*4.14. Immunofluorescence, Cell Size and Stress Fiber Analysis*

12Z cells were seeded in 6-well plates and subject to siRNA knockdown for a nontargeting control, *LINC01133a* and *LINC01133b* oligos, as described above. The cells were trypsinized 48 h after transfection, 20,000 cells from each treatment re-plated on 8-well chamber slides, and then 24 h later the cells were fixed and processed for immunofluorescence as previously described [60]. Cells were stained for F-actin (Rhodamine conjugated Phalloidin, Invitrogen Cat. R415) and counterstained for DAPI. Cells were then imaged with a Leica SP8 confocal microscope using an ×63 glycerol objective and images processed using ImageJ.

We measured cell area in ImageJ using a previously described protocol (https://www. youtube.com/watch?v=IeicxaeMUwA) (accessed on 15 March 2021). First, we used the line tool to draw a line over a scale bar on one image and selected "measure" under the analyze menu. Next, we selected "set scale" under the analyze menu, and entered the number of pixels for 20 µM, a known distance of 20, and µM as the unit of length, and ticked the "Global" box so that this scale would be applied to all images. To measure the cross-sectional area of cells we chose the free-hand selection tool, mapped the outline of the first cells with the computer mouse, and pressed "measure" under the analyze menu. We then repeated this for all cells in each image.

We used ImageJ to measure fluorescence intensity in control and *LINC01133a* knockdown cells followed an established technique https://theolb.readthedocs.io/en/latest/ imaging/measuring-cell-fluorescence-using-imagej.html (accessed on 15 March 2021) [61]. Briefly, we used drawing tools to select cells, chose "area integrated intensity" and "mean grey value" in the "set measurements" menu, and then "measure" from the analyze menu. We then measured an area near the cell with no fluorescence as a background control. We then repeated this process until all cells in the field had been measured. We then calculated the corrected total cell fluorescence (CTCF) for each cell in each image using the following formula (Equation (1)):

CTCF = Integrated Density − (area of selected cell × mean fluorescence background) (1)
