Journal: Oncogene

Article Title: Oncosuppressive miRNAs loaded in lipid nanoparticles potentiate targeted therapies in BRAF-mutant melanoma by inhibiting core escape pathways of resistance

doi: 10.1038/s41388-022-02547-9

Figure Lengend Snippet: A Venn Diagram showing the intersected data obtained by RNA-seq, GSEA and cytokinome analyses for the identification of VEGFA, TGFβ1, CCL5 and CXCL2. B Quantification of miR-199b-5p and miR-204-5p by using qRT–PCR in A375 and M14 BRAFi-resistant cells vs. sensitive counterparts. C Elisa assays measuring VEGFA, TGFβ1, CCL5 and CXCL2 soluble levels in cell media (CM) deriving from A375 (upper panels) and M14 (lower panels). For these experiments cells have been serum starved for 24 h and then CM have been collected; results were determined by measuring absorbance at 450 nm into a microplate reader. D Representative results of miR-204-5p and miR-199b-5p expression from matched formalin-fixed paraffin-embedded (FFPE) melanoma samples before initiation of targeted therapy (Pre) and after disease progression (PD). E VEGFA, TGFβ1, CCL5 and CXCL2 mRNA expression levels (Up-genes) measured by qRT-PCR in PD vs. Pre-therapy samples ( n = 14). F Spearman correlation calculated using qRT-PCR data of the two Down-miRs (miR-204-5p and miR-199b-5p) and the four Up-genes in PD (right panel) and in Pre-therapy (left panel) samples. * p < 0.05; ** p < 0.01; and *** p < 0.001. qRT-PCR data are represented as the mean ( n = 3) ±SD; Elisa results are expressed as mean of at least three independent experiments ± SEM.

Article Snippet: For luciferase assays, the plasmids containing the 3′UTR relative to CCL5 (SC210875) or CXCL2 (SC209527) have been purchased by Origene (Rockville, MD, USA) and evaluated by Dual-Luciferase® Reporter Assay System (Promega).

Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Formalin-fixed Paraffin-Embedded

Journal: Oncogene

Article Title: Oncosuppressive miRNAs loaded in lipid nanoparticles potentiate targeted therapies in BRAF-mutant melanoma by inhibiting core escape pathways of resistance

doi: 10.1038/s41388-022-02547-9

Figure Lengend Snippet: A Schematic illustration of the biological assays carried out upon melanoma cells exposure to LNPs. B Quantification of miR-199b-5p and miR-204-5p by using qRT–PCR in A375 and M14 BRAFi-resistant cells upon 48 h of exposure to LNP-Scr or LNP-miRs. C Elisa assays measuring VEGFA, TGFβ1, CCL5 and CXCL2 soluble levels in CM coming from A375 res (upper panels) and M14 res (lower panels) cells upon LNPs’ exposure (48 h). For these experiments cells have been serum starved for 24 h and then CM have been collected; results were determined by measuring absorbance at 450 nm into a microplate reader. D Luciferase reporter assays of the constructs containing CCL5 and CXCL2 3’UTRs co-transfected with the indicated miRNAs alone or in combination for 48 h have been performed in HEK293 cells. CCL5 and CXCL2 reporter plasmids were transfected at 500 ng; pLX313-Renilla plasmid has been used to normalize results at 50 ng. E Cell viability evaluation by measuring ATP content in A375 and M14 BRAFi-resistant cells left untreated, treated with the sole Dabrafenib (BRAFi, 500 nM) or in combination with the MEKi, i.e. Trametinib at 10 nM (MAPKi) in the presence of LNP-Scr or LNP-miR (30 μg each). * p < 0.05; ** p < 0.01; and *** p < 0.001. qRT-PCR data are represented as mean ( n = 3) ± SD; Elisa, luciferase and cell viability results are expressed as the mean of at least three independent experiments ±SEM.

Article Snippet: For luciferase assays, the plasmids containing the 3′UTR relative to CCL5 (SC210875) or CXCL2 (SC209527) have been purchased by Origene (Rockville, MD, USA) and evaluated by Dual-Luciferase® Reporter Assay System (Promega).

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Luciferase, Construct, Transfection, Plasmid Preparation

Journal: Oncogene

Article Title: Oncosuppressive miRNAs loaded in lipid nanoparticles potentiate targeted therapies in BRAF-mutant melanoma by inhibiting core escape pathways of resistance

doi: 10.1038/s41388-022-02547-9

Figure Lengend Snippet: Kaplan–Meier curves estimating the clinical relevance of M2 (left graphs) or M1 (right graphs) macrophage infiltrates in association with patient clinical outcome alone ( A ) or in combination ( B ) with Up-genes (i.e. VEGFA, TGFβ1, CCL5 and CXCL2). The hazard ratio, p values for Cox models and the log-rank p values are shown on the Kaplan–Meier plots. The hazard ratio, Cox models and the log-rank p values were evaluated to plot KM curves.

Article Snippet: For luciferase assays, the plasmids containing the 3′UTR relative to CCL5 (SC210875) or CXCL2 (SC209527) have been purchased by Origene (Rockville, MD, USA) and evaluated by Dual-Luciferase® Reporter Assay System (Promega).

Techniques:

Journal: The EMBO Journal

Article Title: Staphylococcus aureus Staphopain A inhibits CXCR2-dependent neutrophil activation and chemotaxis

doi: 10.1038/emboj.2012.212

Figure Lengend Snippet: Staphopain A inhibits CXCR2-mediated calcium mobilization of neutrophils. Fluo-3-labelled neutrophils were preincubated with buffer or 0.5 μM Staphopain A with or without 1 μM Staphostatin A, for 15 min at 37°C. After washing, cells were stimulated with different concentrations of CXCL1 ( A ), CXCL7 ( B ), CXCL8 ( C ), fMLF ( D ), C5a ( E ) or a fixed concentration (3 × 10 −9 M) of CXCL2, CXCL3, CXCL5 and CXCL6 ( F ). To determine the IC 50 (50% of inhibition), Fluo-3-labelled neutrophils were preincubated with different concentrations of Staphopain A for 15 min at 37°C and subsequently stimulated with 3 × 10 −9 M CXCL1 ( G ) or 1 × 10 −8 M CXCL7 ( H ). The IC 50 for CXCL1 and CXCL7 was calculated with the formulas y =−4 E +10 −7 x +4.1314 and y =−3 E +10 −7 x +4.0248, respectively. All figures represent the mean±s.e. of three separate experiments using different donors. The relative calcium mobilization was calculated by dividing the fluorescence after stimulation by the baseline fluorescence. * P <0.05 versus buffer; ** P <0.01 for Staphopain A versus buffer (two-tailed Student's t -test).

Article Snippet: Recombinant mouse KC and human CXCL8 were purchased from Tebu-Bio, recombinant human CXCL2, CXCL3, CXCL5, CXCL6 and CXCL7 from R&D systems. fMLF and recombinant C5a were obtained from Sigma-Aldrich.

Techniques: Concentration Assay, Inhibition, Fluorescence, Two Tailed Test

Journal: Frontiers in Pharmacology

Article Title: Cisplatin Promotes the Efficacy of Immune Checkpoint Inhibitor Therapy by Inducing Ferroptosis and Activating Neutrophils

doi: 10.3389/fphar.2022.870178

Figure Lengend Snippet: N1-polarized neutrophils induced by cisplatin-mediated tumor ferroptosis exerted an anti-tumor effect. (A) Relative mRNA expression of CXCL1 and CXCL2 was tested by RT-qPCR assay in A549 cells treated with 5 μM DDP or rescued by 10 μM ferrostatin-1. (B) and (C) Concentration of CXCL1 (B) and CXCL2 (C) released by A549 cells in culture medium, which was tested by ELISA assay. A549 cell were treated with 5 μM DDP or rescued by 10 μM ferrostatin-1. (D) and (E) Relative mRNA expression of neutrophil cytotoxic markers (D) and pro-inflammatory markers (E) were tested by RT-qPCR assays in neutrophils co-cultured with A549 cells, which were pretreated with 5 μM DDP or rescued by ferrostatin-1. (F) Cell death ratio of A549 cells treated with DDP or rescued by ferrostatin-1 before and after being co-cultured with neutrophils. (G) Quantification of data in (F) . DDP pretreatment remarkably increases the cytotoxic effect of neutrophils which would be rescued by ferrostatin-1. Bar graphs represent the mean ± SD of indicated samples. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: ELISA was performed using human interferon gamma (IFN-γ) (430107; BioLegend, San Diego, CA, United States), human C-X-C Motif Chemokine Ligand 1 (CXCL1) (RAB0116; Sigma-Aldrich, St. Louis, MO, United States), and human C-X-C motif chemokine ligand 2 (CXCL2) (DY276-05; R&D Systems, Minneapolis, MN, United States), according to the manufacturers’ protocols.

Techniques: Expressing, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay, Cell Culture