Journal: Cancer Medicine

Article Title: Clinicopathological significance of microRNA‐21 in extracellular vesicles of pleural lavage fluid of lung adenocarcinoma and its functions inducing the mesothelial to mesenchymal transition

doi: 10.1002/cam4.2928

Figure Lengend Snippet: Mesothelial to mesenchymal transition and its function equivalent to cancer‐ associated fibroblasts induced by microRNA‐21 in extracellular vesicles: A, Separate cocultured studies of mesothelial cells (Met‐5A) or lymphatic endothelial cells (HLECs) with mouse fibroblast cell line (NIH3T3) producing microRNA‐21 mimic in extracellular vesicles (NIH3T3‐miR‐21‐mimic) and NIH3T3‐miR‐control. B, Met‐5A cocultured with NIH3T3‐miR‐21‐mimic demonstrates decreased E‐cadherin and increased vimentin and αSMA immunoreactivity. HLECs cocultured with NIH3T3‐miR‐21‐mimic also demonstrate decreased E‐cadherin and increased αSMA immunoreactivity (bar, 50 µm). C, Fluorescence signal intensities of each cell are also measured and they show statistically significant differences (E‐cad, E‐cadherin; Vim, vimentin; SMA, αSMA). D, Schematic representation of protocol for isolated extracellular vesicles (EVs) from NIH3T3‐miR‐21‐mimic or NIH3T3‐miR‐control directly applying to Met‐5A. E, F, Met‐5A incubated with EVs from NIH3T3‐miR‐21‐mimic showing decreased E‐cadherin and increased vimentin and αSMA immunoreactivity (E bar, 20 µm; F *, P < .05; **, P < .01). G, Nuclear and cytoplasmic processing of microRNA (miRNA). The pre‐miRNA is cleaved by the RNase III enzyme (Dicer), incorporated into the RNA‐induced silencing complex (RISC), and becomes mature miRNA in the cytoplasm. H, I, Met‐5A overexpressing pre‐miR‐21 showing decreased E‐cadherin and increased vimentin and αSMA immunoreactivity (H bar, 20 µm; I ** P < .01). J, Wound healing assay and (K) collagen gel contraction assay of Met‐5A overexpressing pre‐miR‐21 and control (* P < .05; ** P < .01)

Article Snippet: Primary cultured human lymphatic endothelial cells (HLECs) were purchased from ScienCell Research Laboratories and grown in endothelial cell medium (ScienCell Research Laboratories) according to the manufacturer's instructions.

Techniques: Control, Fluorescence, Isolation, Incubation, Wound Healing Assay, Collagen Gel Contraction Assay

Journal: Frontiers in Oncology

Article Title: LncRNA-AC009948.5 promotes invasion and metastasis of lung adenocarcinoma by binding to miR-186-5p

doi: 10.3389/fonc.2022.949951

Figure Lengend Snippet: Expression of LncRNA-AC009948.5 is up-regulated in lung adenocarcinoma cells and tissues, and patients with a high level of LncRNA-AC009948.5 demonstrate poor prognosis. (A) Schematic flowchart showed the overlapping of LncRNA could combine with miR-186-5p and the upregulated and LncRNAs collection in LncRNA microarray data (GSE115734) (filtered by fold change ≥2 and p-value ≤ 0.05). (B) Volcano plots show that thousands of LncRNAs are significantly different by using lncRNA expression thresholds of more than twofold change with P< 0.05 in GSE115734. (C) Clustered heatmap of significant differentially expressed LncRNAs in GSE115734. Each sample contained a mixture of three repeats, red represents high expression, and green represents low expression. (D) Expression of LncRNA-AC009948.5 in 80 lung adenocarcinoma tissues and paired non-tumor ones analyzed by qRT-PCR. GAPDH was used as an internal control. The results were expressed as Log2. (E) Expression of LncRNA-AC009948.5 in human normal lung epithelial cells (BEAS-2B) and human lung adenocarcinoma cell lines (H1299, H226, A549, and H1975). GAPDH was used as an internal control. Data represent the mean ± S. D, *P<0.05 versus on BEAS-2B cells. The results were repeated three times independently. (F) Kaplan-Meier analysis for overall survival based on differential expression levels of LncRNA-AC009948.5 in patients with lung adenocarcinoma. (G) Kaplan-Meier analysis for recurrence-free survival based on different expression levels of LncRNA-AC009948.5 in patients with lung adenocarcinoma.

Article Snippet: Normal human lung epithelial cells of BEAS-2B, along with four human lung adenocarcinoma cell lines of H1299, H226, A549 and H1975, and human embryonic kidney cells (HEK-293T) were purchased from the ATCC (Manassas, VA, USA).

Techniques: Expressing, Microarray, Quantitative RT-PCR, Control, Quantitative Proteomics

Journal: JCI Insight

Article Title: Mycobacterium tuberculosis cords within lymphatic endothelial cells to evade host immunity

doi: 10.1172/jci.insight.136937

Figure Lengend Snippet: (A) Images of primary hLECs infected with GFP expressing M. tuberculosis for 2 to 72 hours. Over time, M. tuberculosis grows and forms large intracellular cords. Nuclei are stained with DAPI (blue) and F-actin is stained by rhodamine phalloidin (red). (B) 3D reconstruction of Z-stacks taken of an intracellular cord from (A). Various angles are shown to confirm that the cord is completely encapsulated within the host cell. (C) Measurement of the intracellular cords over time in hLECs (using the Feret diameter; see Supplemental Figure 1) showing that the cords elongated up to a maximum of 150 μm. The numbers of bacterial clusters analyzed were 418 (2 hours), 233 (24 hours), 814 (48 hours), and 618 (72 hours), obtained from 3 independent experiments. One-way ANOVA with Tukey’s multiple comparisons tests: ***P < 0.001. (D) Image of A549 cells infected with M. tuberculosis-EGFP for 72 hours showing an intracellular cord looping around the nucleus. Nuclei are stained with DAPI (blue) and F-actin is stained with rhodamine phalloidin (red). (E) Intracellular cord formation after 72 hours was also observed in hLECs infected with representative strains from 3 other M. tuberculosis lineages: N0072 (lineage 1), N0145 (lineage 2), and N0024 (lineage 3). Images displayed in D and E are representative of at least 3 independent experiments. (F) Tissue section of a granuloma present in a human lymph stained for AFB. Zoomed region shows association of M. tuberculosis cords with cells (black boxes). Representative histological sections from human patients after lymph node tissue resection surgery were stained for PDPN, M. tuberculosis, and nuclei (DAPI). Scale bar: 1 mm. White boxes delimit the zoomed regions displayed on the right-hand side. Arrows indicate the presence of M. tuberculosis cords within PDPN+ cells. Scale bar: 20 μm. hLECs, human lymphatic endothelial cells; AFB, acid fast bacilli; PDPN, podoplanin.

Article Snippet: Cells Primary hLECs taken from inguinal lymph nodes (ScienCell Research Laboratories, catalog 2500) were cultured according to the manufacturer’s instructions up to passage 5 as described fully in ref. 19 .

Techniques: Infection, Expressing, Staining

Journal: Biomaterials

Article Title: Human organotypic lymphatic vessel model elucidates microenvironment-dependent signaling and barrier function

doi: 10.1016/j.biomaterials.2019.119225

Figure Lengend Snippet: Vessel structure and markers. a Confocal image of a lymphatic vessel showing patent tubular structure. b Immunofluorescent of lymphatic marker - LYVE1, endothelial cell marker - CD31, and endothelial cell junctions - CD31, VE-cadherin, and ZO-1. c Flow cytometry quantification of CD31, LYVE1, podoplanin, and PROX1 for both blood (HUVEC) and lymphatic (HLEC) endothelial cells used in the study. HUVECs express CD31, LYVE1, and PROX1, but not podoplanin. HLECs express all four markers. d Cells in the endothelium align in the direction of fluid flow from daily medium exchanges as compared to cells maintained in excess medium without flow (static condition). e Histograms of cell alignment for static and flow conditions. A higher number of cells in the flow condition align in the direction of flow (angle of 90 degrees). Histogram frequency data was generated by combining values of n = 4 individual vessels for each condition.

Article Snippet: To generate a lymphatic vessel, collagen type I gel is polymerized around the lumen rod, the rod is removed leaving an empty lumen, and the subsequent lumen is seeded with primary human lymphatic endothelial cells (HLECs) isolated from the lymph node (ScienCell) forming vessels with diameters in the range of 200-250 μm ( and , and Fig. S1 ).

Techniques: Marker, Flow Cytometry, Generated

Journal:

Article Title: Bacterial Peptide Recognition and Immune Activation Facilitated by Human Peptide Transporter PEPT2

doi: 10.1165/rcmb.2008-0059OC

Figure Lengend Snippet: γ-iE-DAP activates innate immunity in lung epithelia. Primary hLECs were cultured with the bacteria muropeptides MDP or γ-iE-DAP for 24 hours, and then cell culture supernatants were analyzed for IL-6 and IL-8 release. As shown, γ-iE-DAP induced the release of (A) IL-6 and (B) IL-8, whereas MDP did not. Next, we repeated the same experiment but in the presence or absence of the competitive PEPT2 transport inhibitor Gly-Sar for 4 hours. The constituents were then removed and cells were cultured in normal medium for an additional 20 hours. Supernatants were collected and again analyzed for (C) IL-6 or (D) IL-8. As shown, GlySar significantly reduced the amount of both IL-6 and IL-8 release only in cultures exposed to γ-iE-DAP, whereas MDP- or GlySar-treated cultures were unaffected. Data are expressed as mean values ± σ of n = 3 (P ≤ 0.05). To verify these findings, we overexpressed human PEPT2 in BEAS-2B cultures and then evaluated IL-6 after exposure to MDP, γ-iE-DAP, or the nonimmunoreactive enantiomer α-iE-DAP. (E) Compared with PEPT2 transfected untreated cultures, only the cultures exposed to γ-iE-DAP exhibited an increase in IL-6 release.

Article Snippet: Western Analysis of Primary hLECs Cell lysis buffer (Cell Signaling, Beverly, MA) with 1 mM PMSF and 1× Complete Mini protease inhibitor cocktail (Roche, Indianapolis, IN) was directly added to the apical surface of the transwell chamber to lyse cells.

Techniques: Cell Culture, Bacteria, Transfection