Journal: Science Advances

Article Title: Immunosuppressive macrophages determine the effect of cellular senescence on tumor progression

doi: 10.1126/sciadv.adx2988

Figure Lengend Snippet: ( A ) Tumor samples from mice treated with vehicle or AP21967 at 9 weeks were homogenized, and the levels of the indicated cytokines were analyzed using a cytokine array. The log 2 FC (AP21967/vehicle) for each cytokine is presented. ENA-78, epithelial-derived neutrophil-activating peptide 78; GROα, growth-regulated oncogene-alpha. ( B ) Levels of CCL2 and CXCL1 were quantified by ELISA from the same tumor homogenates as in (A). ( C ) UMAP plots showing the expression of the most up-regulated cytokines (CCL2, CCL3, and CCL7) and their receptors (CCR1, CCR2, and CCR5) in different cell populations. Relevant cell clusters are indicated. N, neutrophils; MØ, macrophages; E, endothelial cells; F, fibroblasts. ( D ) Flow cytometry analysis of myeloid cells purified from tumors obtained from SuSe mice treated with vehicle or AP21967. We identified TAMs and cells positive for the myeloid marker CD11B and for the macrophage marker F4/80. ( E ) The same cells as in (D) were cultured for 24 hours—in the presence of vehicle or anti-CCL2—and the presence of CCL2 in the conditioned medium was determined by ELISA. ( F ) Schematic representation of the specificities of receptors for CCL2, CCL3, and CCL7. ( G ) Flow cytometry analysis of CCR2 in the same cells as in (D). FSH-H, forward scatter height.

Article Snippet: For selective CCL2 blocking, 4-week-old PyMT/SuSe mice were treated every 3 days with anti-CCL2 antibody (#BE0185, BioXCell,) at a concentration of 2 μg/g of body weight.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Purification, Marker, Cell Culture

Journal: Theranostics

Article Title: Nanogel-mediated delivery of oncomodulin secreted from regeneration-associated macrophages promotes sensory axon regeneration in the spinal cord.

doi: 10.7150/thno.73386

Figure Lengend Snippet: Figure 3. CCL2/CCR2 chemokine signaling regulates ONCM expression in macrophages. A, Representative images of neurite outgrowth in DRG neuron cultures treated with macrophage conditioned medium (MCM) obtained from neuron-macrophage cocultures using WT or CCR2-deficient (CCR2-/-) neurons (N) or macrophages (M). All cocultures were treated with either PBS or db-cAMP (cAMP), and the MCMs were collected for 72 h. DRG neurons and their neurites were visualized by immunofluorescence staining for beta3 tubulin. Scale bars represent 100 µm. B, Quantification graphs of neurite outgrowth in the presence of MCM from cultures of the different neuron-macrophage genotype combinations treated with PBS or cAMP. N = 4 independent cultures using independent MCMs for each condition. ***p < 0.001 and *p < 0.05 by unpaired t test. C, ELISA measurement of the ONCM concentration in the cell culture media obtained from different neuron-macrophage genotype combinations treated with PBS or cAMP. N = 4 independent cultures using independent MCMs for each condition. ***p < 0.001 and *p < 0.05 by unpaired t test. D, ELISA measurement of ONCM levels in DRGs from WT and CCR2-/- mice 0 (CTL) and 7 d after sciatic nerve injury. n = 4 animals for each group. ***p < 0.001 and *p < 0.05 by unpaired t test. E, Experimental schematic diagram depicting the experimental processes. F, Representative images of neurons cultured from L5 DRGs freshly dissected from WT or ONCM-/- mice subjected 2 weeks previously to intraganglionic injection of AAV5-GFP or AAV5-CCL2. The culture period was 15 h and DRG neurons and their neurites were visualized by immunofluorescence staining for beta3 tubulin. Scale bars represent 100µm. G, H, Quantification graphs comparing the mean neurite length (G) and the protein level of ONCM (H). N = 3 animals for each group. ***p < 0.001 and **p < 0.01 by unpaired t test.

Article Snippet: Preparation and injection of recombinant adeno-associated virus A recombinant adeno-associated virus (AAV) vector containing a CCL2 expression cassette was generated by replacing the GFP cassette of pAAV-CAG-GFP (Addgene) with the full-length cDNA for CCL2 (OriGene).

Techniques: Expressing, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture, Injection

Journal: Cell Death & Disease

Article Title: REDD1 expression in podocytes facilitates renal inflammation and pyroptosis in streptozotocin-induced diabetic nephropathy

doi: 10.1038/s41419-025-07396-4

Figure Lengend Snippet: Diabetes was induced in REDD1 +/+ and REDD1 −/− mice by streptozotocin (STZ) administration. Non-diabetic control mice received vehicle (Veh). A REDD1 protein was evaluated in kidney cortical tissue homogenates by western blotting. Representative blots are shown. Molecular mass in kDa is indicated at right of each blot. B Correlation between fasting blood glucose and urine ACR is shown for REDD1 +/+ mice ( blue ; Pearson r = 0.72; p < 0.0001) and REDD1 −/− mice ( red ; Pearson r = 0.55; p = 0.029). C Ccl2 mRNA expression was quantified in kidney homogenates by qPCR. D CCL2 protein abundance was quantified in kidney homogenates by western blotting. E Il1b mRNA expression was quantified in kidney homogenates by qPCR. F IL-1β protein levels were determined in kidney homogenates by western blotting and quantified by ELISA. Individual data points are plotted with values presented as means ± SD ( n = 4–6). Differences between groups were identified by two-way ANOVA. * p < 0.05 versus Veh; # p < 0.05 versus REDD1 +/+ . n.d., not detected.

Article Snippet: CCL2 recombinant protein (R&D systems, Minneapolis, MN, USA) was subjected to western blotting and CCL2 protein in cell (10 5 cells) and tissue lysates were quantified (Fig. S ).

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

Journal: Cell Death & Disease

Article Title: REDD1 expression in podocytes facilitates renal inflammation and pyroptosis in streptozotocin-induced diabetic nephropathy

doi: 10.1038/s41419-025-07396-4

Figure Lengend Snippet: A , B Diabetes was induced in REDD1 +/+ and REDD1 −/− mice by administration of streptozotocin (STZ). Non-diabetic control mice received vehicle (Veh). A Nuclear isolates were prepared from kidney homogenates. NF-κB and Lamin B were examined in nuclear isolates by western blotting and NF-κB activity was quantified by DNA-binding ELISA. Representative blots are shown with protein molecular mass in kDa indicated at right of each blot. B REDD1 ( red ) and Nephrin ( green ) were visualized in kidneys by immunofluorescence microscopy. White box indicates area shown at increased magnification. Representative micrographs are shown (scale bar 50 μm). C – I Wild-type (WT) and REDD1 knockout (KO) CIHP-1 were exposed to culture media containing either 30 mM glucose (HG) or 5 mM glucose plus 25 mM mannitol (OC) for 48 h. NF-κB phosphorylation at S536 and REDD1 protein abundance was determined in cell lysates by western blotting ( C ). Nuclear localization of NF-κB p65 ( white arrowheads ) was evaluated by immunofluorescence ( D ). Nuclei were visualized with DAPI (scale bar 25 μm). NF-κB activity was measured in lysates from cells expressing NF-κB firefly luciferase/ Renilla luciferase reporter plasmids by dual luciferase assay ( E ). Relative expression of IL1B and CCL2 mRNA were determined by qPCR ( F ). IL-1β secreted into culture media was determined by ELISA ( G ). Chromatin immunoprecipitation (ChIP)-PCR analysis was carried out in WT and REDD1 KO podocytes to determine binding of p65 NF-κB to the promoter region of the CCL2 gene ( H ). CCL2 protein levels were determined in cell lysates by western blotting ( I ). J NF-κB p65 phosphorylation and NF-κB luciferase reporter activity was evaluated in REDD1 KO cells expressing either an empty vector control (EV) or hemagglutinin (HA)-tagged REDD1. Individual data points are presented as means ± SD ( n = 4–6). Differences between groups were identified by two-way ANOVA. * p < 0.05 versus Veh or NG; # p < 0.05 versus REDD1 +/+ , WT, or EV.

Article Snippet: CCL2 recombinant protein (R&D systems, Minneapolis, MN, USA) was subjected to western blotting and CCL2 protein in cell (10 5 cells) and tissue lysates were quantified (Fig. S ).

Techniques: Control, Western Blot, Activity Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Microscopy, Knock-Out, Phospho-proteomics, Quantitative Proteomics, Expressing, Luciferase, Chromatin Immunoprecipitation, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: REDD1 expression in podocytes facilitates renal inflammation and pyroptosis in streptozotocin-induced diabetic nephropathy

doi: 10.1038/s41419-025-07396-4

Figure Lengend Snippet: A , B Differentiated wild-type (WT) and REDD1 knockout (KO) CIHP-1 were exposed to culture media containing either 30 mM glucose (HG) or 5 mM glucose plus 25 mM mannitol as an osmotic control (OC) for 48 h. Transwell migration assay was used to evaluate chemotaxis in a co-culture model with CIHP-1 and THP-1 macrophages ( A ). Macrophages were stained with crystal violet and cells that migrated across the Transwell were counted ( B ). C Cre-lox recombination was used to achieve conditional podocyte-specific REDD1 knockout (REDD1 PodKO). D – H Diabetes was induced in REDD1 fl/fl and REDD1 PodKO mice by streptozotocin (STZ) administration. Non-diabetic groups were administered a vehicle (Veh) control. All assessments were performed after 16 weeks of diabetes. Urine albumin to creatinine ratio (ACR) was determined (D). Kidney sections from diabetic and non-diabetic mice were immunolabeled for REDD1 ( red ) and the podocyte marker Nephrin ( green ) ( E ). Protein abundance of CCL2 was determined in renal homogenates by western blotting ( F ). Representative blots are shown with protein molecular mass in kDa indicated at right of each blot. Kidney sections were immunolabelled for F4/80 ( red ) and nuclei were counterstained with Hoechst 33342 ( blue ) ( G ). Representative micrographs (scale bar 50 µm) are shown. Immune cell populations of CD11b + F4/80+ macrophages ( H ) and CD86 + M1 macrophages ( I ) were determined by flow cytometry. Individual data points are plotted. Significance was analyzed by two-way ANOVA and pairwise comparisons were made using the Tukey’s test for multiple comparisons. * p < 0.05 versus OC or Veh; #, p < 0.05 versus WT or REDD1 fl/fl .

Article Snippet: CCL2 recombinant protein (R&D systems, Minneapolis, MN, USA) was subjected to western blotting and CCL2 protein in cell (10 5 cells) and tissue lysates were quantified (Fig. S ).

Techniques: Knock-Out, Control, Transwell Migration Assay, Chemotaxis Assay, Co-Culture Assay, Staining, Immunolabeling, Marker, Quantitative Proteomics, Western Blot, Flow Cytometry