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: 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