Fig 2: Radiation-mediated CRMP4 downregulation inhibits the mitochondrial membrane depolarization followed by apoptosis induction. Radiation- or Ca2+ ionophore A23187-mediated improper intracellular Ca2+ influx collapses the cellular Ca2+-mediated pathway followed by MMP depolarization. Apoptosis resulting from radiation or A23187 treatment is diminished by CRMP4-deficiency and low levels of the intracellular Ca2+ chelator, BAPTA-AM (<5 µM). Conversly, significant cell death can be triggered by CRMP4-deficiency and a high dose of BAPTA-AM (>10 µM). This suggests that CRMP4 plays an important role in the Ca2+-mediated cell death pathway. CRMP4, collapsin response mediator protein 4; MMP, mitochondrial membrane potential; PARP, polyADP-ribose polymerase; IR, ionizing radiation.

Fig 3: CRMP4-deficiency inhibits mitochondrial membrane depolarization under radiation exposure. (A) Inhibition of cytochrome c (CytC) release and PARP cleavage (C-PARP) in IR-resistant cells. After cells were exposed to 5 Gy of radiation for 0–72 h, cytosolic fractions were isolated and western blotting was conducted. (B) Inhibition of cytochrome c release and PARP cleavage in shCRMP4 cells. After cells were exposed to 5 Gy of radiation, cytosolic fractions were isolated and western blotting was conducted. (C) MMP depolarization was significantly increased in parental cells but not in ionizing radiation (IR)-resistant cells. After cells were exposed to 5 Gy of radiation, cells were stained with DIOC6(3) (3,3′-dihexyloxacarbocyanine iodide) fluorescent dye and analyzed by flow cytometry. The quantitative graph is shown on the right. Student's t-test was performed. *P<0.05. (D) After cells were exposed to 5 Gy of radiation for 72 h, cells were analyzed by flow cytometry using DiOC6(3). MMP depolarization was strongly increased in shControl cells, but it was weakly increased in shCRMP4 cells. Student's t-test was performed. *P<0.05. CRMP4, collapsin response mediator protein 4; MMP, mitochondrial membrane potential; PARP, polyADP-ribose polymerase.

Fig 4: The Ca2+ chelator, BAPTA-AM, downregulates CRMP4 and inhibits MMP depolarization. (A) CRMP4 downregulation by BAPTA-AM treatment. SW620 cells were treated with BAPTA-AM (0–20 µM) for 24 h, and western blotting was conducted. (B) A23187-mediated cytochrome c (CytC) release was inhibited by BAPTA-AM. SW620 cells were treated with A23187 (+, 1 µM; ++, 2 µM) in the absence or presence of BAPTA-AM (5 µM) for 12 h, and mitochondrial and cytosolic fractions were isolated for western blotting. (C) Radiation-mediated PARP cleavage (C-PARP) was blocked by BAPTA-AM. Cells were treated with BAPTA-AM (5 µM) and/or radiation (5 Gy), and after 72 h, cell lysates were analyzed by western blotting. (D) Radiation-mediated MMP depolarization was inhibited by BAPTA-AM. Cells were treated with BAPTA-AM (5 µM) and/or radiation (5 Gy), and after 72 h, cells were stained with DiOC6(3) for flow cytometry. The quantitative graph is shown on the right. Student's t-test was performed. *P<0.05. CRMP4, collapsin response mediator protein 4; MMP, mitochondrial membrane potential.

Fig 5: CRMP4-deficiency inhibits the Ca2+-mediated cell death pathway. (A) Intracellular Ca2+ level was not affected by CRMP4-deficiency. Cells were irradiated with 5 Gy of radiation for 72 h, treated with Fluo-3 AM for Ca2+ detection, and analyzed by flow cytometry. Histograms of intracellular Ca2+ content are shown on the right. N.S, not significant. (B) Increased cell survival by cyclosporine A (CsA) in radiation-treated cells. Cells were treated with or without CsA (5 µM) for 1 h and exposed to 5 Gy of radiation, and after 14 days, cells were stained with 0.1% crystal violet. (C) CRMP4-deficiency inhibited A23187-mediated cell death. Several cells were treated with the Ca2+ ionophore A23187 for 24 h, and their proliferation was measured by a plate reader using the WST-1 reagent. The survival rate is expressed as the % of control cells. Student's t-test was performed. *P<0.05, **P<0.01. (D) Cytochrome c (CytC) release inhibition in CRMP4-downregulated cells. After cells were treated with A23187 (0–2 µM) for 12 h, the cytosolic fractions were isolated and western blotting was conducted. CRMP4-deficient ionizing radiation (IR)-resistant and shCRMP4 cells showed a decreased cytochrome c release. (E) MMP depolarization was increased in shControl cells but not in shCRMP4 cells. After cells were treated with A23187 for 24 h, cells were stained with DIOC6(3) and analyzed by flow cytometry. The quantitative graph is shown on the right. Student's t-test was performed. *P<0.05. CRMP4, collapsin response mediator protein 4; MMP, mitochondrial membrane potential.