DOI: 10.5061/dryad.z612jm6r4
title: Autophagolysosomal exocytosis inverts Src kinase and other N-myristoylated proteins onto the cell surface in cancer

description: Overexpression of the protoconogene Src is common to a wide variety of
 cancers. Here, we demonstrate that Src is noncanonically inverted onto the
 cell surface in cancer both in vitro and in vivo. We identify
 autophagolysosomal exocytosis (ALE) as a secretory mechanism prominent in
 cancer cell lines and show that Src is the prototypical example for a
 family of membrane-anchored proteins that are inverted by this process.
 Furthermore, we show that this extracellular membrane-associated Src
 (eSrc) is present in primary tumors and that anti-Src antibody-based
 therapies mediate tumor cell killing in vitro and in vivo. Collectively,
 this work presents a secretory mechanism by which intracellular
 N-myristoylated proteins, prototypically Src, are inverted on the cell
 surface in cancer and can be targeted with antibody therapeutics.
description: # Autophagolysosomal exocytosis inverts Src kinase and other
 N-myristoylated proteins onto the cell surface in cancer Dataset DOI:
 [10.5061/dryad.z612jm6r4](https://doi.org/10.5061/dryad.z612jm6r4) ##
 Description of the data and file structure Files
 "Acquisitions_2023-08-03_EK-170_IMP1088_siRNA_anti-Src800_anti-tubulin680.zip 110.88 KB", "Acquisitions_2023-08-30_EK190_SRC_siRNA.zip 114.04 KB", and "Acquisitions_2023-09-20_EK188_ATG7IN2_and_EK190_SRC_siRNA.zip 245.13 KB" are fluorescence immunoblots collected on a LiCOR Odyssey CLx (use courtesy of the Renslo Lab at UCSF). In-file metadata have the wrong timestamps due to the fact that the operating computer in the Renslo Lab runs an outdated version of Windows. Dates recorded are listed in the titles. Data are exported by LiCOR's Image Studio 6 as .zip files and can be directly imported into Image Studio Lite (free version) for analysis and processing. File "Select_Images_corresponding_to_Figure_3E.zip 366.64 MB" contains microscopy images of cells stained for LC3 (green) and Src (red), counterstained with DAPI (blue), and brightfield (black/white). Data were acquired on a Crest LFOV Spinning Disk/ C2 Confocal microscope in the Center for Advanced Light Microscopy at UCSF. Data were acquired on the Nikon on-board software. Images were analyzed and processed in ImageJ/FIJI (freely available: [https://imagej.net/software/fiji/downloads](https://imagej.net/software/fiji/downloads)). ### Files and variables **Naming Conventions:** Files with titles beginning "Acquisition_" are titled Acquisition [date acquired] [notebook page] [treatment]. These files are .zip files, but are meant to be directly imported into LiCOR's Image Studio 6 or Image Studio Lite (free version) for interpretation. The folder Select_Images_corresponding_to_Figure_3E.zip is a .zip file that can be uncompressed traditionally. This folder contains .nd2 files, an extension from the Nikon acquisition software that can be opened with ImageJ or FIJI. The naming convention for the files in the subfolder corresponds to [notebook page] [treatment] [field of view] (e.g., EK213_DMSO001 is notebook page EK213, DMSO treated, FOV 001). Single color controls from the experiment are annotated [notebook page] [color] [treatment] [field of view]. #### File: Acquisitions_2023-09-20_EK188_ATG7IN2_and_EK190_SRC_siRNA.zip **Description:** Contains fluorescence immunoblots for Src and tubulin of cell treated with the autophagosomal biogenesis inhibitor ATG7-IN-2. Ladder and lane annotations can be found in Figure S11D. Chamaeleon duo protein ladder is visualized on the far left. Src is visualized in the 800 nm channel and tubulin is visualized in the 700 nm channel. #### File: Acquisitions_2023-08-30_EK190_SRC_siRNA.zip **Description:** Contains fluorescence immunoblots for Src and tubulin of cells treated with either a negative control siRNA or an siRNA targeting the gene *SRC*. Ladder and lane annotations can be found in Figure S4C. Chamaeleon duo protein ladder is visualized on the far left. Src is visualized in the 800 nm channel and tubulin is visualized in the 700 nm channel. #### File: Acquisitions_2023-08-03_EK-170_IMP1088_siRNA_anti-Src800_anti-tubulin680.zip **Description:** Contains fluorescence immunoblots for Src and tubulin of cells treated with N-myristoyaltransferase inhibitor IMP-1088 or vehicle. Ladder and lane annotations can be found in Figure S8B. Chamaeleon duo protein ladder is visualized on the far left. Src is visualized in the 800 nm channel and tubulin is visualized in the 700 nm channel. #### File: Select_Images_corresponding_to_Figure_3E.zip **Description:** Additional images used in the quantitation of LC3/Src colocalization in Figure 3. Files include microscopy images of HCC1569 cells pretreated with the small molecule inhibitor IMP-1088 or a vehicle (dimethylsulfoxide, DMSO) control, and swollen with chloroquine prior to treatment. Cells were labeled with three markers, corresponding to: Src (protein of interest) is in the red/647 channel; LC3 (autophagosomal marker) is in the green/488 channel; DAPI (nuclear counterstain) is in the blue channel. Brightfields are also included. ## Access information Data was derived from the following sources: * Wells Lab, UCSF (experimental) * Renslo Lab, UCSF (instrument) * Center for Advanced Light Microscopy, UCSF (instrument)