8 well microscopy slides

All media were supplemented with 10% fetal calf serum (PAN Biotech, Aidenbach, Germany) and glutamax (Thermofisher Scientific, Waltham, MA, USA). All cell lines were incubated at 37 °C in 5% CO₂ in a humidified atmosphere.
A431 cells were maintained in DMEM (Thermofisher Scientific) supplemented as indicated. For microscopy experiments, cells were seeded at a density of 30,000 cells per well a day before the experiment, or 15,000 cells per well two days before, in 8-well microscopy slides (Ibidi, Gräfelfing, Germany). For binding and internalization assays with radioactive compounds, A431 cells were seeded in 6-well plates at a density of 300,000 cells/well, 2 days before the experiment.
HEK293T cells (ATCC) were maintained in DMEM, supplemented as indicated. For microscopy experiments, HEK293T cells were seeded at a density of 60,000 cells per well one day before the experiment, or 30,000 cells two days before. For binding and internalization assays with radioactive compounds, HEK293T cells were seeded in 6-well plates at a density of 300,000 cells/well, 2 days before the experiment.
SKOV3 cells were maintained in DMEM (Thermofisher Scientific) supplemented as indicated. For microscopy experiments, SKOV3 cells were seeded at a density of 40,000 cells/well one day before the experiment, or 20,000 cells/well two days before in 8-well microscopy slides (Ibidi).

HeLa cells were seeded, grown on coverslips or 8-well microscopy slides (ibidi) and transfected with either wild type or mutant Nix or Bnip3 together with wild type or mutant LC3B constructs using JetPRIME transfection reagent according to manufacturer’s instructions. 24 hrs post transfection cells were fixed in 2% or 4% paraformaldehyde and permeabilized with 0,1% Triton X-100 solution. Cells were blocked in 5% BSA/PBS/0.1% Triton X-100 for 1 hr at room temperature or 4 °C overnight. Primary and secondary antibodies were diluted in blocking solution and washed in 0,1% Triton X-100/PBS. Coverslips were mounted in Prolong mounting media (Invitrogen). Cells in 8-well microscopy slides were placed in PBS following staining. Cells were imaged using a Zeiss AxioVision or DeltaVision RT microscope system (Applied Precision). Quantification of Nix-LC3B colocalization was performed as follows: for each Nix construct, LC3B positive dots (signals) were numbered in 100 cells. Only clear and well-defined LC3B signals are taken into consideration, while weak and oversized signals were excluded from the analysis.