Journal: Journal of Oncology

Article Title: Cancer-Associated Fibroblast-Derived Exosomal miRNA-320a Promotes Macrophage M2 Polarization In Vitro by Regulating PTEN/PI3K γ Signaling in Pancreatic Cancer

doi: 10.1155/2022/9514697

Figure Lengend Snippet: CAFs-derived conditioned medium (CAFs-CM) facilitated macrophage M2 polarization. (a) qPCR analysis of mRNA expression of classic M2 signature markers (CD163, CD206, and IL-10) and classic M1 signature markers (HLA-DR and iNOS) in U937 cells cultured with HPDE6-C7-CM, PANC-CM, NF3-CM, NF6-CM, CAF3-CM, or CAF6-CM. (b) qPCR analysis of mRNA expression of CD163, CD206, and IL-10 in U937 cells cultured with NFs-CM, CAFs-CM, or CAFs/GW4869-CM. (c) Western blot analysis of CD163, CD206, and IL-10 protein expression in U937 cells cultured with NFs-CM, CAFs-CM, or CAFs/GW4869-CM. (d) Quantitative analysis of CD163, CD206, and IL-10 protein expression in U937 cells cultured with NFs-CM, CAFs-CM, or CAFs/GW4869-CM. (e) The effect of NFs-CM, CAFs-CM, or CAFs/GW4869-CM on proliferation of pancreatic cancer cells by CCK-8 assay. (f) The effect of NFs-CM, CAFs-CM, or CAFs/GW4869-CM on pancreatic cancer cells invasion was determined by the Transwell assay. Representative photographs (magnification, 100) and the number of invaded cells are displayed. ∗∗ p < 0.01. # vs CAFs-CM. qPCR, quantitative real-time PCR; CM, conditioned medium; NFs, normal fibroblasts; CAFs, cancer-associated fibroblasts.

Article Snippet: Human monocytic cell line U937 was purchased from Merck Millipore (Temecula, California, USA) and cultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 10 nM 12-o-tetradecanoyl-phorbol-13-acetate (TPA).

Techniques: Derivative Assay, Expressing, Cell Culture, Western Blot, CCK-8 Assay, Transwell Assay, Real-time Polymerase Chain Reaction

Journal: Journal of Oncology

Article Title: Cancer-Associated Fibroblast-Derived Exosomal miRNA-320a Promotes Macrophage M2 Polarization In Vitro by Regulating PTEN/PI3K γ Signaling in Pancreatic Cancer

doi: 10.1155/2022/9514697

Figure Lengend Snippet: CAFs-derived exosomes (CAFs-Exo) facilitated macrophage M2 polarization. (a) Electron micrograph analysis of exosomes collected from CAFs-CM (bar, 500 nm). (b) Size distribution and concentration range characterization of exosomes collected from CAFs-CM assayed with qNano. (c) Western blot analysis of protein expression of CD63, CD81, and HSP70 (markers of exosomes) in exosomes collected from CAFs-CM assayed. (d) qPCR analysis of CD163, CD206, and IL-10 mRNA expression in U937 cells cultured with NFs-Exo or CAFs-Exo. (e) Western blot analysis of protein expression of CD163, CD206, and IL-10 in U937 cells cultured with NFs-Exo or CAFs-Exo. (f) Quantitative analysis of CD163, CD206, and IL-10 protein expression in U937 cells cultured with NFs-Exo or CAFs-Exo. ∗∗ p < 0.01. qPCR, quantitative real-time PCR; CM, conditioned medium; NFs, normal fibroblasts; CAFs, cancer-associated fibroblasts; Exo, exosome.

Article Snippet: Human monocytic cell line U937 was purchased from Merck Millipore (Temecula, California, USA) and cultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 10 nM 12-o-tetradecanoyl-phorbol-13-acetate (TPA).

Techniques: Derivative Assay, Concentration Assay, Western Blot, Expressing, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Journal of Oncology

Article Title: Cancer-Associated Fibroblast-Derived Exosomal miRNA-320a Promotes Macrophage M2 Polarization In Vitro by Regulating PTEN/PI3K γ Signaling in Pancreatic Cancer

doi: 10.1155/2022/9514697

Figure Lengend Snippet: Exosomes mediated the transfer of miRNA-320a from CAFs to macrophages. qPCR analysis of miR-106b, miR-148a, miR-125b, miR-320c, miR-320a, miR-1285, miR-422a, miR-29a, and miR-378d mRNA expression in NFs and CAFs (a) or NFs-Exo and CAFs-Exo (b). (c, d) qPCR analysis of miR-320a mRNA expression in 7 pancreatic cancer tissues-derived CAFs and 7 pancreatic cancer tissues-derived NFs or 7 pancreatic cancer tissues-derived CAFs-Exo and 7 pancreatic cancer tissues-derived NFs-Exo. (e–g) qPCR analysis of miR-320a mRNA expression in miRNA-320a-overexpressed CAFs (e), corresponding CAFs-Exo (f), and U937 cells after treatment with miRNA-320a-overexpressed CAFs-Exo (g). (h) Fluorescence microscope analysis of FAM-tagged miRNA-320a in U937cells treated with CAFs-Exo/FAM-miRNA-320a. ∗∗ p < 0.01. qPCR, quantitative real-time PCR; CM, conditioned medium; NFs, normal fibroblasts; CAFs, cancer-associated fibroblasts; Exo, exosome; CAFs-Exo/FAM-miRNA-320a, FAM-miRNA-320a-overexpressed CAFs-Exo.

Article Snippet: Human monocytic cell line U937 was purchased from Merck Millipore (Temecula, California, USA) and cultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 10 nM 12-o-tetradecanoyl-phorbol-13-acetate (TPA).

Techniques: Expressing, Derivative Assay, Fluorescence, Microscopy, Real-time Polymerase Chain Reaction

Journal: Journal of Oncology

Article Title: Cancer-Associated Fibroblast-Derived Exosomal miRNA-320a Promotes Macrophage M2 Polarization In Vitro by Regulating PTEN/PI3K γ Signaling in Pancreatic Cancer

doi: 10.1155/2022/9514697

Figure Lengend Snippet: miRNA-320a facilitated macrophage M2 polarization. qPCR analysis of classic M2 signature markers (CD163, CD206, and IL-10) and classic M1 signature markers (HLA-DR and iNOS) mRNA expression in U937 cells cultured with miRNA-320a mimics (a) or inhibitor (b). Western blot (c) and quantitative analysis (d) of CD163, CD206, and IL-10 protein expression in U937 cells cultured with miRNA-320a mimics. (e) The effect of U937/miR-320a-CM on pancreatic cancer cell proliferation by CCK-8 assay. (f, g) The effect of U937/miR-320a-CM on pancreatic cancer cells invasion was determined by the Transwell assay. Representative photographs (magnification, 100) and the number of invaded cells are displayed. ∗∗ p < 0.01. qPCR, quantitative real-time PCR; CM, conditioned medium; NFs, normal fibroblasts; CAFs, cancer-associated fibroblasts; U937/miR-320a-CM, miRNA-320a-overexpressed U937-derived CM.

Article Snippet: Human monocytic cell line U937 was purchased from Merck Millipore (Temecula, California, USA) and cultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 10 nM 12-o-tetradecanoyl-phorbol-13-acetate (TPA).

Techniques: Expressing, Cell Culture, Western Blot, CCK-8 Assay, Transwell Assay, Real-time Polymerase Chain Reaction, Derivative Assay

Journal: Journal of Oncology

Article Title: Cancer-Associated Fibroblast-Derived Exosomal miRNA-320a Promotes Macrophage M2 Polarization In Vitro by Regulating PTEN/PI3K γ Signaling in Pancreatic Cancer

doi: 10.1155/2022/9514697

Figure Lengend Snippet: miRNA-320a functions by targeting PTEN/PI3K γ signaling. (a) Schematic representation of the miR-320a site in PTEN-3′UTR. (b, c) Luciferase activity was assayed in PCa cells co-transfected with miR-320a and luciferase reporters containing PTEN-3′UTR. Data are presented as the relative ratio of firefly luciferase activity to renilla luciferase activity. Western blot (d) and quantitative analysis (e) of PTEN, PI3K γ , AKT, and p-AKT protein expression in miRNA-320a-overexpressed U937 cells. Western blot (f) and quantitative analysis (g) of CD163 and CD206 protein expression in U937 cells overexpressed with miRNA-320a in the presence or absence of PI3K γ siRNA. ∗∗ p < 0.01. # vs miR-320a mimic group. qPCR, quantitative real-time PCR.

Article Snippet: Human monocytic cell line U937 was purchased from Merck Millipore (Temecula, California, USA) and cultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 10 nM 12-o-tetradecanoyl-phorbol-13-acetate (TPA).

Techniques: Luciferase, Activity Assay, Transfection, Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: RNA

Article Title: IRES-dependent translation of egr2 is induced under inflammatory conditions

doi: 10.1261/rna.033019.112

Figure Lengend Snippet: Experimental setup. U937 cells were differentiated with 10 nM TPA for 48 h or left untreated. Then, cells were trypsinized, washed with PBS, and reseeded in fresh medium. After 24 h, the conditioned medium from activated U937 monocyte-derived macrophages (CM) or undifferentiated U937 monocytes (Ctr) was harvested. MCF7 cells were incubated for 4 h with CM or Ctr. Subsequently, the respective cellular lysates were subjected to polysomal fractionation. Equal aliquots of RNA isolated from single fractions were analyzed using denaturing agarose gel electrophoresis to verify 28S and 18S rRNA distribution as indicators for ribosome distribution. Pooled polysomal fractions 6–10 and total RNA were analyzed using whole genome microarrays. Comparing polysomal changes (CM vs. Ctr) with total changes (CM vs. Ctr) revealed translationally regulated genes.

Article Snippet: Briefly, we used supernatants of activated U937 monocyte-derived macrophages (CM) which we have previously shown to contain significantly elevated protein levels of the proinflammatory mediators TNFα, IL-6, and IL-8 as compared to supernatants of undifferentiated U937 monocytes (Ctr) ( Schmid et al. 2011 ).

Techniques: Derivative Assay, Incubation, Fractionation, Isolation, Agarose Gel Electrophoresis

Journal: Cancer Science

Article Title: High mobility group box‐1‐inducible melanoma inhibitory activity is associated with nodal metastasis and lymphangiogenesis in oral squamous cell carcinoma

doi: 10.1111/j.1349-7006.2008.00894.x

Figure Lengend Snippet: Expression of melanoma inhibitory activity (MIA), high mobility group box‐1 (HMGB1) and nuclear factor kB (NFkB) in human oral squamous cell carcinoma (OSCC) cells. MIA expression was compared with the expressions of HMGB1, NFkBp65 and coprecipitation between NFkBp65 and HMGB1 in HSC3, HSC4 human OSCC cells and U937 monocytic cells. Expressions of MIA, HMGB1 and NFkBp65 were examined by immunoblotting. Tubulin was served as a loading control. Co‐precipitation of HMGB1 and NFkB was examined by immunoprecipitation. A precipitant with anti‐HMGB1 antibody was detected with anti‐NFkBp65 antibody by immunoblotting. Reverse immunoprecipitation was also examined. A precipitant with anti‐NFkBp65 antibody was detected with anti‐HMGB1 antibody.

Article Snippet: No metastatic sublines are derived from HSC4 cells. ( 31 ) Controlled cell line was used for U937 (monocytic leukemia cell line, purchased from Dainihon Pharmaceutical, Tokyo, Japan).

Techniques: Expressing, Activity Assay, Western Blot, Control, Immunoprecipitation

Journal: Cancer Science

Article Title: High mobility group box‐1‐inducible melanoma inhibitory activity is associated with nodal metastasis and lymphangiogenesis in oral squamous cell carcinoma

doi: 10.1111/j.1349-7006.2008.00894.x

Figure Lengend Snippet: Melanoma inhibitory activity (MIA) intracellular signaling and vascular endothelial growth factor (VEGF)‐C and VEGF‐D expression in HSC3 cells. (a) Expression of VEGF‐C and VEGF‐D was examined by reverse transcriptase–polymerase chain reaction (RT‐PCR) in HSC3, HSC4 and U937 cells. β‐actin was served as a loading control. (b) Effects of anti‐MIA antibody on phosphorylation of extracellular signal‐related kinase (ERK)1/2 and p38 and expressions of integrin α5β1, VEGF‐C and VEGF‐D in HSC3 human oral squamous cell carcinoma (OSCC) cells. Protein levels of MIA, ERK1/2, phosphorylated ERK1/2 (pERK1/2), p38, phosphorylated p38 (pp38), VEGF‐C and VEGF‐D were examined by immunoblotting in HSC3 human OSCC cells treated with MIA antibody. Tubulin was served as a loading control. (c) The effect of inhibition of p38 on expression of VEGF‐C and VEGF‐D. HSC3 cells were treated with p38 inhibitor (SB239063). Expression of VEGF‐C and VEGF‐D was examined by RT‐PCR. β‐actin was served as a loading control.

Article Snippet: No metastatic sublines are derived from HSC4 cells. ( 31 ) Controlled cell line was used for U937 (monocytic leukemia cell line, purchased from Dainihon Pharmaceutical, Tokyo, Japan).

Techniques: Activity Assay, Expressing, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Control, Phospho-proteomics, Western Blot, Inhibition

Journal: Blood Cancer Journal

Article Title: Bispecific antibody releasing-mesenchymal stromal cell machinery for retargeting T cells towards acute myeloid leukemia blasts

doi: 10.1038/bcj.2015.73

Figure Lengend Snippet: Efficient T-cell mediated killing of target cell lines with varying CD33 expression levels is induced in the presence of bsAb-releasing hMSCs. ( a ) HEK293T, OCI-AML3, U937 and MOLM-13 were analyzed for CD33 surface expression levels by staining with anti-CD33/PE mAb (in black) or matched isotype control Ab (in gray), respectively. Numbers represent mean fluorescence intensity (MFI) of total cells. ( b ) In a standard chromium release assay 51 Cr labeled CD33 + MOLM-13 cells and CD33 - HEK293T cells were incubated with freshly isolated T cells at effector-to-target (e:t) cell ratio of 5:1 for 20 h with decreasing concentrations of the purified bsAb CD33–CD3. Mean±s.d. of two independent donors is shown. ( c ) Specific cell lysis of AML cell lines U937 (upper) and MOLM-13 (lower) measured with standard chromium release assay. Freshly isolated CD3 + T cells were co-cultured for 10 and 20 h with 51 Cr labeled CD33 + target cells at an e:t cell ratio of 5:1 in the presence of hMSC lines seeded at different concentrations 48 h before adding effector T cells and target cells. Data are presented as means±s.d. from two or three different donors, respectively. ( d ) Decreasing densities of 51 Cr labeled gene-modified hMSCs were co-cultured with PBMCs in the presence or absence of CD33 + MOLM-13 cells at an e:t ratio of 5:1. After 20 h of co-incubation the specific hMSCs lysis was examined via chromium release assay. Data shown as mean±s.d. from two independent donors.

Article Snippet: The human AML cell lines U937 (ACC 5) and MOLM-13 (ACC 554) were cultured in complete RPMI 1640 medium (Biochrom AG, Berlin, Germany).

Techniques: Expressing, Staining, Control, Fluorescence, Release Assay, Labeling, Incubation, Isolation, Purification, Lysis, Cell Culture, Modification