Journal: PLoS Biology

Article Title: Role of the AP-5 adaptor protein complex in late endosome-to-Golgi retrieval

doi: 10.1371/journal.pbio.2004411

Figure Lengend Snippet: (A) Immunofluorescence microscopy of control or AP5Z1 knockout cells depleted of VPS35 and pulse chased with anti-CIMPR, as shown in . Individually, the knockout of AP5Z1 or the knockdown of retromer caused a reduction in the retrieval of CIMPR back to the TGN region; this was further exacerbated when the knockout and knockdown were combined. The dotted lines indicate the boundaries of each cell. Scale bar: 20 μm. (B) Quantification of the retrieval defect of CIMPR, using the CX7 automated microscope and a Colocalisation Bioapplication. The increase in CIMPR (Total Object Count) that failed to be retrieved back to the TGN region was 1.48 ± 0.10 for VPS35 kd, 1.26 ± 0.07 for KO1, and 1.97 ± 0.14 for the combined KO1 plus VPS35 kd. More than 1,500 cells were scored per knockdown condition (7 independent repeats; error bars indicate SEM). The raw data can be found in . (C) Whole cell lysates from control and AP5Z1 knockout lines. The steady-state levels of CIMPR are not significantly affected by loss of AP-5 or depletion of VPS35. In addition, the surface expression of CIMPR did not change substantially in the knockdown and knockout cells when compared to control, as determined by flow cytometry (VPS35kd 1.22 ± 0.36, KO1 1.12 ± 0.18, KO1+VPS35kd 1.25 ± 0.44). AP, adaptor protein; CHC, clathrin heavy chain; CIMPR, cation-independent mannose 6-phosphate receptor; con, control; KO, knockout; TGN, trans-Golgi network.

Article Snippet: To quantify increased fluorescent spot size or retrieval deficits, we used automated high content screening (HCS) microscopes—either an ArrayScan VTI microscope (Cellomics/Thermo Scientific) or its upgrade, a Cell Insight CX7 Microscope (Thermo Scientific).

Techniques: Immunofluorescence, Microscopy, Knock-Out, Expressing, Flow Cytometry

Journal: PLoS Biology

Article Title: Role of the AP-5 adaptor protein complex in late endosome-to-Golgi retrieval

doi: 10.1371/journal.pbio.2004411

Figure Lengend Snippet: (A) Immunofluorescence microscopy of cells treated with monensin for 90 min followed by a 2.25-h washout. Individually, the knockdown of retromer or the knockout of AP5Z1 caused a reduction in the retrieval of GOLIM4 back to the Golgi; this was exacerbated when the knockout and knockdown were combined. Scale bar: 20 μm. (B) Quantification of the retrieval defect of GOLIM4 was performed using a CX7 automated microscope and a Colocalisation Bioapplication with an adapted protocol that was originally designed for quantifying CIMPR retrieval (see ). The increase in GOLIM4 (Total Object Area) that failed to be retrieved back to the Golgi region was 1.40 ± 0.11 for VPS35 kd, 1.51 ± 0.12 for AP5Z1_KO1 knockout, and 2.20 ± 0.08 for VPS35 kd + AP5Z1 KO. More than 1,500 cells were scored per knockdown condition (3 independent repeats; error bars indicate SEM). The raw data can be found in . AP, adaptor protein; CIMPR, cation-independent mannose 6-phosphate receptor; con, control; KO, knockout; Mon, monensin; WO, washout.

Article Snippet: To quantify increased fluorescent spot size or retrieval deficits, we used automated high content screening (HCS) microscopes—either an ArrayScan VTI microscope (Cellomics/Thermo Scientific) or its upgrade, a Cell Insight CX7 Microscope (Thermo Scientific).

Techniques: Immunofluorescence, Microscopy, Knock-Out