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. 2021 Feb 1;24(2):102128. doi: 10.1016/j.isci.2021.102128

Figure 1.

Figure 1

Inhibition of de novo fatty acid synthesis reduces cell growth and MYC expression and induces differentiation in neuroblastoma cells, related to Figure S1 and Table S1

(A) Fatty acid synthesis pathway and inhibitors used in this study. See main text for details. TCA cycle: tricarboxylic acid cycle; ACLY: ATP-citrate lyase; ACACA: acetyl-CoA carboxylase; FASN; fatty acid synthase.

(B) Cell proliferation curves of the indicated MYCN-amplified (SK-N-BE(2), IMR-32, and KCN-n69) and non-MYCN-amplified (SH-SY5Y and SK-N-SH) neuroblastoma cell lines as quantified by cell counting at the indicated time points. Data are presented as mean ± SD of three independent experiments, % of day 0.

(C) Western blot analysis of MYCN protein expression in the indicated cell lines and time points upon fatty acid synthesis inhibition.

D: DMSO; T: TOFA; C: Cerulenin; O: Orlistat; S: Soraphen A; U: UB006. α-tubulin or β-actin were used as loading controls as indicated.

(D) Immunofluorescence staining of SK-N-BE(2) cells treated for 7 days with vehicle or the indicated inhibitors. Blue: DAPI; green: Tubulin B3; red: SCG2. Scale bar indicates 50 μm.

(E) Immunofluorescence staining of KCN-n69 cells treated for 7 days with vehicle or the indicated inhibitors. Blue: DAPI; green: Tubulin B3; red: SCG2. Scale bar indicates 50 μm.

(F) Immunofluorescence staining of IMR-32 cells treated for 7 days with vehicle or the indicated inhibitors. Blue: DAPI; green: Tubulin B3; red: SCG2. Scale bar indicates 100 μm.

(G) mRNA fold expression of the indicated genes in SK-N-BE(2) treated for 7 days with vehicle or the indicated inhibitors, as quantified by qPCR.

(H) Immunofluorescence staining of PDX-derived cell cultures, LU-NB-2 and LU-NB-3, treated for 7 days with vehicle or the indicated inhibitors. Blue: DAPI; green: Tubulin B3; red: SCG2. Scale bar indicates 100 μm.

(I) Western blot analysis of SCG2 and MYCN protein expression in LU-NB-2 and LU-NB-3 after 7 days upon fatty acid synthesis inhibition. (D) DMSO; T: TOFA; U: UB006. β-actin was used as loading control as indicated.

Data are presented as mean ± SD of three independent experiments; statistical analysis: t test of each inhibitor compared with vehicle, with ∗, ∗∗, and ∗∗∗ indicating p < 0.05, p < 0.01, and p < 0.001, respectively. Western blot and microscopic images are representative of at least three independent experiments.