Cell culture, transfection, and drug treatments

Day 1: Cell Plating

1. 35-mm glass-bottom microscope dishes (Cell Vis #D35201.5N) were coated with 10 mg/ml of fibronectin for 5 hours at 37°C. 
2. After 5 hours, the fibronectin solution was removed, the microscope dishes were rinsed with 1mL PBS to remove excess fibronectin.
3. U-2 OS cells were seeded at 0.5 × 10⁵ cells/ml using 2mL Full McCoy's media (+10% FBS and pen/strep).

Day 2: Transfection

1. Cells were transfected with 100ng of GFP-G3BP1 and 75ng of mCh-KDEL expression plasmids using Lipofectamine 3000 (according to manufacturer's protocol).
2. Transfection mix was removed after 5 hours at 37°C.
3. Cells were rinsed with 1mL of Full McCoy's media.
4. Cells were replenished 2mL Full McCoy's media.

Day 3: Live-cell imaging

1. Warm the incubator on the spinning disc confocal microscope to 37°C with 5% CO₂. 
2. Cells were treated with 0.5mM NaAsO₂ for 1 hour to induce the formation of stress granules. 
3. Remove media, rinse with full media, then replenish with 2mL full media to washout NaAsO₂ and initiate the stress granule disassembly process. Incubate for 40 minutes.
4. Identify cells with suitable expression of GFP-G3BP1 and mCh-KDEL prior to the addition of ISRIB.
5. Spike in ISRIB to a final concentration of 200nM.
   1. ISRIB accelerates the disassembly process (~10 minutes). Begin imaging quickly after ISRIB addition.
6. Begin capturing 2 minute time-lapse movies with frames captured every 5 seconds of the previously identified cells.

Analysis of SG fission and ER localization

1. Open time-lapse movies in ImageJ and manually identify stress granule fission events.
2. Once fission events are identified.
3. Use the segmented line tool. Double-click to increase the thickness of the line to 10. 
4. Draw a segmented line across the longitude-axis of the stress granule through the fission site.
5. Measure and record the intensity along the segmented line for the green channel.  
6. Measure and record the intensity along the segmented line for the red channel.  
7. Plot and graph the intensity measurements in Excel, graphpad, etc.

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