Increased abundance of ER sheets upon loss of INPP5K or ARL6IP1. (A) Western blots of WT HeLa cells transfected with the indicated siRNAs showing depletion of INPP5K or ARL6IP1 proteins. (B) Top row: Representative confocal images of HeLa cells transfected with the indicated siRNAs and expressing the ER marker EGFP-Sec61β. High magnification of the regions enclosed by dashed boxes in the top row are shown in the bottom row. Scale bars: 5 µm in the top row, 2 µm in the bottom row. Solid red lines denote the edge of the cell. Note the predominant presence of ER tubules in the control cell, but the predominance of ER sheets throughout the cytoplasm of INPP5K or ARL6IP1 siRNA-treated cells. (C) Occupancy of the ER (both tubules and sheets) per unit area of each cell analyzed based on the analysis described in B for cells treated with control or INPP5K siRNA and expressing the indicated EGFP-INPP5K constructs. Automatic thresholding was applied (see Materials and methods for details) to select the total area occupied by the ER network (delineated by solid lines). The occupancy of the ER network per unit cell area serves as a proxy for the abundance of peripheral ER sheets and was calculated by dividing the pixel area occupied by the ER by that of the total cell area. Pooled data from three independent experiments are presented as scattered dots (solid black bars indicating the mean ± SD) and analyzed by two-tailed t-tests. n = 28 cells for control or INPP5K siRNA, 25 cells for INPP5K siRNA + EGFP-INPP5K^WT, 25 cells for INPP5K + EGFP-INPP5K^D192A, 21 cells for INPP5K + EGFP-INPP5K^D361G. ****, p < 0.0001; n.s., p > 0.05 (INPP5K siRNA + EGFP-INPP5K^D192A vs. INPP5K siRNA, p = 0.0541; INPP5K siRNA + EGFP-INPP5K^D361G vs. INPP5K siRNA, p = 0.057).