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: 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: 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: Journal of Biomedicine and Biotechnology

Article Title: Hunting for Serine 276-Phosphorylated p65

doi: 10.1155/2010/275892

Figure Lengend Snippet: Anti-phospho-p65 Ser276 (Cell Signaling no. 3037) detects atypical (with MWs of 80 and 130 kDa), inducible immunoreactivities in different cell types, induced with different NF- κ B-activating agents. Cells were stimulated for increasing times (L929sA, A549, L363) or for 30 min (Raw264.7, C2C12, 1321N1) with TNF- α (2000 IU/mL), LPS (1 μ g/mL), PMA (10 ng/mL), forskolin (forsk, 10 μ M) or isoproterenol (iso, 10 μ M). For blocking experiments, lysates were loaded on one gel in duplicate and after transfer, blots were cut in half. Immunodetection was performed in parallel, using Cell Signaling no. 3037 or Cell Signaling no. 3037 preincubated for at least 30 minutes with double volume of blocking peptide (BLOCK). Arrow 1 indicates an immunoreactive band with an MW of 130 kDa; arrow 2 indicates an immunoreactive band of 80 kDa.

Article Snippet: To investigate whether other available antibodies would perhaps be more useful to detect Ser276 phosphorylated p65 via Western Blotting, we investigated phosphorylation of p65 at Ser276 using two anti-P-p65 Ser276 antibodies distributed by independent companies (no. 11011 from SAB and no. 100-401-264 from Rockland).

Techniques: Blocking Assay, Immunodetection

Journal: Journal of Biomedicine and Biotechnology

Article Title: Hunting for Serine 276-Phosphorylated p65

doi: 10.1155/2010/275892

Figure Lengend Snippet: The Cell Signaling no. 3037 anti-P-p65 Ser276-immunoreactive band persists in cells where p65 is silenced via an siRNA approach. 1321N1 cells were transiently transfected with control or p65 siRNA as described in . Cells were induced for 90 min with TNF- α (2000 IU/mL), iso (10 μ M), or a combination of both stimuli. Presence of Ser276-phosphorylated p65 in cell lysates was analysed by Western Blot using Cell Signaling no. 3037 (A1). 130 and 80 kDa immunoreactivities are indicated by arrows. Western Blot to detect anti-P-p65Ser536 (65 kDa arrow) was performed on the same lysates (B1). Blots A1 and B1 were extensively washed and reprobed with anti-p65 to investigate efficiency of p65 knock-down. Anti-p65 detected bands are marked with arrows (A2, B2). Finally, blots were reprobed with anti-tubulin to check loading efficiency. Anti-tubulin-detected bands are marked with arrows (A3, B3).

Article Snippet: To investigate whether other available antibodies would perhaps be more useful to detect Ser276 phosphorylated p65 via Western Blotting, we investigated phosphorylation of p65 at Ser276 using two anti-P-p65 Ser276 antibodies distributed by independent companies (no. 11011 from SAB and no. 100-401-264 from Rockland).

Techniques: Transfection, Control, Western Blot, Knockdown

Journal: Journal of Biomedicine and Biotechnology

Article Title: Hunting for Serine 276-Phosphorylated p65

doi: 10.1155/2010/275892

Figure Lengend Snippet: Four independent anti-P-p65 Ser276 antibodies detect inducible bands, with MWs of 80 and 130 kDa, that do not disappear upon p65 knock-down, but are inhibited when PKAc α is silenced. 1321N1 cells were transiently transfected with control, p65 or PKAc α siRNA, as described in . Cells were induced for 90 min with TNF- α (2000 IU/mL), iso (10 μ M) or a combination of both stimuli. Presence of Ser276-phosphorylated p65 in cell lysates was analysed by Western Blot using SAB no. 11011 (A1), Rockland no. 100-401-264 (B1), Cell Signaling no. 3037 (B3), or a homemade anti-P-p65 Ser276 antibody (C1). 130 and 80 kDa immunoreactivities are indicated by arrows (1 and 2, resp.). Blots were extensively washed and reprobed with a mixture of anti-p65 and anti-PKAc α to investigate knock-down efficiency. Anti-p65-detected bands are marked by the upper arrow, anti-PKAc α -reactive bands are marked by the lower arrow (A2, B2, C2). Tubulin loading controls are added as Supplementary Figure 1.

Article Snippet: To investigate whether other available antibodies would perhaps be more useful to detect Ser276 phosphorylated p65 via Western Blotting, we investigated phosphorylation of p65 at Ser276 using two anti-P-p65 Ser276 antibodies distributed by independent companies (no. 11011 from SAB and no. 100-401-264 from Rockland).

Techniques: Knockdown, Transfection, Control, Western Blot

Journal: Journal of Biomedicine and Biotechnology

Article Title: Hunting for Serine 276-Phosphorylated p65

doi: 10.1155/2010/275892

Figure Lengend Snippet: Presence of anti-P-p65 Ser276-reactive bands in p65 deficient MEF cells. p65 knock-out MEF cells (−/−), or p65 −/− MEF cells reconstituted with wild type p65 (p65 wt) or p65 with Ser276 mutated to alanine (p65 S/A), were induced with forskolin (forsk, 10 μ M) for 0, 30, and 60 min. Presence of Ser276-phosphorylated p65 in cell lysates was analysed by Western Blot using Cell Signaling no. 3037 (A1) or SAB no. 11011 (B1). Blots were extensively washed and reprobed with anti-p65 to confirm p65 absence in −/− MEFs, and p65 presence in wt and S/A reconstituted MEFs (A2 and B2, arrows indicate anti-p65 detected bands).

Article Snippet: To investigate whether other available antibodies would perhaps be more useful to detect Ser276 phosphorylated p65 via Western Blotting, we investigated phosphorylation of p65 at Ser276 using two anti-P-p65 Ser276 antibodies distributed by independent companies (no. 11011 from SAB and no. 100-401-264 from Rockland).

Techniques: Knock-Out, Western Blot

Journal: Journal of Biomedicine and Biotechnology

Article Title: Hunting for Serine 276-Phosphorylated p65

doi: 10.1155/2010/275892

Figure Lengend Snippet: The detected 130 kDa and 80 kDa immunoreactivities are not NF- κ B p105/p50 or c-Rel. 1321N1 cells were transiently transfected with control, p105/p50 or c-Rel siRNA as described in . Cells were induced for 30 min with TNF- α (2000 IU/mL) or iso (10 μ M). Presence of Ser276-phosphorylated p65 in cell lysates was analysed by Western Blot using Cell Signaling no. 3037 (A1, B1). 130 and 80 kDa immunoreactivities are indicated by arrows (1 and 2 resp.). Because of the overlap of the 130 kDa and the p105 immunoreactive bands, blot A was first stripped and reprobed with anti-p105/p50 (A2). p105 immunoreactivity is indicated by the upper arrow; p50 immunoreactivity is indicated by the lower arrow. Blot B was extensively washed and reprobed (without stripping) with anti-c-Rel (B2). c-Rel immunoreactivity is indicated by an arrow. Tubulin was used as a loading control (A3, B3).

Article Snippet: To investigate whether other available antibodies would perhaps be more useful to detect Ser276 phosphorylated p65 via Western Blotting, we investigated phosphorylation of p65 at Ser276 using two anti-P-p65 Ser276 antibodies distributed by independent companies (no. 11011 from SAB and no. 100-401-264 from Rockland).

Techniques: Transfection, Control, Western Blot, Stripping Membranes

Journal: Journal of Biomedicine and Biotechnology

Article Title: Hunting for Serine 276-Phosphorylated p65

doi: 10.1155/2010/275892

Figure Lengend Snippet: In vitro recognition of the phosphorylated Serine 276 residue of p65 by Cell Signaling no. 3037. Recombinant wt p65-GST or SC mutant p65-GST fusion proteins were in vitro phosphorylated using recombinant MSK-1. 250 ng of the recombinant proteins were spotted on a nitrocellulose membrane and subjected to Western Blotting with Ser276 phospho-specific p65 antibody, either preincubated with 10 μ g/mL phosphorylated peptide that was used to generate the antibody (upper panel) or not. 1 μ g of recombinant protein was separated by SDS-PAGE after the in vitro kinase assays performed either in the presence of active MSK-1 and/or 10 μ M H89 (lower panel) or not.

Article Snippet: To investigate whether other available antibodies would perhaps be more useful to detect Ser276 phosphorylated p65 via Western Blotting, we investigated phosphorylation of p65 at Ser276 using two anti-P-p65 Ser276 antibodies distributed by independent companies (no. 11011 from SAB and no. 100-401-264 from Rockland).

Techniques: In Vitro, Residue, Recombinant, Mutagenesis, Membrane, Western Blot, SDS Page