Journal: Clinical and Molecular Hepatology

Article Title: Ursolic acid targets secreted phosphoprotein 1 to regulate Th17 cells against metabolic dysfunction-associated steatotic liver disease

doi: 10.3350/cmh.2024.0047

Figure Lengend Snippet: Identification of SPP1 as a direct ursolic acid-binding protein. (A) Bioinformatic analysis was conducted in this study, 106 DEGs were identified from two microarrays, which notably delineated the intricate comparative stages between NAFLD and NASH, and a volcano plot was plotted. A PPI network was constructed for these DEGs using the STRING database, providing insights into the functional relationships between identified proteins. And centrality analysis, pivotal for discerning nodes of paramount importance within the network, was systematically executed based on the intricate interaction relationships between nodes. A pathway enrichment was performed to acquire a holistic understanding of the biological functions and pathways associated with the DEGs. (B) A schematic diagram showed that biotin-labeled ursolic acid was co-incubated with this array, followed by signal interpretation using Cy3-streptavidin. (C) 502 targets were identified that interacted directly with ursolic acid, and intersection analysis with the previously identified DEGs retained five candidates, wherein the differentially binding proteins were presented in the proteomics chip and heatmap. (D) The SPR analysis was produced using a biacore sensor chip, wherein the molecular dynamics analysis revealed that ursolic acid and SPP1 exhibited high-affinity interaction, with the equilibrium dissociation constant of 1.705E-5. (E) A pull-down assay was performed to further verify the binding affinity between ursolic acid and SPP1 in vivo. (F) The molecular docking simulation showed a mutual binding between ursolic acid and SPP1, with a binding energy of –5.8. SPP1, secreted phosphoprotein 1; DEGs, differentially expressed genes; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; STRING, Search Tool for the Retrieval of Interacting Genes; SPR, surface plasmon resonance; KD, knockdown.

Article Snippet: Primary antibodies against ITGB1 (1:2,000, 34971), ITGB3 (1:2,000, 13166), p-ERK (1:2,000, 4370), ERK (1:2,000, 4695) and β-actin (1:2,000, 4970) were obtained from Cell Signaling Technology, against CD44 (1:1,000, ab189524) were purchased from Abcam, and against SPP1 (1:2,000, sc-21742) were purchased from Santa Cruz Biotechnology.

Techniques: Binding Assay, Construct, Functional Assay, Labeling, Incubation, Produced, Pull Down Assay, In Vivo, SPR Assay, Knockdown

Journal: Clinical and Molecular Hepatology

Article Title: Ursolic acid targets secreted phosphoprotein 1 to regulate Th17 cells against metabolic dysfunction-associated steatotic liver disease

doi: 10.3350/cmh.2024.0047

Figure Lengend Snippet: SPP1 gene knockdown affords protective effects against HFD-induced MASLD. (A) Schematic diagram revealed the protocol of SPP1 knockdown. Specifically, AAV2/8 vectors harboring a CMV-driven shRNA targeting SPP1 were employed for in vivo gene delivery. Western blot analyses demonstrated a marked reduction in SPP1 protein expression in the liver tissues from these KD mice, thus affirming the successful knockdown. Subsequently, mice were fed with HFD to construct the MASLD mice model. (B) Body weights were recorded weekly. (C) Liver weights were measured by the termination of this experiment at the twelfth week, and liver indexes were calculated by the ratio of liver to body weight. (D) Serum and hepatic lipid concentrations were determined by an automatic biochemical analyzer. (E) IPGTT and ITT were performed to monitor glucose tolerance and insulin resistance. (F) Histological staining of H&E and oil red O in liver tissues was performed to observe the inflammatory cytokine infiltration and lipid droplet accumulation. (G) Serum inflammatory cytokines were determined to detect inflammatory reactions. SPP1, secreted phosphoprotein 1; HFD, high-fat diets; MASLD, metabolic dysfunction-associated steatotic liver disease; AAV, adeno-associated virus; KD, knockdown; IPGTT, intraperitoneal glucose tolerance tests; ITT, insulin tolerance tests; SD, standard deviation. Data are represented as mean±SD. n=3-8. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Primary antibodies against ITGB1 (1:2,000, 34971), ITGB3 (1:2,000, 13166), p-ERK (1:2,000, 4370), ERK (1:2,000, 4695) and β-actin (1:2,000, 4970) were obtained from Cell Signaling Technology, against CD44 (1:1,000, ab189524) were purchased from Abcam, and against SPP1 (1:2,000, sc-21742) were purchased from Santa Cruz Biotechnology.

Techniques: Knockdown, shRNA, In Vivo, Western Blot, Expressing, Construct, Staining, Virus, Standard Deviation

Journal: Clinical and Molecular Hepatology

Article Title: Ursolic acid targets secreted phosphoprotein 1 to regulate Th17 cells against metabolic dysfunction-associated steatotic liver disease

doi: 10.3350/cmh.2024.0047

Figure Lengend Snippet: SPP1 promotes Th17 cell differentiation via interactions with ITGB1 and CD44. (A) Flow cytometry revealed a dose-dependent facilitation of Th17 differentiation by SPP1, with a concentration of 0.2 μg/mL emerging as optimal for ensuing experimental endeavors. Th17 cells were identified as CD3+CD4+IL-17A+ cells, and these results were represented as the percentage of Th17 cells among CD3+ T cells. (B) Flow cytometry indicated that ursolic acid dose-dependently inhibited SPP1-induced Th17 cell differentiation, with a concentration of 5 μM emerging as optimal for subsequent investigative pursuits. (C) Western blot analyses were conducted to detect the phosphorylation level of ERK protein. (D) SPP1 KD decreased the ERK phosphorylation level and substantially curtailed Th17 cell differentiation. (E) Co-immunoprecipitation analyses suggested strong interactions between SPP1 and both ITGB1 and CD44. (F) Both ITGB inhibitors and CD44 antagonists suppressed the SPP1-driven Th17 cell differentiation, with an even more pronounced inhibitory effect upon their combined application. SPP1, secreted phosphoprotein 1; ITGB1, integrin β1; CD44A, CD44 antagonists; ERK, extracellular signal-regulated kinase; SD, standard deviation. Data are represented as mean±SD. n=3. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Primary antibodies against ITGB1 (1:2,000, 34971), ITGB3 (1:2,000, 13166), p-ERK (1:2,000, 4370), ERK (1:2,000, 4695) and β-actin (1:2,000, 4970) were obtained from Cell Signaling Technology, against CD44 (1:1,000, ab189524) were purchased from Abcam, and against SPP1 (1:2,000, sc-21742) were purchased from Santa Cruz Biotechnology.

Techniques: Cell Differentiation, Flow Cytometry, Concentration Assay, Western Blot, Phospho-proteomics, Immunoprecipitation, Standard Deviation

Journal: Clinical and Molecular Hepatology

Article Title: Ursolic acid targets secreted phosphoprotein 1 to regulate Th17 cells against metabolic dysfunction-associated steatotic liver disease

doi: 10.3350/cmh.2024.0047

Figure Lengend Snippet: Ursolic acid modulates SPP1-mediated Th17 cell differentiation to ameliorate MASLD. (A–C) Mice were derived from the experiments of SPP1 KD, wherein (A) presented that the protein levels of ITGB1 and CD44 expressed in the pull-down products were conspicuously surged by HFD administration, whereas a precipitous decline in their expression was observed in the liver tissue lysates procured from the SPP1 KD mice, while (B) demonstrated that such alterations were concomitant with trends in the phosphorylation level of ERK protein; and concurrently, (C) displayed a similar trend in hepatic Th17 cell populations. (D, E) Mice were derived from the first part of experiments, those were fed with HFD and different concentrations of ursolic acid, wherein (D) suggested that ursolic acid dose-dependently ameliorated the escalated Th17 cell populations induced by the high-fat dietary regimen, and (E) revealed consistent protein levels of SPP1, ITGB1, CD44, as well as the phosphorylation level of ERK. Data are represented as mean±SD. n=3-6. * P <0.05, ** P <0.01, *** P <0.001. SPP1, secreted phosphoprotein 1; MASLD, metabolic dysfunction-associated steatotic liver disease; KD, knockdown; ITGB1, integrin β1; CD44A, CD44 antagonists; HFD, highfat diets; ERK, extracellular signal-regulated kinase; SD, standard deviation.

Article Snippet: Primary antibodies against ITGB1 (1:2,000, 34971), ITGB3 (1:2,000, 13166), p-ERK (1:2,000, 4370), ERK (1:2,000, 4695) and β-actin (1:2,000, 4970) were obtained from Cell Signaling Technology, against CD44 (1:1,000, ab189524) were purchased from Abcam, and against SPP1 (1:2,000, sc-21742) were purchased from Santa Cruz Biotechnology.

Techniques: Cell Differentiation, Derivative Assay, Expressing, Phospho-proteomics, Knockdown, Standard Deviation

Journal: Biomedicines

Article Title: Exploring the Osteogenic Potential of Zinc-Doped Magnesium Phosphate Cement (ZMPC): A Novel Material for Orthopedic Bone Defect Repair.

doi: 10.3390/biomedicines12020344

Figure Lengend Snippet: Figure 6. (A,B) Osteogenic differentiation on the samples. The osteogenic gene markers BMP2, OCN RUNX2 and ALP were determined using quantitative PCR and western blotting. (C,D) Quantitative analysis of western blotting and fluorescence intensity. (E) Immunofluorescence images stained with OPN (red) and ALP (green). The scale is 50 µm. * p < 0.05, ** p < 0.01, *** p < 0.001,**** p < 0.0001.

Article Snippet: Immunofluorescence Microscopy Cells (4 × 104) were cultured in confocal dishes, fixed with 4% formaldehyde for 20 min, and permeabilized with 0.1% Triton X-100 for 20 min. After blocking with 5% BSA (in TBST), the cells were incubated with the primary antibodies against OPN (boster, Wuhan, PB0589, 1:200) and ALP (absin, Shanghai, abs125381, 1:200) at 4 ◦C overnight.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Fluorescence, Immunofluorescence, Staining

Journal: Oncology Letters

Article Title: Single-cell RNA sequencing analysis of intrahepatic cholangiocarcinoma reveals SPP1 facilitates disease progression via interaction with CD4 + T cells

doi: 10.3892/ol.2025.15135

Figure Lengend Snippet: Differential gene expression of ICC tumor cells. (A) Normal liver cells and tumor cells isolated from 4 patients with ICC utilizing a graph-based Louvain clustering algorithm on the K-Nearest Neighbors graph. (B) Specific marker gene expression of four tumors and normal liver cells. (C) The four ICC tumor cells were classified into specific CNV groups based on their identified CNV patterns. (D) Kyoto Encyclopedia of Genes and Genomes pathway analysis of differentially expressed genes of ICC tumor cells. The circle size represents the level of statistical significance, and the circle color represents the intensity of the interaction. (E) Volcano plot displaying the 176 significantly upregulated and 228 downregulated genes. (F) SPP1 is overexpressed in ICC tumor cells. (G) SPP1 upregulation was observed in ICC tumors when compared with both non-tumor tissues and liver tumors based on data obtained from the Metabolic gEne RApid Visualizer website. (H) Increased expression levels of SPP1 are correlated with a poor prognosis for patients with ICC. ICC, intrahepatic cholangiocarcinoma; SPP1, secreted phosphoprotein 1; CNV, copy number variant; NLT, normal liver tissue; HCC, hepatocellular carcinoma; HR, hazard ratio; UMAP, Uniform Manifold Approximation and Projection.

Article Snippet: Subsequently, the tissues were blocked with 3% BSA in PBS for 30 min at room temperature and incubated overnight at 4°C with antibody against human SPP1 (1:100; cat. no. ER1802-16; Hangzhou HuaAn Biotechnology Co., Ltd.).

Techniques: Gene Expression, Isolation, Marker, Expressing, Variant Assay

Journal: Oncology Letters

Article Title: Single-cell RNA sequencing analysis of intrahepatic cholangiocarcinoma reveals SPP1 facilitates disease progression via interaction with CD4 + T cells

doi: 10.3892/ol.2025.15135

Figure Lengend Snippet: Distinguishing T-cell subsets in ICC tumor tissues between the early and late stages of disease. (A) Identification of ICC tumor cells in the early and late stage of disease. (B) Expression of SPP1 is higher in late-stage ICC tumor tissues compared with early-stage tumor tissues. ***P<0.001. (C) A total of seven subsets of T cells in the late stages were identified utilizing a graph-based Louvain clustering algorithm applied to the KNN graph. (D) A total of seven subsets of T cells in the early stages were identified utilizing a graph-based Louvain clustering algorithm applied to the KNN graph. (E) Ratio of Treg cells to Tcm cells in early- and late-stage ICC. (F) Pseudotime analysis of T-cell diffusion patterns across various stages of ICC. ICC, intrahepatic cholangiocarcinoma; SPP1, secreted phosphoprotein 1; KNN, K-Nearest Neighbors; Treg, regulatory T cells; Tcm, central memory T cells; UMAP, Uniform Manifold Approximation and Projection.

Article Snippet: Subsequently, the tissues were blocked with 3% BSA in PBS for 30 min at room temperature and incubated overnight at 4°C with antibody against human SPP1 (1:100; cat. no. ER1802-16; Hangzhou HuaAn Biotechnology Co., Ltd.).

Techniques: Expressing, Diffusion-based Assay

Journal: Oncology Letters

Article Title: Single-cell RNA sequencing analysis of intrahepatic cholangiocarcinoma reveals SPP1 facilitates disease progression via interaction with CD4 + T cells

doi: 10.3892/ol.2025.15135

Figure Lengend Snippet: Interactions between tumor cells and different immune cell types. (A) Bubble plot representing ligand-receptor interactions between tumor cells and immune cell clusters in ICC. (B) Details of interaction between tumor cells and immune cells in ligand-receptor pair of SPP1-CD44. (C) Details of interaction between tumor cells and immune cells in ligand-receptor pair of MIF-(CD74+CXCR4). (D) Details of interaction between tumor cells and immune cells in ligand-receptor pair of CXCL12-CXCR4. (E) Details of interaction between tumor cells and immune cells in ligand-receptor pair of CCL3-CCR1. ICC, intrahepatic cholangiocarcinoma; MIF, macrophage migration inhibitory factor; SPP1, secreted phosphoprotein 1; CXCR4, C-X-C chemokine receptor type 4; CCR4, C-C chemokine receptor type 1; CCL3, C-C motif chemokine ligand 3; DC, dendritic cell; NK, natural killer.

Article Snippet: Subsequently, the tissues were blocked with 3% BSA in PBS for 30 min at room temperature and incubated overnight at 4°C with antibody against human SPP1 (1:100; cat. no. ER1802-16; Hangzhou HuaAn Biotechnology Co., Ltd.).

Techniques: Migration

Journal: Journal of Neuroinflammation

Article Title: St6gal1-mediated sialylation protects retinal ganglion cells by restraining microglial phagocytosis

doi: 10.1186/s12974-026-03736-z

Figure Lengend Snippet: αRGCs maintain higher levels of sialyltransferases. A Dot plot showing the expression of four sialyltransferases in different RGC subtypes. The size of each circle represents the percentage of RGC subtypes expressing the gene, and the color indicates the expression level. B Line chart showing St6gal1 expression dynamics for each RGC subtype at different time points after ONC. C Representative retinal sections showing SNA expression in RGCs at the indicated time points after ONC. D Quantification of relative SNA immunostaining intensity in RGCs. n = 3 retinas per group. One-way analysis of variance (ANOVA) with Turkey’s multiple comparisons test. E Representative retinal sections showing ST6Gal1 expressions in retinas at indicated time points after ONC. F Quantification of relative ST6Gal1 immunostaining intensity in GCL. n = 6 retinas per group. One-way analysis of variance (ANOVA) with Turkey’s multiple comparisons test. G Representative retinal sections showing ST6Gal1 expressions in SPP1 + αRGCs and non-αRGCs at indicated time points after ONC. H-J Quantification of relative ST6Gal1 immunostaining intensity in SPP1 + αRGCs and non-αRGCs. n = 5 retinas per group. Unpaired t test. * P < 0.05, ** P < 0.01, ns, not significant. All error bars represent means ± SEM. Scale bar, 50 μm (main views) and 10 μm (enlarged views)

Article Snippet: Primary antibodies used were directed against SPP1 (1:100, AF808, R&D Systems), ST6Gal1 (1:100, 14355–1-AP, Proteintech),TUJ1 (1:500, 801202, Biolegend), RBPMS (1:500, GTX118619, GeneTex), melanopsin (1:1000, AB-N39, Advanced Targeting Systems), pCREB (1:100, ab32096, Abcam), pCaMKII (1:200, ab32678, Abcam), pS6 (1:100, 2211S, Cell Signaling Technology), pSTAT3 (1:100, 9145S, Cell Signaling Technology), pAKT (1:100, 4060S, Cell Signaling Technology), p–c-Jun (1:100, 2361S, Cell Signaling Technology), IBA1 (1:500, 019–19741, Wako).

Techniques: Expressing, Immunostaining

Journal: Journal of Neuroinflammation

Article Title: St6gal1-mediated sialylation protects retinal ganglion cells by restraining microglial phagocytosis

doi: 10.1186/s12974-026-03736-z

Figure Lengend Snippet: Sialylation blockade exacerbates RGC death after ONC injury. A Representative retinal sections showing SNA expressions in RGCs treated with 3Fax-PN. B Quantification of relative SNA immunostaining intensity in RGCs. n = 5 retinas per group. C Schematic illustration of 3Fax-PN treatment and ONC and timeline. D Representative retinal flat mounts showing TUJ1 + surviving RGCs in differentially treated retinas at 7 dpc. E Quantification of RGC survival rate at 7 dpc. n = 5 retinas per group. F Representative retinal flat mounts showing RBPMS + RGCs and SPP1 + /RBPMS + αRGCs in differentially treated retinas at 14 dpc. G Quantification of RGC survival rate at 14 dpc. n = 5 retinas per group. H-I Quantification of percentages of surviving αRGC and non-αRGC in total surviving RGCs. n = 5 retinas per group. All statistical analyses were performed using unpaired t test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, not significant. All error bars represent means ± SEM. Scale bar, 50 μm

Article Snippet: Primary antibodies used were directed against SPP1 (1:100, AF808, R&D Systems), ST6Gal1 (1:100, 14355–1-AP, Proteintech),TUJ1 (1:500, 801202, Biolegend), RBPMS (1:500, GTX118619, GeneTex), melanopsin (1:1000, AB-N39, Advanced Targeting Systems), pCREB (1:100, ab32096, Abcam), pCaMKII (1:200, ab32678, Abcam), pS6 (1:100, 2211S, Cell Signaling Technology), pSTAT3 (1:100, 9145S, Cell Signaling Technology), pAKT (1:100, 4060S, Cell Signaling Technology), p–c-Jun (1:100, 2361S, Cell Signaling Technology), IBA1 (1:500, 019–19741, Wako).

Techniques: Immunostaining

Journal: Journal of Neuroinflammation

Article Title: St6gal1-mediated sialylation protects retinal ganglion cells by restraining microglial phagocytosis

doi: 10.1186/s12974-026-03736-z

Figure Lengend Snippet: ST6Gal1 overexpression confers neuroprotection. A Schematic illustration of AAV2- St6gal1 injection and ONC and timeline. B Representative retinal flat mounts showing RBPMS + surviving RGCs in differentially treated retinas at 14 dpc. C Quantification of RGC survival rate. n = 7 retinas per group. D Representative retinal flat mounts showing SPP1 + surviving αRGCs in differentially treated retinas at 14 dpc. E–F Quantification of survival rates of αRGC and non-αRGC. n = 5 retinas per group. G Representative retinal flat mounts showing melanopsin + surviving ipRGCs in differentially treated retinas at 14 dpc. H Quantification of survival rates of ipRGC. n = 7 retinas per group. I Schematic illustration of AAV2- St6gal1 injection and I/R and timeline. J Representative retinal flat mounts showing RBPMS + surviving RGCs in differentially treated retinas at 7 days post injury. K-M Quantification of RGC survival rate in whole-mount, central, and peripheral retinas. n = 5 retinas per group. N Schematic illustration of AAV2- St6gal1 injection and NMDA injection and timeline of experiments. O Representative retinal flat mounts showing RBPMS + surviving RGCs in differentially treated retinas at 7 days after NMDA induced injury. P Quantification of RGC survival rates. n = 5 retinas per group. Q Representative responses of PERG recordings 7 days after NMDA induced injury. R Quantification of PERG amplitudes. n = 6 eyes per group. All statistical analyses were performed using unpaired t test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, not significant. All error bars represent means ± SEM. Scale bar, 50 μm

Article Snippet: Primary antibodies used were directed against SPP1 (1:100, AF808, R&D Systems), ST6Gal1 (1:100, 14355–1-AP, Proteintech),TUJ1 (1:500, 801202, Biolegend), RBPMS (1:500, GTX118619, GeneTex), melanopsin (1:1000, AB-N39, Advanced Targeting Systems), pCREB (1:100, ab32096, Abcam), pCaMKII (1:200, ab32678, Abcam), pS6 (1:100, 2211S, Cell Signaling Technology), pSTAT3 (1:100, 9145S, Cell Signaling Technology), pAKT (1:100, 4060S, Cell Signaling Technology), p–c-Jun (1:100, 2361S, Cell Signaling Technology), IBA1 (1:500, 019–19741, Wako).

Techniques: Over Expression, Injection

Journal: Journal of Neuroinflammation

Article Title: St6gal1-mediated sialylation protects retinal ganglion cells by restraining microglial phagocytosis

doi: 10.1186/s12974-026-03736-z

Figure Lengend Snippet: ST6Gal1 overexpression enhances optic nerve regeneration. A Schematic illustration of AAV2- St6gal1 injection and timeline of experiments. B Maximum projections of CTB-labeled regenerating axons. Asterisks indicate crush site. Scale bar, 250 μm. C Quantification of regenerating axons in differentially treated groups. n = 3 to 10 nerves per group. Two-way analysis of variance (ANOVA) with Turkey’s multiple comparisons test. D Representative retinal sections showing phosphorylation levels of CREB, CAMKII, S6, STAT3, AKT, c-Jun in TUJ1 + /SPP1 + RGCs from differentially treated groups at 3 dpc. Scale bar, 50 μm. E–G Quantification of relative fluorescent intensities of pCREB in total RGCs ( E ), αRGCs ( F ) and non-αRGCs ( G ). n = 5 retinas per group. Unpaired t test. H–L Quantification of relative fluorescent intensities of pCAMKII ( H ), pSTAT3 ( J ), pAKT ( K ), p-c-Jun ( L ) in total RGCs and percentage of pS6 + RGCs ( I ). n = 5 retinas per group. Unpaired t test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, not significant. All error bars represent means ± SEM

Article Snippet: Primary antibodies used were directed against SPP1 (1:100, AF808, R&D Systems), ST6Gal1 (1:100, 14355–1-AP, Proteintech),TUJ1 (1:500, 801202, Biolegend), RBPMS (1:500, GTX118619, GeneTex), melanopsin (1:1000, AB-N39, Advanced Targeting Systems), pCREB (1:100, ab32096, Abcam), pCaMKII (1:200, ab32678, Abcam), pS6 (1:100, 2211S, Cell Signaling Technology), pSTAT3 (1:100, 9145S, Cell Signaling Technology), pAKT (1:100, 4060S, Cell Signaling Technology), p–c-Jun (1:100, 2361S, Cell Signaling Technology), IBA1 (1:500, 019–19741, Wako).

Techniques: Over Expression, Injection, Labeling, Phospho-proteomics

Journal: Oncotarget

Article Title: Fibrotic microenvironment promotes the metastatic seeding of tumor cells via activating the fibronectin 1/secreted phosphoprotein 1-integrin signaling

doi: 10.18632/oncotarget.10157

Figure Lengend Snippet: ( A , B ) FN1 but not SPP1 silencing in fibrotic lung-derived fibroblasts attenuated the ability of CM-FLF to chemoattract tumor cells. The fibrotic lung-derived fibroblasts were transfected with the indicated siRNAs for 48 hours, followed by refreshment with 1% FBS-supplemented RPMI 1640. The conditioned medium was harvested 24 hours later, and then applied to the lower chamber of transwell. ( C , D ) FN1 and SPP1 silencing in fibrotic lung-derived fibroblasts abrogated the anti-apoptosis activity of CM-FLF. The fibrotic lung-derived fibroblasts were transfected with the indicated siRNAs for 48 hours, followed by refreshment with serum-free RPMI 1640. The conditioned medium was harvested 24 hours later, and applied to incubation with Hepa1-6 (C) and 4T1 (D) for 36 hours before DAPI staining. iMAX, treatment with transfection reagent RNAiMAX. NC, transfection with negative control duplex for siRNAs. Data are derived from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant.

Article Snippet: Sections were incubated at 4°C overnight with mouse monoclonal antibody against SPP1 (1:500, ZM-0174, ZSGB-BIO, China) or rabbit polyclonal antibody against GFP (1:200, LS-C154219, LSBio, WA, USA), FSP1 (1:400, ZA-0257, ZSGB-BIO, China) and FN1 (1:200, ZA-0106, ZSGB-BIO, China), then immunostained using ChemMate DAKO EnVision Detection Kit, Peroxidase/DAB, Rabbit/Mouse (DakoCytomation, Glostrup, Denmark).

Techniques: Derivative Assay, Transfection, Activity Assay, Incubation, Staining, Negative Control

Journal: Journal of Translational Medicine

Article Title: Temporal dynamics of the multi-omic response reveals the modulation of macrophage subsets post-myocardial infarction

doi: 10.1186/s12967-025-06726-6

Figure Lengend Snippet: Immunofluorescence of signature genes of macrophage subsets post-MI. A – D Representative images of immunostaining for the macrophage marker F4/80 (red) and signature genes Spp1 (green, A), Apoe (green, B), Trem2 (green, C) and Arg1 (green, D) in mice heart after MI. Nuclear was counterstained with DAPI (blue). White arrows indicate double-postive macrophages in the heart. Scale bar = 20μm. E–H Quantification of Spp1 + /F4/80 + ( E ), Apoe + /F4/80 + ( F ), Trem2 + /F4/80 + ( G ) and Arg1 + /F4/80 + ( H ) macrophage proportion

Article Snippet: Following blocked with 5% non-fat milk(Sangon Biotech Co.,Ltd, A600669-0250) in TBST (Sangon Biotech Co.,Ltd, G901BA0002), the membrane was incubated with antibodies against Spp1 (Santa, sc-21742, 1:1000), Apoe (HUABIO, HA723216, 1:1000), Trem2 (CST, 91068T, 1:1000), Arg1 (HUABIO, 66129-1-Ig, 1:1000) at 4 °C overnight, and then with HRP-conjugated secondary antibody at room temperature for 1 h. The membrane was soaked with ECL (New Cell & Molecular Biotech Co.,Ltd, P10300 ) and exposed with the Imaging System.

Techniques: Immunofluorescence, Immunostaining, Marker

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: VSIG4 + tumor-associated macrophages mediate neutrophil infiltration and impair antigen-specific immunity in aggressive cancers through epigenetic regulation of SPP1

doi: 10.1186/s13046-025-03303-z

Figure Lengend Snippet: VSIG4 + TAMs regulating SPP1 signaling to recruit neutrophil and shaped the immunosuppressive microenvironment. ( A ) The differences of SPP1 signaling in cell-cell communication after VSIG4 knockout. ( B ) The proportions of tumor-infiltrating neutrophils (CD11b + Ly6G + ) were measured by flow cytometry in wild-type ( n = 6) and VSIG4-KO ( n = 6) mice bearing mATC-derived tumor. ( C ) Gene set enrichment analysis to evaluate the scores of indicated pathways in different TAM subpopulations in human ATC samples. ( D ) The correlation between VSIG4 and SPP1 in PDAC was retrieved from GEPIA database. ( E ) Pan-cancer analysis of the correlation between VSIG4 and SPP1 in myeloid cells using TISCH2 database. ( F ) The expressions of Spp1 after VSIG4 knockout in TAMs. ( G - I ) Co-culture of mATC with BMDM isolated from VSIG4-KO and WT mice to examine the expressions of Spp1 by RT-qPCR and western blotting ( n = 3 for each group). Data are presented as mean ± S.D. ( J - M ) The mATC tumor-bearing mice were intraperitoneally treated with 10 mg/kg anti-VSIG4 and/or anti-SPP1 as single agent and combination therapy every 3 days, respectively. Tumors were isolated on day 35. The tumor growth curve, body weight, and tumor weight of each group were recorded. ( N - S ) The proportions of TILs in ATC tumor-bearing mice measured by flow cytometry in IgG isotype ( n = 6) and anti-SPP1 groups ( n = 6). Data are presented as mean ± S.E.M. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Samples were blocked by 5% skim milk at room temperature for 1 h. Primary antibodies against Spp1 (Affinity, AF0227), Stat1 (Proteintech, 10144-2-AP), Stat3 (Proteintech, 10253-2-AP), p-Stat1 (Proteintech, HKP0249), p-Stat3 (Proteintech, HKP0250), p-Stat5 (Proteintech, YP0253), p-Stat6 (Proteintech, YP0256), L-Lactyl-Histone H3 (Lys18) (PTMBio, PTM-1427RM) were incubated at 4 °C.

Techniques: Knock-Out, Flow Cytometry, Derivative Assay, Co-Culture Assay, Isolation, Quantitative RT-PCR, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: VSIG4 + tumor-associated macrophages mediate neutrophil infiltration and impair antigen-specific immunity in aggressive cancers through epigenetic regulation of SPP1

doi: 10.1186/s13046-025-03303-z

Figure Lengend Snippet: VSIG4 promoted SPP1 expression through enhancing the histone H3 lysine 18 lactylation and promoting the transcription activity of STAT3. ( A ) Co-culture of mATC with BMDMs isolated from VSIG4-KO and WT mice to examine the lactate concentration. ( B ) Isolation of TAMs from mATC-derived tumors in VSIG4-KO ( n = 3) and WT mice ( n = 3) on day 32 to examine the lactate concentration. Data are presented as mean ± S.E.M. ( C - D ) Co-culture of mATC with BMDMs isolated from VSIG4-KO and WT mice to examine the expressions of histone H3 lysine 18 lactylation (H3K18la). ( E ) Genome browser track analysis using dataset GSE192358 showed the H3K18la lactylation levels in the binding region of Spp1 with or without the lactate treatment, where the left area of the dotted line represented the binding region of H3K18la on the Spp1 promotor. The mRNA ( F ) and protein ( G - H ) level of Spp1 in BMDM with or without the lactate treatment ( n = 3). ( I - J ) The expression of Stat1, pStat1, Stat3, pStat3, and pStat6 in BMDMs after VSIG4 knockout ( n = 3). ( K ) The JASPAR database predicted the presence of STAT3 binding sites in the SPP1 promoter region. The mRNA ( L ) and protein ( M - N ) level of Spp1 in BMDMs after STAT3 inhibitor Stattic treatment ( n = 3). ( O ) The transcriptional activity of STAT3 on SPP1 was determined by dual luciferase reporter gene assay ( n = 3). Data are presented as mean ± S.D. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Samples were blocked by 5% skim milk at room temperature for 1 h. Primary antibodies against Spp1 (Affinity, AF0227), Stat1 (Proteintech, 10144-2-AP), Stat3 (Proteintech, 10253-2-AP), p-Stat1 (Proteintech, HKP0249), p-Stat3 (Proteintech, HKP0250), p-Stat5 (Proteintech, YP0253), p-Stat6 (Proteintech, YP0256), L-Lactyl-Histone H3 (Lys18) (PTMBio, PTM-1427RM) were incubated at 4 °C.

Techniques: Expressing, Activity Assay, Co-Culture Assay, Isolation, Concentration Assay, Derivative Assay, Binding Assay, Knock-Out, Luciferase, Reporter Gene Assay