Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Sphingomyelin is sorted at the trans Golgi network into a distinct class of secretory vesicle.

doi: 10.1073/pnas.1602875113

Figure Lengend Snippet: Fig. 4. Eqt-SM and GPI are cosorted into secretory vesicles. (A) Trafficking of GPI and CD8α reporter proteins. HeLa cells were transfected with plasmids that direct expression of mKate2-FM4-GPI or mKate2-FM4-CD8α. In the ab- sence of the disaggregating molecule (“ER retain”) mKate2-FM4-GPI and mKate2-FM4-CD8α are retained in the ER. Incubation of cells with the dis- aggregating molecule (“release 45’”) releases GPI and CD8α from the ER, and they are subsequently trafficked to the cell surface. The micrographs show cells incubated with solubilizer for 45 min. (B and C) Example TIRFM time-lapse gal- lery of Eqt-SM, Eqt-sol, and GPI (B) and mKate2-FM4-CD8α (C) exocytic events. GPI and CD8α reporters were coexpressed in cells with Eqt-SM-pHlourin or Eqt- sol-pHlourin and released from the ER of cells by the addition of solubilizer drug. Exocytic events were recorded by TIRFM beginning 30 min after ER release. The graphs show fluorescence intensity in each channel over time. (D) Summary of Eqt vesicle cargo content and GPI-containing vesicle content. The propor- tions of exocytic vesicles containing the indicated cargos (mean ± SD) are listed. The number of fusion events scored for each condition are as follows: Eqt-SM+CD8, n = 146; Eqt-SM+GPI, n = 157; Eqt-sol+CD8, n = 153; Eqt-sol+GPI, n = 136; GPI+Eqt-SM, n = 85; GPI+Eqt-sol, n = 65.

Article Snippet: On the addition of D/D solubilizer (Clontech), both reporters were released from the ER, trafficked to the Golgi apparatus, and then delivered to the plasma membrane (Fig. 4A).

Techniques: Transfection, Expressing, Incubation, Fluorescence

Journal:

Article Title: Nrdp1-mediated regulation of ErbB3 expression by the androgen receptor in androgen-dependent but not castrate-resistant prostate cancer cells

doi: 10.1158/0008-5472.CAN-09-4440

Figure Lengend Snippet: (A, B) CWR22 xenograft tumors were established by subcutaneous injections of cell suspensions (2.5 × 106 cells in Matrigel; 1:1, v/v) bilaterally into the flanks of 4–5-week-old nu/nu athymic male mice (n=10) previously implanted with sustained-release testosterone pellets. When palpable tumors were observed, animals were treated with (i) vehicle (peanut oil) or (ii) the AR antagonist bicalutamide (n=5/group). After two weeks on this treatment, the mice were euthanized, tumors were harvested and divided into sections that were paraffin-embedded snap-frozen in liquid nitrogen. Paraffin-embedded tumors were analyzed by immunohistochemistry for ErbB3 whereas frozen tumors were excised, lysed and protein levels were determined by Western blotting. (A) Western blotting revealed that ErbB3 levels increased while Nrdp1 levels decreased with bicalutamide treatment. (B) Immunohistochemistry demonstrating that ErbB3 levels increased in tumors extracted from the bicalutamide-fed mice. Brown staining denotes ErbB3 while blue represents counterstaining with hematoxylin. Negative control showed no ErbB3 staining, the xenograft from the vehicle-fed mouse shown showed lower ErbB3 expression (scored +1) compared to the bicalutamide-treated one (scored +3). (C) In contrast, CWR22Rv1, a CRPC cell line derived from a CWR22 relapsed tumor grown in a castrated mouse, failed to respond to bicalutamide and in these cells, Nrdp1 and ErbB3 were not androgen regulated. (D) Scheme describing androgen regulation of Nrdp1 transcription in androgen-dependent but not androgen-independent cells.

Article Snippet: Suspensions of CWR22 cells were made in 50% Matrigel solubilized basement membrane (BD Biosciences, Bedford, MA) and xenografts were established by subcutaneous injections of 2.5 × 10 6 cells/site into both flanks.

Techniques: Immunohistochemistry, Western Blot, Staining, Negative Control, Expressing, Derivative Assay