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. Author manuscript; available in PMC: 2021 Dec 1.
Published in final edited form as: Cancer Res. 2021 Feb 18;81(11):2995–3007. doi: 10.1158/0008-5472.CAN-20-1890

Figure 2. GAS7 expression is suppressed by high levels of MYCN expression.

Figure 2.

(A) Relative expression of GAS7 versus ACTIN in SHEP-Tet/21N human NB cell line in the presence or absence of doxycycline induction by semiquantitative RT-PCR analysis. The data are presented as means ± SD of triplicate experiments; *p < 0.05 and ***p < 0.001 by two-tailed t test.

(B) Top: Relative expression of gas7 versus elfa in the FACS-sorted EGFP-positive PSNS cells from control dβh-EGFP or dβh-EGFP-MYCN (MYCN) transgenic fish by semiquantitative RT-PCR analysis. The data are presented as means ± SD of triplicate experiments; *p < 0.05 by two-tailed t test. Bottom: Electrophoresis of MYCN and EGFP genotyping confirms the genotypes of transgenic embryos subjected to qRT-PCR analysis.

(C) Dual cross-linking ChIP-PCR with SHEP-Tet/21N human NB cell line showing co-occupancy of SP1 and MYCN at two regions (II and III) of the GAS7 promoter (region I serves as a negative control). Arrow marks the transcription start site. Experiments were performed in duplicate; and error bars represent SEM.

(D) Relative luciferase activity in SHEP-Tet/21N human NB cell line transfected with luciferase reporter vector containing the GAS7 promoter region in the presence or absence of doxycycline (Dox). The data are presented as means ± SEM of duplicate experiments; ***p=0.0009 by two-tailed t test.

(E-F) Relative expression of SP1 (E) or GAS7 (F) versus ACTIN in SHEP-Tet/21N human NB cell line transfected with control siRNA (Ctl siRNA) or three independent SP1 siRNAs (SP1 siRNA1, siRNA2 or siRNA3) by semiquantitative RT-PCR analysis. The data are presented as means ± SD of triplicate experiments; ***p < 0.001 by two-tailed t test.