(A–G) J774 cells were incubated with Alexa546-agLDL (red in D–F) for the indicated time periods either on ice (A, D) or at 37°C (B, C and E, F), fixed, and labeled with filipin (A–C) and Alexa488-phalloidin (green in D–F). Arrows show agLDL surrounded by F-actin-rich structures. Arrowheads show agLDL that is not surrounded by F-actin. (G) Quantification of average filipin intensity associated with agLDL touching cells and with agLDL not interacting with cells (extracellular), the latter reflecting the baseline FC content of agLDL. For statistical tests values were compared to cells incubated on ice. (H–N) J774 cells were either left untreated (H, K) or pretreated with toxin B (I, L) or C3 transferase (J, M), then incubated with Alexa546-agLDL (red in K–M) in the presence of inhibitors, fixed, and labeled with filipin (H–J) and Alexa488-phalloidin (green in K–M). Arrows show agLDL surrounded by F-actin-rich structures. Arrowheads show agLDL that is not surrounded by F-actin. (N) Quantification of average filipin intensity associated with agLDL touching cells and agLDL not interacting with cells. For statistical tests values were compared to non-treated cells. Error bars, SEM for 10 fields containing >100 cells. * p<0.05, ** - p<0.01, n.s. – non-significant difference. All images were taken on a wide-field fluorescence microscope. Scale bar: 10μm.

J774 cells were incubated with agLDL reconstituted with either CE (A, B, E) or cholesteryl ether (C, D, F). After 30 minutes incubation, cells were fixed and labeled with filipin (B, D) or Alexa488-phalloidin (green in E, F). Arrowheads show agLDL-macrophage contact areas. (G) Fluorescence power of Alexa488-phalloidin associated with agLDL in contact with cells was quantified for cholesteryl ether- and CE-containing agLDL. Error bars, SEM for 10 fields with >100 cells. ** p<0.01. Scale bars: 10μm. Images (A–D) were taken on a wide-field fluorescence microscope, (E, F) – single confocal slice superimposed with DIC.

J774 cells were incubated with Alexa546-agLDL (red in C, D) in the presence (B, D) or absence (A, C) of bafilomycin A1, fixed, and labeled with filipin (A, B) and Alexa488-phalloidin (green in C, D). (E) Quantification of the fluorescence power of F-actin around agLDL; nt – not treated, bm – bafilomycin A1 treated. (F) Quantification of the average filipin intensity in parts of agLDL touching cells. For statistics values were compared to non-treated cells. Error bars: SEM for 10 fields containing >100 cells. ** p<0.01. Scale bar: 10μm. Images were taken on a wide-field fluorescence microscope.

J774 cells were incubated with Alexa546-agLDL for 30 minutes. Samples were either fixed and labeled for F-actin with fluorescent phalloidin (A, B) or labeled with fluorescent CtB before fixation (C, D). (A) Merged images showing F-actin (green) and agLDL (red). Arrowheads, agLDL associated with F-actin-rich structures. Inset, an enlarged view of an F-actin structure around agLDL. (B) Transmitted-light DIC image. (C, C') Alexa-488 CtB labeling showed that agLDL-containing compartments are connected to the cell surface. Arrowheads, compartments containing agLDL (red) also label with CtB (green). (D) Transmitted-light DIC image. Images in A, C are single confocal slices. C' is an axial slice through the 17μm thick confocal stack at the position of the green line in C. Axial-step is 0.45μm. Scale bars: 10μm.

(A) LAL protein levels in control and siRNA transfected cells. All lanes are from the same blot but are shown in two panels to remove extraneous lanes. (B, C) Transfected cells were incubated with Alexa546-agLDL (red) and labeled with Alexa488-phalloidin (green). Arrowheads, agLDL-macrophage contact areas. (D) Fluorescence power of Alexa488-phalloidin around agLDL was quantified for non-transfected cells, cells transfected with non-targeting siRNA, cells transfected with 2 different LAL-specific siRNAs, and cells treated with phLAL after LAL knock-down. For statistical analysis values were compared to non-targeting siRNA-transfected cells. Error bars: SEM for 14 fields containing >150 cells. ** p<0.01, n.s. – not significant. Scale bar: 15μm. Images are single confocal slices superimposed with DIC.

Blue balls, agLDL. Red dots and lines, FC. Purple-shaded areas, acidified compartments. Green hatches, F-actin. Lys, lysosome.