Journal: Comprehensive Physiology

Article Title: Vildagliptin and Omarigliptin Differentially Bind to DPP‐4 Homodimers and Modulate Osteoclast‐Mediated Bone Resorption

doi: 10.1002/cph4.70103

Figure Lengend Snippet: (A) A schematic picture shows the experimental plan for osteoblast viability and mRNA expression study. (B–C) Cell viability of primary osteoblasts after exposure to 1, 10, 100 μM vildagliptin or omarigliptin. mRNA expression of osteoblast‐specific genes, that is, (D–E) Runx2, (F–G) alkaline phosphatase (ALP), (H–I) osteocalcin, and osteoclastogenic factors, that is, (J–K) macrophage‐colony stimulating factor (M‐CSF), (L–M) receptor activator of nuclear factor‐κB ligand (RANKL) in primary osteoblasts after treatment with 1, 10, 100 μM vildagliptin or omarigliptin for 5 days. n = 5–6; * p < 0.05, ** p < 0.01 vs. vehicle‐treated group (Veh) by one‐way analysis of variance (ANOVA) with Dunnett's multiple comparisons test.

Article Snippet: The osteoclast precursors were maintained in α‐MEM supplemented with 10% FBS and 10 ng/mL macrophage colony‐stimulating factor (M‐CSF) (catalog no. 216‐MC‐025; R&D Systems, Minneapolis, MN, USA) for 3 days.

Techniques: Expressing

Journal: Comprehensive Physiology

Article Title: Vildagliptin and Omarigliptin Differentially Bind to DPP‐4 Homodimers and Modulate Osteoclast‐Mediated Bone Resorption

doi: 10.1002/cph4.70103

Figure Lengend Snippet: Representative images of primary osteoclast captured by holotomographic microscope. Osteoclast morphology on (A) x‐y, (B) x‐z, (C) y‐z axis at day 7 after culture bone marrow cells with 10 ng/mL M‐CSF and RANKL. (D) Refractive index (RI) distribution in primary osteoclasts, (E) three‐dimentional (3D) rendered image of RI distribution. Blue is DAPI stained nuclei. Red is pseudocolor labeling for the remaining of intracellular compartments. (F) Correlation between number of nuclei and cell volume of multinucleated osteoclasts.

Article Snippet: The osteoclast precursors were maintained in α‐MEM supplemented with 10% FBS and 10 ng/mL macrophage colony‐stimulating factor (M‐CSF) (catalog no. 216‐MC‐025; R&D Systems, Minneapolis, MN, USA) for 3 days.

Techniques: Microscopy, Refractive Index, Staining, Labeling

Journal: Neuro-Oncology

Article Title: Therapy-induced senescent glioblastoma cells sustain a procancer immune microenvironment by activating DDX58-mediated STAT1 signaling

doi: 10.1093/neuonc/noaf107

Figure Lengend Snippet: DDX58 regulated the protein stability of STAT1 via the ubiquitin E3 ligase TRIM21 ( A ). Western blot analysis of the DDX58, P21, and STAT1 proteins and their phosphorylation in DOX- or TMZ-induced senescent LN229 cells transduced with DDX58-targeting siRNAs. ( B-C ). Western blot analysis of the P21, DDX58, and STAT1 proteins and their phosphorylation in LN229 (B) and U87MG (C) cells overexpressing DDX58. ( D ). Protein stability assays to assess the effect of DDX58 on the STAT1 protein. DDX58-knockdown senescent LN229 cells were treated with cycloheximide (50 μg/mL) for up to 9 h, and STAT1 and DDX58 expression was tested via western blotting. ( E ). LN229 cells were transfected with DDX58 siRNA and then treated with MG132 (20 μM) for 5 h. ( F ). The binding between DDX58 and STAT1 was examined by co-IP and western blotting. ( G ). LN229 cells were transiently transfected with DDX58 siRNA and then treated with TMZ or DMSO, and the changes in the ubiquitin level of STAT1 in LN229 cells were examined by co-IP and western blotting. All the samples were treated with 20 µM MG132 for 2 h. ( H ). Regulators involved in the regulation of STAT1 ubiquitination were screened by transient transfection of LN229 cells with the His-STAT1 pcDNA 4.0 plasmid. One sample was treated with 50 μM TMZ for 4 days. All the cells were examined via co-IP and western blotting ( I ). LN229 cells were transiently transfected with the His-STAT1 plasmid, and changes in the STAT1 binding to TRIM21 in LN229 cells overexpressing DDX58 were examined via co-IP and western blotting. ( J ). Schematic diagram of the DDX58/RIG-I protein domains. P21 and STAT1 protein expression and phosphorylation by overexpressing the CARD, CTD, and helicase domains of DDX58 in LN229 and 293FT cells. ( K ). THP-1 macrophages were cocultured with CM from LN229 cells (CON335, DDX58 overexpressing, DDX58 overexpressing plus fludarabine, 50 ng/mL CSF-1) for 48 h. Scale bars: 1.5 mm, 150 μm. ( L ). Statistical analysis of the data in (K). The samples were analyzed in triplicate with 3 fields per well; **** p < 0.0001 by one-way ANOVA with Tukey’s multiple comparison test. ( M ). ELISA analysis of CSF1 in LN229 cells overexpressing DDX58 or treated with fludarabine. Comparisons were performed with two-tailed Student’s t tests. * p < 0.01, *** p < 0.001. All the data are presented as the means ± SDs.

Article Snippet: Secreted CSF1/M-CSF protein levels were measured via a human macrophage colony-stimulating factor (M-CSF) ELISA kit (CUSABIO CSB-E04658h).

Techniques: Ubiquitin Proteomics, Western Blot, Phospho-proteomics, Transduction, Knockdown, Expressing, Transfection, Binding Assay, Co-Immunoprecipitation Assay, Plasmid Preparation, Comparison, Enzyme-linked Immunosorbent Assay, Two Tailed Test