Journal: Cancer Research

Article Title: Liver Colonization by Colorectal Cancer Metastases Requires YAP-Controlled Plasticity at the Micrometastatic Stage

doi: 10.1158/0008-5472.CAN-21-0933

Figure Lengend Snippet: Dynamic Yap activity is required for the outgrowth of colorectal cancer organoids. A, Diminishing expression of micro-organoid–associated genes during the first 7 days of A/K/P/S organoid outgrowth. Gene expression, represented as mean + SEM, is normalized to day 1. B–D, Single A/K/P/S cells treated with 500 nmol/L XMU-MP-1 (MST1/2 inhibitor) for 3 or 7 days. B, Schematic of experimental setup (top) and representative organoid overview after 7 days of culture (bottom). Scale bars, 100 µm. C, Relative fraction of EdU-incorporating cells. D, Flow analysis of STAR levels. C and D, Data is normalized to DMSO control. E–H, Ten-day-old A/K/P/S organoids were treated with 500 nmol/L XMU-MP-1 and analyzed after 96 hours by flow cytometry. E, Experimental setup. F, Relative viability assessed by DAPI. G, Relative change in STAR populations. H, Relative fraction of EdU-incorporating cells. F–H, Data are normalized to their respective DMSO control. I–J, Three µmol/L verteporfin was added to single A/K/P/S cells for 48 hours prior to wash out. Organoids were analyzed after 7 days. I, Schematic of experimental setup (top) and representative organoid overview after 7 days of culture (bottom). Scale bars, 100 µm. J, Relative viability as assessed by CellTiter-Glo. Data are normalized to DMSO control. n.s., nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: For analysis, 50% v/v CellTiter-Glo reagent (Promega) was added to each well.

Techniques: Activity Assay, Expressing, Control, Flow Cytometry

Journal: Cell chemical biology

Article Title: Chlorcyclizine Inhibits Viral Fusion of Hepatitis C Virus Entry by Directly Targeting HCV Envelope Glycoprotein 1

doi: 10.1016/j.chembiol.2020.04.006

Figure Lengend Snippet: A. Structures and dose-response curves of (S)-CCZ, CCZ-diazirine, CCZ-diazirine-biotin (CCZ-DB) and diazirine-biotin (DB) control. CCZ-diazirine and CCZ-diazirine-biotin were active in inhibiting HCV in the HCV infection assay, with EC50 of 25.0 nM and 19.7 nM, respectively. The DB control has the diazirine-biotin moieties but was not active against HCV. B. Cross-linking of CCZ-DB with recombinant HCV E1/E2 protein. Purified recombinant E1/E2 protein (genotype 1a) was incubated with various compounds described above at room temperature for 1 h, subjected to UV cross-linking and then purified by Neutravidin beads followed by Western blot with anti-E1 antibody. Recombinant E1/E2 protein was included on the blot as a reference. In one sample, a 100-fold higher concentration of (S)-CCZ (100 μM) was added to the CCZ-DB cross-linking condition. C. Cross-linking of CCZ-DB with E1 protein of HCV genotype 1a-infected cells. Huh7.5.1 cells were infected with high-titer HCVcc genotype 1a virus in the presence of the various compounds at 37°C for 1 h, subjected to UV cross-linking and then lysed for purification by Neutravidin beads followed by Western blot with anti-E1 antibody. The same conditions were tested as the HCV recombinant E1/E2 protein above. A high-titer stock of the HCV genotype 1a virus was also run on the blot to demonstrate the presence of E1 protein. The results are representative of three separate experiments.

Article Snippet: Affinity Pull-Down of CCZ-DB Cross-Linked Proteins and Western Blot Analysis 50 μL of Pierce NeutrAvidin agarose beads (Thermo Fischer Scientific, Waltham, MA, USA) were spun down, the supernatant was discarded, and the beads were washed twice with 1 mL of PBS.

Techniques: Infection, Recombinant, Purification, Incubation, Western Blot, Concentration Assay

Journal: Cell chemical biology

Article Title: Chlorcyclizine Inhibits Viral Fusion of Hepatitis C Virus Entry by Directly Targeting HCV Envelope Glycoprotein 1

doi: 10.1016/j.chembiol.2020.04.006

Figure Lengend Snippet: Key Resources Table

Article Snippet: Affinity Pull-Down of CCZ-DB Cross-Linked Proteins and Western Blot Analysis 50 μL of Pierce NeutrAvidin agarose beads (Thermo Fischer Scientific, Waltham, MA, USA) were spun down, the supernatant was discarded, and the beads were washed twice with 1 mL of PBS.

Techniques: Recombinant, Plasmid Preparation, Luciferase, Purification, Mutagenesis, Clone Assay, Western Blot, Software, Sequencing

Journal: The Journal of Experimental Medicine

Article Title: Rab6-dependent retrograde traffic of LAT controls immune synapse formation and T cell activation

doi: 10.1084/jem.20162042

Figure Lengend Snippet: LAT recruitment to the immune synapse and late T cell activation are deficient in Rab6 cKO CD4 + T lymphocytes. (A) Immunoblot analysis of Rab6 and gp96 (loading control) expression in CD4 + T cells (left) or total kidney cells (right) isolated from control (WT) and Rab6 cKO mice. Molecular mass is indicated in kilodaltons. (B and C) Quantification by TIRFM of the number of LAT (B) or ZAP70 (C) microclusters recruited to the immune synapse in CD4 + T cells incubated on poly- l -lysine alone or anti-CD3+anti-CD28 mAb (1 or 10 µg/ml). Bars, 5 µm. (D–F) Flow cytometry analysis of the proliferation of CD4 + T cells isolated from control (Ctrl) or Rab6 cKO mice labeled with CTV proliferation dye and stimulated for 5 d by different numbers of irradiated allogeneic Balb/C splenocytes (stimulatory cells) in a mixed lymphocyte reaction. (D) Representative experiment. (E) Percentages of proliferating CD4 + T cells from five Rab6 cKO or control mice. (F) Rab6-deficient CD4 + T cells labeled with the CFSE proliferation dye were either left alone (dark colors) or mixed with (1:1) control CD4 + T cells labeled with CTV (light colors). Analysis of the percentages of proliferating CD4 + T cells exposed for 5 d to different numbers of irradiated allogeneic Balb/C splenocytes was performed as in D. (G) Percentages of proliferating CD4 + T cells isolated from control or Rab6 cKO mice left untreated (left) or stimulated for 5 d with low concentrations of PMA/ionomycin (7.5 × 10 −7 M and 2 × 10 −8 M; right). *, P < 0.05; **, P < 0.01; ****, P < 0.0001; one-way ANOVA in B and C; two-way ANOVA in E; mean and SEM in D. Horizontal lines represent median in B and C. Data from three experiments in B, from two experiments in C, and from four experiments in E. Data representative from three experiments in A, four in D, two in F, and three in G.

Article Snippet: For mixed lymphocyte reaction analysis, 50 µl of anti-CD4 APC (1:150, 553051; BD PharMingen) and anti–MHC-II APC-eFluor 780 (1:250, 47-5321-82; eBioscience) were directly added 30 min before fixation.

Techniques: Activation Assay, Western Blot, Expressing, Isolation, Incubation, Flow Cytometry, Labeling, Irradiation