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: 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