Journal: Human Molecular Genetics

Article Title: A meckelin–filamin A interaction mediates ciliogenesis

doi: 10.1093/hmg/ddr557

Figure Lengend Snippet: Loss of meckelin or filamin A causes deregulation of Wnt signalling. (A) TopFlash assays to measure levels of activated β-catenin, and hence canonical Wnt signalling, following co-transfection of immortalized normal control (control fibs, blue bars) and MKS3-mutated dermal fibroblasts (MKS3 fibs, red bars; see Fig. 3C) with reporter constructs and empty vector, wild-type HA-meckelin, p.F919del mutant HA-meckelin constructs and c myc-filamin A, as indicated. The empty vector results combine the data from transfections with pCMV-HA and pCMV-c myc. Activity is expressed as ratios of luciferase reporter construct expression, normalized for loading by measurement of a Renilla construct expression. The responses are shown to 0.5 × L cell control conditioned media (control), and conditioned media containing expressed Wnt3A and/or Wnt5A. Values shown are means of at least four independent replicates, with error bars indicating SEM. Statistical significance of pair-wise comparisons are shown (*P < 0.01 and **P < 0.001, Student's t-test). (B) TopFlash reporter assays as in (A) for control fibroblasts (control fibs, blue bars) and FLNA-mutated fibroblasts (FLNA fibs, green bars). Values shown are means of three independent replicates. Statistical significance of pair-wise comparisons are shown (*P < 0.05, error bars = SEM, **P < 0.01, Student's t-test).

Article Snippet: For luciferase assays of canonical Wnt activity, we grew fibroblasts in 12-well plates and co-transfected with 0.5 μg of Topflash firefly luciferase construct (or Fopflash, as a negative control), 0.5 μg of expression constructs (pCMV HA-meckelin, pCMV c myc-filamin A or empty pCMV-HA/pCMV c myc vector) and 0.1 μg of pRL-TK (Promega Corp., WI, USA; Renilla luciferase construct used as an internal control reporter).

Techniques: Cotransfection, Control, Construct, Plasmid Preparation, Mutagenesis, Transfection, Activity Assay, Luciferase, Expressing

Journal: Virus research

Article Title: The canonical Wnt/β-catenin signaling pathway stimulates herpes simplex virus 1 productive infection

doi: 10.1016/j.virusres.2018.07.020

Figure Lengend Snippet: HLF cells in 12-well plates were transfected with 0.6 μg of TOPflash or FOPflash luciferase reporter construct and 0.02 μg of the Renilla reporter construct. Renilla reporter construct was used as an internal control to allow normalization of promoter activity. At 36 h after transfection, cells were infected with HSV-1 (Panels A-D) or UV-inactivated HSV-1 (Panels E and F) at the indicated MOI. At 16 h after infection, dual luciferase activity was measured. Where indicated (Panels D and E), the designated concentration of iCRT14 was added immediately after infection. At 16 h after infection, dual luciferase activity was measured. These results are the average of three independent experiments. An asterisk denotes significant differences (P < 0.05) in promoter activity compared to the indicated control by the Student t-test.

Article Snippet: To detect the effect of HSV-1 infection on β-catenin dependent transcription, HFL, Vero, or Neuro-2 A cells were cotransfected with the Super 8x TOPflash plasmid and a plasmid encoding Renilla luciferase under the control of a minimal herpesvirus promoter (Promega) using Lipofectamine ® 3000 Transfection Reagent (Invitrogen, L3000075).

Techniques: Transfection, Luciferase, Construct, Control, Activity Assay, Infection, Concentration Assay

Journal: Virus research

Article Title: The canonical Wnt/β-catenin signaling pathway stimulates herpes simplex virus 1 productive infection

doi: 10.1016/j.virusres.2018.07.020

Figure Lengend Snippet: Vero cells were grown in 12-well dishes and then transfected with 0.4 μg of TOPflash or FOPflash luciferase reporter construct and 0.01 μg of the Renilla reporter construct. Renilla reporter construct was used as an internal control to allow normalization of promoter activity. At 36 h after transfection, cells were infected with HSV-1 (Panels A and B) or UV-inactivated HSV-1 (Panels E and F) using the indicated MOI. At 16 h after infection, dual luciferase activity was measured.

Article Snippet: To detect the effect of HSV-1 infection on β-catenin dependent transcription, HFL, Vero, or Neuro-2 A cells were cotransfected with the Super 8x TOPflash plasmid and a plasmid encoding Renilla luciferase under the control of a minimal herpesvirus promoter (Promega) using Lipofectamine ® 3000 Transfection Reagent (Invitrogen, L3000075).

Techniques: Transfection, Luciferase, Construct, Control, Activity Assay, Infection

Journal: Virus research

Article Title: The canonical Wnt/β-catenin signaling pathway stimulates herpes simplex virus 1 productive infection

doi: 10.1016/j.virusres.2018.07.020

Figure Lengend Snippet: Panel A: HLF cells were cotransfected with 0.3 μg of the TOPflash or FOPflash luciferase reporter, and 0.02 μg of Renilla reporter construct. Where indicated, 0.5 μg of β-catenin (S33Y) and 1 μg of HSV-1 VP16 construct were used to examine the effects that VP16 have on β-catenin dependent transcription in HLF cells. Neuro-2 A cells were cotransfected with 0.1 μg of the TOPflash luciferase reporter and 0.01 μg of Renilla reporter construct. Where indicated, 0.3 μg of activated β-catenin (S33Y) (Panel B) or wild type β-catenin (Panel C) and 1 μg of VP16 at the designated concentration was used to examine the effects that VP16 had on β-catenin dependent transcription in Neuro-2 A cells.

Article Snippet: To detect the effect of HSV-1 infection on β-catenin dependent transcription, HFL, Vero, or Neuro-2 A cells were cotransfected with the Super 8x TOPflash plasmid and a plasmid encoding Renilla luciferase under the control of a minimal herpesvirus promoter (Promega) using Lipofectamine ® 3000 Transfection Reagent (Invitrogen, L3000075).

Techniques: Luciferase, Construct, Concentration Assay

Journal: eLife

Article Title: FAK activity sustains intrinsic and acquired ovarian cancer resistance to platinum chemotherapy

doi: 10.7554/eLife.47327

Figure Lengend Snippet: ( A ) Immunoblotting of KMF and CRISPR-mediated FAK KO clones KT3 and KT13 cell lysates for FAK, Pyk2, β-catenin, and actin. ( B ) KMF and FAK KO KT13 cell viability treated with DMSO (control) or CP (1 µM) after 72 hr as measured by Alamar Blue. Values are means (± SEM, *p<0.05, **p<0.01, ***p<0.001, one-way ANOVA with Fisher’s LSD multiple comparison test) for three independent experiments. ( C ) β-catenin transcriptional reporter activity (TOPFlash) in transfected KMF and KT13 FAK KO cells + /- GSK3β inhibitor. Values are arbitrary units (***p<0.001, unpaired T-test, two independent experiments). ( D ) Immunoblotting for pY397 FAK, FAK, and actin in lysates of KMF, FAK KO, GFP-FAK-WT, and GFP-FAK-R454 re-expressing cells. ( E ) Top 10 proteomic differences (fold-change) detected by mass spectroscopy of membrane associated proteins in KT13 FAK KO, GFP-FAK-WT, and GFP-FAK R454 re-expressing cells. ( F ) Immunoblotting for β-catenin and actin in lysates of KT13 FAK KO cells stably expressing GFP-FAK-WT, GFP-FAK-R454, or β-catenin (ΔGSK). ( Gand H ) XTT metabolic activity (panel G) or tumorsphere formation (panel H) of KMF, KT13 FAK KO, or the indicated reconstituted cells in PromoCell after 5 days. Values are means (± SEM, *p<0.05, ***p<0.001, one-way ANOVA with a Tukey’s multiple comparisons test) from 2 (panel G) or 3 (panel H) independent experiments. 10.7554/eLife.47327.027 Figure 8—source data 1. KMF FAK KO clone KT13 exome sequencing variants. 10.7554/eLife.47327.028 Figure 8—source data 2. Summary of mass spectrometry-detected proteomic changes between KMF FAK KO, FAK-WT, and FAK kinase-inactive (K454R) re-expressing cells grown as tumorspheres.

Article Snippet: For transient transfection of a β-catenin TOPFlash reporter, 3.5e4 cells were seeded in triplicate in 24-well plates and co-transfected with M50 Super 8x TOPFlash with firefly and Renilla luciferase expression vectors using Fugene HD (Promega).

Techniques: Western Blot, CRISPR, Clone Assay, Control, Comparison, Activity Assay, Transfection, Expressing, Mass Spectrometry, Membrane, Stable Transfection, Sequencing

Journal: eLife

Article Title: FAK activity sustains intrinsic and acquired ovarian cancer resistance to platinum chemotherapy

doi: 10.7554/eLife.47327

Figure Lengend Snippet:

Article Snippet: For transient transfection of a β-catenin TOPFlash reporter, 3.5e4 cells were seeded in triplicate in 24-well plates and co-transfected with M50 Super 8x TOPFlash with firefly and Renilla luciferase expression vectors using Fugene HD (Promega).

Techniques: Transfection, Protease Inhibitor, Recombinant, Proliferation Assay, Reporter Assay, SYBR Green Assay, Reverse Transcription, ALDH Detection Assay, Isolation, Plasmid Preparation, Cloning, Expressing