Journal: Advanced Science

Article Title: VDLIN: A Deep Learning‐Based Platform for Methylcobalamin‐Inspired Immunomodulatory Compound Screening

doi: 10.1002/advs.202413775

Figure Lengend Snippet: Co7 induces Ifnb1 expression via the TLR4 signaling pathway. A) Volcano plot illustrating the distribution of differentially expressed genes (DEGs) between the Co7 and DMSO treatment groups after 3 h in RAW 264.7 cells. Fold changes are presented as log 2 transformations. Red dots represent DEGs upregulated in the Co7 group. B) Protein‐protein interaction (PPI) network analysis of differentially expressed genes (DEGs) in the Co7‐treated group compared to the DMSO group, revealing significant enrichment in pathways associated with the innate immune response and type I interferon signaling. C) KEGG pathway enrichment analysis of DEGs induced by Co7, highlighting associations with innate immune response pathways. D) Co7 (50 µmol/L) exhibiting strong antiviral effects against VSV in RAW 264.7 and HT29 cells. E) Co7 significantly reduced the inflammatory response induced by LPS, VSV, EMCV, and HSV in RAW 264.7 cells. F) Volcano plot representing the differential gene expression analysis between Co7‐ and LPS‐treated RAW 264.7 cells after 3 h of treatment. Fold changes are displayed as log 2 transformations. Red dots indicate genes upregulated in the Co7 group, while blue dots represent downregulated genes compared to LPS treatment. G) Western blot analysis demonstrating that Co7 inhibited the expression of iNOS and COX2, as well as the phosphorylation of NF‐κB‐P65 at the protein level in RAW 264.7 cells. H) Co7 significantly reduced the mortality rate in mice (n = 10 per group) following LPS challenge (20 mg/kg), compared to the PBS control group. RT‐qPCR data were presented as means ± SEM from three independent experiments. Statistical significance was determined using one‐way ANOVA with Bonferroni's multiple comparisons test (left three panels in E), paired‐samples t‐test (D, right panel of EMCV and HSV in E), or the log‐rank test (H). * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Each inhibitor was dissolved in anhydrous DMSO and diluted to its respective working concentration: C29 (10 μM, S6597, Selleck) served as a TLR2 inhibitor; Procyanidin B1 (30 μM, HY‐N0795, MedChemExpress) acted as a TLR4 inhibitor; MyD88‐IN‐1 (30 μM, HY‐149992, MedChemExpress) was used to inhibit MyD88; Pepinh‐TRIF TFA (30 μM, HY‐P2565, MedChemExpress) functioned as a TRIF inhibitor; and GSK8612 (5 μM, T5540, TargetMol) inhibited TBK1/IKKε.

Techniques: Expressing, Gene Expression, Western Blot, Phospho-proteomics, Control, Quantitative RT-PCR

Journal: Aging (Albany NY)

Article Title: Geniposide-mediated protection against amyloid deposition and behavioral impairment correlates with downregulation of mTOR signaling and enhanced autophagy in a mouse model of Alzheimer's disease

doi: 10.18632/aging.101759

Figure Lengend Snippet: Geniposide treatment decreases mTOR activation markers in brains of APP/PS1 mice. Hippocampal expression of Akt, mTOR, and 4E-BP1, and their respective phosphorylated forms was detected by western blot. The expression of p-Akt ( A ) and p-mTOR ( B ) was enhanced in APP/PS1 mice compared to WT, and geniposide attenuated this increase. The expression of p-4E-BP1 ( C ) in APP/PS1 mice was reduced compared to WT, and geniposide partly restored this decrease. Data are presented as mean ± SEM (n = 6). *** p < 0.001, ** p < 0.01, * p < 0.05 vs. WT; # p < 0.05 vs. APP/PS1 mice (one-way ANOVA, Tukey's Multiple Comparison Test). WT: wild-type mice. GP: geniposide.

Article Snippet: The membranes were blocked in 5% bovine serum albumin in TBST (Tris-buffered saline with 0.05% Tween-20) for 1h, and incubated overnight at 4°C with primary antibodies directed against: Akt (1:1,000), p-Akt (1:2,000), mTOR (1:1,000), p-mTOR (1:1,000), 4E-BP1 (1:1,000), or p-4E-BP1 (1:2,000), followed by incubation at 4°C for 2h with a goat-anti-rabbit IgG-horseradish peroxidase-conjugated secondary antibody (Boster, Wuhan, China).

Techniques: Activation Assay, Expressing, Western Blot, Comparison

Journal: American Journal of Cancer Research

Article Title: NRSN2 promotes osteosarcoma cell proliferation and growth through PI3K/Akt/MTOR and Wnt/β-catenin signaling

doi:

Figure Lengend Snippet: NRSN2 regulates PI3K/Akt/GSK3β axis and Wnt/β-catenin signaling in osteosarcoma cells. A. The level of phosphorylated Akt, mTOR, p-GSK3β and nuclear β-catenin (nu-β-catenin) are positively correlated with the level of NRSN2 in U2OS and MG63 cells. B. Luciferase reporter assays revealed that NRSN2 could regulate Wnt/β-catenin signaling in U2OS and MG63 cells. C. Knockdown NRSN2 inhibits the expression of CCND1 and c-myc in U2OS cells. D. Overexpression of NRSN2 elevates the mRNA levels of CCND1 and c-myc in MG63 cells. E. The pro-proliferation effect was reversed when treated with IWR-1-endo, a inhibitor of β-catenin. *p<0.05, **p<0.01.

Article Snippet: The following antibodies were used in this study: NRSN2 (1:1000, Proteintech), GAPDH (1:5000, Proteintech), p-Akt, Akt, p-mTOR, mTOR, p-GSK3β, GSK3β, β-catenin and the second antibodies were purchased from Cell Signaling Technology and with 1:1000 dilutions.

Techniques: Luciferase, Expressing, Over Expression

Journal: The American Journal of Pathology

Article Title: Receptor-Interacting Serine/Threonine-Protein Kinase 3 (RIPK3)–Mixed Lineage Kinase Domain-Like Protein (MLKL)–Mediated Necroptosis Contributes to Ischemia-Reperfusion Injury of Steatotic Livers

doi: 10.1016/j.ajpath.2019.03.010

Figure Lengend Snippet: Increases of key necroptosis proteins in steatotic livers. Wild-type mice were fed with chow diet (CD) or Western diet (WD) for 5 to 12 weeks. Liver steatosis, as assessed by staining with hematoxylin and eosin (H&E; A) and oil red O (B). C: Western blot analysis for receptor-interacting serine/threonine-protein kinase (Ripk)-1, Ripk3, and mixed lineage kinase domain-like protein (Mlkl) after total protein extraction from liver homogenates. D: Densitometry analysis. Data are expressed as means ± SEM. n = 3 per group. ∗P < 0.05 and ∗∗P < 0.01 versus CD.

Article Snippet: Antibodies used in this study were Mlkl and β-actin (catalog numbers SAB1302339 and A5441, respectively; Sigma-Aldrich, St. Louis, MO); glyceraldehyde phosphate dehydrogenase (Gapdh), Ripk1, and phosphorylated (p)-Mlkl (catalog numbers 2118, 3493, and 62233; Cell Signaling Technology, Beverly, MA); pMlkl, sodium potassium ATPase, and proteasome subunits (Psm)-α2 and -β5 (catalog numbers ab66596, ab76020, ab109525, and ab3330; Abcam, Cambridge, MA); Ripk3 (catalog number 2283; ProSci, Poway, CA); myeloperoxidase (Mpo; catalog number PP023AA; Biocare Medical, Concord, CA); and 26S proteasome regulatory subunit 4 (Psmc1; catalog number A303-821A; Bethyl Laboratories, Montgomery, TX).

Techniques: Western Blot, Staining, Protein Extraction

Journal: The American Journal of Pathology

Article Title: Receptor-Interacting Serine/Threonine-Protein Kinase 3 (RIPK3)–Mixed Lineage Kinase Domain-Like Protein (MLKL)–Mediated Necroptosis Contributes to Ischemia-Reperfusion Injury of Steatotic Livers

doi: 10.1016/j.ajpath.2019.03.010

Figure Lengend Snippet: Hepatic ischemia-reperfusion (IR) injury in Mlkl wild-type (WT) and knockout (KO) mice fed with chow diet (CD) or Western diet (WD). A–C:Mlkl KO and matched WT mice were fed with CD or WD for 8 weeks and were subjected to 45 minutes of ischemia and 6 and 24 hours of reperfusion. Liver injury as assessed by staining with hematoxylin and eosin (A) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL; B), with the area of necrosis with TUNEL-positive staining measured (C). D: Serum alanine aminotransferase (Alt) levels. E: Expression of mixed lineage kinase domain-like protein (Mlkl) as detected by Western blot and densitometry analysis after extraction of plasma membrane fractions from livers. F: Monomer, oligomer, and polymer of Mlkl on Western blot and densitometry analysis after total protein extraction from liver homogenates and gel electrophoresis under nonreducing conditions. Data are expressed as means ± SEM. n = 4 to 7 per group. ∗P < 0.05 versus WT.

Article Snippet: Antibodies used in this study were Mlkl and β-actin (catalog numbers SAB1302339 and A5441, respectively; Sigma-Aldrich, St. Louis, MO); glyceraldehyde phosphate dehydrogenase (Gapdh), Ripk1, and phosphorylated (p)-Mlkl (catalog numbers 2118, 3493, and 62233; Cell Signaling Technology, Beverly, MA); pMlkl, sodium potassium ATPase, and proteasome subunits (Psm)-α2 and -β5 (catalog numbers ab66596, ab76020, ab109525, and ab3330; Abcam, Cambridge, MA); Ripk3 (catalog number 2283; ProSci, Poway, CA); myeloperoxidase (Mpo; catalog number PP023AA; Biocare Medical, Concord, CA); and 26S proteasome regulatory subunit 4 (Psmc1; catalog number A303-821A; Bethyl Laboratories, Montgomery, TX).

Techniques: Knock-Out, Western Blot, Staining, TUNEL Assay, Expressing, Extraction, Clinical Proteomics, Membrane, Polymer, Protein Extraction, Nucleic Acid Electrophoresis

Journal: The American Journal of Pathology

Article Title: Receptor-Interacting Serine/Threonine-Protein Kinase 3 (RIPK3)–Mixed Lineage Kinase Domain-Like Protein (MLKL)–Mediated Necroptosis Contributes to Ischemia-Reperfusion Injury of Steatotic Livers

doi: 10.1016/j.ajpath.2019.03.010

Figure Lengend Snippet: Detection of neutrophil infiltration in Mlkl wild-type (WT) and knockout (KO) mouse livers after ischemia-reperfusion (IR) surgery. Mlkl KO and matched WT mice were fed with chow diet (CD) or Western diet (WD) and subjected to 45 minutes of ischemia and 6 and 24 hours of reperfusion. A: Neutrophil staining with myeloperoxidase on liver sections after 6 and 24 hours of reperfusion. B: Mpo-positive cells were counted in 20 high-power fields (HPFs). C: mRNA level of a neutrophil marker Ly6G as analyzed by real-time RT-PCR. Data are expressed as means ± SEM. n = 4 to 7 per group. ∗P < 0.05 versus WT.

Article Snippet: Antibodies used in this study were Mlkl and β-actin (catalog numbers SAB1302339 and A5441, respectively; Sigma-Aldrich, St. Louis, MO); glyceraldehyde phosphate dehydrogenase (Gapdh), Ripk1, and phosphorylated (p)-Mlkl (catalog numbers 2118, 3493, and 62233; Cell Signaling Technology, Beverly, MA); pMlkl, sodium potassium ATPase, and proteasome subunits (Psm)-α2 and -β5 (catalog numbers ab66596, ab76020, ab109525, and ab3330; Abcam, Cambridge, MA); Ripk3 (catalog number 2283; ProSci, Poway, CA); myeloperoxidase (Mpo; catalog number PP023AA; Biocare Medical, Concord, CA); and 26S proteasome regulatory subunit 4 (Psmc1; catalog number A303-821A; Bethyl Laboratories, Montgomery, TX).

Techniques: Knock-Out, Western Blot, Staining, Marker, Quantitative RT-PCR

Journal: The American Journal of Pathology

Article Title: Receptor-Interacting Serine/Threonine-Protein Kinase 3 (RIPK3)–Mixed Lineage Kinase Domain-Like Protein (MLKL)–Mediated Necroptosis Contributes to Ischemia-Reperfusion Injury of Steatotic Livers

doi: 10.1016/j.ajpath.2019.03.010

Figure Lengend Snippet: Detection of inflammation in ischemia-reperfusion (IR) livers of Ripk3 knockout (KO) mice. mRNA levels of cytokines tumor necrosis factor (Tnf)–α (A), Il-1β (B), and Il-6 (C), and chemokine macrophage inflammatory protein (Mip)-2 (D) as analyzed by real-time PCR. Total protein and plasma membrane fractions were extracted from livers. Equal amounts of proteins from the samples of the same groups were combined. E–G: Expression levels of mixed lineage kinase domain-like protein (Mlkl), receptor-interacting serine/threonine-protein kinase (Ripk)-1, and Ripk3 in total lysate (E); plasma membrane Mlkl (F); and oligomer and polymer of Mlkl (G) as detected by Western blot analysis. Data are expressed as means ± SEM. n = 4 to 7 per group. ∗P < 0.05 versus wild type (WT). #Nonspecific band. CD, chow diet; Gapdh, glyceraldehyde phosphate dehydrogenase; WD, Western diet.

Article Snippet: Antibodies used in this study were Mlkl and β-actin (catalog numbers SAB1302339 and A5441, respectively; Sigma-Aldrich, St. Louis, MO); glyceraldehyde phosphate dehydrogenase (Gapdh), Ripk1, and phosphorylated (p)-Mlkl (catalog numbers 2118, 3493, and 62233; Cell Signaling Technology, Beverly, MA); pMlkl, sodium potassium ATPase, and proteasome subunits (Psm)-α2 and -β5 (catalog numbers ab66596, ab76020, ab109525, and ab3330; Abcam, Cambridge, MA); Ripk3 (catalog number 2283; ProSci, Poway, CA); myeloperoxidase (Mpo; catalog number PP023AA; Biocare Medical, Concord, CA); and 26S proteasome regulatory subunit 4 (Psmc1; catalog number A303-821A; Bethyl Laboratories, Montgomery, TX).

Techniques: Knock-Out, Real-time Polymerase Chain Reaction, Clinical Proteomics, Membrane, Expressing, Polymer, Western Blot