Journal: The Journal of infectious diseases

Article Title: Osteopontin impairs host defense during pneumococcal pneumonia.

doi: 10.1093/infdis/jir185

Figure Lengend Snippet: Figure 1. Pulmonary and plasma osteopontin concentrations are elevated during pneumococcal pneumonia. Osteopontin concentrations in (A) lung and (B) plasma before and 6, 24, and 48 h after infection with 104 colony-forming units of Streptococcus pneumoniae. Data are expressed as mean 6 standard error of the mean (SEM); n 5 8 mice per group. Asterisk, P , .05; double asterisk, P , .01; triple asterisk, P , .001, compared with t 5 0. Osteopontin concentrations in culture supernatants after incubation of (C) MH-S cells and (D) primary alveolar macrophages with medium or growth- arrested S. pneumoniae (multiplicity of infection, 1:6 and 1:60 for MH-S cells; 1:20 and 1:200 for primary alveolar macrophages) for 4 h (MH-S cells) or 20 h (primary alveolar macrophages). Data are expressed as mean 6 SEM; n 5 3 per group. Asterisk, P ,.05, compared with medium. OPN, osteopontin.

Article Snippet: Effect of Osteopontin on S. pneumoniae Viability, Phagocytosis, and Phagolysosomal Fusion S. pneumoniae or Staphylococcus (S.) aureus (Newman strain) (1 3 106 bacteria/mL) was incubated in sterile normal saline in the presence of 0.8–800 ng/mL recombinant mouse osteopontin (rOPN;,1.0 endotoxin units per 1 lg as determined by the LAL assay; R&D Systems), 800 ng/mL boiled rOPN (30 min at 100 C), 800 ng/mL bovine serum albumin (BSA), or normal saline only at 37 C for 6 h. At indicated time points the number of bacteria was determined.

Techniques: Clinical Proteomics, Infection, Incubation

Journal: The Journal of infectious diseases

Article Title: Osteopontin impairs host defense during pneumococcal pneumonia.

doi: 10.1093/infdis/jir185

Figure Lengend Snippet: Figure 2. Prolonged survival and reduced bacterial growth in osteopontin knockout (KO) mice. A, Percentage survival of wild-type (WT) mice (filled symbols) and osteopontin KO mice (open symbols) after intranasal infection with 104 colony-forming units (CFU) of Streptococcus pneumoniae (n 5 14 mice per group). P value indicates the difference between groups. WT (gray) and osteopontin KO (white) mice were infected with 104 CFU of S. pneumoniae, and bacterial loads were determined 6, 24, and 48 h after infection in (B) lung, (C) blood, and (D) spleen. Data are expressed as box-and- whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile, and largest observation; n 5 8 mice per group; asterisk, P ,.05; double asterisk, P ,.01; triple asterisk, P ,.001, compared with WT mice. Note to panels C and D: none of the mice in either group displayed positive blood or spleen culture results 6 h after infection; at 24 h, S. pneumoniae could be cultured from samples of the blood of only 3 of 8 osteopontin KO mice, compared with 7 of 8 WT mice and from the spleen tissue of only 1 of 7 osteopontin KO mice, compared with 7 of 8 WT mice (P , .05 and P , .01, respectively). OPN, osteopontin.

Article Snippet: Effect of Osteopontin on S. pneumoniae Viability, Phagocytosis, and Phagolysosomal Fusion S. pneumoniae or Staphylococcus (S.) aureus (Newman strain) (1 3 106 bacteria/mL) was incubated in sterile normal saline in the presence of 0.8–800 ng/mL recombinant mouse osteopontin (rOPN;,1.0 endotoxin units per 1 lg as determined by the LAL assay; R&D Systems), 800 ng/mL boiled rOPN (30 min at 100 C), 800 ng/mL bovine serum albumin (BSA), or normal saline only at 37 C for 6 h. At indicated time points the number of bacteria was determined.

Techniques: Knock-Out, Infection, Whisker Assay, Cell Culture

Journal: The Journal of infectious diseases

Article Title: Osteopontin impairs host defense during pneumococcal pneumonia.

doi: 10.1093/infdis/jir185

Figure Lengend Snippet: Figure 3. Decreased lung histopathology in osteopontin knockout (KO) mice. Representative lung histology of wild-type (WT) (A, D, G) and osteopontin KO (B, E, H ) mice at 6 h (A–C ), 24 h (D–F ), and 48 h (G–I) after intranasal infection with 104 CFU of Streptococcus pneumoniae. The lung sections are representative for 8 mice per group per time point. Hematoxilin and eosin staining, original magnification, 310. Inflammation scores are expressed as mean 6 standard error of the mean (WT mice, black bars; osteopontin KO mice, white bars; n 5 8 mice per group). Double asterisk, P ,.01, compared with WT mice. OPN, osteopontin.

Article Snippet: Effect of Osteopontin on S. pneumoniae Viability, Phagocytosis, and Phagolysosomal Fusion S. pneumoniae or Staphylococcus (S.) aureus (Newman strain) (1 3 106 bacteria/mL) was incubated in sterile normal saline in the presence of 0.8–800 ng/mL recombinant mouse osteopontin (rOPN;,1.0 endotoxin units per 1 lg as determined by the LAL assay; R&D Systems), 800 ng/mL boiled rOPN (30 min at 100 C), 800 ng/mL bovine serum albumin (BSA), or normal saline only at 37 C for 6 h. At indicated time points the number of bacteria was determined.

Techniques: Histopathology, Knock-Out, Infection, Staining

Journal: The Journal of infectious diseases

Article Title: Osteopontin impairs host defense during pneumococcal pneumonia.

doi: 10.1093/infdis/jir185

Figure Lengend Snippet: Figure 4. Osteopontin stabilizes Streptococcus pneumoniae viability in vitro. A, S. pneumoniae in saline (106 colony-forming units [CFU]/mL) was incubated with increasing doses (0.8–800 ng/mL) of recombinant osteopontin (black symbols) or saline (white symbols), and the viability of S. pneumoniae was determined over 6 h at 37C. B, S. pneumoniae in saline (106 CFU/mL) was incubated with 800 ng/mL recombinant osteopontin (filled squares), 800 ng/mL boiled recombinant osteopontin (open squares), 800 ng/mL bovine serum albumin (triangles), or saline (circles), and the viability of S. pneumoniae was determined over 6 h at 37C. Dashed lines depict detection limits. C, Osteopontin binds to S. pneumoniae. Enzyme-linked immunosorbent assay plates were coated or not coated with 1 3 108 CFU/mL S. pneumoniae type 3 (ATCC 6303) or serotype 2 (D39); coating with anti-osteopontin IgG was used as positive control. Binding was assessed using biotin-labeled recombinant mouse osteopontin. Data are means 6 standard error (n 5 4–6). Double asterisk, P , .01 vs buffer. OPN, osteopontin.

Article Snippet: Effect of Osteopontin on S. pneumoniae Viability, Phagocytosis, and Phagolysosomal Fusion S. pneumoniae or Staphylococcus (S.) aureus (Newman strain) (1 3 106 bacteria/mL) was incubated in sterile normal saline in the presence of 0.8–800 ng/mL recombinant mouse osteopontin (rOPN;,1.0 endotoxin units per 1 lg as determined by the LAL assay; R&D Systems), 800 ng/mL boiled rOPN (30 min at 100 C), 800 ng/mL bovine serum albumin (BSA), or normal saline only at 37 C for 6 h. At indicated time points the number of bacteria was determined.

Techniques: In Vitro, Saline, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Positive Control, Binding Assay, Labeling

Journal: The Journal of infectious diseases

Article Title: Osteopontin impairs host defense during pneumococcal pneumonia.

doi: 10.1093/infdis/jir185

Figure Lengend Snippet: Figure 5. Similar bacterial growth during pneumococcal sepsis. Bacterial loads in (A) blood, (B) lung, (C) liver, and (D) spleen from wild-type (WT; gray) and osteopontin knockout (OPN KO; white) mice at 24 and 48 h after intravenous injection with 105 colony-forming units (CFU) of Streptococcus pneumoniae. Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile, and largest observation; n 5 8 mice per group. Dashed line depicts detection limit.

Article Snippet: Effect of Osteopontin on S. pneumoniae Viability, Phagocytosis, and Phagolysosomal Fusion S. pneumoniae or Staphylococcus (S.) aureus (Newman strain) (1 3 106 bacteria/mL) was incubated in sterile normal saline in the presence of 0.8–800 ng/mL recombinant mouse osteopontin (rOPN;,1.0 endotoxin units per 1 lg as determined by the LAL assay; R&D Systems), 800 ng/mL boiled rOPN (30 min at 100 C), 800 ng/mL bovine serum albumin (BSA), or normal saline only at 37 C for 6 h. At indicated time points the number of bacteria was determined.

Techniques: Knock-Out, Injection, Whisker Assay

Journal: International immunopharmacology

Article Title: Tumor cell derived osteopontin and prostaglandin E2 synergistically promote the expansion of myeloid derived suppressor cells during the tumor immune escape phase.

doi: 10.1016/j.intimp.2024.111584

Figure Lengend Snippet: Fig. 5. Increased production of osteopontin (OPN) and prostaglandin E2 (PGE2) in EL4luc2 cells and associated tumor model. (a and b), Expression of angiogenic factors and cytokines produced by EL4luc2 cells was screened using proteome profiler kits for mouse angiogenesis factors (a) and cytokines/chemokines (b). (c-e), levels of OPN and PGE2, and IL-17 were measured in the control culture medium (white bars: c, d, e) EL4 cells-derived cell culture medium (black bars: c, d, e) and sera from tumor free (control) mice (white bars: c, d, e), and EL4luc2 tumor-bearing mice (black bars: c, d, e), respectively. Statistical analysis was performed using the Mann-Whitney U test (*P ≤0.05).

Article Snippet: The levels of PGE2, OPN and IL-17 in the cell culture supernatants and serum samples were quantified using commercially available ELISA kits (Mouse Prostaglandin E2 (PGE2) ELISA Kit, MyBioSource, Inc. San Diego, CA, USA, Mouse/Rat Osteopontin (OPN) Quantikine ELISA Kit, R&D Systems, Abingdon, UK, and ELISA Flex: Mouse IL-17A (HRP), Mabtech AB, Nacka Strand, Sweden), in accordance with the manufacturers’ instructions.

Techniques: Expressing, Produced, Control, Derivative Assay, Cell Culture, MANN-WHITNEY

Journal: International immunopharmacology

Article Title: Tumor cell derived osteopontin and prostaglandin E2 synergistically promote the expansion of myeloid derived suppressor cells during the tumor immune escape phase.

doi: 10.1016/j.intimp.2024.111584

Figure Lengend Snippet: Fig. 6. Osteopontin (OPN) and prostaglandin E2 (PGE2) synergistically induce myelopoiesis with immunosuppressive activity. (a-d) Freshly flushed bone marrow (BM) cells were cultured in a 24-well cell culture plate for 72 h in the presence of various combinations of PGE2, OPN, and IL-17. Subsequently, cell viability (a and d) and immunophenotypic analysis of myeloid cells (b and e) flow cytometry analyses were conducted on the cultured BM cells using trypan blue exclusion test and flow cytometry, respectively. Representative photographs of myeloid cell populations are displayed above respective bars (b and e). c) Conditioned medial levels of arginase were quantified using colorimetric assays after treating BM cells with PGE2 and OPN. f) Effect of pioglitazone on the inhibition of proliferation of EL4 cells. Statistical analysis was performed using ANOVA test (*P ≤0.05, **P < 0.01, ***P < 0.01).

Article Snippet: The levels of PGE2, OPN and IL-17 in the cell culture supernatants and serum samples were quantified using commercially available ELISA kits (Mouse Prostaglandin E2 (PGE2) ELISA Kit, MyBioSource, Inc. San Diego, CA, USA, Mouse/Rat Osteopontin (OPN) Quantikine ELISA Kit, R&D Systems, Abingdon, UK, and ELISA Flex: Mouse IL-17A (HRP), Mabtech AB, Nacka Strand, Sweden), in accordance with the manufacturers’ instructions.

Techniques: Activity Assay, Cell Culture, Flow Cytometry, Inhibition

Journal: Frontiers in Cardiovascular Medicine

Article Title: Serotonin transporter downregulation is associated with aortic stenosis, and early profibrotic remodeling is mitigated by pharmacological inhibition of HTR2B receptor

doi: 10.3389/fcvm.2026.1729078

Figure Lengend Snippet: Effects of AngII on human AVICs with SERT knockdown. (A) Gene expression of SERT, HTR2B, HTR2A in AngII-treated compared to Nontreated (NT) Ctrl AVICs. (B) SERT KD by siRNA lead to increased expression of HTR2A gene expression. (C) HTR2A and HTR2B expression with siSERT or siSERT combined with AngII treatment. (D) COL1A1, SPP1, RUNX2, TGFβ1 expression in response to AngII treatment in human AVICs compared to Nontreated group. (E) COL1A1, RUNX2, TGFβ1 expression treated with siSERT alone or siSERT combined with AngII treatment. All gene expression results were calculated by the 2 −ΔΔCT method, n ≥ 4 per group. Error bars indicate SEM. P-value vs. NT. *indicates p-value <0.1, **indicates p-value <0.05, and ***indicates p -value <0.01 by Student's t -test or one-way ANOVA with post-hoc Dunnett's test.

Article Snippet: SPP1 , Hs00959010_m1.

Techniques: Knockdown, Gene Expression, Expressing

Journal: Smart Materials in Medicine

Article Title: Eggshell-derived amorphous calcium phosphate: Synthesis, characterization and bio-functions as bone graft materials in novel 3D osteoblastic spheroids model

doi: 10.1016/j.smaim.2023.04.001

Figure Lengend Snippet: Fig. 10. Confocal microscopic inspection of OPN and collagen I expression of MC-3T3-E1 cells in 3D spheroids with ACP particles embedded and 2D culture system, respectively. (A) Left panel: OPN and collagen I expression in different culture media in 3D reconstructed images; Right panel: maximal fluorescent projection of OPN and collagen I in 3D spheroids as shown in left penal; (B) OPN and collagen I expression in different culture media in a 2D culture system. Scale bar ¼ 100 μm.

Article Snippet: In brief, cells and spheroids were cultured for 7 days and then fixed with 4 wt % PFA for 20 min. After washing in PBS thrice, samples were treated with 1% (v/v) TritionX-100 (X100, Sigma-Aldrich, USA) for 20 min, followed by blocking in 1 wt % BSA/PBS solution for 1 h. Then, the samples were incubated in goat-anti-mouse OPN primary antibody (Ab) (AF808, R&D systems, USA) and rabbit-anti-mouse collagen I Ab (ab21286, Abcam, USA) at 4 C overnight.

Techniques: Expressing

Journal: Aging Cell

Article Title: Deficiency of Microglial‐Derived Spp1 Exacerbates Age‐Related Memory Decline by Impairing Mitochondrial Complex I Function

doi: 10.1111/acel.70378

Figure Lengend Snippet: Microglial Spp1 deficiency led to age‐dependent memory deficits. (A) Western blot analysis of Spp1 protein in the hippocampus of young (31–43 years; mean 38.3) and old humans (88–95 years; mean 90.6) ( n = 3 per group). (B) Representative images showing the co‐localization of Spp1 and the microglial marker (IBA1) in brain sections from young and aged humans. Dashed white boxes indicate magnified images of individual cells, and donut chart point to IBA1 + Spp1 +/− cells, Scale bars = 10 μm ( n = 3 per group). (C) Representative images showing the co‐localization of Spp1 mRNA and the microglial marker (IBA1) in brain sections from young (3 M) and aged (22 M) mice. Dashed white boxes indicate magnified images of individual cells, and donut chart indicate the proportion IBA1 + Spp1 mRNA +/− cells, Scale bars = 10 μm ( n = 3 per group). (D) Representative images showing the co‐localization of Spp1 and the microglial marker (IBA1) in brain sections from young (3 M), and aged (22 M) mice. Dashed white boxes indicate magnified images of individual cells, and donut chart point to IBA1 + Spp1 +/− cells, Scale bars = 10 μm ( n = 3 per group). (E) Schematic diagram of the experimental design. (F) Percentage of correct alternations in the Y‐maze test of adult ( n = 12) and aged ( n = 13) Spp1 fl/fl mice and Spp1‐cKO mice. (G/J) Learning curves during Morris Water Maze (MWM) training for adult (G) ( n = 12) and aged (J) ( n = 13) Spp1 fl/fl and Spp1‐cKO mice, measured by latency to find the platform. (H/K) Representative path plots showing the search patterns of adult (H) and aged (K) Spp1 fl/fl and Spp1‐cKO mice during the MWM probe trial. (I) Number of platform crossings during the MWM probe trial of adult ( n = 12) and aged ( n = 13) Spp1 fl/fl mice and Spp1‐cKO mice. (L) Time spent in the target quadrant during the MWM probe trial of adult ( n = 12) and aged ( n = 13) Spp1 fl/fl mice and Spp1‐cKO mice. Data are presented as the mean ± standard error of the mean (SEM). Two‐way repeated measures ANOVA was used to compare the latency to platform acquisition during the learning curves (G/J). Other data were analyzed using unpaired two‐tailed t ‐tests. * p < 0.05, ** p < 0.01, *** p < 0.001, ns = no significant difference. Figure was created using BioRender.

Article Snippet: The Cx3cr1 ‐CreERT2 (Stock No. C001247) and Spp1 ‐flox (Spp1 fl/fl ) (Stock No. S‐CKO‐05240) mice were obtained from Cyagen Biosciences Co. Ltd. (China).

Techniques: Western Blot, Marker, Two Tailed Test

Journal: Aging Cell

Article Title: Deficiency of Microglial‐Derived Spp1 Exacerbates Age‐Related Memory Decline by Impairing Mitochondrial Complex I Function

doi: 10.1111/acel.70378

Figure Lengend Snippet: Deficiency of Spp1 impaired microglial phagocytic function. (A) Heatmap showing differentially expressed genes (DEGs) between two groups of aged MG with high ( n = 3) and low ( n = 4) phagocytic activity. Red indicates upregulated genes, while blue indicates downregulated genes. (B) Representative images of microglial uptake of myelin‐555 in aged mice. Yellow arrows indicate Spp1‐positive microglia phagocytosing myelin‐555; blue arrows indicate Spp1‐negative microglia phagocytosing myelin‐555. Scale bars = 15 μm ( n = 6 mice per group). (C, D) Representative images showing phagocytosis of myelin‐555 by primary microglia isolated from postnatal day 3 (P3) Spp1 fl/fl and Spp1‐cKO mice; white dashed lines indicating magnified images of individual MG. Scale bars = 15 μm. (E) %Myelin + cell (of cell population) = number of phagocytic cells/total cell count ( n = 3 per group). (F) Relative myelin‐555 intensity of phagocytic cells = total fluorescence intensity/total number of phagocytic cells ( n = 3 per group). (G, H) Myelin‐555 was stereotactically injected into the hippocampus of aged Spp1 fl/fl and Spp1‐cKO mice to assess MG phagocytosis. Scale bars, 10 μm (low magnification) and 5 μm (high magnification). (I) Proportion of myelin + MG ( n = 4 per group). (J) Analysis of myelin volume within MG ( n = 4 per group). Data are presented as the mean ± standard error of the mean (SEM). Data were analyzed by unpaired two‐tailed t ‐tests. * p < 0.05, ** p < 0.01, *** p < 0.001. Mouse/Cell pattern was created using BioRender.

Article Snippet: The Cx3cr1 ‐CreERT2 (Stock No. C001247) and Spp1 ‐flox (Spp1 fl/fl ) (Stock No. S‐CKO‐05240) mice were obtained from Cyagen Biosciences Co. Ltd. (China).

Techniques: Activity Assay, Isolation, Cell Characterization, Fluorescence, Injection, Two Tailed Test

Journal: Aging Cell

Article Title: Deficiency of Microglial‐Derived Spp1 Exacerbates Age‐Related Memory Decline by Impairing Mitochondrial Complex I Function

doi: 10.1111/acel.70378

Figure Lengend Snippet: Deficiency of Spp1 disrupted microglia oxidative phosphorylation. (A) Heatmap of GSVA analysis comparing gene set enrichment between primary microglia isolated from postnatal day 3 (P3) Spp1 fl/fl and Spp1‐cKO mice; blue indicates downregulation, and red indicates upregulation. (B) Quantification of ATP levels in primary microglia isolated from postnatal day 3 (P3) Spp1 fl/fl and Spp1‐cKO mice ( n = 6 per group). (C) Measurement of Seahorse XF mitochondrial stress OCR in primary microglia from postnatal day 3 (P3) Spp1 fl/fl and Spp1‐cKO mice. (D) Basal OCR ( n = 3 per group). (E) Maximum OCR ( n = 3 per group). (F) Heatmap of GSVA pathway scores showing significant downregulation of Oxidative Phosphorylation in the microglial cluster of aged (18‐month‐old) Spp1‐cKO mice compared to controls. (G) Subsets of high SPP1‐expressing (SPP1 high MG) and low SPP1‐expressing (SPP1 low MG) in aged human brains ( GSE157827 ). (H) Oxidative phosphorylation pathway scores in SPP1 high and SPP1 low MG in aged human brains. (I) Violin plot showing AUCell scores for SPP1 high MG and SPP1 low MG cells. Data are presented as the mean ± standard error of the mean (SEM). Statistical significance was determined using unpaired two‐tailed t ‐tests for (B, D, E) and Wilcoxon rank‐sum test for (I). FCCP, Carbonyl cyanide 4‐trifluoromethoxyphenylhydrazone; Anti & Rot, Antimycin A and Rotenone; 2‐DG, 2‐Deoxy‐D‐glucose. * p < 0.05, ** p < 0.01, ns = no significant difference. Human/Mouse/Cell pattern was created using BioRender.

Article Snippet: The Cx3cr1 ‐CreERT2 (Stock No. C001247) and Spp1 ‐flox (Spp1 fl/fl ) (Stock No. S‐CKO‐05240) mice were obtained from Cyagen Biosciences Co. Ltd. (China).

Techniques: Phospho-proteomics, Isolation, Expressing, Two Tailed Test

Journal: Aging Cell

Article Title: Deficiency of Microglial‐Derived Spp1 Exacerbates Age‐Related Memory Decline by Impairing Mitochondrial Complex I Function

doi: 10.1111/acel.70378

Figure Lengend Snippet: Deficiency of Spp1 in microglia disrupted mitochondrial complex I‐dependent oxidative phosphorylation. (A) Heatmap showing changes in key OXPHOS‐related genes in primary microglia isolated from postnatal day 3 (P3) Spp1 fl/fl and Spp1‐cKO mice. (B) Workflow diagram of the Oroboros O2k multi‐dimensional energy metabolism analysis system used to measure OCR in BV2 cell homogenates. (C) Basal OCR in BV2 cells ( n = 3 per group). (D‐F) Mitochondrial respiration states in BV2 cells: Complex I respiration (D), complex II respiration (E), and complex IV respiration (F) ( n = 3 per group). (G) Histogram showing the relative mitochondrial complex I enzyme activity ( n = 4 per group). (H) Western blot analysis showing changes in protein expression levels of Spp1 and Ndufs2 after Spp1 knockdown in BV2 cells ( n = 3 per group). (I) Workflow diagram of the Oroboros O2k multi‐dimensional energy metabolism analysis system used to measure OCR in hippocampal tissue from Spp1 fl/fl and Spp1‐cKO mice. (J) Basal OCR in hippocampal tissue from Spp1 fl/fl and Spp1‐cKO mice ( n = 4 per group). (K–M) Mitochondrial respiration states in hippocampal tissue from Spp1 fl/fl and Spp1‐cKO mice: Complex I respiration (K), complex II respiration (L), and complex IV respiration (M) ( n = 4 per group). (N) Schematic diagram showing Spp1 deficiency disrupting OXPHOS via compromised complex I activity. Data are presented as the mean ± standard error of the mean (SEM). Data were analyzed by unpaired two‐tailed t ‐tests. * p < 0.05, ** p < 0.01, *** p < 0.001, ns = no significant difference. Mouse patterns and experimental apparatus were created using BioRender.

Article Snippet: The Cx3cr1 ‐CreERT2 (Stock No. C001247) and Spp1 ‐flox (Spp1 fl/fl ) (Stock No. S‐CKO‐05240) mice were obtained from Cyagen Biosciences Co. Ltd. (China).

Techniques: Phospho-proteomics, Isolation, Activity Assay, Western Blot, Expressing, Knockdown, Two Tailed Test

Journal: Aging Cell

Article Title: Deficiency of Microglial‐Derived Spp1 Exacerbates Age‐Related Memory Decline by Impairing Mitochondrial Complex I Function

doi: 10.1111/acel.70378

Figure Lengend Snippet: Spp1 deficiency inhibited AKT signaling pathway and administration of AKT agonist reversed manifestation. (A) KEGG pathway analysis of differentially expressed genes from sequencing data of primary microglia isolated from postnatal day 3 (P3) Spp1 fl/fl and Spp1‐cKO mice. (B) Heatmap showing GSVA enrichment scores for the “PI3K‐Akt signaling” pathway in primary microglia isolated from postnatal day 3 (P3) Spp1 fl/fl and Spp1‐cKO mice. Each column represents one RNA‐seq sample (red indicating up‐regulation, and blue indicating down‐regulation). (C) scRNA‐seq analysis showing the downregulation of PI3K–Akt signaling pathway in the microglial cluster of 18‐month‐old Spp1‐cKO mice compared to controls. (D) Western blot analysis showing the changes in protein expression levels of p‐AKT, AKT after Spp1 knockdown in BV2 cells ( n = 3 per group). (E) ATP levels ( n = 4 per group). (F) Mitochondrial complex I‐dependent oxygen consumption rate (OCR) ( n = 3 per group). (G) Histogram showing the relative mitochondrial complex I enzyme activity ( n = 4 per group). (H) %Myelin + cell (of cell population) = number of phagocytic cells/total cell count ( n = 4 per group). (I) ATP levels in the hippocampus of Spp1 fl/fl + solvent, Spp1‐cKO + solvent, and Spp1‐cKO + SC79 mice ( n = 3 per group). (J) Western blot analysis showing the changes in protein expression levels of p‐AKT, AKT, and Ndufs2 ( n = 3 per group). (K) Percentage of correct alternations in the Y‐maze test. (L) Learning curves during MWM training, measured by latency to find the platform. (M) Time spent in the target quadrant during the probe phase of the MWM. Data are presented as the mean ± standard error of the mean (SEM). Two‐way repeated measures ANOVA was used to compare the latency to find the platform in the learning curves (L). Figure D was analyzed using two‐tailed t ‐tests. Other data were analyzed using one‐way ANOVA and LSD post hoc test. * p < 0.05, ** p < 0.01, ns = no significant difference. Mouse pattern was created using BioRender.

Article Snippet: The Cx3cr1 ‐CreERT2 (Stock No. C001247) and Spp1 ‐flox (Spp1 fl/fl ) (Stock No. S‐CKO‐05240) mice were obtained from Cyagen Biosciences Co. Ltd. (China).

Techniques: Sequencing, Isolation, RNA Sequencing, Western Blot, Expressing, Knockdown, Activity Assay, Cell Characterization, Solvent, Two Tailed Test

Journal: Aging Cell

Article Title: Deficiency of Microglial‐Derived Spp1 Exacerbates Age‐Related Memory Decline by Impairing Mitochondrial Complex I Function

doi: 10.1111/acel.70378

Figure Lengend Snippet: Microglia‐specific Spp1 overexpression restores ATP production, phagocytosis, and memory in Spp1‐deficient models. (A) Representative confocal images of Spp1 immunofluorescence in BV2 cells: Control (NC), Spp1 knockdown (sh‐Spp1), and sh‐Spp1 rescued by AAV‐Spp1. Scale bars = 10 μm. (B) Quantification of relative Spp1 fluorescence intensity ( n = 3 per group). (C) Representative bright‐field/fluorescence overlays of myelin‐555 uptake (red) by BV2 cells under the three conditions. Scale bars = 10 μm. (D) %Myelin + cell (of cell population) = number of phagocytic cells/total cell count ( n = 3 per group). (E) ATP levels normalized to total protein in control (NC), Spp1 knockdown (sh‐Spp1), and sh‐Spp1 cells rescued by AAV‐Spp1 ( n = 3 per group). (F) Mitochondrial complex I activity in control (NC), Spp1 knockdown (sh‐Spp1), and sh‐Spp1 cells rescued by AAV‐Spp1 ( n = 3 per group). (G) Top: Schematic diagram illustrating the experimental strategy for microglia‐specific Spp1 overexpression. Bottom: Relative fluorescence intensity of Spp1 immunostaining in IBA1 + microglia ( n = 3 mice per group). (H) Representative immunofluorescence images showing the expression of AAV‐driven EGFP in IBA1 + microglia in the hippocampus. Scale bars = 10 μm. (I) Y‐maze spontaneous alternation rate ( n = 8 mice per group). (J) Learning curves during MWM training, measured by latency to find the platform ( n = 8 mice per group). (K) Representative path plots showing the search patterns during the MWM probe trial. (L) Time spent in the target quadrant during the probe phase of the MWM. Data are presented as the mean ± standard error of the mean (SEM). Two‐way repeated measures ANOVA was used to compare the latency to find the platform in the learning curves (J). Other data were analyzed using one‐way ANOVA and LSD post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001. Brain pattern was created using BioRender.

Article Snippet: The Cx3cr1 ‐CreERT2 (Stock No. C001247) and Spp1 ‐flox (Spp1 fl/fl ) (Stock No. S‐CKO‐05240) mice were obtained from Cyagen Biosciences Co. Ltd. (China).

Techniques: Over Expression, Immunofluorescence, Control, Knockdown, Fluorescence, Cell Characterization, Activity Assay, Immunostaining, Expressing