Journal: Journal for immunotherapy of cancer

Article Title: Subcutaneous checkpoint inhibition is equivalent to systemic delivery when combined with nelitolimod delivered via pressure-enabled drug delivery for depletion of intrahepatic myeloid-derived suppressor cells and control of liver metastases.

doi: 10.1136/jitc-2024-008837

Figure Lengend Snippet: Figure 2 Modulation of liver myeloid and lymphoid compartments by nelitolimod via pressure-enabled drug delivery was preserved in combination with Sys or SQ checkpoint inhibitor. Liver of tumor-bearing mice were harvested 10 days post- treatment. CD45+ cells were isolated from non-parenchymal cells. (A) MDSC cell population (CD11b+Gr1+), (B) monocytic MDSCs (M-MDSC; CD11b+Ly6C+/hiLy6G−/lo), (C) dendritic (CD11c+) cells, (D) B cells (B220+), (E) T cells (CD3+) and (F) M1- like macrophage (F/4/80+CD38+EGR2−) were quantified by flow cytometry. (G) i (a–h) Tumors were isolated from each group, OCT-mounted tissues were sectioned, fixed, and stained for CD3 (green), CD8 (red), CD11b (green), and Gr1 (red). (G) ii–iii Quantification of CD11b+Gr1+ MDSCs and CD3+CD8+T cells and from tumors of mice were performed across five fields/mouse and n=3 mice were used per group. Scale (20 µm). Animal data were presented as mean±SEM from and n was mentioned in the individual graph. One-way analysis of variance was performed to determine statistical differences among multiple groups. MDSC, myeloid-derived suppressor cells; M-MDSC, monocytic MDSC; G-MDSC, granulocytic MDSC; OCT, Optimal temperature cutting compound; DAPI, 4′,6-Diamidino-2-phenylindole; SQ, subcutaneous; Sys, systemic; Veh, vehicle.

Article Snippet: Primary antibodies and their corresponding clones used for immunofluorescence (IF) staining: CD11b; E6E1M (Cell Signaling, Massachusetts, USA), GR1; RB6- 8C5 (BioLegend, California, USA), CD3; SP7 (Novus Biologicals, USA) and CD8a; 53–6.7 μ (BioLegend, California, USA).

Techniques: Isolation, Flow Cytometry, Staining, Derivative Assay

Journal: Biomedicines

Article Title: Targeting the Glutamine Transporter SLC1A5 Enhances Sensitivity of Acute Myeloid Leukemia to MLN4924

doi: 10.3390/biomedicines14030667

Figure Lengend Snippet: Effect of MLN4924 in combination with SLC1A5 inhibitor (V9302) on AML cell proliferation and cell differentiation. ( A – C ) Evaluate the interference effect of shRNA on SLC1A5 in AML cells through RT-qPCR and Western blot. ( D , E ) The effect of SLC1A5 knockdown on the sensitivity to MLN4924 of AML cells was detected through CCK8 assay (24 h after treatment). ( F , G ) Kasumi-1 and MOLM-13 cells were treated with V9302 for 24 h and IC50 was detected through CCK8 assay. ( H – M ) CCK8 assay was used to verify the effect of MLN4924 combined with V9302 on AML cell viability. ( N – V ) CD11b, CD14, and CD16 expression on AML cell surface treated with MLN4924 and V9302 for 48 h was detected through flow cytometry. ( W ) Wright–Giemsa staining to detect morphological changes of AML cells treated with MLN4924 and V9302 for 24 h captured using an oil immersion lens (1000×). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Antibody against CD11b (Anti-Human CD11b Mouse IgG2a Recombinant Antibody, FITC-65582) was purchased from Proteintech (China).

Techniques: Cell Differentiation, shRNA, Quantitative RT-PCR, Western Blot, Knockdown, CCK-8 Assay, Expressing, Flow Cytometry, Staining

Journal: Journal of Biological Chemistry

Article Title: Cholesterol-binding protein TSPO2 coordinates maturation and proliferation of terminally differentiating erythroblasts

doi: 10.1074/jbc.ra119.011679

Figure Lengend Snippet: Figure 3. TSPO2 mutations causative of the HK RBC phenotype are detrimental to the TSPO2 function. A, copy numbers of transfected cTSPO2 cDNAs (indicated by open bars) and the contents of cTSPO2 mRNA (indicated by gray bars) in stably transfected K562 cells were estimated by quantitative PCR and quantitative RT-PCR, respectively, for the wild-type (WT), C40Y (C40Y), and VFT (VFT) cTSPO2. The cellular contents of 16-kDa cTSPO2 were quantitated by densitometric scanning of the immunoblots using the anti-cTSPO2 antibody and normalized with actin (43 kDa, indicated by black bars). A representative immunoblot from 3 independent experiments is shown in the right panel (an entire blot is shown in Fig. S1). Data are expressed as the means ± S.D. (n =3). ** P < 0.01. The migrating positions of the size markers are shown in kDa. B, fluorescent signals of incorporated NBD-cholesterol (NBD- cholesterol), non-esterified cholesterol stained with filipin III (Filipin), cholesteryl esters (CEs) stained with Nile Red (Nile Red), and incorporated FITC-transferrin (FITC-Tf) in K562 cells stably transfected with the empty vector (Control) and wild-type (WT), C40Y (C40Y), or VFT (VFT) cTSPO2 cDNA are shown. Bars, 20 μm. C, lipid droplets stained with Nile Red in B were counted and the numbers of vesicles larger than 1 μm in the major axis were compared. Data are expressed as the means ± S.D. (n = 22~28). ***P < 0.001. D, cholesterol accumulation in the ER was assessed by an LDLR-promoter luciferase assay. Activities relative to the control cells are expressed as the means ± S.D. (n =3). ** P < 0.01, ***P < 0.001. E, total cholesterol contents in the cells are expressed as the means ± S.D. (n =3).

Article Snippet: Other antibodies used are anti-canine Na,K-ATPase α-subunit (12), anti-human stomatin (13), phycoerythrin (PE)-conjugated anti-mouse CD71 (BD Pharmingen), PE-Cy7-conjugated anti-mouse CD44 and fluorescein isothiocyanate (FITC)-conjugated anti-mouse TER119 (both from Tonbo Biosciences, Japan), allophycocyanin (APC)-conjugated anti-mouse CD45.2, Gr1, and CD11b (all from BioLegend).

Techniques: Transfection, Stable Transfection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Staining, Plasmid Preparation, Control, Luciferase

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: The Thyroid Hormone Analog GC-1 Mitigates Acute Lung Injury by Inhibiting M1 Macrophage Polarization.

doi: 10.1002/advs.202401931

Figure Lengend Snippet: Figure 2. GC-1 selectively inhibited the M1 macrophage polarization. A) Representative images of IHC staining for macrophage marker F4/80, M1 macrophage marker CD86, or M2 macrophage marker CD206 in lung sections of LPS-induced ALI model mice (n = 3), Scale bars: 100 and 20 μm (insets). B) Quantitative statistical results of IHC-stained positive cells (n = 3). C) Representative pictures of F4/80, CD86, and CD206 IF staining in lung sections from ALI mice induced by HCl (n = 3), Scale bars: 50 μm. Quantitative statistical results of fluorescence intensity from D) F4/80+CD86+ macrophages and E) F4/80+CD206+ macrophages (n = 3). F) Representative flow cytometry plots showing the proportions of M1 (CD11b+CD86+CD206−) and M2 (CD11b+CD86−CD206+) macrophages (n = 3). THP-1 cells were treated with LPS (100 ng mL−1) alone or in combination with GC-1 (100 nm) for 24 h after PMA induction. G) CD11b+CD86+CD206−M1 macrophages and H) CD11b+CD86−CD206+ M2 macrophages quantitative statistical results (n = 3). I) Immunoblot analysis of lysates from THP-1 cells (n = 3). J) Immunoblot gray values statistical quantification (n = 3). The values are shown as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns = not significant.

Article Snippet: Flow Cytometry: According to the instructions, APC Anti-Human CD206 Antibody (E-AB-F1161E, Elabscience Biotechnology), PE AntiHuman CD86 Antibody (E-AB-F1012D, Elabscience Biotechnology), and FITC Anti-Human CD11b Antibody (E-AB-F1081C, Elabscience Biotechnology) were separately added.

Techniques: Immunohistochemistry, Marker, Staining, Cytometry, Western Blot

Journal: Stem Cell Research & Therapy

Article Title: Single-cell RNA sequencing of endometrium uncovers dynamic characteristics and dysregulation of perivascular CD9 + SUSD2 + cells in thin endometrium

doi: 10.1186/s13287-025-04658-y

Figure Lengend Snippet: Single-cell transcriptomic data revealed that CD9 + SUSD2 + cells were likely to be the progenitors in the endometrium. A UMAP of cells with the associated cell types in samples among proliferative ( n = 3), secretory ( n = 3), and thin endometrium ( n = 3). Peri, perivascular cell; Str, stromal cell; pStr, proliferative stromal cell. B Expression of classical marker genes of each cell type in the endometrial samples. C Velocities derived from the dynamical model for endometrial samples are visualized as streamlines in a UMAP-based embedding. D Bar plot shows the representative GO terms of significant gene markers for perivascular CD9 + SUSD2 + cells. E Flow cytometry revealed that perivascular CD9 + SUSD2 + cells were positive for CD73, CD90, and CD105, but negative for HLA-DR, CD34, CD45, CD11b, and CD19. Negative staining results were also generated for corresponding isotype control antibodies. Grey peaks show the isotype control and red peaks display the indicated marker

Article Snippet: Subsequently, the cells were incubated in the dark with fluorescein isothiocyanate (FITC)-labeled anti-CD73 (Cat# 10904-MM07-F, Sino Biological, China), CD90 (Cat# 16897-MM10-F, Sino Biological, China), CD105 (Cat# 561443, BD Pharmingen, USA), CD34 (Cat# 68035-XM01-F, Sino Biological, China), CD45 (Cat# 10086-MM05-F, Sino Biological, China), CD11b (Cat# 03211-50, BioGems, USA), CD19 (Cat# 11880-MM17-F, Sino Biological, China), HLA-DR (Cat# 68038-XM01-F, Sino Biological, China), and Isotype Control FITC (Cat# 44212-50, BioGems, USA) at room temperature for 30 min.

Techniques: Expressing, Marker, Derivative Assay, Flow Cytometry, Negative Staining, Generated, Control