-RelA immunoblot showing levels of native and ubiquitinylated RelA in response to aspirin (10 mmol/L, 2 h) treatment. Native RelA controls for protein loading. Numbers indicate the mean relative intensity (RI) of high molecular weight RelA (compared with native RelA; quantified using ImageJ; n = 3). C, immunocytochemical staining determined the nuclear distribution of RelA. The percentage of cells showing nucleolar RelA was calculated as above. Inset, Western blot analysis determined the levels of COMMD1 with (+) and without (−) aspirin (10 mmol/L, 2 h) treatment. Actin was used as a control for protein loading. D, SW480 cells were transfected as indicated, then either untreated or treated with aspirin (5 mmol/L) for 24 h. Apoptotic cells were visualized using fluorescent microscopy and the percentage of apoptotic cells within the total cell population was calculated. Columns, average fold increase in apoptosis in response to aspirin compared with the equivalent nontreated control (n = 3); bars, SE.

Article Snippet: The following primary antibodies were used: p65(C-20), p65(F-6), nucleolin (C23), fibrillarin, glutathione S-transferase (GST), and green fluorescent protein (GFP; all from Santa Cruz); His [generated in-house (purified form)]; ubiquitin (rabbit antibody; Dako Cytomation); ubiquitin (mouse monoclonal; Stressgen); filamin A (Bethyl Laboratories); phospho-p65 (Ser468) and phospho-p65 (Ser536) (Cell Signaling Technology).

Techniques: Translocation Assay, Transfection, Control, Plasmid Preparation, Marker, Western Blot, High Molecular Weight, Staining, Microscopy

Journal: Cancer Research

Article Title: Nucleolar Targeting of RelA(p65) Is Regulated by COMMD1-Dependent Ubiquitination

doi: 10.1158/0008-5472.can-09-1397

Figure Lengend Snippet: Figure 6. Aspirin modulates the COMMD1-RelA interaction. A, SW480 cells were treated with 10 mmol/L aspirin for the times indicated and COMMD1 protein levels determined by Western blot analysis. Actin was used as a control for protein loading. B, SW480 cells stably expressing GFP-RelA were transfected with either GST-COMMD1 or an empty vector control (Vec-GST). Twenty-four hours after transfection, cells were treated with 10 mmol/L aspirin for the indicated times, and GST-tagged proteins precipitated from whole-cell lysates using glutathione Sepharose (GSH beads). Anti-RelA Western blot analysis (WB) of precipitated proteins confirmed an interaction between COMMD1 and RelA in response to aspirin treatment. Gels were reprobed with GST to examine the levels of precipitated GST-COMMD1 and GST-control. GFP-RelA and GST-COMMD1 levels in input samples are shown. C, SW480 cells were treated with either aspirin (10 mmol/L) or MG132 (25 μmol/L) for 2 h in the presence or absence of calyculin A (50 nmol/L). Anti-RelA immunoblot shows native and ubiquitinated (Ub) protein in whole-cell extracts. The levels of phosphorylated RelA (S468 and S563) were also determined by Western blot analysis. Filamin was used as a control for protein loading. D, model for nucleolar translocation of RelA and NF-κB–regulated apoptosis in colorectal cancer cells. See Discussion for details.

Article Snippet: The following primary antibodies were used: p65(C-20), p65(F-6), nucleolin (C23), fibrillarin, glutathione S-transferase (GST), and green fluorescent protein (GFP; all from Santa Cruz); His [generated in-house (purified form)]; ubiquitin (rabbit antibody; Dako Cytomation); ubiquitin (mouse monoclonal; Stressgen); filamin A (Bethyl Laboratories); phospho-p65 (Ser468) and phospho-p65 (Ser536) (Cell Signaling Technology).

Techniques: Western Blot, Control, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Translocation Assay

Journal: bioRxiv

Article Title: Interferon regulatory factor 3 upregulates the Treg recruitment factor CCL22 in response to double-stranded DNA in cancer cells

doi: 10.1101/2022.03.08.483519

Figure Lengend Snippet: A , HeLa cells were treated with a mock water control or 0.6 μM of the TBK1/IKKe inhibitor MRT67307 for 1.5 hours, then treated with TNFα (8 ng/mL) or PMA (10 ng/mL), or untreated (UN) and harvested after 5 minutes (TNFα) or 20 minutes (PMA and UN). Lysates (25 μg) were resolved by SDS-PAGE PAGE and probed for phospho-p65 (S536) and p65. The image shown is representative of at least two independent experiments. B-C , HeLa cells were treated with a mock water control or 0.6 μM of the TBK1/IKKe inhibitor MRT67307 for 1.5 hours, then transfected with 10 μM 2’3’-cGAM(PS) 2 (Rp/Sp) and Trans IT-LT1 at a 1:1 μg/μL ratio, or dsDNA (2 μg/mL) and Trans IT-LT1 at a 1:2 μg/μL ratio, or untreated (UN). Cells were harvested 5 hours after treatment, and lysates were resolved with SDS-PAGE and probed for p-p65 (S536) or p65 as in A (25 μg lysate loaded) or for p-IRF3 (S386) and IRF3 (100 μg lysate loaded). Image shown is representative of at least two independent experiments.

Article Snippet: Primary antibodies to the following human proteins were used: phospho-STING (S366, Cell Signaling Technology, cat. 19781S); RELA/p65 (R&D, cat. AF5078-SP); phospho-RELA/p65 (S536, R&D, cat. MAB72261-SP); IRF3 (R&D, cat. AF4019-SP); phospho-IRF3 (S386, Cell Signaling Technology, cat. 37829T); and beta-tubulin loading control (Abcam, cat. ab6046).

Techniques: SDS Page, Transfection

Journal: bioRxiv

Article Title: Interferon regulatory factor 3 upregulates the Treg recruitment factor CCL22 in response to double-stranded DNA in cancer cells

doi: 10.1101/2022.03.08.483519

Figure Lengend Snippet: A , HeLa cells were seeded in 12-well plates to achieve ∼ 65% confluency in 24 hours, at which time cells were treated with either 8 ng/mL TNFα or a water vehicle control. Cells were harvested 24 hours after treatment, and RTqPCR was performed. Resulting levels of CCL22 mRNA are shown. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with an unpaired, two-tail t test; error bars represent standard deviations. B , HeLa cells were seeded and grown as for A and treated with either 10 ng/mL PMA or a 0.0004% DMSO vehicle control. Significance testing was performed with a one-way ANOVA and Tukey’s pairwise comparison; error bars represent standard deviations. C , HeLa cells expressing no shRNA (NS) or stably expressing a non-targeting shRNA (NT) or shRNAs against RELA/p65 (p65-1 or p65-2) were harvested for RTqPCR; levels of RELA/p65 RNA are shown relative to untreated (NS) cells. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with a one-way ANOVA and Tukey’s pairwise comparison; error bars represent standard deviations. D , Lysates (20 μg) from HeLa cells carrying no shRNA (NS), non-targeting shRNA (NT) or shRNAs against RELA/p65 (p65-1 or p65-2) were resolved using SDS-PAGE and probed for RELA/p65 and beta-tubulin. The image shown is representative of at least three independent experiments. E , HeLa cells described in C and D were transfected with a mock control or dsDNA (2 μg/mL) with Trans IT-LT1 at a 1:2 ratio and harvested after 48 hours for RTqPCR. Resulting fold change of CCL22 mRNA is shown relative to the mock control for each individual cell line. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with a one-way ANOVA and Dunnett’s pairwise comparison of each shRNA group to the control non-targeting group; note that this analysis was performed alongside the IRF3 shRNA groups from in order that all non-targeting control experiments be included; error bars represent standard deviations. F , HeLa cells carrying the shRNAs described above were transfected as in E in parallel experiments using a GFP expression plasmid (2 μg/mL, Trans IT-LT1 1:2 ratio) and imaged 48 hours after transfection. Brightfield (BF) shows the confluency of cells in the same field of view as GFP.

Article Snippet: Primary antibodies to the following human proteins were used: phospho-STING (S366, Cell Signaling Technology, cat. 19781S); RELA/p65 (R&D, cat. AF5078-SP); phospho-RELA/p65 (S536, R&D, cat. MAB72261-SP); IRF3 (R&D, cat. AF4019-SP); phospho-IRF3 (S386, Cell Signaling Technology, cat. 37829T); and beta-tubulin loading control (Abcam, cat. ab6046).

Techniques: Derivative Assay, Expressing, shRNA, Stable Transfection, SDS Page, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: Interferon regulatory factor 3 upregulates the Treg recruitment factor CCL22 in response to double-stranded DNA in cancer cells

doi: 10.1101/2022.03.08.483519

Figure Lengend Snippet: A , HeLa cells expressing no shRNA (NS) or stably expressing a non-targeting shRNA (NT) or shRNAs against IRF3 (IRF3-1 or IRF3-2) were harvested for RTqPCR; levels of IRF3 RNA are shown relative to untreated (NS) cells. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with a one-way ANOVA and Tukey’s pairwise comparison; error bars represent standard deviations. B , Lysates (20 μg) from HeLa cells carrying no shRNA (NS), non-targeting shRNA (NT) or shRNAs against RELA/p65 (p65-1 or p65-2) were resolved using SDS-PAGE and probed for RELA/p65 and beta-tubulin. The image shown is representative of at least three independent experiments. C , HeLa cells described in A and B were transfected with a mock control or dsDNA (2 μg/mL) with Trans IT-LT1 at a 1:2 ratio and harvested after 48 hours for RTqPCR. Resulting fold change of CCL22 mRNA is shown relative to the mock control for each individual cell line. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with a one-way ANOVA and Dunnett’s pairwise comparison of each shRNA group to the control non-targeting group; note that this analysis was performed alongside the RELA/p65 shRNA groups from in order that all non-targeting control experiments be included; error bars represent standard deviations. D , HeLa cells carrying the shRNAs described above were transfected as in C in parallel experiments using a GFP expression plasmid (2 μg/mL, Trans IT-LT1, 1:2 ratio) and imaged 48 hours after transfection. Brightfield (BF) shows the confluency of cells in the same field of view as GFP. E-F , HeLa cells ( E ) and MCF7 cells ( F ) were transfected with a mock control or 2 μg/mL of empty plasmid (EV) or the constitutively active IRF3-5D with Trans IT-LT1 (HeLa) at a 1:2 ratio or TransfeX (MCF7) at a 1:4 ratio. Cells were harvested 48 hours after transfection, and RTqPCR was performed. Resulting fold change of CCL22 mRNA relative to the mock control is shown. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with an unpaired, one-tailed t test; error bars represent standard deviations.

Article Snippet: Primary antibodies to the following human proteins were used: phospho-STING (S366, Cell Signaling Technology, cat. 19781S); RELA/p65 (R&D, cat. AF5078-SP); phospho-RELA/p65 (S536, R&D, cat. MAB72261-SP); IRF3 (R&D, cat. AF4019-SP); phospho-IRF3 (S386, Cell Signaling Technology, cat. 37829T); and beta-tubulin loading control (Abcam, cat. ab6046).

Techniques: Expressing, shRNA, Stable Transfection, Derivative Assay, SDS Page, Transfection, Plasmid Preparation, One-tailed Test

Journal: Cancers

Article Title: Transfection with GLS2 Glutaminase (GAB) Sensitizes Human Glioblastoma Cell Lines to Oxidative Stress by a Common Mechanism Involving Suppression of the PI3K/AKT Pathway

doi: 10.3390/cancers11010115

Figure Lengend Snippet: Transfection with GAB increases the sensitivity of GBM cell lines T98G, U87MG, and LN229 to H 2 O 2 -induced oxidative stress. Upon H 2 O 2 treatment, the GAB-transfected cells of all three cell lines present decreased levels of phosphorylated PI3K and PDK1 compared to the pcDNA-transfected counterparts. Hypothetically, changes in the activity of the PI3K signaling cascade may ensue modulation of RTKs by GAB, but experimental evidence to support this notion is absent. GAB-transfected T98G and U87MG cells display decreased AKT phosphorylation on both Thr308 and Ser473, reduced NF-κB phosphorylation, and increased activity of caspase 3 and 7, suggesting that caspase dependent apoptosis contributes to their death. By contrast, in the LN229 cell line, the lack of changes in the phosphorylation level of NF-κB and in caspase 3 and 7 activities indicates that the mechanism underlying GAB-mediated death is other than caspase dependent apoptosis. Note that in contrast to GAB-transfected T98G and U87MG cells, GAB-transfected LN229 cells present increased AKT phosphorylation on Ser473: The implications (if any) of this difference for the nature of cell death are not known.

Article Snippet: The following antibodies were used: p-AKT (Thr308) (#4056, Cell Signaling, Danvers, MA, USA), p-AKT (Ser473) (#4060, Cell Signaling), AKT (#4691, Cell Signaling), p-PDK1 (Ser241) (#ab109460, Abcam), PDK1 (#17086-1-AP, ProteinTech, Chicago, IL, USA), p-PI3K (Tyr199) (#4228, Cell signaling), PI3K (#11889, Cell Signaling), p-NF-κB (Ser536) (#MAB72261, Novus Biologicals, Littleton, CO, USA), and NF-κB (#4764, Cell Signaling) according to the manufacturer’s instruction.

Techniques: Transfection, Activity Assay, Phospho-proteomics

Journal: Scientific Reports

Article Title: Promotion of the inflammatory response in mid colon of complement component 3 knockout mice

doi: 10.1038/s41598-022-05708-8

Figure Lengend Snippet: Expression levels of members in the NF-κB signaling pathway. ( A ) Expression levels of NF-κB-p65 and IκB-α proteins were determined by Western blot analysis using specific primary antibody and HRP-labeled anti-rabbit IgG antibody. ( B ) Band intensities were determined using an imaging densitometer, and protein expressions were calculated relative to the intensity of β-actin. Tissue samples were collected from 3 to 5 mice per group, and each lysate was analyzed in duplicate for Western blot (final n = 6–10). Data are reported as the mean ± SD. * indicates p < 0.05 compared to the WT mice.

Article Snippet: Proteins (30 μg) were then separated by 4–20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) for 2 h, and subsequently transferred to nitrocellulose membranes for 3 h at 40 V. Each membrane was then incubated separately, overnight at 4 °C, with the following primary antibodies: anti-C3 (ab200999, Abcam Com.), anti-C3aR (bs-2955R, Bioss Inc.), anti-CR1 (LS-C777464, LSBio Inc.), anti-COX-2 (12282, Cell Signaling Technology Inc., Danvers, MA, USA), anti-NLRP3 (15101, Cell Signaling Technology Inc.), anti-Cas 1 (24232, Cell Signaling Technology Inc.), anti-ASC (67824, Cell Signaling Technology Inc.), anti-iNOS (PA3-0304, Thermo Fisher Scientific Inc.), anti-ERK1/2 (9102, Cell Signaling Technology Inc.), anti-p-ERK (E-4) (9101, Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-JNK (9252, Cell Signaling Technology Inc.), anti-p-JNK (9251, Cell Signaling Technology Inc.), anti-p38 (9212, Cell Signaling Technology Inc.), anti-p-p38 (9211, Cell Signaling Technology Inc.), anti-p-NF-κB-p65 (A00284T254, Boster Bio Inc., Pleasanton, CA, USA), anti-IκB (9242, Cell Signaling Technology Inc.), anti-p-IκB (9246S, Cell Signaling Technology Inc.), anti-IL-6 (SC-1265, Santa Cruz Biotechnology Inc.), anti-C5 (ab11898, Abcam Com.), anti-thrombin (ab92621, Abcam Com.), anti-E-cadherin (24E10, Cell Signaling Technology Inc.) or anti-β-actin (4967, Sigma-Aldrich Co.).

Techniques: Expressing, Western Blot, Labeling, Imaging

Journal: Heliyon

Article Title: Zhilong Huoxue Tongyu capsule inhibits rabbit model of hyperlipidemia and atherosclerosis through NF-κB/NLRP3 signaling pathway

doi: 10.1016/j.heliyon.2023.e20026

Figure Lengend Snippet: Details of antibodies.

Article Snippet: p -NFκB , St John's Laboratory, UK , stj28018 , WB.

Techniques:

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