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. 2020 Dec 8;11(12):1041. doi: 10.1038/s41419-020-03258-3

Fig. 1. Generation of a cell line model to identify CICD regulators.

Fig. 1

HeLa cells were stably transfected with either a non-targeting shRNA (shCont) or a shRNA targeting Apaf-1 (shApaf-1). A immunoblotting for Apaf-1 in whole cell lysates isolated from both cell types (hsp60 was used as a loading control), B protein extracts were isolated from HeLa shCont or shApaf-1 cells and apoptosome formation was measured in vitro upon addition of cytochrome c, C HeLa shCont or shApaf-1 cells were treated with Actinomycin D (1 µM, as an apoptosis induction) for the indicated times and DEVDase activity was measured. D HeLa shCont were treated for 3 days with Act D (1 µM, to induce apoptosis) or with Act D (1 µM) + qVD-OPH (20 µM) to induce CICD. HeLa shApaf-1 cells were treated for 3 days with Act D (1 µM). qVD-OPH was not added to HeLa shApaf-1 cells as those cells cannot activate caspases upon Act D treatment (see B and C). Cell death was analyzed by flow cytometry using a Propidium Iodide (PI) staining. E HeLa shCont or shApaf-1 cells were treated with Actinomycin D + qVD-OPH (1 μM or 20 μM respectively) as a CICD stimulus for the indicated times. Isolation of cytosolic (cyto) and microsomal fractions (micro, which contains mitochondria in particular) was assessed by cell fractionation. MOMP, assessed by looking at the relocalization of Cytochrome c (Cyto C) and Smac from the mitochondria to the cytosol upon CICD treatment, was measured by immunoblotting. hsp60 was used as a control for the presence of mitochondria in the microsomal fraction. Data are expressed as mean ± s.d (n = 3), immunoblots are representative of 3 or more individual experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 according to a two-way ANOVA. N.S: non-significant.