Supplementary Figure 6: Deficiency of ATG5 halts RIPK1-dependent mitophagy during ECM-detachment and mitochondrial ROS generation during ECM-detachment is a consequence of RIPK1-mediated mitophagy.

(a) Immunoblotting against ATG5-ATG12 conjugate and GAPDH was used to confirm the ATG5 knockout clone in MCF-10A cells using CRISPR/Cas9. Experiments were performed a minimum of three independent times with similar results. (b) MCF-10A EV, ATG5KO, and HA-ATG5-R/E cells were grown in Att or Det for 24h treated with DMSO or Nec1 (10μM). Cell viability was measured using AlamarBlue. n=3 independent biological samples. (c) MCF-10A EV, ATG5KO, and HA-ATG5-R/E cells were grown in Att or Det for 24h treated with DMSO or Nec1 (10μM). Lysates were immunoblotted for Tom20 and β actin. Experiments were performed a minimum of three independent times with similar results. (d) 10A-Bcl2 cells were grown in Att or Det for 24h treated with DMSO, Nec1 (10μM), BAFA (10nM), MitoTEMPO (10μM) or CCCP (10μM). Cells were then cytospun onto slides, fixed, and stained with MitoSOX Red (5μM) (red) and DAPI (blue). Representative images of each condition are shown (40x). (e) Relative fluorescence measurements for each condition in (d) were determined using Fiji. For fluorescence calculations, n = 5 images. (f) H460 cells were grown in Att or Det for 24h treated with DMSO, Nec1 (10μM), BAFA (10nM), MitoTEMPO (10μM) or CCCP (10μM). Cells were then cytospun onto slides, fixed, and stained with MitoSOX Red (5μM) (red) and DAPI (blue). Representative images of each condition are shown (40x). (g) Relative fluorescence measurements for each condition in (f) were determined using Fiji. For fluorescence calculations, n = 5 images. All results are presented as means +/- SEM and statistical significance was calculated using a Student’s two tailed t-test. p<0.05 is statistically significant. N.S., not significant. Scale bars, 100 μm. Unprocessed original scans of blots are available in Supplementary Fig. 9.