Additional file 1 of Targeting MYC-enhanced glycolysis for the treatment of small cell lung cancer
Creators: name: Cargill, Kasey R.
name: Stewart, C. Allison
name: Park, Elizabeth M.
name: Ramkumar, Kavya
name: Gay, Carl M.
name: Cardnell, Robert J.
name: Wang, Qi
name: Diao, Lixia
name: Shen, Li
name: Fan, You-Hong
name: Chan, Wai Kin
name: Lorenzi, Philip L.
name: Oliver, Trudy G.
name: Wang, Jing
name: Byers, Lauren A.
Publisher: name: figshare
Year: 2021
Subjects: subject: Biochemistry
subject: Space Science
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Pharmacology
subject: Biological Sciences not elsewhere classified
subject: Mathematical Sciences not elsewhere classified
subject: Cancer
subject: Computational Biology

description: Additional file 1: Supplementary Table 1: List of antibodies. Comprehensive list of all antibodies, dyes, and fluorescent analogues utilized in this investigation. Supplementary Table 2: List of siRNA. Comprehensive list of the siRNA utilized in this investigation. Supplementary Table 3: Seahorse extracellular flux reagents. Assay kit reagents used for the glycolysis stress test and mito stress test during Seahorse extracellular flux analysis. Supplementary Figure 1: Metabolic characterization of MYC-expressing tumors and cell lines. A) There are 364 genes commonly down regulated in the MYCHigh subset of the George et al, Gay et al, and Sato et al datasets [11, 37, 38]. B) 19.7% of the down regulated genes are linked to a metabolic process. C) The top GO terms for the down regulated genes are not implicated in metabolic pathways. D) HK2, PFKFB3, and LDHA gene expression are decreased in MYCHigh patient sample s[38]. E) Proline significantly and positively correlates to MYC expressio n[39]. F) Arachidonyl-carnitine significantly and negatively correlates to MYC expressio n[39]. G) Metabolites isolated from normal lung tissue and spontaneous RPM lung tumors revealed 57 out of 108 significantly altered metabolites. H-I) Lactate and proline are significantly increased in RPM tumors. J-K) Pyruvate and carnitine are significantly reduced in RPM tumors. Supplementary Figure 2: PFK158 and glucose restriction reduce viability and proliferation. A) Immunoblot of cell lines classified as MYC, MYCL, or MYCN showing protein expression of MYC and LDHA with HSP90 loading control. B) Representative MYCLow (DMS79) and MYCHigh (H446) apoptotic flow plots after treatment with 2.5μM PFK158. C) The percent of apoptotic RP and RPM cells increases with higher doses of PFK158. D) The percent proliferation of RP and RPM cells is significantly decreased after 2.5μM PFK158 treatment. E) The percent proliferation of MYCLow (H2029, DMS79, H526) and MYCHigh (H847, H146, H82, H446) cells is significantly decreased after 2.5μM PFK158 treatment. F-G) Glucose restriction reduces viability and proliferation in MYCHigh (H146, H446, H847, H865) cell lines but not MYCLow (H526, H2029, DMS79, H1836) cell lines. H) Immunoblots of H524 cell line transfected with SCR siRNA, MYC siRNA, and PFKFB3 siRNA showing lower MYC, HK2, PFKFB3, and LDHA protein expression with vinculin loading control. I) The percent of viable cells among siRNA treated H524 cells is not significantly altered. J) The percent proliferation cells among siRNA treated H524 cells is not significantly altered. K) Immunoblots of H82 cell line transfected with SCR siRNA, MYC siRNA, and PFKFB3 siRNA showing lower MYC, HK2, PFKFB3, and LDHA protein expression with vinculin loading control. L) The percent of viable cells among siRNA treated H82 cells is not significantly altered. M) The percent proliferation cells among siRNA treated H82 cells is not significantly altered. (*P&lt;0.05; **P&lt;0.01; ****P&lt;0.001). Supplementary Figure 3: Targeting glycolysis reduces ATP generation and lactate secretion. A) Intracellular lactate trends downward in MYCHigh (NJH29, H446, H82, H1930, H1048, H841) and MYCLow (H1092, H1436, DMS79, H526) cells treated with 2.5μM PFK158. B) Glucose uptake in significantly lower in H524 cells treated with siRNA against MYC and PFKFB3 compared to a SCR control. C) Extracellular lactate is significantly reduced in H524 cells treated with siRNA against MYC and PFKFB3 compared to a SCR control. D) Glucose uptake in significantly lower in H82 cells treated with siRNA against MYC and PFKFB3 compared to a SCR control. E) Extracellular lactate is significantly reduced in H82 cells treated with siRNA against MYC and PFKFB3 compared to a SCR control. F) IC50 values of cell lines treated with the glycolysis inhibitor YN1. G) There are no significant differences in glucose uptake after YN1 treatment; MYCLow (H1436, DMS79, H1092, H526, H1522) cell lines; MYCHigh (H446, H82, NJH29, H841, H524) cell lines. H) Extracellular lactate is significantly reduced after YN1 treatment in MYCHigh cell lines; MYCLow (H1436, DMS79, H1092, H526, H1522) cell lines; MYCHigh (H446, H82, NJH29, H841, H524) cell lines. (*P&lt;0.05; **P&lt;0.01; ***P&lt;0.005; ****P&lt;0.001). Supplementary Figure 4: PFK158 treatment in H446 and DMS79 tumors. A) DMS79 tumor growth curve showing no statistical difference. B-C) H&amp;E of DMS79 tumors showing similar pathology. D-E) Body weight curves of DMS79 and H446 xenografts with no statistical differences. F) 17.9% of genes that are significantly altered between vehicle and PFK148-treated H446 xenografts are linked to a metabolic process. G) Several of the top GO terms related to the significantly altered genes between the vehicle and PFK158-treated H446 animals. H-I) The glucose-to-lactate metabolite ratio in H446 and DMS79 tumors. J) Oxygen consumption rate of H446 xenograft tumors (N=1) treated with vehicle or PFK158 analyzed in six technical replicates. K) Extracellular acidification rate of H446 xenograft tumors (N=1) treated with vehicle or PFK158 analyzed in six technical replicates.