Journal: Research

Article Title: Pim1 Serves as a Therapeutic Target for Inflammatory Arthritis via Mitochondrial Metabolism and Th17 Cell Differentiation

doi: 10.34133/research.1137

Figure Lengend Snippet: Pim1 promotes Th17 cell differentiation in vitro. (A) Relative mRNA levels of Pim1 during Th17 cell differentiation ( n = 9). (B) Relative protein levels of pSTAT3, RORγt, and Pim1 during Th17 cell differentiation ( n = 9). (C and D) Frequency of Th17 cells among CD4 + cells after AZD1208 treatment ( n = 9). (E) Relative protein levels of Pim1 among CD4 + T cells overexpressing (OE) vector or Pim1 ( n = 9). (F) Frequency of Th17 cells in CD4 + cells after overexpressing vector or Pim1 ( n = 9). (G) Relative protein levels of RORγt and pSTAT3 after AZD1208 treatment or Pim1 overexpression ( n = 9). (H and I) Relative mRNA levels of Th17-cell-associated pathogenic genes after AZD1208 treatment (H) or Pim1 overexpression (I) ( n = 9). (J and K) Concentration of IL-17A, IL-17F, IL-22, and GM-CSF in the cell supernatant after AZD1208 treatment (J) or Pim1 overexpression (K). (L) Frequencies of Th1, Th2, and Treg cells among CD4 + T cells after treatment with AZD1208 ( n = 9). The data were statistically analyzed via one-way ANOVA, followed by Bonferroni’s post hoc comparisons (A to D) and paired t test (E to L).

Article Snippet: Human IL-17A enzyme-linked immunosorbent assay (ELISA) kit (Elabscience, #E-EL-H5812), human IL-17F ELISA kit (Elabscience, #E-EL-H4193), human IL-22 ELISA kit (Elabscience, #E-EL-H0106), and human GM-CSF ELISA kit (Elabscience, #E-EL-H0081) were used.

Techniques: Cell Differentiation, In Vitro, Plasmid Preparation, Over Expression, Concentration 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

Journal: iScience

Article Title: Identification of potential biomarkers and therapeutic targets for antineutrophil cytoplasmic antibody-associated glomerulonephritis

doi: 10.1016/j.isci.2023.108157

Figure Lengend Snippet:

Article Snippet: Human M-CSF ELISA Kit , Boster , EK0444.

Techniques: Enzyme-linked Immunosorbent Assay, Software

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: iScience

Article Title: Identification of potential biomarkers and therapeutic targets for antineutrophil cytoplasmic antibody-associated glomerulonephritis

doi: 10.1016/j.isci.2023.108157

Figure Lengend Snippet:

Article Snippet: The plasma proteins levels of CSF-1R (Boster, EK0807), C1Q (Invitrogen, BMS209), CSF-1 (Boster, EK0444), IL-34 (Boster, EK1365) and CRP (Boster, EK1316) were measured using the Quantikine enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Software