Supplementary Figure 3: RIPK3 nor MLKL are required for ECM-detachment induced RIPK1 signalling. | Nature Cell Biology

Supplementary Figure 3: RIPK3 nor MLKL are required for ECM-detachment induced RIPK1 signalling.

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

Supplementary Figure 3

(a, b) MCF-10A pLKO (a,b), shRIPK3 (a) and shMLKL (b) cells were grown in Att and Det for 48h. Caspase-3/7 activation was measured 48h after plating in Att and Det using the CaspaseGlo 3/7 assay. Caspase activation is represented as a ratio of Det value over Att value. n=3 independent biological samples. (c) Immunoblotting against RIPK3 and β actin was used to confirm the RIPK3 knockdown (shRIPK3) in H460 cells grown in Det for 24h. Experiments were performed a minimum of three independent times with similar results. (d-f) Cell viability (d), caspase activation (e), and anchorage-independent growth in soft agar (f) was measured in Att and Det H460 pLKO and shRIPK3 cells at 24h. Viability was measured using AlamarBlue, caspase activation using CaspaseGlo 3/7 assay, and after 7 days, images of soft agar growth taken following INT-violet staining. Colony count was determined using ImageJ. n=6 independent biological replicates for soft agar and n=3 independent biological samples for viability and caspase measurements. (g) Immunoblotting against MLKL and β actin was used to confirm the MLKL knockdown (shMLKL) in H460 cells grown in Det for 24h. Experiments were performed a minimum of three independent times with similar results. (h-j) Cell viability (h), caspase activation (i), and anchorage-independent growth in soft agar (j) was measured in Att and Det H460 pLKO and shMLKL cells at 24h. Viability was measured using AlamarBlue, caspase activation using CaspaseGlo 3/7 assay, and after 7 days, images of soft agar growth taken following INT-violet staining. Colony count was determined using ImageJ. n=6 biological independent biological replicates for soft agar and n=3 independent biological samples for viability and caspase measurements. All results are presented as means +/- SEM and statistical significance was calculated using a Student’s two tailed t-test. N.S., not significant. Unprocessed original scans of blots are available in Supplementary Fig. 9

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