Glass coverslip bottomed plastic dishes

Control and TAGLN-KO cells were plated in glass coverslip-bottomed plastic dishes (Mat Tek Co, Ashland, MA) containing 2.5 x 10 5 cells/dish and grown in complete growth media for 48 h to allow recovery of LDLR on the cell surface. Cells were then washed twice with ice cold PBS and incubated for 4 h in pre-chilled DMEM supplemented with 0.1% BSA at 4°C with 50 µg/ml LDL containing 20% DiI-LDL (Molecular Probes, Eugene, OR). Next, cells were thoroughly washed with ice-cold PBS and fluorescent LDL bound on the cell surface was imaged with a Zeiss 880 confocal microscope (Jena, Germany) using a Zeiss 40x Plan-Apochromat objective lens (N.A. 1.3). DiI-LDL (Molecular Probes, Eugene, OR) fluorescence in labelled cells was acquired using 561nm excitation, an emission bandwidth of 565-700nm, a pinhole set to 1 A.U., a pixel format that varied with optical zoom to provide a lateral pixel size of 110nm, and an interslice thickness of 640nm (total slice number/ stack varied with cell height). In addition, a portion of the cells, after thorough washing with cold PBS, were incubated at 37°C for 2 hours in complete growth media to allow LDL internalization, and then imaged as described above.