Journal: Cell reports

Article Title: Disseminated Melanoma Cells Transdifferentiate into Endothelial Cells in Intravascular Niches at Metastatic Sites.

doi: 10.1016/j.celrep.2020.107765

Figure Lengend Snippet: Figure 4. EndT and EndMT of Metastatic Melanoma Cells in Mouse (A) Whole-mount staining of pulmonary artery with GFP+ cells located at the endothelium (Videos S1, S2, and S3). Bars: 10 mm. (B and C) EndT occurred in lymph nodes. EndT occurred in LYVE-1+ lymphatic vessles (B) and CD31+ blood vessels (C) in lymph nodes. Bars: 20 mm. (D) LYVE-1 immunostaining on lung cryosections. Bar: 20 mm. (E–J) Immunostaining indicated EndT is transient during tumor progression. At primary tumor sites, GFP+ cells were CD31-, VE-cadherin+ (E and H), while in the lung blood vessels, GFP+ cells were both CD31+ and VE-cadherin+ (F and I). GFP+ cells lost both endothelial markers when populating metastases in the lung (G and J). Bars: 20 mm. (K) CD31+/a-SMA+/GFP+ cell (arrowhead) inside the alveolar capillary. CD31+/aSMA+/GFP cell (arrow) near the GFP+ cell showed the same phenotype. It is possible that this is an authentic vascular endothelial cell undergoing EndMT or a metastatic cell that was not labeled with GFP. Bars: 10 mm. (A–K) Mice, n R 3. (L) Correlation between the number of GFP+ cells per vessel and the number of metastasis foci or the size of metastasis foci per mouse. The number of GFP+ cells/vessel vs. number of metastasis foci, r = 0.7950, p = 0.03*; number of GFP+ cells/vessel vs. metastasis size, r = 0.8012, *p = 0.03. Mice, n = 7.

Article Snippet: Human pulmonary artery endothelial cells (PAECs; ATCC PCS-100-022) were cultured according to the supplier’s instructions.

Techniques: Staining, Immunostaining, Labeling

Journal: Cell reports

Article Title: Disseminated Melanoma Cells Transdifferentiate into Endothelial Cells in Intravascular Niches at Metastatic Sites.

doi: 10.1016/j.celrep.2020.107765

Figure Lengend Snippet: Figure 5. In Vitro System of Melanoma Cell EndT (A–C) Melanoma/endothelial marker immunostaining of Q-YUWERA cells (cells with green dots) cultured alone (A, VE-cadherin showed background staining in nuclei) or 5 days after co-culturing with PAECs (B and C). CD31 and HMB45 double staining in (B), VE-cadherin and HMB45 double staining in (C). Bars: 20 mm. Independent experiments, n R 3. (D) Imaging flow cytometry confirmed the expression of CD31 by individual YUWERA (APC+) cells after the co-culture. BF, bright field; SSC, side scatter; APC, CellTrace far red-labeled YUWERA cells. (E) YUWERA cells were labeled with a CFSE proliferation kit and co-cultured with PAECs for 5 days with 300 nM sunitinib treatment or control solution. Cells were subjected to CD31 staining followed by flow cytometry analysis. Independent experiments, n = 3; replicates, n = 3 in each experiment. 300 nM sunitinib treatment significantly increased the percentage of CD31+ YUWERA cells of the overall YUWERA cells in the co-culture (control group 2.878% ± 0.1309%, sunitinib group 4.677% ± 0.3125%,*p = 0.0357, shown as mean ± SEM by a Mann-Whitney test).

Article Snippet: Human pulmonary artery endothelial cells (PAECs; ATCC PCS-100-022) were cultured according to the supplier’s instructions.

Techniques: In Vitro, Marker, Immunostaining, Cell Culture, Staining, Double Staining, Imaging, Cytometry, Expressing, Co-Culture Assay, Labeling, Control, MANN-WHITNEY

Journal: Cell reports

Article Title: Disseminated Melanoma Cells Transdifferentiate into Endothelial Cells in Intravascular Niches at Metastatic Sites.

doi: 10.1016/j.celrep.2020.107765

Figure Lengend Snippet: Figure 6. EndT Inferred from Single-Cell RNA-Seq Data and Immunofluorescence in Metastatic Human Melanoma Biopsies (A) tSNE plot from single-cell RNA-seq showing metastatic melanoma cells of different patients (23). Malignant melanoma cells were selected based on aberrant copy number profiles, which are mutually exclusive from authentic endothelial cells, and plotted based on their pigmentation activity score, based on AUCell (37). The zoom shows cells of a melanoma patient that does not show pigmentation activity but had rare cells with high expressional activity for an endothelial gene signature. (B–I) Immunofluorescence on metastatic melanoma biopsies from BRAFV600E-harboring patients. (B and C) Representative double-immunofluorescence mi- crographs for BRAFV600E and CD31 of metastatic melanoma biopsies in the lung (B) and in the brain (C). (D and E) BRAFV600E+/CD31+ cells (arrows) localized inside the vasculature of metastatic melanoma biopsies in the lung (D) and in the brain (E). Bars: 50 mm. (F and G) Representative triple-immunofluorescence micrographs for BRAFV600E, MITF and CD31 of metastatic melanoma biopsies in the lung F) and in the brain (G). (H and I) BRAFV600E+/CD31+ cells inside the vasculature of metastatic melanoma biopsies in the lung (H) and in the brain (I) were negative for melanocytic marker MITF (arrows). Bars: 50 mm.

Article Snippet: Human pulmonary artery endothelial cells (PAECs; ATCC PCS-100-022) were cultured according to the supplier’s instructions.

Techniques: RNA Sequencing, Activity Assay, Marker

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: GSK-3Beta-Dependent Activation of GEF-H1/ROCK Signaling Promotes LPS-Induced Lung Vascular Endothelial Barrier Dysfunction and Acute Lung Injury

doi: 10.3389/fcimb.2017.00357

Figure Lengend Snippet: LPS induces GSK-3beta activation in dose- and time-dependent manners in HPMECs. Expression of P-GSK-3beta and GSK-3beta was detected after incubation with different concentrations of LPS for 1 h (A) . The expression of P-GSK-3beta was represented as a histogram according to band intensities (B) . Expression of P-GSK-3beta and GSK-3beta was examined at indicated time points after stimulation with LPS (0.1 μg/ml) in HPMECs (C) . The Western blotting results are presented as a histogram showing the band intensity values (D) . * P < 0.05 vs. LPS un-treatment group.

Article Snippet: Primary human pulmonary micro-vascular endothelial cells (HPMECs) were obtained ScienCell Research Laboratories and maintained in ScienCell Endothelial Cell Medium in a humidified 37°C, 5% CO 2 incubator.

Techniques: Activation Assay, Expressing, Incubation, Western Blot

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: GSK-3Beta-Dependent Activation of GEF-H1/ROCK Signaling Promotes LPS-Induced Lung Vascular Endothelial Barrier Dysfunction and Acute Lung Injury

doi: 10.3389/fcimb.2017.00357

Figure Lengend Snippet: Involvement of GSK-3beta in LPS-induced GEF-H1/ROCK signaling activation. HPMECs were incubated with LPS (0.1 μg/ml) at different indicated times, and the GEF-H1 and myosin-associated phosphatase type 1 (P-MYPT 1: the substrate of ROCK) were detected by Western blot assay (A) . The expression of GEF-H1 and P-MYPT 1 were represented as a histogram according to band intensities (B) . * < 0.05 vs. LPS un-treatment group. Inhibition effect of GSK-3beta activity in HPMECs was analyzed by Western blot (C,D) . * P < 0.05 vs. the negative control group, # P < 0.05 vs. the corresponding LPS treatment group. HPMECs were pretreated with SB-216763 (20 μM) for 1 h and then were exposed to LPS (0.1 μg/ml) for 1 h. The expression of GEF-H1 and P-MYPT 1 were determined by Western blot (E) . The Western blotting results are presented as a histogram showing the band intensity values (F) . * P < 0.05 vs. the negative control group, # P < 0.05 vs. the corresponding LPS treatment group.

Article Snippet: Primary human pulmonary micro-vascular endothelial cells (HPMECs) were obtained ScienCell Research Laboratories and maintained in ScienCell Endothelial Cell Medium in a humidified 37°C, 5% CO 2 incubator.

Techniques: Activation Assay, Incubation, Western Blot, Expressing, Inhibition, Activity Assay, Negative Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: GSK-3Beta-Dependent Activation of GEF-H1/ROCK Signaling Promotes LPS-Induced Lung Vascular Endothelial Barrier Dysfunction and Acute Lung Injury

doi: 10.3389/fcimb.2017.00357

Figure Lengend Snippet: GSK-3beta signaling is involved in LPS-induced HPMECs barrier disruption. The HPMECs were plated on the gold microelectrodes. When HPMECs formed monolayers and reached stable TER values, the SB-216763 (20 μM) was added. After 1 h, the medium or LPS (0.1 μg/ml) was added for another 6 h. The HPMEC monolayers permeability was determined by real-time TER measurement (A) . The results of the 3 h LPS stimulation were represented as a histogram in (B) according to the TER curves. * P < 0.05 vs. negative control. # P < 0.05 vs. corresponding LPS-stimulated group.

Article Snippet: Primary human pulmonary micro-vascular endothelial cells (HPMECs) were obtained ScienCell Research Laboratories and maintained in ScienCell Endothelial Cell Medium in a humidified 37°C, 5% CO 2 incubator.

Techniques: Disruption, Permeability, Negative Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: GSK-3Beta-Dependent Activation of GEF-H1/ROCK Signaling Promotes LPS-Induced Lung Vascular Endothelial Barrier Dysfunction and Acute Lung Injury

doi: 10.3389/fcimb.2017.00357

Figure Lengend Snippet: LPS induces degradation of beta-catenin and ZO-1 in HPMECs monolayer. LPS (0.1 μg/ml) induced down-regulation of ZO-1 expression and increase of phosphorylated degradation of beta-catenin in a time-dependent manner (A) . The Western blotting results are presented as a histogram showing the band intensity values (B) . * P < 0.05 vs. LPS un-treatment group.

Article Snippet: Primary human pulmonary micro-vascular endothelial cells (HPMECs) were obtained ScienCell Research Laboratories and maintained in ScienCell Endothelial Cell Medium in a humidified 37°C, 5% CO 2 incubator.

Techniques: Expressing, Western Blot

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: GSK-3Beta-Dependent Activation of GEF-H1/ROCK Signaling Promotes LPS-Induced Lung Vascular Endothelial Barrier Dysfunction and Acute Lung Injury

doi: 10.3389/fcimb.2017.00357

Figure Lengend Snippet: GSK-3beta/GEF-H1/ROCK signaling is required for LPS-induced degradation of beta-catenin and ZO-1. After transfection with GEF-H1 siRNA and Control siRNA for 48 h, HPMECs were treated with SB-216763 (20 μM) and/or Y-27632 (10 μM) for another 1 h prior to LPS stimulation (0.1 μg/ml) for 3 h. The expression of ZO-1 was determined by immunoblotting, and GAPDH protein was used as loading control (A) . The Western blotting results are presented as a histogram showing the band intensity values (B) . The expression of P-beta-catenin was determined by immunoblotting, and GSK-3beta and GAPDH proteins were used as control (C) . The Western blotting results are presented as a histogram showing the band intensity values (D) . * P < 0.05 vs. negative control. # P < 0.05 vs. corresponding LPS-stimulated group. NS, no significance.

Article Snippet: Primary human pulmonary micro-vascular endothelial cells (HPMECs) were obtained ScienCell Research Laboratories and maintained in ScienCell Endothelial Cell Medium in a humidified 37°C, 5% CO 2 incubator.

Techniques: Transfection, Control, Expressing, Western Blot, Negative Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: GSK-3Beta-Dependent Activation of GEF-H1/ROCK Signaling Promotes LPS-Induced Lung Vascular Endothelial Barrier Dysfunction and Acute Lung Injury

doi: 10.3389/fcimb.2017.00357

Figure Lengend Snippet: GSK-3beta/GEF-H1/ROCK pathway is involved in LPS-induced HPMECs barrier disruption by beta-catenin and ZO-1. HPMECs monolayer was pretreated with SB-216763 (20 μM) (A) , GEF-H1 siRNA (B) , or Y-27632 (10 μM) (C) , for indicated times and then was exposed to LPS (0.1 μg/ml) for 3 h before fixation and staining with anti-beta-catenin and anti-ZO-1 antibody as described in Materials and Methods. Beta-catenin (green) and ZO-1 (green) were visualized by immunofluorescence microscopy. Red arrows not only represent the expression of beta-catenin and ZO-1 in the membrane of HPMECs but also represent the cell-cell gaps formation in the ECs monolayer.

Article Snippet: Primary human pulmonary micro-vascular endothelial cells (HPMECs) were obtained ScienCell Research Laboratories and maintained in ScienCell Endothelial Cell Medium in a humidified 37°C, 5% CO 2 incubator.

Techniques: Disruption, Staining, Immunofluorescence, Microscopy, Expressing, Membrane