Journal: Oncogenesis

Article Title: microRNA-378a-5p iS a novel positive regulator of melanoma progression

doi: 10.1038/s41389-020-0203-6

Figure Lengend Snippet: a In vitro cell migration and invasion of M14 and A375 melanoma cells transiently transfected with mimic miRNA scramble control (mimic Ctrl) or mimic miR-378. Values are expressed as number of migrated/invaded cells ± standard deviation. b Western blot analysis of MMP2 protein expression in M14 cells transiently transfected with mimic Ctrl or mimic miR-378. c Quantification of capillary-like structures formation in M14 cells transiently transfected with mimic Ctrl or mimic miR-378. Values are expressed as number of intersection points/field ± standard deviation. d VEGF secretion by M14 and A375 cells transfected with mimic Ctrl or mimic miR-378. Results are reported as fold over control. e Quantification of capillary-like structures formation in M14 cells transiently transfected with mimic Ctrl or mimic miR-378 incubated in the absence (no Ab) or presence of specific human VEGF- (anti-VEGF) or IL-8- (anti-IL-8) -neutralizing antibodies (0.2 ug/mL). Results are reported as number of intersection points/field (average ± SD). f qRT-PCR analysis of uPAR mRNA expression in M14 cells transfected with mimic Ctrl or mimic miR-378. The results are reported as fold induction ± SEM in cells transfected with mimic miR-378 relative to control ones. a , c - f Statistical analysis was performed applying t -test. ***p < 0.001, *p < 0.05. g Western blot analysis of uPAR protein expression in M14 and A375 cells transiently transfected with mimic miR-378 or with miR-378 inhibitor (anti-miR-378) and the relative miRNA scramble control (mimic Ctrl; anti-miR-Ctrl). b , g Representative images of one out of three independent experiments are reported. HSP72/73 and α-tubulin were used as loading and transferring control.

Article Snippet: Transfected 2 × 10 5 melanoma cells were seeded in serum-free medium onto the gelled BME and incubated at 37 °C for 18 h. Cells were incubated alone or in the presence of specific human IL-8- or VEGF-neutralizing antibodies (0.2 μg/mL, CXCL8-MAB208 and VEGF-MAB293, R&D Systems) and capillary-like structures (CLS) evaluated.

Techniques: In Vitro, Migration, Transfection, Control, Standard Deviation, Western Blot, Expressing, Incubation, Quantitative RT-PCR, Transferring

Journal: Oncogenesis

Article Title: microRNA-378a-5p iS a novel positive regulator of melanoma progression

doi: 10.1038/s41389-020-0203-6

Figure Lengend Snippet: a Endothelial capillary tube-like network formation evaluated in HUVEC seeded on Cultrex BME and exposed to conditioned medium (CM) derived from mimic control (mimic Ctrl) or mimic miR-378 overexpressing M14 cells. The average ± SD of three independent experiments performed in triplicate is reported. Statistical analysis was performed applying t -test. * p < 0.05. b Representative images of one representative experiment performed as reported in a . c Box and Whisker plot showing results quantification of the haemoglobin content in the Matrigel plugs containing CM from M14 cells transiently transfected with mimic Ctrl or mimic miR-378. In the negative and positive controls, the CM was replaced with serum-free medium or VEGF, respectively. Statistical analysis was performed applying Mann-Whitney test, * p < 0.05. d Images of one representative experiment performed as reported in c .

Article Snippet: Transfected 2 × 10 5 melanoma cells were seeded in serum-free medium onto the gelled BME and incubated at 37 °C for 18 h. Cells were incubated alone or in the presence of specific human IL-8- or VEGF-neutralizing antibodies (0.2 μg/mL, CXCL8-MAB208 and VEGF-MAB293, R&D Systems) and capillary-like structures (CLS) evaluated.

Techniques: Derivative Assay, Control, Whisker Assay, Transfection, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: M-CSF Induces Vascular Endothelial Growth Factor Production and Angiogenic Activity From Human Monocytes

doi: 10.4049/jimmunol.171.5.2637

Figure Lengend Snippet: Kinetic analysis of VEGF production by M-CSF-stimulated monocytes. VEGF detected in supernatants over 5 days. Monocytes (5 × 106/condition) were left unstimulated (☐) or were stimulated with M-CSF (100 ng/ml; ■) for the indicated time points, and VEGF in the cell-free supernatants was assayed by ELISA (picograms per milliliter). M-CSF induced more VEGF production from monocytes at 5 days than that in unstimulated, time-matched cells (*, p = 0.08 vs VEGF released from M-CSF-stimulated monocytes on day 3;**, p < 0.05 vs VEGF released from M-CSF-stimulated monocytes on day 1; ***, p < 0.003 vs VEGF released from M-CSF-stimulated monocytes on day 2; ♦, p < 0.001 vs VEGF released from M-CSF-stimulated monocytes on day 1; ♦ ♦, p < 0.001 vs VEGF released from M-CSF-stimulated monocytes on days 1 and 2). Data represent the mean ± SEM from six independent experiments.

Article Snippet: Anti-human VEGF neutralizing Ab and anti-human M-CSF Ab were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: M-CSF Induces Vascular Endothelial Growth Factor Production and Angiogenic Activity From Human Monocytes

doi: 10.4049/jimmunol.171.5.2637

Figure Lengend Snippet: M-CSF induces the expression of VEGF mRNA in human monocytes. Total RNA was isolated from monocytes (5 × 106/condition) stimulated by M-CSF (100 ng/ml) for 1, 2, 3, 4, and 5 days (■) as detected by real-time PCR. M-CSF induced more VEGF mRNA at each time point and peaked on day 3 as assessed by ANOVA with post hoc testing vs unstimulated control samples at the same time (p < 0.05 for VEGF mRNA level vs unstimulated monocytes on day 3; p < 0.005 for day 3 VEGF mRNA compared with M-CSF-stimulated monocytes on day 1; p < 0.003 for day 3 VEGF mRNA compared with M-CSF-stimulated monocytes on day 5). Data represent the mean ± SEM from six independent studies.

Article Snippet: Anti-human VEGF neutralizing Ab and anti-human M-CSF Ab were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA).

Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: M-CSF Induces Vascular Endothelial Growth Factor Production and Angiogenic Activity From Human Monocytes

doi: 10.4049/jimmunol.171.5.2637

Figure Lengend Snippet: There is no evidence of paracrine factors inducing VEGF in M-CSF-stimulated monocyte supernatants. A, Monocytes (5 × 106/condition) were either left unstimulated (☐) or were stimulated with M-CSF (100 ng/ml; ■) for 72 h. The cell-free supernatants of these monocytes were subsequently immunodepleted of VEGF and M-CSF using specific neutralizing Abs, and the Abs were then removed by incubation with protein G agarose. The resulting supernatants were directly added to freshly isolated monocytes for an additional 72 h. Afterward, the supernatants from these monocytes were assayed for VEGF by ELISA. There was no significant difference in VEGF levels produced by the two samples after depletion (p = 0.405). Data represent the mean ± SEM calculated from four independent studies. B, Recombinant human VEGF (150 pg/ml) was supplemented back into the monocyte supernatants that were either left unstimulated (Non-stim.+ rhVEGF) or were stimulated with M-CSF (100 ng/ml; M-CSF-stim.+ rhVEGF) from A that were previously immunodepleted of VEGF and M-CSF using either anti-VEGF or anti-M-CSF neutralizing Abs (*, p < 0.001 for samples containing recombinant human VEGF added to depleted, unstimulated supernatants vs depleted, unstimulated supernatants alone; **, p < 0.001 for samples containing rhVEGF added to depleted, M-CSF-stimulated supernatants vs depleted, M-CSF-stimulated supernatants alone). These data represent the mean ± SEM calculated from four independent studies.

Article Snippet: Anti-human VEGF neutralizing Ab and anti-human M-CSF Ab were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA).

Techniques: Incubation, Isolation, Enzyme-linked Immunosorbent Assay, Produced, Recombinant

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: M-CSF Induces Vascular Endothelial Growth Factor Production and Angiogenic Activity From Human Monocytes

doi: 10.4049/jimmunol.171.5.2637

Figure Lengend Snippet: VEGF within the supernatants of M-CSF-stimulated monocytes induces network tube formation by HUVECs. A, HUVEC (1.5 × 105) were grown on Matrigel as follows: A) HUVECs with RPMI medium (1 ml) alone for 20 h (HUVECs alone); B) HUVECs plus rhMCSF (100 ng/ml); C) HUVECs plus rhVEGF (10 ng/ml); D) HUVECs, rhVEGF (10 ng/ml), and isogenic IgG Ab (0.6 μg/ml; rhVEGF + IgG Ab); E) HUVECs, rhVEGF (10 ng/ml), and anti-VEGF neutralizing IgG Ab (0.6 μg/ml; rhVEGF + anti-VEGF Ab); F) HUVECs and 1 ml of supernatants from monocytes left unstimulated for 72 h (Non-stimulated sups); G) HUVECs and supernatants from 72-h M-CSF (100 ng/ml)-stimulated monocytes (1 ml; M-CSF sups); H) HUVECs, M-CSF-stimulated supernatants (1 ml), and isogenic IgG Ab (1 μg/ml; M-CSF sups + IgG); and I) HUVECs, M-CSF-stimulated supernatants (1 ml), and anti-VEGF neutralizing IgG Ab (0.6 μg/ml; M-CSF sups + anti-VEGF Ab). Pictures were taken after 20-h incubation. B, Networks of tube formation from HUVECs (1 × 105/condition) that were stimulated as described above were counted in a blinded manner by adding the sum of three different fields in the well for each condition and counting completely enclosed networks. Error bars represent the mean ± SEM calculated from three independent studies. Recombinant human VEGF with or without isogenic IgG control induced endothelial tube formation (*, p < 0.05 vs unstimulated HUVECs). Similarly, supernatants from monocytes stimulated with M-CSF induced endothelial cells to form tubes (***, p < 0.01 vs unstimulated HUVECs) that were reduced by anti-VEGF, but not by isogenic control Abs (p < 0.02).

Article Snippet: Anti-human VEGF neutralizing Ab and anti-human M-CSF Ab were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA).

Techniques: Incubation, Recombinant, Control