Journal: Gastroenterology Report

Article Title: TRUB1 is a novel biomarker for promoting malignancy in colorectal cancer via NFκB signaling

doi: 10.1093/gastro/goaf027

Figure Lengend Snippet: Potential downstream targets of TRUB1 in CRC. (A) Scatter plot showing genes with significant changes in RNA-seq after TRUB1 knock-down; (B) GSEA analysis showing enrichment of TNFα signaling via NFκB in cells with high TRUB1 expression; (C) Western blot results for TRUB1, P65, and pP65 in control and TRUB1-knock-down HCT116 cells; (D) intersection analysis of gene sets between HCT116-sh-TRUB1-1 and HCT116-sh-TRUB1-2 (FC ≥ 1.5, FDR ≤ 0.05); (E) intersection analysis of TNFα signaling via NFκB gene sets between HCT116-sh-TRUB1-1 and HCT116-sh-TRUB1-2 ( P ≤ 0.05); (F) intersection analysis of gene sets between GSEA_NFκB signaling (61 genes), RNA-seq (261 genes), and apoptosis via NFκB (33 genes); (G) expression of TRUB1 and BIRC3 mRNA in control and TRUB1-knock-down HCT116 cells. GSEA = Gene Set Enrichment Analysis, pP65 = phosphorylated P65, BIRC3 = Baculoviral IAP repeat-containing protein 3.

Article Snippet: The antibodies that were used in the Western blot were TRUB1 (1:2,000; #PA536003; Thermo, Waltham, MA, USA); TRUB2 (1:2,000; #K110029P; Solarbio, Beijing, China); p65 (1:2,000; #a22331; ABclonal, Woburn, MA, USA); phosphorylated-P65 (pP65; 1:2,000; #af2006; Affinity, Nottingham, UK); GAPDH (1:1,000; #BM3876; BOSTER, Pleasanton, CA, USA); Cytochrome C (1:2,000; #A13430; ABclonal); Bcl 2 (1:2,000; #A0208; ABclonal); Caspase-8 (1:2,000; #A0215; ABclonal); Caspase-3 (1:2,000; #A02156; ABclonal); Caspase-9 (1:2,000; #A11910; ABclonal); cleaved Caspase-3 (1:2,000; #9661 T; CST, Danvers, MA, USA); and β-Actin (1:2,000; #AC038; ABclonal).

Techniques: RNA Sequencing, Knockdown, Expressing, Western Blot, Control

Journal: Frontiers in Nutrition

Article Title: Curcumin attenuates liver injury by modulating the AGE–RAGE axis and metabolic homeostasis in high-fat diet/streptozotocin-induced type 2 diabetic mice

doi: 10.3389/fnut.2025.1710380

Figure Lengend Snippet: CUR modulates AGE/RAGE signaling and its downstream targets PI3K/Akt and NFκB pathways. (A,B) The levels of plasma (A) and hepatic AGEs (B) were measured by ELISA ( n = 5). (C) The mRNA expression levels of RAGE in the liver were analysed ( n = 3). (D–F) Representative bands and quantitative expression levels of p-PI3K, PI3K, p-Akt, Akt, p-NFκB p65, and NFκB p65 proteins in the liver from Western blot were presented. β-actin was served as an internal reference ( n = 3). For quantitative analysis (D–F) , the band intensity of each target protein was normalized to its corresponding internal control. The normalized values were presented relative to the mean of the NC group, which was set to 1. * p < 0.05, ** p < 0.01 and *** p < 0.001; ns, not significant difference.

Article Snippet: Antibodies against PI3K (CAT#AF6241), phospho-PI3K (Tyr 607 ) (CAT#AF3241), Akt (CAT#AF6261), phospho-Akt (Ser 473 ) (CAT#AF0016), phospho-NFκB p65 (Ser 536 ) (CAT#AF2006), TNF- α (CAT#AF7014), and IL-1 β (CAT#AF5103) were purchased from Affinity Biosciences (OH, USA).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Control

Journal: Heliyon

Article Title: Bardoxolone methyl ameliorates osteoarthritis by inhibiting osteoclastogenesis and protecting the extracellular matrix against degradation

doi: 10.1016/j.heliyon.2023.e13080

Figure Lengend Snippet: CDDO-Me inhibits with the NF-κB pathways in BMMs and ATDC5 cells. (A) Western blots analysis of NF-κB p65, p- NF-κB p65 in OCs pretreated with CDDO-Me (31.25 nM and 62.5 nM), followed by stimulation with or without RANKL (50 ng/mL) for 30min. (B) The quantification of NF-κB p65, p- NF-κB p65, theβ-actin was selected as the control protein, n = 3. (C) Western blots analysis of NF-κB p65, p- NF-κB p65 in ATDC5 pretreated with CDDO-Me (0.2 μM, 0.4 μM, and 0.6 μM), followed by stimulation with or without IL-1β (10 ng/mL) for 30 min. See supplementary material for non-adjusted images. (D) The quantification of NF-κB p65, p–NF–κB p65. The β-actin was selected as the control protein, n = 3. (E) Immunofluorescence staining was performed to analyze the nuclear translocation of p–NF–κB p65 upon IL-1βstimulation in ATDC5 cells, scale bar, 20 μm. (F) Nuclear fluorescence intensity/cytoplasmic fluorescence intensity. n = 3. *P < 0.05 and **P < 0.01 compared with control group. # P < 0.05, ## P < 0.01 and ### P < 0.001 compared with IL-1βgroup or RANKL group.

Article Snippet: The membranes were incubated overnight at 4 °C with the following primary antibodies: anti-MMP13 (1:1000, Cat.# AF5355, Affinity), anti-MMP3 (1:1000, Cat.# AF0217, Affinity), anti-Aggrecan (1:1000, sc-166,951, Santa Cruz), anti-Sox9 (1:1000, ab185966, Abcam), anti-p65 (1:1000, Cat.# BF0382, Affinity), anti-phospho-p65 (1:1000, Cat.# AF2006, Affinity), anti-Nrf2 (1:1000, Cat.# AF0639, Affinity), anti–HO–1 (1:1000, ab52947, Abcam), anti- c -Fos (1:1000, Cat.# AF0132, Affinity), anti-NFATc1 (1:1000, sc-7294, Santa Cruz), anti-CTSK (1:1000, sc-48353, Santa Cruz), anti-Integrinβ3 (sc-365679, Santa Cruz), anti-GAPDH (1:1000, Cat. AC001, Abconal), and anti-βactin (1:1000, Cat. AC026, Abconal).

Techniques: Western Blot, Immunofluorescence, Staining, Translocation Assay, Fluorescence

Journal: Heliyon

Article Title: Bardoxolone methyl ameliorates osteoarthritis by inhibiting osteoclastogenesis and protecting the extracellular matrix against degradation

doi: 10.1016/j.heliyon.2023.e13080

Figure Lengend Snippet: CDDO-Me prevents subchondral bone loss and alleviates OA development in DMM mice, n = 6. (A) 3D micro-CT images of subchondral bone after sham operation or DMM surgery, scale bar, 1 mm. (B) HE staining, scale bar, 100 μm. Safranin O staining, scale bar, 100 μm (top panelsnjjj); 50 μm (bottom panels). (C) Tartrate-resistant acid phosphatase (TRAP) staining, scale bar, 100 μm (top panels); 50 μm (bottom panels). (D) Immunohistochemical staining of Col2a1, MMP-13, p–NF–κB p65 and Nrf2 expression, scale bar, 50 μm. (E) Quantitative micro-CT analysis of tibial subchondral bone of bone volume fraction (BV/TV), bone surface/volume ratio (BS/BV), structure model index (SMI), trabecular number (Tb.N), subchondral trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp). (F) Osteoarthritis Research Society International (OARSI) score indicates cartilage degeneration after the DMM operation. (G) Quantitative analysis of osteoclast surface per millimeter bone perimeter (Oc.s/BS) indicates subchondral bone remodeling. (H)The quantitative analysis of positive cells of Col2a1, MMP-13, p–NF–κB p65 and Nrf2 in each group. Low CDDO-Me (10 mg.kg-1), High CDDO-Me (50 mg.kg-1). *P < 0.05, ***P < 0.01 and ****P < 0.0001 compared with sham group. # P < 0.05, ## P < 0.01, ### P < 0.001 and #### P < 0.0001 compared with DMM group.

Article Snippet: The membranes were incubated overnight at 4 °C with the following primary antibodies: anti-MMP13 (1:1000, Cat.# AF5355, Affinity), anti-MMP3 (1:1000, Cat.# AF0217, Affinity), anti-Aggrecan (1:1000, sc-166,951, Santa Cruz), anti-Sox9 (1:1000, ab185966, Abcam), anti-p65 (1:1000, Cat.# BF0382, Affinity), anti-phospho-p65 (1:1000, Cat.# AF2006, Affinity), anti-Nrf2 (1:1000, Cat.# AF0639, Affinity), anti–HO–1 (1:1000, ab52947, Abcam), anti- c -Fos (1:1000, Cat.# AF0132, Affinity), anti-NFATc1 (1:1000, sc-7294, Santa Cruz), anti-CTSK (1:1000, sc-48353, Santa Cruz), anti-Integrinβ3 (sc-365679, Santa Cruz), anti-GAPDH (1:1000, Cat. AC001, Abconal), and anti-βactin (1:1000, Cat. AC026, Abconal).

Techniques: Micro-CT, Staining, Immunohistochemical staining, Expressing

Journal: Biomedicines

Article Title: Electroacupuncture Attenuates Intestinal Barrier Disruption via the α7nAChR-Mediated HO-1/p38 MAPK/NF-κB Pathway in a Mouse Model of Metabolic Dysfunction-Associated Fatty Liver Disease: A Randomized, Single-Blind, Controlled Trial

doi: 10.3390/biomedicines13040802

Figure Lengend Snippet: EA mitigated MAFLD via the involvement of the α7nAChR-mediated HO-1/p38 MAPK/NF-κB pathway. ( A – E ) Relative protein contents of p-p38 MAPK, p38 MAPK, p-p65, p65, α7nAChR, and HO-1 were evaluated with Western blot. ( F ) The relative mRNA content of HO-1 was evaluated with RT-qPCR. ( G ) Immunofluorescence assay with p-p38 MAPK (green) and DAPI (blue) in the intestine tissues (scale bar = 100 μm). ( H ) Immunohistochemistry assays with p-p65 and HO-1 in the intestine tissues (scale bar = 100 μm). Data were obtained from at least three independent assays in several groups of mice. * p < 0.05, and *** p < 0.001 vs. CON. & p < 0.05, && p < 0.01, and &&& p < 0.001 vs. HFD. # p < 0.05, ## p < 0.01, and ### p < 0.001 vs. EA.

Article Snippet: During the process of immunohistochemistry staining, after the blockage in 5% bovine serum albumin (BSA, Solarbio, Beijing, China), slices were incubated with primary antibody against α-SMA (14395-1-AP, Proteintech, Rosemont, IL, USA), phospho-NF-kB p65 (AF2006, Affinity, Changzhou, China), and HO-1 (10701-1-AP, Proteintech, Rosemont, IL, USA) at 4 °C.

Techniques: Western Blot, Quantitative RT-PCR, Immunofluorescence, Immunohistochemistry

Journal: Biomedicines

Article Title: Electroacupuncture Attenuates Intestinal Barrier Disruption via the α7nAChR-Mediated HO-1/p38 MAPK/NF-κB Pathway in a Mouse Model of Metabolic Dysfunction-Associated Fatty Liver Disease: A Randomized, Single-Blind, Controlled Trial

doi: 10.3390/biomedicines13040802

Figure Lengend Snippet: Schematic presentation of the underlying mechanisms of EA at ST-36 on the gut barrier in MAFLD. EA treatment activates the vagus nerve-mediated CAP, in which ACh released by vagal efferent fibers binds to α7nAChR on macrophages to promote anti-inflammation effects. It enhances HO-1 expression, thereby suppressing the phosphorylation of downstream targets p38 MAPK and NF-κB p65, ultimately repairing gut leakage (including enhanced expressions of tight junctions and decreased gut permeability) and reducing inflammation to ameliorate the pathogenesis of HFD-induced MAFLD via the gut–liver axis.

Article Snippet: During the process of immunohistochemistry staining, after the blockage in 5% bovine serum albumin (BSA, Solarbio, Beijing, China), slices were incubated with primary antibody against α-SMA (14395-1-AP, Proteintech, Rosemont, IL, USA), phospho-NF-kB p65 (AF2006, Affinity, Changzhou, China), and HO-1 (10701-1-AP, Proteintech, Rosemont, IL, USA) at 4 °C.

Techniques: Expressing, Phospho-proteomics, Permeability