Journal: Cancer Cell

Article Title: Cancer cell autophagy, reprogrammed macrophages, and remodeled vasculature in glioblastoma triggers tumor immunity

doi: 10.1016/j.ccell.2022.08.014

Figure Lengend Snippet: IM + anti-VEGF prolongs survival of GBM mice and is immunostimulatory (A) Schematic of the long-term therapeutic trials in the lentiviral-induced mouse model of glioma. (B) Representative images of H&E-stained tissue sections from a tumor that developed in an end-stage LVRshp53 animal. Scale bar, 30 μm. Representative of whole-slide images of three tumors. (C) Survival of tumor-bearing LVRshp53 animals subjected to the indicated treatments. Control (Ctrl) (n = 10), anti-VEGF (n = 7), IM + anti-VEGF (n = 7), IM + anti-VEGF + CDL (n = 8). (D) Normalized bioluminescence in LVRshp53 animals treated as indicated for 2 weeks. (E) Survival of PDG animals subjected to the indicated treatments. Ctrl (n = 9), IM + anti-VEGF (n = 10). (F) Representative images of CD8 (green) and DAPI nuclear staining (blue). Scale bar, 50 μm. Image is illustrative of the analysis shown in (H). (G) High-magnification images of CD8 T cells in a Ctrl versus an IM + anti-VEGF-treated tumor. Scale bar, 50 mm (H) Quantification of CD8 T cells in LVRshp53 tumors treated as indicated for 12 days. Each dot indicates the average of 8–12 immuno-stained tumor tissue sections from one mouse. (I) Flow cytometry analysis of CD8 T cells in LVRshp53 tumors treated as indicated for 12 days. Cells were gated as CD45 + CD3 + CD8 + . Ctrl (n = 15), anti-VEGF (n = 8), IM (n = 10), IM + anti-VEGF (n = 10). (J and K) Representative images (J) and quantification (K) of CD4 T cells in whole LVRshp53 tumor tissue section. Animals were treated for 12 days. Scale bar, 50 μm. Ctrl (n = 6), anti-VEGF (n = 4), IM (n = 4), IM + anti-VEGF (n = 6). (L) Assessment of the functional contributions of CD8 and CD4 T cells to survival benefit. Ctrl (n = 6), αCD8 + αCD4 (n = 4), IM + anti-VEGF (n = 5), IM + anti-VEGF + αCD8 + αCD4 (n = 5), IM + anti-VEGF + αCD8 (n = 5). (M) Normalized bioluminescence in LVRshp53 mice treated as indicated in (L). (Para break) Data in all quantitative panels are shown as mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, no statistical significance. For survival analyses, Mantel-Cox test was performed. Other analyses by Mann-Whitney or one-way ANOVA tests.

Article Snippet: InVivo MAb anti-mouse CD4 (clone GK1.5) , BioXCell , Cat #: BE0003-1; RRID: AB_1107636.

Techniques: Staining, Control, Flow Cytometry, Functional Assay, MANN-WHITNEY

Journal: Cancer Cell

Article Title: Cancer cell autophagy, reprogrammed macrophages, and remodeled vasculature in glioblastoma triggers tumor immunity

doi: 10.1016/j.ccell.2022.08.014

Figure Lengend Snippet: CD8 and CD4 T cells are activated upon IM + anti-VEGF treatment (A) Flow cytometry analysis of effector T cells (CD62L-CD44 + ). Ctrl (n = 6), IM (n = 4), anti-VEGF (n = 7), and IM + anti-VEGF (n = 7). (B) FACS analysis of IFNγ intracellular staining in fixed and permeabilized CD8+T cells. Ctrl (n = 16), IM (n = 10), anti-VEGF (n = 12), IM + anti-VEGF (n = 12). (C and D) Flow cytometry analysis of GzB (C) and TNFα (D) intracellular staining in CD8 T cells. Ctrl (n = 15), IM (n = 10), anti-VEGF (n = 8), IM + anti-VEGF (n = 8). (E) Functional importance of IFNγ for the survival of LVRshp53 animals subjected to the indicated treatments. Ctrl (n = 9), anti-IFNγ (n = 5), IM + anti-VEGF (n = 7), IM + anti-VEGF + anti-IFNγ (n = 6). Statistical analysis by Mantel-Cox test. (F, G, and H) Flow cytometry analysis of Ki67 (F), pSTAT5 (G), and TCF1 (H) in CD8 T cells. Ctrl (n = 7), IM (n = 4), anti-VEGF (n = 9), and IM + anti-VEGF (n = 10). (I) Representative image of HIF-1α (red) and CD8 (green) in the anti-VEGF-treated tumor. Image is illustrative of the analysis performed in (J). Scale bar, 50 mm (J) Quantification of the proximity of CD8 T cells to hypoxic regions in the entire area of full sections of GBM tumors. The zones were divided into 0 μm (i.e., within the HIF-1α+ zone), >0 and <5 μm, and >5 μm separating T cells and HIF-1α+ regions. Ctrl (n = 11), IM (n = 4), anti-VEGF (n = 4), IM + anti-VEGF (n = 5). (K) Flow cytometry analysis of intracellular HIF-1α expression in fixed and permeabilized CD8 T cells. Ctrl (n = 5), IM (n = 4), anti-VEGF (n = 8), anti-VEGFR2 (n = 5). (L) Flow cytometry analysis of intracellular FOXP3 expression in CD4 T cells. Ctrl (n = 5), IM (n = 5), anti-VEGF (n = 4), IM + anti-VEGF (n = 8). (M) Flow cytometry analysis of intracellular TGFβ expression in CD4 T cells. Ctrl (n = 5), IM (n = 5), anti-VEGF (n = 4), IM + anti-VEGF (n = 7). (N and O) Flow cytometry analysis of SLAMF7 (N) and GzB (O) expression in CD4 T cells. Ctrl (n = 8), IM (n = 5), anti-VEGF (n = 5), IM + anti-VEGF (n = 7). (Para break) Data in all quantitative panels are shown as mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, no statistical significance. Statistical analysis by one-way ANOVA, unless otherwise indicated.

Article Snippet: InVivo MAb anti-mouse CD4 (clone GK1.5) , BioXCell , Cat #: BE0003-1; RRID: AB_1107636.

Techniques: Flow Cytometry, Staining, Functional Assay, Expressing

Journal: Cancer Cell

Article Title: Cancer cell autophagy, reprogrammed macrophages, and remodeled vasculature in glioblastoma triggers tumor immunity

doi: 10.1016/j.ccell.2022.08.014

Figure Lengend Snippet: Imipramine downregulates an M2-like program in TAMs (A) Western blot analysis of ARG1 and IL-10 in single tumors treated or not with IM or anti-VEGF. (B) Flow cytometry analysis of ARG1 and IL-10 expression in GBM tumors treated for 1 week. Ctrl (n = 9 tumors), IM (n = 6), anti-VEGF (n = 6), anti-VEGFR2 (n = 6), Axitinib (n = 6). Macrophages were gated as CD45 + CD11b+Ly6C-Ly6G−. (C) Expression of ARG1 and IL-10 in microglia (CD49d−) and MDMs (CD49d+) assessed by FACS in untreated tumors (n = 5). (D) Expression of MHC-II within microglia as assessed by flow cytometry. Ctrl (n = 4 tumors) and IM + anti-VEGF (n = 4). (E) Ex vivo co-cultures of tumoral CD11b cells and activated splenic CFSE-labeled CD8 or CD4 T cells. Each dot represents the average of two or three technical replicates. T cells alone (n = 4), Ctrl co-culture (n = 5), anti-VEGF (n = 3), IM (n = 4), IM + anti-VEGF (n = 4). (F) Analysis of the M2-like program in cytokine-polarized macrophages as assessed by qRT-PCR analysis of Ctrl and IM-treated M2-like BMDMs. Expression is normalized to 18S statistics by Welch’s t test. Each dot represents an individual sample. Data are representative of three independent experiments. (G) Analysis of the M1-like program in BMDMs assessed by FACS. Each dot represents an individual replicate. (H) Expression of Hrh1 mRNA normalized to 18S in ex vivo M1-and M2-polarized BMDMs, either untreated or IM treated for 24 h. Each dot represents an individual sample. Data are representative of three independent experiments. (I) mRNA expression of Hrh1 in FACS-sorted microglia or MDMs from Ctrl and IM-treated tumors. (J) mRNA expression of Arg1 , Chil3 , and Il10 in M2-polarized macrophages that were transfected with si Ctrl or two different si Hrh1 constructs. Cells were treated with 40 μm IM for 24 h. Data are representative of two independent experiments. (K) Western blot analysis of MRC1 and ARG1 expression of siRNA-transfected M2 BMDMs. Data are representative of two independent experiments. (L) CD8 and CD4 T cell proliferation during co-culture with tumoral CD11b cells isolated from untreated (n = 2) or TFP-treated tumors (n = 4). (M) mRNA expression of Hrh1 , Arg1 , and MMP2 in CD11b cells isolated from tumors treated with IM (n = 4), TFP (n = 4), or untreated Ctrl (n = 5). (N) Phagocytosis assay involving sorted microglia and MDMs from untreated or IM-treated tumors assayed with green pHrodo S. aureus bioparticles. Data presented as mean fluorescence intensity (MFI) of pHrodo/live cells. (Para break) Data in all quantitative panels are presented as mean ± SEM ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, no statistical significance. Statistical analysis by Mann-Whitney test or one-way ANOVA, unless otherwise stated.

Article Snippet: InVivo MAb anti-mouse CD4 (clone GK1.5) , BioXCell , Cat #: BE0003-1; RRID: AB_1107636.

Techniques: Western Blot, Flow Cytometry, Expressing, Ex Vivo, Labeling, Co-Culture Assay, Quantitative RT-PCR, Transfection, Construct, Isolation, Phagocytosis Assay, Fluorescence, MANN-WHITNEY

Journal: Cancer Cell

Article Title: Cancer cell autophagy, reprogrammed macrophages, and remodeled vasculature in glioblastoma triggers tumor immunity

doi: 10.1016/j.ccell.2022.08.014

Figure Lengend Snippet: Macrophage-derived CXCR3 ligands are required for the therapeutic benefit conveyed by the combinatorial regimen of IM + anti-VEGF (A) Cxcl10 and Cxcl9 expression in bulk tumors. mRNA expression is shown relative to Gapdh . Ctrl (n = 6), anti-VEGF (n = 9), IM (n = 6), IM + anti-VEGF (n = 8). (B) Representative image of CXCL10 (magenta), F4/80 (red), and DAPI (blue) staining of LVRshp53 tumors treated with IM + anti-VEGF. Scale bar, 50 μm. Images are illustrative of five to six fields in tissue sections from three different tumors. (C) CXCL9 expression in TAMs in untreated (n = 6) or tumors treated with anti-VEGF (n = 4), IM (n = 4), or IM + anti-VEGF (n = 8) revealed by flow cytometry. (D) CXCL9 expression in MDMs and microglia, evaluated as in (C). (E) mRNA Cxcl9 and Cxcl10 expression assessed in bulk tumors treated with IM + anti-VEGF (n = 10) ± αCD49d (n = 7) to selectively deplete MDMs but not microglia. Expression is normalized to Gapdh housekeeping gene. (F) Assessing the contribution of CXCR3 function to the survival of LVRshp53 animals subjected to the indicated treatments. Treatment cohorts: αCXCR3 (n = 6), IM + anti-VEGF + αCXCR3 (n = 6), IM + anti-VEGF (n = 5). (G) Representative images of CD8 T cells aimed to assess the effects of αCXCR3. Representative of whole-slide image analysis of three tumors per treatment. Scale bar, 50 mm (H) Flow cytometry analysis of CD8 T cells in tumors subjected to indicated treatments. (I) Ex vivo co-culture of tumor-derived CD11b cells and CFSE-labeled CD8 or CD4 T cells. Myeloid cells were isolated from tumors treated with IM + anti-VEGF (n = 4), IM + anti-VEGF + αCXCR3 (n = 4), or untreated Ctrl (n = 5). Each dot represents an average of two or three technical replicates. (J) Minimal effect on IFNγ secretion by CD8 T cells in tumors treated with αCXCR3, IM + anti-VEGF, or the triple combination. (K and L) No effect of αCXCR3 on (K) GzB or (L) TNFα secretion by CD8 T cells co-treated with IM + anti-VEGF. (M) Quantification of immunostaining for HEVs in tumors treated with IM + anti-VEGF (n = 8) or IM + anti-VEGF + αCXCR3 (n = 4). The data are shown as number of HEVs per square millimeter of tumor tissue. (Para break) Data in all quantitative panels are presented as mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, no statistical significance. Statistical analysis by Mann-Whitney test or one-way ANOVA, unless otherwise stated.

Article Snippet: InVivo MAb anti-mouse CD4 (clone GK1.5) , BioXCell , Cat #: BE0003-1; RRID: AB_1107636.

Techniques: Derivative Assay, Expressing, Staining, Flow Cytometry, Ex Vivo, Co-Culture Assay, Labeling, Isolation, Immunostaining, MANN-WHITNEY

Journal: Cancer Cell

Article Title: Cancer cell autophagy, reprogrammed macrophages, and remodeled vasculature in glioblastoma triggers tumor immunity

doi: 10.1016/j.ccell.2022.08.014

Figure Lengend Snippet: PD-L1 is induced in relapsing tumors and its blockade potentiates T cell function to prolong survival benefit in GBM mice (A) FACS analysis of PD-L1 in the live cell compartment of tumors treated as indicated. Ctrl (n = 4 tumors), responding to IM + anti-VEGF (n = 4), and relapsing from IM + anti-VEGF (n = 8). Responding tumors were collected after 12 days of treatment. Relapsing tumors were collected when mice became symptomatic or when tumors started to re-grow following a stable phase. (B) Representative image of immunostaining to reveal PD-L1 (red), CD45 (green), and DAPI nuclei (blue) in relapsing tumors after IM + anti-VEGF. Scale bar, 50 μm. Assessed in four relapsing tumors, n = 8–10 fields imaged per tumor. (C) Percentage of PD-L1-positive live cells comparing the CD45 − and CD45 + compartments of n = 4 relapsing GBM tumors as revealed by flow cytometry. (D) PD-L1 expression in the CD11b− and CD11b+ compartments of CD45 + cells in n = 8 tumors assessed by flow cytometry. (E) Percentage of PD-L1-positive TAMs assessed by FACS. Ctrl (n = 4), responding tumor (n = 4), relapsing tumor (n = 8). (F) Representative immunostaining to reveal PD-L1 expression in TAMs. CD206 (magenta), PD-L1 (red), and DAPI in n = 3 relapsing tumors, 8–10 images per tumor. Scale bar, 50 μm. (G) Expression of PD-L1 in MDMs and microglia of n = 8 relapsing tumors assessed by flow cytometry. (H) MHC-II expression in microglia comparing responding (n = 4) and non-responding tumors (n = 4), assessed by flow cytometry. (I and J) Representative images (I) and quantification (J) of CD8 T cells in untreated (n = 3), responding (n = 4), and relapsing (n = 3) tumors under IM + anti-VEGF treatment. CD8 (magenta) and DAPI-stained nuclei. Scale bar, 50 μm. Each dot indicates the total number of CD8 T cells in an entire tissue section from a tumor. (K) Abundance of CD8 T cells from tumors treated short term with IM + anti-VEGF (n = 5) or IM + anti-VEGF + αPD-L1 (n = 5), assessed by flow cytometry. (L–N) GzB (L), IFNγ (M), and TNFα (N) expression in CD8 T cells from tumors treated as in (K). (O) Assessment of the benefits of early versus late incorporation of anti-PD-L1. Ctrl (n = 5), IM + anti-VEGF (n = 7), IM + anti-VEGF + late anti-PD-L1 (n = 4), IM + anti-VEGF + early anti-PD-L1 (n = 7), anti-VEGF + anti-PD-L1 (n = 6). (P) The combination of a TCA (e.g., imipramine) and VEGF/VEGFR inhibitors induces autophagy in cancer cells and remodels the tumor vasculature, conveying survival benefit for mice bearing GBM. Imipramine reprograms M2-like TAMs to more pro-inflammatory phenotype, via inhibition of histamine receptor signaling. Consequent to the dual treatment, CD8 and CD4 T cells are recruited and activated to evoke their cytotoxic effects. The inclusion of anti-PD-L1 in the therapeutic regimen helps sustain the immune response and increases survival benefit. (Para break) Data in all quantitative panels are presented as mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, no statistical significance. Statistical analysis by Mann-Whitney test or one-way ANOVA, unless otherwise stated.

Article Snippet: InVivo MAb anti-mouse CD4 (clone GK1.5) , BioXCell , Cat #: BE0003-1; RRID: AB_1107636.

Techniques: Cell Function Assay, Immunostaining, Flow Cytometry, Expressing, Staining, Inhibition, MANN-WHITNEY

Journal: Cancer Cell

Article Title: Cancer cell autophagy, reprogrammed macrophages, and remodeled vasculature in glioblastoma triggers tumor immunity

doi: 10.1016/j.ccell.2022.08.014

Figure Lengend Snippet:

Article Snippet: InVivo MAb anti-mouse CD4 (clone GK1.5) , BioXCell , Cat #: BE0003-1; RRID: AB_1107636.

Techniques: Purification, Plasmid Preparation, Virus, Recombinant, Activation Assay, Staining, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Fluorescence, cAMP Assay, Competitive ELISA, Selection, Cell Isolation, Derivative Assay, Negative Control, shRNA, Software

Journal: OncoImmunology

Article Title: Anti-CD137 enhances anti-CD20 therapy of systemic B-cell lymphoma with altered immune homeostasis but negligible toxicity

doi: 10.1080/2162402x.2016.1192740

Figure Lengend Snippet: Figure 1. Treatment with aCD20 and aCD137 enhances survival against B lymphoma. (A) Tumor injection and treatment design. 1 £ 105 BL3750 cells were injected intra- venously at day (D) 0. Control Ig (2A3, 250 mg i.p.) or anti-CD20 (clone 5D2, 250 mg i.p.) was administered on day 4, and anti-CD137 (clone 3H3, 100 mg i.p.) either alone or in combination on day 5 in C57BL6 wild-type (WT) and various gene-targeted mice (FcRIII¡/¡, FcRIV¡/¡, and FcgR¡/¡). (B) Results are pooled from three independent experiments (n (2A3) D 24; n (5D2) D 31; n (3H3) D 12; n (5D2C3H3) D 34). Statistical analysis was calculated using Mantel–Cox test. p < 0.01 and p < 0.001 as indi- cated. (C) Results representative from one experiment, n D 6 each group. p < 0.05 and p < 0.01 as indicated. (D) Results representative from one experiment, n D 6 each group. (E) Results are pooled from two independent experiments (n (2A3) D 14; n (5D2) D 15; n (3H3) D 6; n (5D2C3H3) D 16). Statistical analysis was calculated using Mantel–Cox test. p < 0.01 as indicated. (F) Tumor inoculation (1 £ 105 BL3750 cells, i.v.) on D0 was followed by administration of anti-CD20 (clone 5D2, 250 mg i. p.) on day 4, and anti-CD137 (clone 3H3, 100 mg i.p.) at day 5, either alone or in combination in C57BL6 WT mice. Antibody treatment was then applied weekly until sacri- fice. Data shown is pooled from two independent experiments (n (2A3) D 16, n (5D2) D 6, n (3H3) D 6, n (5D2C3H3) D 16). p < 0.001, and p < 0.001 as indicated. (G) WT mice survival after single treatment with anti-CD20 (clone 5D2, 250 mg i.p. 20 d after BL3750 inoculation) and anti-CD137 (clone 3H3, 100 mg i.p. 21 d after BL3750 inoculation). Results are representative of one experiment, n D 6, each group. p < 0.01 as indicated.

Article Snippet: On day 5 post BL3750 lymphoma inoculation, anti-CD137 mAb (3H3, Bio X Cell) was administered (100 mg i.p. per injection).

Techniques: Injection, Control

Journal: OncoImmunology

Article Title: Anti-CD137 enhances anti-CD20 therapy of systemic B-cell lymphoma with altered immune homeostasis but negligible toxicity

doi: 10.1080/2162402x.2016.1192740

Figure Lengend Snippet: Figure 2. Treatment with aCD20 and aCD137 reduces blood B-cell numbers in mice bearing B-cell lymphoma. Blood samples were collected for hemogram assessment according to the experimental design shown in (A). 1 £ 105 BL3750 cells were injected i.v. at D0, and followed by treatment with control Ig (2A3, 250 mg i.p.) or anti- CD20 (clone 5D2, 250 mg i.p.) on day 4, and anti-CD137 (clone 3H3, 100 mg i.p.) either alone or in combination on day 5 in C57BL6 WT or various FcR-deficient mice, as indicated. Peripheral blood lymphocyte (B), and B cell numbers (C). Results are representative of one experiment n D 6 per group. Statistical analysis was calculated using Multiple T tests using the Holm–Sidak Method for multiple comparisons, where p < 0.05 was considered statistically significant between respective anti-CD137 (clone 3H3), anti-CD20 (clone 5D2), 5D2 C 3H3 groups versus (2A3) isotype control-treated mice.

Article Snippet: On day 5 post BL3750 lymphoma inoculation, anti-CD137 mAb (3H3, Bio X Cell) was administered (100 mg i.p. per injection).

Techniques: Injection, Control

Journal: OncoImmunology

Article Title: Anti-CD137 enhances anti-CD20 therapy of systemic B-cell lymphoma with altered immune homeostasis but negligible toxicity

doi: 10.1080/2162402x.2016.1192740

Figure Lengend Snippet: Figure 3. Treatment with aCD20 and aCD137 induces BAFF and cytokines in plasma with or without B lymphoma inoculation. Control Ig (2A3, 250 mg i.p.) or anti-CD20 (clone 5D2, 250 mg i.p.) was administered on day (D) 0, and anti-CD137 (clone 3H3, 100 mg i.p.) either alone or in combination on D1 in C57BL6 wild-type (WT) or gene- targeted mice. In some experiments (B, D), 1 £ 105 BL3750 cells were injected intravenously on day (D) 0, and control Ig (2A3, 250 mg i.p.) or anti-CD20 (clone 5D2, 250 mg i.p.) was administered on day (D) 4, and anti-CD137 (clone 3H3, 100 mg i.p.) either alone or in combination on D5 in WT or gene-targeted mice. (A) and (B) BAFF assessment from plasma of mice at D2, D5, D8 and D15 post anti-CD20 treatment. Data is representative of two experiment, n D 5 each group (n D 10 total).

Article Snippet: On day 5 post BL3750 lymphoma inoculation, anti-CD137 mAb (3H3, Bio X Cell) was administered (100 mg i.p. per injection).

Techniques: Clinical Proteomics, Control, Injection

Journal: Biochemistry and Biophysics Reports

Article Title: HUC-MSC-derived exosomal miR-16-5p attenuates inflammation via dual suppression of M1 macrophage polarization and Th1 differentiation

doi: 10.1016/j.bbrep.2025.102078

Figure Lengend Snippet: miR-16-5p mimics blocks M1 macrophage polarization and Th1 cell differentiation. A: Raw264.7 cells were transfected with miR-16-5p mimics or NC mimics and subsequently induced to M1 macrophage polarization. After 24 h of culture, the percentage of CD11b + iNOS + cells in each group were measured by flow cytometry. B: Data are presented as mean ± standard deviation ( x ‾ ± s), with n = 3. p < 0.0001 compared with M1 group and NC mimics group. C: Naïve CD4 + T cells were induced to Th1 cells and then transfected with miR-16-5p or NC mimic. After 5 days of culture, the percentage of CD4 + IFN-γ + cells in each group were measured by flow cytometry. D: Data are presented as mean ± standard deviation ( x ‾ ±s), with n = 3. p = 0.0319, compared with Th1 group p = 0.0237, compared with NC mimics group. E: The inhibitory effect of miR-16-5p transfection on the mRNA levels of iNOS and TNF-α in M1 cells was measured by q-PCR. Data are presented as mean ± standard deviation ( x ‾ ±s), with n = 3. p iNOS <0.0001, p TNF-α = 0.0038, compared with M1 group, p iNOS <0.0001, p TNF-α = 0.0002, compared with NC mimics group. F: The mRNA expression levels of IFN-γ and STAT4 in different groups were measured by q-PCR. Data are presented as mean ± standard deviation ( x ‾ ±s), with n = 3. p STAT1 = 0.0018, p I - γ = 0.0032, compared with Th1 group, p STAT1 <0.0001, p I - γ = 0.0286, compared with NC mimics group.

Article Snippet: The single cells were labeled with iNOS (APC-iNOS, #17592082, eBioscience), F4/80 (FITC-F4/80, #35-4801-U100, Tonbo Biosciences), CD11b (PerCP-Cyanine5.5-CD11b, 65-0112-U100, Tonbo Biosciences), CD45(PE-CD45, #147712, Biolegend), CD3 (FITC-CD3, #35-0032-U100, Tonbo Biosciences) and CD4 (PE-CD4, #50-0041-U100, Tonbo Biosciences) respectively.

Techniques: Cell Differentiation, Transfection, Flow Cytometry, Standard Deviation, Expressing

Journal: Biochemistry and Biophysics Reports

Article Title: HUC-MSC-derived exosomal miR-16-5p attenuates inflammation via dual suppression of M1 macrophage polarization and Th1 differentiation

doi: 10.1016/j.bbrep.2025.102078

Figure Lengend Snippet: miR-34a-5p and miR-125b-5p mimics suppress M1 macrophage polarization and Th1 cell differentiation. A: The Raw264.7 cells were transfected miR-34a-5p mimic, miR-125b-5p mimics or NC mimics and subsequently induced to M1 macrophage. After 24 h of culture, the percentage of CD11b + iNOS + cells in various group was measured by flow cytometry. B: Data are presented as mean ± standard deviation ( x ‾ ± s), with n = 3. p M1,34 = 0.0118, p M1,125 = 0.0037, compared with M1 group; p NC,34 = 0.0202, p NC,125 = 0.0060, compared with NC mimics group. C: Naïve CD4 + T cells were induced to Th1 cells and then transfected with miR-34a-5p mimics, miR-125b-5p mimics or NC mimic. After 5 days of culture, the percentage of CD4 + IFN-γ + cells in various groups was measured by flow cytometry. D: Data are presented as mean ± standard deviation ( x ‾ ± s), with n = 3. p Th1,34 = 0.0102, p Th1,125 = 0.0201, compared with Th1 group, p NC,34 = 0.0220, p NC,125 = 0.0447, compared with NC mimics group. E: The inhibitory effects of miR-34a-5p mimics or miR-125b-5p mimics transfection on the mRNA levels of iNOS and TNF-α in M1 cells was measured by q-PCR. Data are presented as mean ± standard deviation ( x ‾ ± s), with n = 3. p 34-iNOS <0.0001, p 125-iNOS <0.0001, p 34-TNF-α = 0.0012, p 125-TNF-α = 0.0117, compared with M1 group, p 34-iNOS = 0.0001, p 125-iNOS = 0.0027, p 34-TNF-α = 0.0039, p 125-TNF-α = 0.0377, compared with NC mimics group. F: The mRNA expression level of I - γ and STAT1 in different groups were measured by q-PCR. x ‾ ± s, with n = 3. p 34-STAT1 <0.0001, p 125-STAT1 <0.0001, p 3 - γ <0.0001, p 1 - γ <0.0001, compared with Th1 group, p 34-STAT1 <0.0001, p 125-STAT1 <0.0001, p 3 - γ = 0.0014, p 1 - γ = 0.0004, compared with NC mimics group.

Article Snippet: The single cells were labeled with iNOS (APC-iNOS, #17592082, eBioscience), F4/80 (FITC-F4/80, #35-4801-U100, Tonbo Biosciences), CD11b (PerCP-Cyanine5.5-CD11b, 65-0112-U100, Tonbo Biosciences), CD45(PE-CD45, #147712, Biolegend), CD3 (FITC-CD3, #35-0032-U100, Tonbo Biosciences) and CD4 (PE-CD4, #50-0041-U100, Tonbo Biosciences) respectively.

Techniques: Cell Differentiation, Transfection, Flow Cytometry, Standard Deviation, Expressing