METAMAQUETAS LA APARICIÓN DE LA VITRINA

For transfections of 3T3 and 3T3-BC5052-/-, the Effectene (Qiagen, Valencia,

CA) method and reagent was used. Briefly, 0.75 x 105 cells were seeded in 0.5 ml of

media in a 24-well dish and transfected at the same time. Plasmid DNA was first mixed with a DNA:Enhancer ratio of 1:5.5 for 5 min and then a DNA:Effectene ratio of 1:7.9 and incubated for 30 min at RT. Specifically, 200 ng of the pGL3-BIC reporter plasmid, 200 ng of pcDNA-based NF-κB expression plasmids, 200 ng of empty vector pcDNA (to increased transfection efficiency), and 30 ng of RSV-Renilla luciferase reporter were mixed with the Enhancer and then with Effectene. The transfection mixtures were then added to the appropriate cells in DMEM/10% FBS, and the final mixture was then plated

in 24-well dishes. The next day, the transfection media was replaced with fresh DMEM/10% FBS. Cells were harvested and lysed 24 h later for luciferase assays.

2.5 Dual Luciferase Assay

COS-1, 3T3, and 3T3-BC5052-/- _{cells were seeded in 24-well tissue culture plates}

as described above. For transfections, 15 ng pRSV-Renilla (normalization plasmid), 200 ng of appropriate Firefly-based luciferase plasmid, 400 ng of pcDNA-bassed expression plasmid, and the appropriate amount of PEI or Effectene was used. The Promega Dual Luciferase Assay System was used to measure luciferase activity. Forty-eight h post- transfection, cells were washed once with 1X PBS (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4) and then lysed directly on the plate with 100 µl 1X Passive Lysis Buffer (provided by Promega) and incubated at 4°C for 20 min on a table top shaker. If cell lysates were to be kept for future experiments, cells were transferred to 1.5-ml microcentrifuge tubes, cell debris was cleared by spinning in a microcentrifuge, and the supernatant was collected and frozen at -80°C. Otherwise, the 24-well plate containing the supernatants was placed on ice and used for luciferase activity determination as follows. Five ul of extract was transferred into a 96-well, white, flat-bottom plate

(Thermo Fisher, Waltham, MA) and mixed with 30 µl of luciferase reagent. Firefly-based luminescence was measured immediately for 10 sec in multilabel plate reader (Perkin Elmer, Waltham, MA). Next, 30 ul of the Stop and Glo reagent was added to each well and the Renilla-based luminescence as recorded. To determine luciferase activity, the Firefly-based luciferase values were divided by Renilla-based luciferase values. Values

(average of three samples) were then normalized to the same control value, so that the control was equal to 1.0 (the average of the three control values). Experiments were performed three times in triplicate and the results represent the mean values +/- SE.

2.6 Retroviral Stock Preparation and Cell Infection

Virus stocks were prepared and used for infection as described (Thompson et al., 2010). For creation of virus stocks directing target gene expression using pMSCV-based plasmids, A293T retroviral packaging cells were transfected in 60-mm dishes with 15 µg of the retroviral expression plasmid and 5 µg of the retroviral packaging plasmid

(pcL10A1) using the PEI method (see above). For creation of virus stocks directing expression of pSIREN-based shRNA plasmids for target gene knock-down, Bosc23 retroviral packaging cells were transfected in 60-mm dishes with 5 µg of the retroviral expression plasmid and 5 µg of each retroviral packaging plasmids pVpack-VSV and pVpack-GP using the PEI method (see above). Two days following transfection of A293T and Bosc23 cells, the supernatants from each dish were harvested, spun in a desktop centrifuge for 5 min at 5,000 rpm to remove remaining cells, and the supernatants were transferred to a new tube.

For infection of target cells, polybrene was then added to a final concentration of

8 µg/ml to the viral supernatant (2 ml) and incubated with 1 x 106 cells of interest in a 14-

ml snap-cap tube (BD Falcon). Cells were incubated with the virus/polybrene mix for 15 min at RT then spun in a desktop centrifuge for ~2 h at 2,500 rpm at RT. Post-spin, the virus/ploybrene mix was carefully aspirated off the cell pellet and the cells were

resuspended in 2 ml of fresh media containing the appropriate concentration of FBS and Pen/Strep, and cells were then placed in tissue culture incubator. Puromycin (Sigma- Aldrich, St. Louis, MO) was added to the cells two days later to a final concentration of 0.5-2.5 µg/ml, as determined by titration (Table 2.4). Cells were selected for 7-21 days in puromycin and the expression of the given protein was determined by Western blotting.

2.7 Soft Agar Colony Formation Assay

Soft agar colony formation assays were performed as described previously (Chin et al., 2009). Agar was prepared by mixing 1 g of BactoAgar (Difco Laboratories, Detroit, MI) in 30 ml of distilled water and then autoclaving for 30 min. For the plating media, 10 ml of the agar mix (equilibrated to 56°C) was added to 90 ml of 37°C DMEM containing 20% FBS. Cells that had been previously counted using a hemocytometer

were initially diluted to 1 x 105 cells/ml, and further diluted to concentrations of 1 x 104

cells/ml and then 1 x 103 _{cells/ml. Equal numbers of cells (250 or 500) were quickly}

added to 5 ml of the plating media, mixed, and placed in 60-mm petri dishes. Dishes were kept at RT for ~15 min until the media was almost solid and then incubated at 37°C in a

humid incubator with 5% CO2. Macroscopic soft agar colonies were counted 14 days

after plating and were performed for both empty plasmid control (MSCV) and gene of

interest (GOI) cell lines. For normalizing soft agar colony formation, 1 x 106 cells were

collected from the above cell counts, whole cell extracts were prepared, and protein concentration was calculated (section 2.10). For each independent experiment, soft agar colony counts were normalized to the protein concentration calculated above as follows:

for MSCV cells, ([MSCV]/[MSCV]) x n colonies; for GOI cells, ([MSCV]/[GOI]) x n colonies. Data are presented as relative soft agar colony formation of five independent experiments performed with 5 plates of cells. Results represent the mean values +/- SE.

2.8 Preparation of Whole Cell Extracts Using Triton-X-100 (AT) Buffer Method

Adherent cells were washed 2X with PBS, scraped in 1 ml of PBS, and collected into a 1.5-ml microcentrifuge tube. Suspension cells were collected into 15-ml centrifuge tubes and pelleted by centrifugation at 3,000 rpm for 5 min at RT. Cells were

resuspended in 1 ml PBS and transferred to a 1.5-ml microcentrifuge tube. Cells were then pelleted by centrifugation at 5,000 rpm for 5 min at 4°C. The PBS was aspirated from the cells and the cell pellet was resuspended in 100-200 µl of AT buffer (20 mM

HEPES [pH 7.9], 1 mM EDTA, 1 mM EGTA, 20 mM NaF, 1 mM Na4P2O7-H20, 1 mM

DTT, 20% w/v glycerol, 1% v/v Triton-X-100, 1 mM Na3VO4, 1 mg/ml of leupeptin,

aprotinin, pepstatin and 1 mM PMSF), depending on the size of the cell pellet, by repeated suspension using a 27.5 gauge needle (Becton Dickinson, Franklin Lakes, NJ). To the cell lysates, 3.1 µl (for 100 µl of AT buffer) or 6.2 µl (for 200 µl of AT buffer) of 5 M NaCl was added to a final concentration of ~150 mM. The samples were centrifuged at 14,000 rpm for 30 min at 4°C, supernatants were collected, and stored at -80°C.

2.9 Preparation of Cytosolic and Nuclear cell Extracts

Adherent cells were washed twice with PBS, gently scraped using a rubber

were transferred from their culture dish into 15 ml centrifuge tubes and pelleted by centrifugation at 3,000 rpm for 5 min at RT. Cells were resuspended in 1 ml PBS and transferred to a 1.5 ml microcentrifuge tube. For both cell types, cells were pelleted by centrifugation at 5,000 rpm for 5 min at 4°C. The supernatant was then aspirated off and the remaining cell pellet was resuspended in 400 µl of Buffer “E” (10 mM HEPES [pH 7.9], 10 mM KCl, 0.1 mM EDTA [pH 8.0], 0.1 mM EGTA [pH 8.0], 1 mM DTT, 1 mM

Na3VO4, 5 µg/ml leupeptin, 62.5 µg/ml aprotinin, 1 µg/ml pepstatin, and 1 mM PMSF),

supplemented with 55 µl of 5% IGEPAL, and cells were vortexed vigorously for 10 sec. Samples were incubated on ice for 15 min, centrifuged at 4°C for 10 min at 14,000 rpm, and the supernatant (cytoplasmic fraction) was carefully removed. The nuclear pellet was then washed once in 100 µl of Buffer “E”, and re-centrifuged as above. The supernatant was once again removed and combined with the rest of the cytoplasmic fraction. The washed pellet was resuspended in 100 µl of Buffer “F” (20 mM HEPES [pH 7.9], 400

mM NaCl, 1 mM EDTA, 1 mM EGTA [pH 8.0], 1 mM DTT, 1 mM Na3VO4, 5 µg/ml

leupeptin, 62.5 µg/ml aprotinin, 1 µg/ml pepstatin, and 1 mM PMSF), vortexed

vigorously for 15 sec, and incubated on ice for 15 min. The sample was then centrifuged for 15 min at 14,000 rpm at 4°C and the supernatant (nuclear fraction) was then carefully removed and stored at -80°C. For long-term storage (>1 week), the fractions were

supplemented with glycerol to 20% (i.e., by adding 140 µl of 100% glycerol to cytoplasmic fractions and 25 µl to nuclear fractions).