Supplementary Figure 5 from Activating Transcription Factor 5 Promotes Neuroblastoma Metastasis by Inducing Anoikis Resistance

Supplementary Figure 5 shows that overexpression of ATF5 promotes anoikis resistance of CHLA-255 in vitro</p

FIGURE 3 from Activating Transcription Factor 5 Promotes Neuroblastoma Metastasis by Inducing Anoikis Resistance

Depletion of ATF5 induces anoikis and decreases metastasis of neuroblastoma cells. A and B, Viability of BE(2)-C and SK-N-DZ suspension cells in poly-HEMA–coated plates, expressing Dox-inducible shATF5-1 or shATF5-2 at different timepoints after Dox treatment. Mean ± SD. C and D, Quantification of anoikis of BE(2)-C and SK-N-DZ suspension cells cultured as in A and B at different timepoints after Dox addition. Mean ± SD. E, Quantification of whole-body bioluminescence flux (photons/second) in mice 24 hours after intracardiac injection of BE(2)-C-shATF5-2 cells, + Dox (n = 9); −Dox, (n = 9). Mice were maintained on drinking water containing Dox (2 mg/mL) from 3 days before injection to the time of euthanasia. F, Bioluminescent images at 24 hours after intracardiac injection under conditions described in E. G, Quantification of bioluminescence of blood from mice collected 12 and 24 hours after intracardiac injection under conditions described in E. H, Quantification of apoptosis of BE(2)-C-shATF5-2 CTCs isolated from mice 12 hours after intracardiac injection and treatment ± Dox (see E and Materials and Methods), + Dox (n = 5), −Dox, (n = 5). I, RT-PCR analyses of ATF5 and ACTB in circulating BE(2)-C-shATF5-2 cells isolated after 12 hours. J, Time course of whole-body bioluminescence flux in mice treated as in E. + Dox (n = 8); −Dox, (n = 9). The mice were monitored for metastatic spread by bioluminescence and euthanized at day 31. K, Quantification of total flux (photons/second) by ex vivo liver bioluminescence at the time of euthanasia (day 31). L, Quantification of bioluminescence in bone marrow homogenates at the time of euthanasia (day 31). *, P P P P < 0.0001.</p

Supplementary Figure 16 from Activating Transcription Factor 5 Promotes Neuroblastoma Metastasis by Inducing Anoikis Resistance

Supplementary Figure 16 shows that CP-d/n-ATF5 inhibits growth of BE(2)-C tumors and increases apoptosis</p

FIGURE 2 from Activating Transcription Factor 5 Promotes Neuroblastoma Metastasis by Inducing Anoikis Resistance

Overexpression of ATF5 promotes anoikis resistance in vitro and in vivo. A and B, ATF5 overexpression, by immunoblot, in suspension culture of BE(2)-C and SH-SY5Y cells, 72 hours after transient transfection with pCCL-GFP or pCCL-ATF5. ACTB was used as a loading control. Densitometric analysis was performed using ImageJ. C and D, BE(2)-C and SH-SY5Y suspension cell viability and anoikis at 12, 24, 48, and 72 hours after transfection, respectively. At each timepoint, the comparison is between cells overexpressing ATF5 and control cells expressing GFP (100%). Mean ± SD. E, Quantification of mouse whole-body bioluminescence flux (photons/second) 12 hours after intracardiac injection of BE(2)-C cells overexpressing ATF5 (n = 8) or GFP (n = 7). F, Images showing increased bioluminescence of animals described in E indicate tumor cell survival 12 hours after intracardiac injection. G, Quantification of bioluminescence of blood collected 12 hours after intracardiac injection as in E. H, Quantification of apoptosis of BE(2)-C CTC isolated at 12 hours, GFP (n = 7); ATF5 (n = 7). *, P P P P < 0.0001.</p

Supplementary Figure 1 from Activating Transcription Factor 5 Promotes Neuroblastoma Metastasis by Inducing Anoikis Resistance

Supplementary Figure 1 shows that ATF5 is expressed in neuroblastoma cell lines.</p

Supplementary Figure 6 from Activating Transcription Factor 5 Promotes Neuroblastoma Metastasis by Inducing Anoikis Resistance

Supplementary Figure 6 shows a decrease in ATF5 after Dox addition under non-adherent suspension conditions.</p

Supplementary Figure 8 from Activating Transcription Factor 5 Promotes Neuroblastoma Metastasis by Inducing Anoikis Resistance

Supplementary Figure S8 shows that BMF overexpression reduces the anchorage-independent viability of BE(2)-C and SK-N-DZ cells.</p

FIGURE 6 from Activating Transcription Factor 5 Promotes Neuroblastoma Metastasis by Inducing Anoikis Resistance

CP-d/n-ATF5 induces anoikis and inhibits neuroblastoma growth and metastasis in vivo. A, Effects of CP-dn-ATF5 on the expression of ATF5 and apoptosis-related BCL-2 proteins in suspension cells. Immunoblot of proapoptotic and antiapoptotic proteins in BE(2)-C and SK-N-DZ suspension cells at 72 hours after CP-d/n-ATF5 treatment. β-Actin was used as a loading control. Densitometric analysis was performed using ImageJ. B, BMF knockdown inhibits CP-d/n-ATF5–induced anoikis of BE(2)-C and SK-N-DZ cells. Adherent cells were treated with or without siBMFs (50 nmol/L) as indicated for 24 hours and then seeded in nonadherent plates. CP-d/n-ATF5 (200 µmol/L) was added, and 72 hours later, anoikis was evaluated. C, Kaplan–Meier analysis of survival of mice bearing BE(2)-C tumors, treated with vehicle (n = 7) or CP-d/n-ATF5 (50 mg/kg; n = 7). Treatment was started 7 days after cell implantation, once daily for the first 3 days, and then twice weekly. P D, Quantification of liver bioluminescence flux (photons/second) by ex vivo imaging at the time of euthanasia from mice bearing BE(2)-C tumors described in C. Vehicle (n = 7), CP-d/n-ATF5 (n = 6). E, Quantification of bioluminescence in bone marrow homogenate measured at the time of euthanasia (RLU/# BM cells, RLU: Relative luciferase unit). Vehicle (n = 7), CP-d/n-ATF5 (n = 6). F, Quantification of CTC measured at the time of euthanasia by bioluminescence of blood. Vehicle (n = 7), CP-d/n-ATF5 (n = 6). G, Twelve hours after intracardiac injection of BE(2)-C cells, blood was collected for (left) quantification of CTC by measurement of blood bioluminescence and (right) for measurement of apoptosis in isolated BE(2)-C CTC. Mice were treated with CP-d/n-ATF5 (n = 9) or vehicle (n = 9) immediately after intracardiac injection. H, BE(2)-C cells were injected intracardially and then treated with vehicle (n = 8) or CP-d/n-ATF5 (n = 10). Whole-body bioluminescence flux was then monitored for subsequent tumor cell growth. I, Representative bioluminescence images showing metastatic growth of BE(2)-C in mice treated with vehicle or CP-dn-ATF5 at day 38 of the experiment described in H. J, Quantification of total flux (photons/second) by ex vivo liver bioluminescence at time of euthanasia (day 38). K, Quantification of bioluminescence in bone marrow homogenate at the time of euthanasia (day 38). L, RT-PCR analysis of ATF5, BMF, and ACTB (internal control) in BE(2)-CTC from K. *, P P P < 0.001.</p

Supplementary Figure 9 from Activating Transcription Factor 5 Promotes Neuroblastoma Metastasis by Inducing Anoikis Resistance

Supplementary Figure 9 shows that BMF knockdown rescues ATF5 loss-induced reduction of anchorage-independent cell viability</p

Supplementary Figure 12 from Activating Transcription Factor 5 Promotes Neuroblastoma Metastasis by Inducing Anoikis Resistance

Supplementary Figure 12 shows that CP-d/n-ATF5 inhibits the growth of MYCN-amplified and MYCN-non-amplified cell lines in a dose-dependent manner</p