Halo-LC3B processing assay to assess autophagy

Generating HeLa cells expressing HaloTag-LC3B using pMRX-IP-HaloTag7-LC3 from Mizushima lab (Addgene #184899; DOI: 10.7554/eLife.78923).

Seed HeLa cells at 100-150K cells/well in 12-well plate one day before.

Next day, incubate cells with complete DMEM medium and 50nanomolar (nM) JF646 HaloTag ligand (Promega) for 1h 0m 0s, and then wash twice with 1xPBS. The non-starved samples can be harvested immediately by trypsinization (step 5).

To induce autophagy by starvation, treat cells with EBSS buffer (Gibco) for desired period.

After the treatment, harvest the cells by trypsinization.

Wash the wells with 1x PBS.

Incubate the wells with 0.5mL trypsin at 37°C for 0h 5m 0s.

Add 0.5mL complete medium into each well.

Transfer cells into pre-chilled Eppendorf tube, spin 2000x g.

Aspirate off the liquid.

Resuspend the cell pellets in 30µL of lysis buffer and incubate On ice for 0h 30m 0s.

Centrifuge the cell lysate at 21000x g. Transfer the cleared lysate into another tube, and measure protein concentration nanodrop spectrophotometer (Thermo Fisher).

For each sample, load 20µg clarified lysates onto NuPAGE 4-12% Bis-Tris Gel (Thermo Fisher).

For in-gel fluorescence imaging, the gel was immediately visualized with ChemiDoc MP imaging system (Bio-Rad) after SDS PAGE. Band intensities are acquired by exciting samples at 546 nm (mCherry signal) and 647 nm (JF646 HaloTag ligand signal).