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: bioRxiv

Article Title: Viral mimicry redirects immunosuppressed colorectal tumour landscapes towards a proinflammatory and CMS1-like regenerative state

doi: 10.1101/2024.11.28.625928

Figure Lengend Snippet: Cell lineage specific response to poly(I:C). (A) Schematic of the experimental outline of the response to poly(I:C). (B) Quantification of cell viability using AnnexinV/PI-stained cells being assessed by flow cytometry at 24hrs and 48hrs (n=3 for each cell line at each concentration). (C-F) Whole lysate western blots of (C) phosphorylated (phospho) STAT1/STAT1 total, (D) phospho NFkB(p65)/ NFkB(p65) total and (E) phospho IkBα/ IkBα total across 6 time points with the absence or addition of poly(I:C). (n=3 for each cell line at each time point). (G) Nuclear and cytoplasmic NFkB (p65) in the epithelial (HCT116) and (H) THP-1 macrophages across 6 time points with the absence or addition of poly(I:C). (Student t-test carried out on Prism: ns: p > 0.05, *: p <= 0.05, **: p <= 0.01, ***: p <= 0.001, ****: p <= 0.0001). Error bars = 1 standard deviation.

Article Snippet: The following Western blot antibodies were used: Phospho NF-κB p65/RelA (S536) (RRID: AB_331284), Phospho IκBα (S32) (RRID: AB_561111), IκBα (RRID: AB_390781), STAT1 (RRID: AB_2198300), STAT2 (RRID: AB_2271323) (all Cell Signalling Technology); GAPDH (Abcam, RRID: AB_2107448), NF-κB p65 (Total RelA) (Santa Cruz Biotechnology, RRID: AB_628017),; Acetyl Histone H3 (EMD Millipore); Caspase-8 (both Enzo Life Sciences) (RRID: AB_2050949), pSTAT1 (Y701) (Invitrogen, ThermoFisher) (RRID: AB_2533113).

Techniques: Staining, Flow Cytometry, Concentration Assay, Western Blot, Standard Deviation

Journal: bioRxiv

Article Title: Viral mimicry redirects immunosuppressed colorectal tumour landscapes towards a proinflammatory and CMS1-like regenerative state

doi: 10.1101/2024.11.28.625928

Figure Lengend Snippet: Transcriptional analysis of in vitro model. (A) Outline of experimental design of the microarray analysis. (n=16 samples, n=4 in each cell line for each condition). (B) Enrichment plots of the gene set enrichment analysis (GSEA) results of Interferon-alpha response and Interferon-gamma response (epithelial;pink and macrophage; purple). (C) Single sample gene set enrichment analysis (ssGSEA), of these same interferon gene sets. (D) Transcription factor activity scores of STAT1 and RELA. (E) Enrichment plots of the GSEA results of TNF-alpha via NFkB for epithelial (pink) and macrophage (purple). (F) Schematic illustrating the development of the poly(I:C) response signatures (PRS), macrophage (n=26 genes), epithelial (n=26 genes) and overlap (n=24 genes). (G) Expression of the PRS genes across the treated and untreated cell lines. (H) Macrophage PRS (left) and Epithelial PRS (right), (I) Macrophage PRS + Epithelial PRS and (J) Overlap PRS was compared across the consensus molecular subtypes (CMS) in transcriptional profiles from a stage II/III colon cancer cohort (GSE39582), (n=258; CMS1 = 49, CMS2 = 75, CMS3 = 35, CMS4 = 58, unknown = 41) (Wilcoxon rank sum test, with CMS1 as the reference group). (K) Schematic highlighting the PRS biology highest in CMS1, followed by CMS4 and then CMS2/3. (Wilcoxon rank sum carried out by ggpubr: ns: p > 0.05, *: p <= 0.05, **: p <= 0.01, ***: p <= 0.001, ****: p <= 0.0001).

Article Snippet: The following Western blot antibodies were used: Phospho NF-κB p65/RelA (S536) (RRID: AB_331284), Phospho IκBα (S32) (RRID: AB_561111), IκBα (RRID: AB_390781), STAT1 (RRID: AB_2198300), STAT2 (RRID: AB_2271323) (all Cell Signalling Technology); GAPDH (Abcam, RRID: AB_2107448), NF-κB p65 (Total RelA) (Santa Cruz Biotechnology, RRID: AB_628017),; Acetyl Histone H3 (EMD Millipore); Caspase-8 (both Enzo Life Sciences) (RRID: AB_2050949), pSTAT1 (Y701) (Invitrogen, ThermoFisher) (RRID: AB_2533113).

Techniques: In Vitro, Microarray, Activity Assay, Expressing

Journal: Cell reports

Article Title: Integration of phospho-signaling and transcriptomics in single cells reveals distinct Th17 cell fates

doi: 10.1016/j.celrep.2025.116006

Figure Lengend Snippet: (A) Schematic overview of antibody-oligonucleotide conjugation and preprocessing for inCITE-seq. (B) Uniform manifold approximation and projection (UMAP) of scRNA-seq data from 2,230 cells annotated according to stimulation conditions. (C) UMAP plots of log normalized counts from inCITE-seq for p-ERK1/2 (T204/Y204), p-FOS (S32), p-STAT3 (Y705), and p-p65 (S536). (D) Comparisons of percent positivity for the four phospho-targets using inCITE-seq versus flow cytometry from the same cultures. Positive gating for inCITE-seq was determined as any cell with >0 phospho-target counts after isotype subtraction and natural log (ln) +1 scaling. (E) Linear regressions for the four phospho-targets comparing percent positivity from inCITE-seq versus flow cytometry. One-way ANOVA with Sidak’s multiple comparisons test. * p < 0.05; ** p ≤ 0.01. Data are represented as mean ± SEM.

Article Snippet: anti-phos-p65 S536 (93H1) , Cell Signaling , 3033.

Techniques: Conjugation Assay, Flow Cytometry