Research ArticleCELL BIOLOGY

Efficient apoptosis requires feedback amplification of upstream apoptotic signals by effector caspase-3 or -7

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Science Advances  31 Jul 2019:
Vol. 5, no. 7, eaau9433
DOI: 10.1126/sciadv.aau9433
  • Fig. 1 Caspase-8 and -9 are both activated during intrinsic and extrinsic apoptosis.

    (A and B). Western blot of NALM6 WT cells treated for the indicated time with (A) Birinapant (50 nM) and hTNF (10 ng/ml) or (B) with ABT263 (5 μM). (C) Western blot of control and CRISPR-knockout caspase-8– or -9–deficient leukemia cells; tubulin (Tub) is shown for loading control. (D and E) Activation of caspase-8 and -9, respectively, using caspase-8– or -9–specific FAM-FLICA stains and flow cytometry at indicated time points after treatment with (D) Birinapant (50 nM) and hTNF (10 ng/ml) (B + T) or (E) ABT263 (5 μM) (ABT) in WT or caspase-8 (C8 ko)– and caspase-9 (C9 ko)–deficient cells. (F) Model of apoptotic response activation of caspase-8 and -9 following extrinsic or intrinsic stimuli. Graphs show the mean ± SEM for three repeated experiments performed in duplicate.

  • Fig. 2 Extrinsic or intrinsic apoptotic inducers differentially trigger mitochondrial depolarization.

    (A to D) Analysis of mitochondria membrane depolarization using TMRE measurements by flow cytometry in WT, caspase-9, and caspase-8 knockout NALM6 cells, upon intrinsic [(A) 5 μM ABT263, quantitation in (B)] or extrinsic trigger [(C) Birinapant (50 nM), TNFa (10 ng/ml), quantitation in (D)]. Graphs show the mean ± SEM for three repeated experiments performed in duplicate. Student’s t test was used for comparison between single time point values as indicated.

  • Fig. 3 Caspase-3 and -7 are redundantly required for intrinsic or extrinsic apoptosis.

    (A to C) Western blot of NALM 6 (A), 658w (B), and Jurkat cells (C) with CRISPR-based deletion of indicated effector caspases to produce single, double, or triple knockouts. (D to F) Cell viability (in % viability compared to control) analyses using CCK-8 of single effector caspase knockouts (KOs) in NALM6 (D), 658w (E), and Jurkat (F) upon incubation with Birinapant (50 nM) and hTNF (10 ng/ml) or ABT263 (5 μM). (G to I) Cell viability analyses (in % viability compared to control) using CCK-8 of indicated double or triple effector caspase knockout in NALM6 (G), 658w (H), and Jurkat (I) upon incubation with Birinapant (50 nM) and hTNF (10 ng/ml) or ABT263 (5 μM). (J) Cell viability using 7-AAD of WT or caspase-3/7 double-knockout NALM6 cells in controls and upon treatment with Birinapant and hTNF or ABT263 for 24 hours. (K) Dotplot indicating IC50 values for indicated WT and knockout cell lines with values for ABT263 given on the y axis and those for Birinapant and hTNF on the x axis. (L) Colony formation assays in Methocult were performed to examine the number of viable cells remaining at 24 to 48 hours after treatment with ABT263 or TNF/SM. Cell viability graphs [shown in (D) to (J)] show the mean ± SEM for three repeated experiments performed in duplicate. Colony-forming units (CFUs) are the average from three experiments each performed in duplicate.

  • Fig. 4 Simultaneous absence of caspase-3 and -7 results in a significant decrease in caspase-8 and -9 activation in extrinsic apoptosis.

    (A to C) Western blot of NALM6 wild-type (WT) and caspase-3, -7, and -6−/− cells as well as caspase-3/7−/− or caspase-3/7/6−/− cells untreated or treated with Birinapant (50 nM) and hTNF (10 ng/ml) for the indicated times. Cells were lysed and processed for Western blot. (A) Disrupting a single effector caspase-3, -7, or -6 results in no difference in caspase-8 or -9 activation. Incubation with caspase-8 antibody shows appearance of intermediate cleaved caspase-8 (41/43 kDa) and active caspase-8 (18 kDa) in WT cells and in caspase-3, -7, or -6−/−. Incubation with caspase-9 antibody shows appearance of cleaved caspase-9 (37/35 kDa) in WT cells and in caspase-3, -7, or -6−/−. Line graphs show the mean ± SEM for two repeated experiments performed in duplicate. (B) Disruption of caspase-3 and -7 simultaneously results in significant delay of the appearance of intermediate cleaved caspase-8 and no appearance of active caspase-8 as well as significant reduction and delay of cleaved caspase-9. Similar results in caspase-3/7/6−/−. (C) Jurkat WT and caspase-3/7−/− cells as well as 658w WT and caspase-3/7−/− cells show analog results. (D to G) NALM6 WT or caspase-3/7−/− cells were left untreated or treated for 22 hours with Birinapant (50 nM) and hTNF (10 ng/ml). Treated WT NALM6 cells show increased cleavage of caspase-8 (E) and caspase-9 (G) substrates over indicated time, whereas caspase-3/7−/− show no increased activity for caspase-8 and -9 substrates. Quantitation of caspase-8 and -9 activity is given (E and G), respectively. Histograms show representative data from at least two experiments performed in duplicate, with the mean from these ± SEM presented in the line graphs.

  • Fig. 5 Simultaneous absence of caspase-3 and -7 is required for significant decrease of caspase-8 and -9 activation in intrinsic apoptosis.

    (A) NALM6 WT or caspase-3/7−/− cells were left untreated or treated for the indicated time with ABT263 5 μM. Cells were lysed and processed for Western blot. (B to E) NALM6 WT or caspase-3/7−/− cells were left untreated or treated 22 hours with ABT263 at a concentration of 5 μM. WT NALM6 cells treated with ABT263 show metabolic activity for caspase-8 (B and C) and caspase-9 (D and E) substrates. Caspase-3/7−/− cells show no increased metabolic activity for caspase-8 (B and C) and caspase-9 (D and E) compared to untreated cells. Quantitation of caspase-8 and -9 activity, respectively. Histograms show representative data from at least two independent experiments performed in duplicate, with the mean from these ± SEM presented in the line graphs.

  • Fig. 6 Caspase-3– and -7–deficient cells maintain mitochondrial membrane polarity following intrinsic or extrinsic apoptotic stimuli.

    (A) TMRE staining and (B) quantification of mean TMRE signal in NALM6 WT and caspase-3/7 knockout cells treated with ABT263 (5 μM) for the indicated time points. TMRE flow cytometry measurements (A and C) and quantification plot (B and D) of NALM6 WT and caspase-3/7 knockout cells treated with Birinapant (50 nM) and TNF (10 ng/ml) for the indicated time points. Cytochrome c release measurement by flow cytometry (E) and its corresponding quantification graph (F) of NALM6 WT and caspase-3/7 knockout cells untreated (control) or treated with ABT263 (5 μM). Flow cytometry graphs are representative images of three independent experiments. Quantification graphs show the mean from three independent experiments ± SEM. (G) Graphical model of feedback activation by caspase-3 and -7 upon apoptotic stimulation. Histograms show representative data from at least three independent experiments performed in duplicate, with the mean from these ± SEM presented in the line or bar graphs.

Supplementary Materials

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Caspase-8 and -9 are specifically required to activate extrinsic and intrinsic apoptosis, respectively.
    • Fig. S2. Caspase-3 and -7 have overlapping roles in executing apoptotic cell death.

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