Supplementary Figure 5: Inhibition of RIPK1 or PGAM5 blocks ECM-detachment-induced mitophagy.

(a) H460 cells were grown in Att or Det for 24h treated with DMSO, Nec1 (10μM), BAFA (10nM) or CCCP (10μM). Cells were then cytospun onto slides, fixed, and stained with Mito-Tracker Red (200nM) (red) and DAPI (blue). Representative images of each condition are shown (40x). (b) Relative fluorescence measurements for each condition in (a) were determined using Fiji. For fluorescence calculations, n = 5 images. (c) H460 pLKO, shRIPK1, shPGAM5, and shPINK1 cells grown in Att or Det for 24h. Cells were then cytospun onto slides, fixed, and stained with Mito-Tracker Red (200nM) (red) and DAPI (blue). Representative images of each condition are shown (40x). (d) Relative fluorescence measurements for each condition in (c) were determined using Fiji. For fluorescence calculations, n = 5 images. (e) 10A-Bcl2 cells were grown in Att or Det for 24h treated with DMSO, Nec1 (10μM), or Nec1 (10μM) and CCCP (10μM). Cell viability was measured using AlamarBlue. Viability measurements are normalized to Att 10A-Bcl2 DMSO. n=3 independent biological samples. 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. Scale bars, 100 μm.