Volume 1, Issue 2, Pages 273-286 (August 2016) Graphene Oxide Nanosheets Stimulate Ruffling and Shedding of Mammalian Cell Plasma Membranes  Chao Sun, Devin L. Wakefield, Yimo Han, David A. Muller, David A. Holowka, Barbara A. Baird, William R. Dichtel  Chem  Volume 1, Issue 2, Pages 273-286 (August 2016) DOI: 10.1016/j.chempr.2016.06.019 Copyright © 2016 Elsevier Inc. Terms and Conditions

Chem 2016 1, 273-286DOI: (10.1016/j.chempr.2016.06.019) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Confocal Micrographs of A555-CTxB-Labeled RBL Cells (A and B) Top (A) and bottom (B) layers of RBL cells that started at a high density and were incubated with GO (100 μg/mL) prior to washing and proliferation. (C and D) Top (C) and bottom (D) layers of RBL cells that started at a high density and were not treated with GO prior to washing and proliferation. (E and F) Top (E) and bottom (F) layers of RBL cells that started at a low density and were incubated with GO (100 μg/mL) prior to washing and proliferation. (G and H) Top (G) and bottom (H) layers of RBL cells that started at a low density and were not treated with GO prior to washing and proliferation. Left, A555-CTxB channel; middle, bright field (BF); right, overlay. Scale bars represent 50 μm. See also Figure S1. Chem 2016 1, 273-286DOI: (10.1016/j.chempr.2016.06.019) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 3D Reconstructions of A555-CTxB-Labeled RBL Cells Incubated with GO-BODIPY (A) Two experimental protocols for RBL cell labeling and GO incubation. Left path: RBL cells were first labeled with A555-CTxB and then incubated with GO-BODIPY for 4 hr Right path: RBL cells were first incubated with GO-BODIPY for 4 hr, after which they were labeled with A555-CTxB. (B and C) 3D reconstructions of RBL cells incubated with GO-BODIPY after A555-CTxB labeling (left path in A). Blue, green, and white arrowheads mark the same cells shown with different 3D perspectives in (B) and (C). (D and E) 3D reconstructions of RBL cells incubated with GO-BODIPY before A555-CTxB labeling (right path in A). Purple and gray arrowheads point to the same cells shown with different 3D perspectives in (D) and (E). These are the only two cells (or part of cells) within the field of view. Other A555-CTxB labeled structures have been identified to be non-cell structures from the bright-field image and F-actin labeling (see below). See also Figures S2 and S3. Chem 2016 1, 273-286DOI: (10.1016/j.chempr.2016.06.019) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 SEM Micrographs of RBL Cells Incubated in the Presence and Absence of GO (A–C) RBL cells were incubated in BSS buffer for 4 hr and then fixed. Scale bars represent (A) 50 μm and (B and C) 10 μm. (D–F) RBL cells were incubated with GO (100 μg/mL) in BSS buffer for 4 hr and then fixed. Scale bars represent (D) 50 μm and (E and F) 10 μm. See also Figure S3. Chem 2016 1, 273-286DOI: (10.1016/j.chempr.2016.06.019) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Confocal Microscopy of Membrane Fragments, GO-BODIPY, and RBL Cells after A555-CTxB and Phalloidin Labeling (A) Confocal micrographs of A555-CTxB, GO-BODIPY, and A647-phalloidin channels, as well as the corresponding bright-field image of RBL cells incubated with GO-BODIPY. White arrowheads in the A555-CTxB channel image indicate membrane fragments with associated GO-BODIPY particles. Scale bar represents 20 μm. (B) Confocal micrographs of A555-CTxB-labeled membrane ruffles still attached to an underlying RBL cell. Scale bar represents 10 μm. (C and D) 3D reconstructions of the RBL cell shown in (B). Overlaid A555-CTxB, GO-BODIPY, and A647-phalloidin channels are shown in (C). The 3D reconstruction shown in (D) is the same as in (C) but now with the A555-CTxB channel removed. See also Figure S5. Chem 2016 1, 273-286DOI: (10.1016/j.chempr.2016.06.019) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Confocal Micrographs of RBL Cells and Membrane Fragments after A488-IgE and Phalloidin Labeling (A) RBL cells incubated with GO (100 μg/mL, 4 hr), washed with BSS, and labeled with A488-IgE were fixed and labeled for F-actin with A647-phalloidin. (B) Membrane fragments observed elsewhere in the same sample. (C) A representative confocal micrograph at lower magnification reveals the abundance of membrane fragments (green) around RBL cells (blue). (D) Membrane fragments from RBL cells treated with 100 μg/mL GO for 4 hr and washed before incubation with unlabeled IgE, followed by the addition of A488-IgE. (E) Membrane fragments from RBL cells treated with 100 μg/mL GO for 4 hr and washed before A488-IgE labeling. Scale bars represent 20 μm. See also Figure S5. Chem 2016 1, 273-286DOI: (10.1016/j.chempr.2016.06.019) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 GO-Induced Membrane Ruffling and Shedding in Other Mammalian Cell Types Confocal micrographs of (A) control MDA-MB-231 human breast cancer cells labeled with A555-CTxB, (B) MDA-MB-231 cells first labeled with A555-CTxB and then incubated with GO for 4 hr, (C) MDA-MB-231 human breast cancer cells incubated with GO for 4 hr and then labeled with A555-CTxB, (D) control NIH-3T3 fibroblast cells labeled with A555-CTxB, (E) NIH-3T3 fibroblast cells first labeled with A555-CTxB and then incubated with GO for 4 hr, and (F) NIH 3T3 cells first incubated with GO for 4 hr and then labeled with A555-CTxB. Scale bars represent 20 μm. Chem 2016 1, 273-286DOI: (10.1016/j.chempr.2016.06.019) Copyright © 2016 Elsevier Inc. Terms and Conditions