Cell lines useful for assessing modulation of autophagy
View Patent ↗Methods for screening for modulators of autophagy are disclosed. Methods for identifying genes whose expression inhibits autophagy, as well as genes whose expression promotes autophagy, are disclosed. Also disclosed are methods for identifying compounds that stimulate autophagy, as well as compounds that inhibit autophagy. Cell lines that may be used in the methods of identification are also disclosed.
1. A cell line comprising an autophagy-defective cell that stably expresses Sequestosome 1 (p62) protein linked to a label molecule.
2. The cell line of claim 1 wherein the cell expresses Enhanced Green Fluorescent Protein (EGFP)-Sequestosome 1 (p62).
3. The cell line of claim 1 wherein the cell has reduced expression of one or more genes selected from the group consisting of Beclin1, Atg5, and Atg7.
4. The cell line of claim 1 wherein the cell is apoptosis-defective.
5. The cell line of claim 1 wherein the cell is a mouse cell.
6. The cell line of claim 1 wherein the cell is an immortalized baby mouse kidney cell.
7. A method for identifying stimulators of autophagy comprising the steps of:
(A) contacting the test cell of claim 1 expressing a marker of protein aggregation with a compound;
(B) subjecting the cell to metabolic stress;
(C) performing analysis on the test cell to determine the level of the marker; and
(D) comparing the level of the marker in the test cell with that of a control cell not contacted with said compound,
wherein a lower level of aggregates comprising the marker in the test cell compared to that demonstrated by the control cell not contacted with said compound indicates that the compound is capable of stimulating autophagy.
8. The method of claim 7 wherein the test cell is apoptosis-defective.
9. The method of claim 7 wherein the test cell is an immortalized baby mouse kidney cell.