Journal: eLife

Article Title: The nanoscale organization of the Nipah virus fusion protein informs new membrane fusion mechanisms

doi: 10.7554/eLife.97017

Figure Lengend Snippet: ( A ) Representative images of 293T cell–cell fusion induced by NiV-G and NiV-F-WT, L53D, V108D, or Q393L. 293T cells were co-transfected with plasmids coding for NiV-G and empty vector (NC) or NiV-F constructs. Cells were fixed at 18 hr post-transfection. Arrows point to syncytia. Scale bar: 10 μm. ( B ) Relative levels of 293T cell–cell fusion in ( A ). Five fields per experiment were counted from three independent experiments. Data are presented as mean ± SEM. p value was obtained by Welch’s test. *p < 0.05; ****p < 0.0001. ( C ) The cell surface expression levels of NiV-F-WT, L53D, V108D, and Q393L on 293T cells were measured by flow cytometry. Mean fluorescence intensity (MFI) values were calculated by FlowJo and were normalized to that of F-WT. Data are presented as mean ± SEM of three independent experiments. Statistical significance was determined by the unpaired t -test with Welch’s correction (ns: p > 0.05). Values were compared to that of the NiV-F-WT. ( D ) NiV-F processing of F-WT, L53D, V108D, Q393L in 293T cells. 293T cells were transfected by F-WT and the mutants. The cell lysates were analyzed on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) followed by western blotting after 28 hr post-transfection. F0 and F2 were probed by M2 monoclonal mouse anti-FLAG antibody. GAPDH was probed by monoclonal mouse anti-GAPDH. ( E ) Relative entry levels of VSV/NiV pseudovirions expressing NiV-G-HA and NiV-F-FLAG (WT; solid black line) or FLAG-tagged NiV-F-L53D (L53D), V108D (V108D), or Q393L (Q393L; dotted red line). The negative control (NC), the recombinant VSV pseudoviruses without glycoproteins, is shown as a dotted gray line. The relative light units (RLUs) of the lysates of infected Vero cells were quantified 18–24 hr post-infection and plotted against the number of viral genomes/ml over 3 logs of viral input. Data shown are mean ± SEM from one representative experiment (of three). ( F ) The result of a representative western blot analysis of VSV/NiV pseudovirions. 4 × 10 8 copies VSV/NiV pseudovirions were separated by a denaturing 10% SDS–PAGE and probed against NiV-G-HA (rabbit anti-HA) and NiV-F-FLAG (mouse anti-Flag). ( G ) The VLPs expressing NiV-M-Bla, G-HA, and the FLAG-tagged WT or mutant NiV-F were allowed to bind to the target HEK293T cells loaded with CCF2-AM dye at 4°C. The Blue/Green (B/G) ratio was measured at 37°C for 4 hr at a 3-min-interval. The average background was subtracted, and the results were normalized to the maximal B/G ratio of WT VLPs. Results from one representative experiment (of three) are shown. ( H ) The result of a representative western blot analysis of NiV VLPs. Equal volumes of VLPs were separated by a denaturing 10% SDS–PAGE and probed against NiV-G (rabbit anti-HA), NiV-F (mouse anti-Flag), and NiV-M (mouse anti-β-lactamase). Figure 3—figure supplement 1—source data 1. Related to . Figure 3—figure supplement 1—source data 2. Related to . Figure 3—figure supplement 1—source data 3. Related to . Figure 3—figure supplement 1—source data 4. Related to . Original files for western blot analysis. Figure 3—figure supplement 1—source data 5. Related to . PPTX files indicating the relevant bands and treatments. Figure 3—figure supplement 1—source data 6. Related to . Figure 3—figure supplement 1—source data 7. Related to . Original files for western blot analysis. Figure 3—figure supplement 1—source data 8. Related to . PPTX files indicating the relevant bands and treatments. Figure 3—figure supplement 1—source data 9. Related to . Figure 3—figure supplement 1—source data 10. Related to . Original files for western blot analysis. Figure 3—figure supplement 1—source data 11. Related to . PPTX files indicating the relevant bands and treatments.

Article Snippet: Cell line ( Homo sapiens ) , HEK293T , ATCC , CRL-3216 , .

Techniques: Transfection, Plasmid Preparation, Construct, Expressing, Flow Cytometry, Fluorescence, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Negative Control, Recombinant, Infection, Mutagenesis

Journal: eLife

Article Title: The nanoscale organization of the Nipah virus fusion protein informs new membrane fusion mechanisms

doi: 10.7554/eLife.97017

Figure Lengend Snippet:

Article Snippet: Cell line ( Homo sapiens ) , HEK293T , ATCC , CRL-3216 , .

Techniques: Recombinant, FLAG-tag, Residue, Plasmid Preparation, Sequencing, Concentration Assay, Imaging, Luciferase, RNA Extraction, Software, Modification, Generated, Microscopy

Journal: Molecular Brain

Article Title: Visualization of spatiotemporal dynamics of human glioma stem cell invasion

doi: 10.1186/s13041-019-0462-3

Figure Lengend Snippet: Characteristics of U87 and hG008 cells. a and f In vitro growth of U87 cells ( a ) and hG008 cells ( f ) expressing ffLuc . Scale bar, 100 μm. b – e and g – j 1 × 10 5 U87 cells or hG008 cells were implanted into the striatum of mouse brain. Representative H&E staining ( b and g ) and fluorescence images ( c and h ) of brain sections at 21 days ( b and c ) and 45 days ( g and h ) after implantation. Scale bar, 100 μm. d and i Kaplan-Meier plots showing survival of mice implanted with U87 cells ( n = 9) ( d ) and hG008 cells ( n = 8) ( i ). e and j Representative whole brain pictures and BLI images at 21 days and 56 days after implantation of U87 cells ( e ) and hG008 cells ( j ), respectively. Colored scale bars in ( e ) and ( j ) represent BLI radiance intensity in photons/second/cm 2 /steradian and photons/minute/cm 2 /steradian, respectively

Article Snippet: The human glioblastoma cell line U87 was obtained from the American Type Culture Collection.

Techniques: In Vitro, Expressing, Staining, Fluorescence

Journal: Molecular Brain

Article Title: Visualization of spatiotemporal dynamics of human glioma stem cell invasion

doi: 10.1186/s13041-019-0462-3

Figure Lengend Snippet: Time-lapse imaging of organotypic brain slice culture. U87 cells were implanted in the right striatum, cortical area, subventricular zone, or corpus callosum. hG008 cells were implanted in the right striatum. Brain tissues from mice 7 days and 45 days after implantation of U87 cells and hG008 cells, respectively, were used for time-lapse imaging of brain slice cultures. See Additional files 2, 3, 4, 5 and 6. a and b Snapshot fluorescence images of the striatum, cortical area, subventricular zone, and corpus callosum with U87 cells ( a ) and hG008 cells ( b ) at the indicated time points are shown. Scale bar, 100 μm. c Quantitative analysis of migration speed and distance of U87 cells and hG008 cells in the striatum. Imaris software was used to track migration of individual cells and calculate the total distance, net distance, and speed of migration. Migration speed was calculated during the period from 48 h to 86 h of culture. Migration distance was measured as the net distance between the initial position after 48 h of culture and the final position after 86 h of culture. Data represent the mean ± SEM (U87, n = 10; hG008, n = 22)

Article Snippet: The human glioblastoma cell line U87 was obtained from the American Type Culture Collection.

Techniques: Imaging, Slice Preparation, Fluorescence, Migration, Software

Journal: Molecular Brain

Article Title: Visualization of spatiotemporal dynamics of human glioma stem cell invasion

doi: 10.1186/s13041-019-0462-3

Figure Lengend Snippet: Migration of hG008 cells in different brain areas of slice culture. a Speed and direction of hG008 cell migration in the cortical area, striatum, and corpus callosum were analyzed using Imaris software during the period from 48 h to 96 h of culture. Data represent the mean ± SEM ( n = 56). b Snapshot images of hG008 cell division in the striatum and cortical area of contralateral side at the indicated time points are shown. Arrows (red, yellow, and blue) indicate dividing cells. c Migration speed was calculated for 12 h before and after cell division using Imaris software. Data represent the mean ± SEM ( n = 12). Snapshot images of cell division are shown. Colored scale bar represents cell migration speed (μm/second). d Migration of hG008 cells along the vertically passing axon bundles in the striatum. Snapshot images after 0 h and 72 h of culture are shown. Areas enclosed by white lines depict axon bundles. Scale bar, 100 μm. e The length of pseudopod of hG008 cells was quantified using ImageJ software during the period from 48 h to 96 h of culture. Data represent the mean ± SEM ( n = 18). Snapshot images of hG008 cells and U87 cells in the corpus callosum at the indicated time points are shown. Scale bar, 100 μm

Article Snippet: The human glioblastoma cell line U87 was obtained from the American Type Culture Collection.

Techniques: Migration, Software

Journal: Cellular and molecular life sciences : CMLS

Article Title: Protein kinase D inhibitor CRT0066101 suppresses bladder cancer growth in vitro and xenografts via blockade of the cell cycle at G2/M

doi: 10.1007/s00018-017-2681-z

Figure Lengend Snippet: CRT0066101 suppresses proliferation, anchorage-independent growth, and migration of bladder carcinoma cells in vitro. a, b Bladder cancer cells (T24, T24T, TCCSUP, and UMUC1) were treated with CRT0066101 at the indicated concentrations. Cell viability was determined at days 0, 2, and 4 by the MTT assay (a). Dose–response effect of CRT0066101 at 4-day treatment (b). Cell viability values are expressed relative to those for cells with no drug exposure. The IC50 values for CRT0066101 at day 4 in the four cell lines were calculated using GraphPad Prism version 6.01 software. c Differential effect of CRT0066101 on cell survival between human uroepithelial cells and bladder tumor cells as determined by using a Celigo image cytometer. The four bladder cancer cell lines and the human uroepithelial cell line SV-HUC were incubated with CRT0066101 at 5 μM for 6 days. Cell viability was simultaneously stained with a mixture of calcein AM, propidium iodide, and Hoechst 33342 reagents for respective staining of live, dead, and all cells, and the percentage of viable cells was quantified with the Celigo imaging cytometer. *P < 0.01 versus SV-HUC cells. d CRT0066101 abrogates bladder carcinoma cell growth in low attachment (GILA). The four bladder cancer cell lines were distributed in a 96-well flat bottom ultra-low attachment microplate and treated with CRT0066101 at the indicated concentrations. After 5 days of incubation, cell viability was measured as described in “Materials and methods”. NC no cells. *P < 0.01 and **P < 0.05 vs. the untreated control group. e, f CRT0066101 reduces the invasive ability of bladder cancer cells. A two-chamber cell invasion assay was used for assessing the effect of CRT0066101 on the invasion capability of bladder carcinoma cells. TCCSUP cells (e) and UMUC1 cells (f) were treated with CRT0066101 at the indicated concentrations, and cell migration/invasion was then assessed after 2 days of incubation at 37 °C in the chamber, as described in “Materials and methods”. The numeric data for the two cell lines are shown in the two column graphs, respectively. *P < 0.01 and **P < 0.05 vs. the untreated control group

Article Snippet: The human bladder carcinoma cell lines SCaBER, 5637, T24, UMUC3, and TCCSUP, and the SV40-transformed human uroepithelial cell line SV-HUC were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Migration, In Vitro, MTT Assay, Software, Cytometry, Incubation, Staining, Imaging, Control, Invasion Assay

Journal: Cellular and molecular life sciences : CMLS

Article Title: Protein kinase D inhibitor CRT0066101 suppresses bladder cancer growth in vitro and xenografts via blockade of the cell cycle at G2/M

doi: 10.1007/s00018-017-2681-z

Figure Lengend Snippet: Immunoblot analysis of PKD1, PKD2, and PKD3 protein levels in human urothelial bladder carcinoma cell lines. Four bladder cancer cell lines (SCaBER, UMUC5, UMUC1, and 5637) were used for analysis of PKD1 protein expression, while 8 bladder tumor cell lines (TCCSUP, T24T, T24, UMUC3, SCaBER, UMUC5, UMUC1, and 5637) were utilized for analysis of PKD2 and PKD3 protein levels. Total cellular protein was extracted from these cell lines, and a total of 80 μg cell extract protein was subjected to SDS-PAGE and immunoblotted with an antibody against PKD1, PKD2, or PKD3, as described in Materials and Methods. β-Actin was used as the control to verify equal protein loading and transfer

Article Snippet: The human bladder carcinoma cell lines SCaBER, 5637, T24, UMUC3, and TCCSUP, and the SV40-transformed human uroepithelial cell line SV-HUC were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Western Blot, Expressing, SDS Page, Control

Journal: Cellular and molecular life sciences : CMLS

Article Title: Protein kinase D inhibitor CRT0066101 suppresses bladder cancer growth in vitro and xenografts via blockade of the cell cycle at G2/M

doi: 10.1007/s00018-017-2681-z

Figure Lengend Snippet: Knockdown of PKD2 expression decreases proliferation, anchorage-independent growth, and invasion of bladder carcinoma cells in vitro. a Six cell lines (T24, T24T, TCCSUP, UMUC1, UMUC3, and UMUC5) were transduced with lentivirus coding for human shRNA sequences for PKD2 (shPKD2#1 and shPKD2#2) or for the control (shNTC). Cell growth and proliferation were assessed by MTT assay at days 0, 2, 4, and 6 after cell plating. P < 0.01 for the shPKD2 groups vs. the control shNTC group at day 6 in all the six cell lines tested. b Depletion of PKD2 abrogates bladder cancer cell growth in low attachment. T24T and UMUC1 cell lines were transduced with lentivirus coding for human shRNA sequences for PKD2 (shPKD2#1 and shPKD2#2) or for the control (shNTC). Cell growth in low attachment was assessed by GILA assay at day 5 after cell plating, as described in “Materials and methods”. *P < 0.01 and **P < 0.05 for the shPKD2 groups vs. the control shNTC group. c, d Knockdown of PKD2 expression compromises the invasive ability of bladder carcinoma cells. TCCSUP cells (c) and UMUC1 cells (d) were transduced with lentivirus coding for human shRNA sequences for PKD2 (shPKD2#1 and shPKD2#2) or for the control (shNTC). A two-chamber cell invasion system was set up, as described in “Materials and methods”, and the number of invading cells was counted in ten random microscopic fields (×40). The numeric data for the two cell lines are shown in the two column graphs, respectively. Values are expressed as mean ± SD. **P < 0.05 and *P < 0.01 for the shPKD2 groups vs. the shNTC group in TCCSUP and UMUC1 cells, respectively

Article Snippet: The human bladder carcinoma cell lines SCaBER, 5637, T24, UMUC3, and TCCSUP, and the SV40-transformed human uroepithelial cell line SV-HUC were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Knockdown, Expressing, In Vitro, Transduction, shRNA, Control, MTT Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Occludin facilitates tumour angiogenesis in bladder cancer by regulating IL8/STAT3 through STAT4

doi: 10.1111/jcmm.17257

Figure Lengend Snippet: Occludin (OCLN) promotes tumour angiogenesis in vitro and in vivo. (a and b) The knockdown efficiency was confirmed by performing (A) Western blotting and (B) RT‐qPCR assays using 5637 and T24 cells. (C), Tube formation by EA.hy926 cells incubated with CM derived from 5637 and T24 OCLN‐silenced bladder cancer (BLCA) cells was assessed using staining with Calcein AM and imaging with a fluorescence microscope. Scale bar = 100 µm. (d and e) The OCLN plasmid was transfected into 5637 and T24 cells; the efficiency of overexpression was analysed using (D) Western blotting and (E) RT‐qPCR. (F) Tube formation by EA.hy926 cells incubated with CM derived from 5637 and T24 OCLN overexpressing cells was assessed using staining with Calcein AM and imaging with a fluorescence microscope. Scale bar = 100 µm. (G) The segment lengths were analysed, and the meshes were quantified using ImageJ software. (H) Matrigel plugs containing 5637 and T24 OCLN stable knockdown cells were removed from BALB/c nude mice. (I) CD31 staining in the indicated cells embedded in Matrigel plugs after growth in BALB/c nude mice. Scale bar = 100 µm. (J) The density of microvessels in Matrigel plugs from BALB/c nude mice injected with the indicated cells. (K) IHC staining showing CD31 levels in clinical patients with high‐/low‐grade BLCA. Scale bar = 100 µm. The results are shown as the mean ± SD. *.01 < p < .05; **.001 < p < .01

Article Snippet: BLCA cell lines, namely T24 (ATCC HTB‐4), 5637 (ATCC HTB‐9) were derived from laboratory retained cells.

Techniques: In Vitro, In Vivo, Knockdown, Western Blot, Quantitative RT-PCR, Incubation, Derivative Assay, Staining, Imaging, Fluorescence, Microscopy, Plasmid Preparation, Transfection, Over Expression, Software, Injection, Immunohistochemistry

Journal: Journal of Cellular and Molecular Medicine

Article Title: Occludin facilitates tumour angiogenesis in bladder cancer by regulating IL8/STAT3 through STAT4

doi: 10.1111/jcmm.17257

Figure Lengend Snippet: Occludin (OCLN) mediates bladder cancer (BLCA) angiogenesis by regulating IL8 expression. (A) Venn diagram showing the differentially expressed genes (DEGs) in the two T24 OCLN knockdown groups compared with the control groups (fold change ≥1, FDR < 0.1, p < .05). (B) Heatmap of the RNA sequencing analysis showing the relative levels of proangiogenic factors. Columns represent probe sets, and rows represent samples receiving the indicated treatments. (C) The relative mRNA levels of proangiogenic factors were detected in control and OCLN shRNA transfected 5637 and T24 cells. (D) The relative IL8 levels in control and OCLN shRNA‐transfected 5637 and T24 cells were measured using an ELISA (pg/ml). (E) The relative IL8 mRNA levels were detected in 5637 and T24 cells following transfection with the vector or OCLN plasmid. (F) Tube formation by EA.hy926 cells cultured with CM derived from 5637 and T24 OCLN‐silenced BLCA cells. IL8 was added, and the cells were stained with Calcein AM and then imaged with a fluorescence microscope. Scale bar = 100 µm. (G) The segment lengths were analysed, and the meshes were quantified using ImageJ software. The results are shown as the mean ± SD. *.01 < p < .05; **.001 < p < .01; *** p < .001

Article Snippet: BLCA cell lines, namely T24 (ATCC HTB‐4), 5637 (ATCC HTB‐9) were derived from laboratory retained cells.

Techniques: Expressing, Knockdown, Control, RNA Sequencing, shRNA, Transfection, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Cell Culture, Derivative Assay, Staining, Fluorescence, Microscopy, Software

Journal: Journal of Cellular and Molecular Medicine

Article Title: Occludin facilitates tumour angiogenesis in bladder cancer by regulating IL8/STAT3 through STAT4

doi: 10.1111/jcmm.17257

Figure Lengend Snippet: IL8/ STAT3 is involved in the process of Occludin (OCLN)‐mediated angiogenesis in bladder cancer (BLCA). (A) p‐STAT3 and STAT3 protein levels were detected in OCLN knockdown 5637 and T24 cells. (B) 5637 and T24 cells were transfected with the OCLN plasmid and treated with or without the STAT3 inhibitor Stattic, and tube formation by EA.hy926 cells incubated with CM derived from the indicated cells was assessed by performing staining with Calcein AM and imagining using a fluorescence microscope. Scale bar = 100 µm. (C) The segment lengths in these images were analysed, and the meshes were quantified using ImageJ software. D, p‐STAT3 and STAT3 protein levels were detected in OCLN knockdown 5637 and T24 cells after IL8 supplementation. (E) p‐STAT3 and STAT3 protein levels were detected in OCLN‐overexpressing 5637 and T24 cells after blocking IL8. (F) 5637 and T24 cells were transfected with the OCLN plasmid or cultured with the IL8‐neutralizing antibody; tube formation by EA.hy926 cells incubated with CM derived from the indicated cells was assessed using staining with Calcein AM and imaging with a fluorescence microscope. Scale bar = 100 µm. G, The segment lengths in these images were analysed, and the meshes were quantified using ImageJ software. The results are shown as the mean ± SD. *.01< p < .05; **.001< p < .01

Article Snippet: BLCA cell lines, namely T24 (ATCC HTB‐4), 5637 (ATCC HTB‐9) were derived from laboratory retained cells.

Techniques: Knockdown, Transfection, Plasmid Preparation, Incubation, Derivative Assay, Staining, Fluorescence, Microscopy, Software, Blocking Assay, Cell Culture, Imaging

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet:

Article Snippet: Cercopithecus aethiops: Vero-CCL81 cells , ATCC , CCL-81.

Techniques: Virus, Recombinant, Transfection, Protease Inhibitor, Magnetic Beads, Electron Microscopy, Reverse Transcription, Cell Viability Assay, Mutagenesis, Cloning, Luciferase, Staining, Labeling, Software, Real-time Polymerase Chain Reaction, Imaging, Laser-Scanning Microscopy, Microscopy