Journal: Cell Reports

Article Title: De novo steroidogenesis in tumor cells drives bone metastasis and osteoclastogenesis

doi: 10.1016/j.celrep.2024.113936

Figure Lengend Snippet: Pregnenolone promotes human osteoclastogenesis and is secreted by human tumor cells (A–C) Representative images (A) and quantification (B and C) of TRAP-stained cell cultures of human blood mononuclear cell-derived OCs cultured for 2 days in the presence of 20 ng/mL recombinant human M-CSF and 20 ng/mL soluble human RANKL and then in the presence of the indicated concentration of pregnenolone or vehicle for 12 days. Scale bars represent 100 μm. (D) Levels of pregnenolone in the supernatants of A375, MDA-MB-435S, A549, NCI-H1299, NCI-H460, MCF-7, MDA-MB-231, DU145, LNCaP, PC-3, Hs895.T, SK-MEL-28, BEAS-2B, NCI-H128, NCI-H2126, HCC70, MCF-10A, MDA-PCa-2b, RWPE-1, and LASCPC-01 cells measured by ELISA. (E) Kaplan-Meier curve of breast-cancer-specific survival for 2,976 patients with high or low expression of Cyp11a1 in the primary tumor. Bar graphs show mean and SD of data from 3 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.002; n.s., not significant.

Article Snippet: Mouse Competitive ELISA Kit M-CSF , Elabscience Biotech. , Cat# E-EL-M2445.

Techniques: Staining, Derivative Assay, Cell Culture, Recombinant, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Cell Reports

Article Title: De novo steroidogenesis in tumor cells drives bone metastasis and osteoclastogenesis

doi: 10.1016/j.celrep.2024.113936

Figure Lengend Snippet:

Article Snippet: Mouse Competitive ELISA Kit M-CSF , Elabscience Biotech. , Cat# E-EL-M2445.

Techniques: Virus, shRNA, Recombinant, Modification, Staining, RNA Library Preparation, TaqMan Assay, Competitive ELISA, RNA Sequencing, Plasmid Preparation, Software, Real-time Polymerase Chain Reaction

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

Journal: Acta biomaterialia

Article Title: Spatial targeting of fibrosis-promoting macrophages with nanoscale metal-organic frameworks for idiopathic pulmonary fibrosis therapy.

doi: 10.1016/j.actbio.2023.12.006

Figure Lengend Snippet: Fig. 1. Overexpression of CSF-1R in CD206+ M2 macrophages. (A) Flow cytometry analysis of CSF-1R and CD206 expression in F4/80+ macrophages. Right panels: Quantified data in sorted F4/80+ macrophages. Results are expressed as means ± SD ( n = 3; ∗∗P < 0.01). (B) Representative results for coimmunostaining of CD206 and CSF-1R in the lung sections from bleomycin-treated mice. Representative images are shown. Bar = 20 μm. (C) Immunohistochemistry staining of CSF-1R and CD206 in the lung sections of IPF patients. Representative images are shown. Bar = 50 μm. (D-F) Single cell atlas of IPF patients according to dataset GSE122960. (D) Upper panels: Cellular populations identified. Lower panels: t-distributed stochastic neighbor embedding (t-SNE) depicting cell clusters originating either from a donor or from IPF patients. (E) Expression of CSF-1R for the cell types defined above each panel. (F) Percentage of cells with non-zero CSF-1R expression. (G) Kaplan– Meier survival analyses of IPF patients based on the expression of CSF-1R according to dataset GSE70866. (H) Immunofluorescence staining of CSF-1R and α-SMA on mouse lung tissues. Representative images are shown. Bar = 50 μm.

Article Snippet: The primary antibodies used were rabbit nti-phospho-CSF-1R, mouse anti- β-actin, and rabbit anti-GAPDH. horseradish peroxidase-conjugated goat anti-mouse/rabbit IgG Boster no. BA1050/BA1056, Wuhan, China, 1:10,0 0 0 dilution) was sed as the secondary antibody.

Techniques: Over Expression, Flow Cytometry, Expressing, Immunohistochemistry, Staining

Journal: Acta biomaterialia

Article Title: Spatial targeting of fibrosis-promoting macrophages with nanoscale metal-organic frameworks for idiopathic pulmonary fibrosis therapy.

doi: 10.1016/j.actbio.2023.12.006

Figure Lengend Snippet: Fig. 5. (A) Schematic diagram of the BLZ-loaded NPs therapy procedure. (B) Inhibitory effects of scrNP-BLZ@Mn-Cur or M2NP-BLZ@Mn-Cur on CSF-1R phosphorylation in lung tissues. (C) Representative flow cytometry results is shown to identify pulmonary macrophage subsets from lung digests in BLM-treated mice. Right panels: Quantified data in sorted F4/80+ macrophages ( n = 3, means ± SD). (D) Cell differential from BAL fluid was counted by Diff-Quik staining ( n = 5; mean ± SD; ∗p < 0.05). (E) The level of TGF- β1, IL-6, IL-11, and TNF- α in lung tissues were determined by ELISA. Results are expressed as means ± SD ( n = 3; ∗∗p < 0.01, ∗p < 0.05).

Article Snippet: The primary antibodies used were rabbit nti-phospho-CSF-1R, mouse anti- β-actin, and rabbit anti-GAPDH. horseradish peroxidase-conjugated goat anti-mouse/rabbit IgG Boster no. BA1050/BA1056, Wuhan, China, 1:10,0 0 0 dilution) was sed as the secondary antibody.

Techniques: Phospho-proteomics, Cytometry, Diff-Quik, Staining, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: FAPα + Macrophages Orchestrate Immune Evasion in Multiple Myeloma by Dual Regulation of PD‐L1 and T Cell Senescence

doi: 10.1002/advs.202506239

Figure Lengend Snippet: FAPα + Macrophages Are Enriched in the Myeloma Microenvironment and Correlate with Disease Progression. (A) Heatmap of gene expression in macrophages from patients with MM (n = 3) or healthy donors (HDs, n = 3). (B,C) Cell percentages of FAPα + macrophages (CD11b + CD14 + ) and FAPα + BMSC (CD45 − CD38 − CD29 + ) in PBMCs or BMMCs from patients with MM (n = 9). (D) Reprehensive immunofluorescence staining (IF) images of CD138, CD68, and FAPα in the BM of a NDMM patient (Magnification ×400. Scale bar, 50 µm). (E–G) Flow cytometry analysis of the cell percentages of FAPα + macrophages and FAPα + BMSCs in BMMCs from patients with MM at different disease stages (n = 27) or HDs (n = 4). (H) Correlation analysis of the cell percentages between CD138 + MM cells and FAPα + macrophages or FAPα + BMSCs in patients with NDMM (n = 15). (I) Reprehensive immunohistochemical staining (IHC) images of CD138 and FAPα in patients with NDMM or MM‐CR (Magnification ×200. Scale bar, 50 µm). (J,K) Western blot (J) and flow cytometry (K) analysis of FAPα expression in macrophages co‐cultured with MM cells (n = 3). (L) TGFβ1 expression in different MM cell lines. (M‐N) TGFβ1‐induced FAPα protein expression in macrophages (Magnification ×400. Scale bar, 50 µm). (O) M‐CSF, TGFβ1, and FAPα levels in BM supernatants from patients with MM at different disease stages (n = 37) using ELISA assay. (P) Correlation analysis of FAPα and TGFβ1 or M‐CSF in BM supernatant from patients with NDMM (n = 17). (Q) Reprehensive IHC images of bone marrow samples from patients with MM (n = 18) (Magnification ×200. Scale bar, 50 µm). (R) IOD values of CD138, CD68, and FAPα in patients with NDMM or RRMM (n = 18). (S) Correlation analysis between CD138 and FAPα in patients with NDMM. (T) Kaplan–Meier curves of PFS and overall OS in the set of patients with NDMM based on FAP protein expression level detected in tumor tissues. The median value of FAP RNA expression in the was 88.26 (IOD). The expression value of the FAP high group (n = 9) was >88.26(IOD) and the FAP low group (n = 9) was <88.26(IOD). Data are presented as mean ± SD. Each dot means independent samples. ns, no significant difference. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Statistical analysis was performed using a 2‐tailed Student's t ‐test in C, E, F, G, K, O, and R, a Pearson correlation in H, P, and S, a log‐rank test in T. MM, multiple myeloma; HD, healthy donors; BMSCs, bone marrow mesenchymal stem cells; PBMCs, peripheral blood mononuclear cells; BMMCs, bone marrow mononuclear cells; BM, bone marrow; NDMM, newly diagnosed MM; RRMM, relapsed or refractory MM; CR, complete response; TGFβ1, Transforming growth factor beta 1; M‐CSF, macrophage colony stimulating factor; IHC, Immunohistochemistry; IOD, Integrated Optical Density; RRMM, Relapsed/Refractory MM; PFS, Progression‐free survival; OS, overall survival.

Article Snippet: The bone marrow supernatants of patients with different MM stages were collected, and different cytokines were detected as described by the respective manufacturers: Human M‐CSF AccuSignal ELISA Kit (Cat #KOA0253, Rockland, USA), Human Seprase/FAP AccuSignal ELISA Kit (Cat #KOA0627, Rockland, USA), Human TGFβ1 ELISA Kit (Cat #1117102) (All from Dakewe Bioengineering Co., Ltd, China).

Techniques: Biomarker Discovery, Gene Expression, Immunofluorescence, Staining, Flow Cytometry, Immunohistochemical staining, Western Blot, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, RNA Expression, Immunohistochemistry