Supplementary Figure 4: PGAM5 is downstream of RIPK1-mediated loss of viability.

(a) Immunoblotting against PGAM5 and β actin was used to confirm the PGAM5 knockdown (shPGAM5) in MCF-10A cells grown in Att for 24h. Experiments were performed a minimum of three independent times with similar results. (b) MCF-10A pLKO and shPGAM5 cells transfected with empty vector (EV) or RIPK1 wildtype (RIPK1 WT) were grown in Att for 24h. Lysates were then immunoblotted for total RIPK1 and β actin. Experiments were performed a minimum of three independent times with similar results. (c, d) Cell viability was measured using AlamarBlue following transfection of MCF-10A pLKO (c) and shPGAM5 (d) cells after being grown in Att for 24h. n=3 independent biological samples. (e) Immunoblotting against PGAM5 and β actin was used to confirm the PGAM5 knockdown (shPGAM5) in H460 cells grown in Att for 24h. Experiments were performed a minimum of three independent times with similar results. (f) H460 pLKO and shPGAM5 cells transfected with empty vector (EV) or RIPK1 wildtype (RIPK1 WT) were grown in Att for 24h. Lysates were immunoblotted for Myc tag, and β actin to confirm the overexpression of Myc-tagged-RIPK1. Experiments were performed a minimum of three independent times with similar results. (g, h) Cell viability was measured using AlamarBlue following transfection H460 pLKO (g) and shPGAM5 (h) cells after being grown in Att for 24h. 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. N.S., not significant. Unprocessed original scans of blots are available in Supplementary Fig. 9.