Supplementary Figure 1: NF-κB promotes CYLD transcription during ECM-detachment and overexpression of RIPK1 lowers cell viability. | Nature Cell Biology

Supplementary Figure 1: NF-κB promotes CYLD transcription during ECM-detachment and overexpression of RIPK1 lowers cell viability.

From: RIPK1-mediated induction of mitophagy compromises the viability of extracellular-matrix-detached cells

Supplementary Figure 1

(a) MCF-10A EV cells were grown in either Att or Det conditions for 24h. Lysates were immunoblotted as noted. (b) Human mammary epithelial cells (HMEC) were grown in either Att or Det conditions for 24h. Lysates were immunoblotted as noted. (c) 10A-Bcl2 cells were grown in either Att or Det conditions for 24h. Lysates were immunoblotted as noted. (d) 10A-Bcl2 cells were grown in Att or Det for 24h. Relative expression of CYLD was measured by qRT-PCR. n=3 independent biological samples. (e, f) 10A-Bcl2 cells were grown in Att or Det for 24h treated with either DMSO or Bay-117082 (10μM). Relative expression of CYLD was measured by qRT-PCR. n=3 independent biological samples (e). Lysates were immunoblotted as noted (f). (g) HMEC cells were grown in Att or Det conditions treated with DMSO or Nec1 (10μM) for 24h. Cell viability was measured using AlamarBlue. n=3 independent biological samples. (h) 10A-Bcl2 cells transfected with empty vector (EV) or RIPK1 wildtype (RIPK1 WT) were grown in Att for 24h. Lysates were immunoblotted as noted. (i) Cell viability was measured using AlamarBlue following transfection of Att 10A-Bcl2 cells after being treated with either DMSO or Nec1 (10μM) for 24h. n=3 independent biological samples. (j) Left panel, Bax/Bak null (Bax(-/-)/Bak(-/-)) MEFs transfected with empty vector (EV), RIPK1 wildtype (RIPK1 WT), or RIPK1 kinase mutant (RIPK1 K45A) were grown in Att for 24h. Lysates were immunoblotted as noted. (k) Cell viability was measured using AlamarBlue following transfection after being grown in Att for 24h. n=3 independent biological samples. (l) BT474, T47D, and MCF7 cells were grown in either Att or Det conditions for 24h. Lysates were immunoblotted as noted. (m) HeLa, HCT116, and H460 cells were grown in either Att or Det conditions for 24h. Lysates were immunoblotted as noted. 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. All western blotting experiments were independently repeated a minimum of three times with similar results. Unprocessed original scans of blots are available in Supplementary Fig. 9.

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