ETFDH loss promotes tumor growth

A) ETFDH protein levels in non-adjacent tissue (NAT) or grade 3 cancerous tissue (Tumor) were determined by immunocytochemistry (IHC) of colorectal cancer tissue microarray. Staining intensity of DAB in arbitrary units (A.U) is shown. Data are presented as means +/- SEM (NAT n=14, Tumor n=13), **p<0.01, unpaired Student’s t test.

B-C) Tumor growth of WT EV, ETFDH KO, and ETFDH Rescue HCT-116 cells following intra-caecal injection (B) and endpoint tumor volumes (C). Growth was assessed by luminescence (photons). Data are presented as means +/- SEM (WT EV n=6, ETFDH KO n=8, ETFDH Rescue n=8), *p<0.05, one-way ANOVA, Tukey’s post-hoc test.

D-E) Tumor growth of WT and ETFDH KO NT2197 cells following mammary fat-pad injection (D) and endpoint tumor volumes (E). Growth was assessed using a caliper. Data are presented as means +/- SEM (WT n=17, ETFDH KO n=17), *p<0.05, unpaired Student’s t test.

Absence of ETFDH promotes mitochondrial metabolism

A-B) Oxygen consumption (A) and extracellular acidification (B) in WT EV, ETFDH KO, and ETFDH Rescue HCT-116 cells were determined using a Seahorse bioanalyzer. Data are normalized to cell count and presented as means +/- SD (n=4), *p<0.05, **p<0.01, one-way ANOVA, Tukey’s post-hoc test.

C) Basal J ATP calculations from WT EV, ETFDH KO, and ETFDH Rescue HCT-116 cells. J ATP ox represents ATP production from oxidative phosphorylation, while J ATP glyc is ATP production from glycolysis. Comparison between J ATP ox (grey bars; top) and J ATP glyc (white bars; bottom) is shown. Data are presented as means +/- SD (n=4), *p<0.05, **p<0.01, one-way ANOVA, Tukey’s post-hoc test.

D-E) Bioenergetic plot for Basal, FCCP, and Monensin J ATP fluxes (D) and bioenergetic capacity (E) of WT EV, ETFDH KO, and ETFDH Rescue HCT-116 cells. Data are presented as means +/- SD (n=4), *p<0.05, one-way ANOVA, Tukey’s post-hoc test.

F) Glutamine uptake and glutamate production in WT and ETFDH KO HCT-116 cells. Results are shown as fold changes of ETFDH KO cells relative to WT cells. Data are presented as means +/- SD (n=3), *p<0.05, paired Student’s t test.

G) Schematic of 13C-glutamine tracing throughout the citric acid cycle (CAC). Isotopomers labelled in green depict 13C-glutamine tracing in the forward direction of the CAC, while those in purple represent reverse tracing.

H) Relative abundance of 13C-labelled metabolites in the forward (green) and reverse (purple) directions from WT (blue) and ETFDH KO (red) NT2197 cells. Data are presented as means +/- SD (n=3), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, paired Student’s t test.

I) Proliferation of WT and ETFDH KO HCT-116 cells grown in the absence or presence of glutamine for 48 hours. Results are shown as fold changes of cell counts under glutamine deprivation relative to those under glutamine repleted conditions. Data are presented as means +/- SD (n=3), *p<0.05, paired Student’s t test.

ETFDH loss bolsters mTORC1 signaling and downregulates 4E-BP1 protein levels.

A) Amino acid profile in WT and ETFDH KO HCT-116 and NT2197 cells. Data are presented as fold change in amino acid levels in ETFDH KO compared to WT cells (n=3).

B) Puromycin incorporation assays in WT and ETFDH KO HCT-116 cells. Levels and phosphorylation status of indicated proteins were determined via western blotting using indicated antibodies. β-Actin was used as a loading control (representative blots of n=3). Quantification of puromycin incorporation is presented as fold change in ETFDH KO vs. WT cells.

C) Levels and phosphorylation status of indicated proteins in WT or ETFDH KO HCT-116 and NT2197 cells were determined by western blotting. β-Actin served as a loading control (representative blots of n=3).

D) Levels and phosphorylation status of indicated proteins in WT EV, ETFDH KO, or ETFDH Rescue HCT-116 and NT2197 cells were determined by western blotting. β-Actin was used as a loading control (representative blots of n=3).

E) Total and phosphoprotein levels in WT or ETFDH KO HCT-116 cells were determined by western blotting using indicated proteins. Cells were serum starved overnight, then depleted of amino acids for indicated timepoints (0, 10, 30, 60 minutes), or stimulated with FBS for 4 hours (Stim). β-Actin was used as loading control (representative blots of n=3).

F) Quantification of pS6(S240/244)/S6 ratio from data presented in panel E. Data are presented as means +/- SD (n=3), *p<0.05, one-way ANOVA, Tukey’s post-hoc test.

G) Quantification of p4E-BP1(S65)/4E-BP1 ratio from data presented in panel E. Data are presented as means +/- SD (n=3), *p<0.05, one-way ANOVA, Tukey’s post-hoc test.

Repression of EIF4EBP1 transcription mediates the effects of ETFDH loss on cancer bioenergetics and tumor growth

A) Levels and phosphorylation status of indicated proteins in WT EV, ETFDH KO, or ETFDH Rescue HCT-116 and NT2197 cells were determined by western blotting. β-Actin was used as a loading control (representative blots of n=3).

B) Levels of indicated protein in λ-phosphatase treated WT or ETFDH KO HCT-116 and NT2197 cell lysates were monitored by western blotting. β-Actin was used as a loading control (representative blots of n=3).

C) Indicated protein levels in WT or ETFDH KO HCT-116 tumors were determined by western blotting. AKT was used as loading control (WT n=8, ETFDH KO n=11).

D-E) Quantification of 4E-BP1 (D) and 4E-BP2 (E) from tumors described in panel C. Data are presented as means +/- SEM (WT n=8, ETFDH KO n=11), ***p<0.001, unpaired Student’s t test.

F) m7GDP pulldown assay in ETFDH KO and ETFDH KO overexpressing 4E-BP1 (ETFDH KO 4E-BP1) NT2197 cells. Specified protein levels in m7GDP pulldown or input were determined by western blot. β-Actin was used as loading control (input) and to exclude contamination in the pulldown material (m7GTP bound) (representative blots of n=3).

G) Oxygen consumption of ETFDH KO and ETFDH KO 4E-BP1 NT2197 cells. Data are normalized to cell count and presented as means +/- SD (n=5), **p<0.01, Student’s t test.

H-I) Tumor growth of ETFDH KO and ETFDH KO 4E-BP1 NT2197 cells following mammary fat-pad injection (H) and endpoint tumor volumes (I). Growth was assessed using caliper measurements. ETFDH KO measurements are the same as in Fig.1D, as these growth curves were obtained in the same experiment. Data are presented as means +/- SEM (ETFDH KO n=17, ETFDH KO 4E-BP1 n=18), **p<0.01, unpaired Student’s t test.

J) Mitochondrial DNA in WT EV, ETFDH KO, and ETFDH Rescue HCT-116 cells was quantified by qPCR. Mitochondrial DNA (mtDNA) content was normalized to genomic DNA (gDNA). Data are presented as fold change relative to WT EV cells +/- SD (n=3), *p<0.05, **p<0.01, one-way ANOVA, Tukey’s post-hoc test.

K) Levels of indicated proteins in WT EV, ETFDH KO, and ETFDH Rescue HCT-116 cells were assessed by western blotting. β-Actin was used as a loading control (representative blots of n=3).

L) EIF4EBP1 and EIF4EBP2 mRNA levels in WT EV, ETFDH KO, and ETFDH Rescue HCT- 116 cells were determined by RT-qPCR. PP1A was used as a house-keeping gene. Data are presented as fold change of EIF4EBP1/PP1A and EIF4EBP2/PP1A ratios relative to WT EV cells (set to 1) +/- SD (n=3), *p<0.05, one-way ANOVA, Tukey’s post-hoc test.

M) Eif4ebp1 and Eif4ebp2 mRNA abundance in WT and ETFDH NT2197 KO cells was determined by RT-qPCR. Actb was used as a house-keeping gene. Data are presented as fold change in Eif4ebp1/Actb and and Eif4ebp2/Actb ratios relative to WT cells +/- SD (n=3), *p<0.05, paired Student’s t test.

Loss of ETFDH increases BCL-6 levels leading to BCL-6-dependent inhibition of EIF4EBP1 transcription.

A) Levels of indicated proteins in WT or ETFDH KO HCT-116 and NT2197 cells were determined by western blotting. β-Actin was used as a loading control (representative blots of n=3).

B) Levels of indicated proteins in ETFDH KO NT2197 cells treated with DMSO or Torin1 (250nM) for 4 hours were determined by western blotting. β-Actin was used as a loading control (representative blots of n=3).

C) BCL6/Bcl6 mRNA abundance in WT and ETFDH KO HCT-116 and NT2197 cells was determined by RT-qPCR. PP1A mRNA was used as a control for HCT-116 experiments, while Actb mRNA was used as a control for NT2197 experiments. Data are presented as fold change in EIF4EBP1/PP1A and EIF4EBP2/PP1A (HCT-116) and Eif4ebp1/Actb and and Eif4ebp2/Actb (NT2197) ratios relative to WT cells +/- SD (n=3), paired Student’s t test.

D) Polysome profiling of WT and ETFDH HCT-116 KO cells. The absorbance at 254nm (AU 254nm) was used to monitor distribution of the 40S-, 60S-ribosomal subunits, monosomes (80S) and polysomes across the gradient. BCL6 mRNA abundance from polysome gradient fractions was obtained using RT-qPCR. Data are shown as a mean percentage of mRNA in each fraction relative to cumulative corresponding mRNA amount across the whole gradient +/-SD (n=3).

E-F) Binding events of BCL-6 to the promoters of EIF4EBP1 and EIF4EBP2 were determined by ChIP-qPCR. IgG was used a negative control. Data are presented as % of input (n=3), *p<0.05, two-way ANOVA, Dunnett’s post-hoc test.

G) The levels of indicated proteins in ETFDH KO NT2197 cells transfected with siRNA targeting BCL-6 (siBCL-6) or control, scrambled siRNA (siCTRL) were determined by western blotting. β-Actin was used as loading control (representative blots of n=2).

H) Levels of indicated proteins in ETFDH KO 4T1 cells infected with shRNAs targeting BCL-6 (shBCL-6 #1 or shBCL-6#2) or control, scrambled shRNA (shCTRL) were monitored by western blotting. β-Actin served as loading control (representative blots of n=3).

I) Proliferation of ETFDH KO shCTRL, shBCL-6 #1, or shBCL-6#2 4T1 cells. Data are presented as cell count means (n=3), *p<0.05, one-way ANOVA, Dunnett’s post-hoc test.

Reduced expression of ETFDH increases mitochondrial metabolism and tumor growth

A) ETFDH mutations from 2683 samples across multiple cancer types. Number of missense and truncating mutations are shown relative to samples with no mutations. Data are acquired from the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes Consortium 34, accessed from the cBioPortal server (www.cbioportal.org).

B) Levels of indicated proteins in WT HCT-116 cells treated with 2.5µM 5-azacytidine (5-aza), 10µM 5-aza-2’ -deoxycytidine (5-aza-2-deoxyC), or a vehicle (DMSO) for 72 hours were determined by western blotting. β-Actin was used as a loading control (representative blots of n=3).

C) Levels and the phosphorylation status of indicated proteins in ETFDH KO NT2197 cells expressing ETFDH harboring (AAG)6, (AAA)4, (AAA)6 tracks were determined by western blotting. β-Actin was used as a loading control (representative blots of n=3).

D) Eif4ebp1 and Eif4ebp2 mRNA abundance in (AAG)6, (AAA)4, and (AAA)6 NT2197 cells were determined by RT-qPCR. Actb was used as a house-keeping gene. Data are presented as fold change in Eif4ebp1/Actb and Eif4ebp2/Actb ratios relative to (AAG)6 NT2197 cells (n=3), *p<0.05, **p<0.01, one-way ANOVA, Dunnett’s post-hoc test.

E) Mitochondrial DNA content in (AAG)6, (AAA)4, and (AAA)6 NT2197 cells were monitored by qPCR. Mitochondrial DNA (mtDNA) content was normalized to genomic DNA (gDNA) content. Data are presented as mean fold change relative to (AAG)6 NT2197 cells -/+ SD (n=3), *p<0.05, **p<0.01, one-way ANOVA, Dunnett’s post-hoc test.

F) Oxygen consumption of (AAG)6, (AAA)4, and (AAA)6 NT2197 cells was determined using Seahorse bioanalyzer. Data are normalized to cell count and presented as means +/- SD (n=4), one-way ANOVA, Dunnett’s post-hoc test.

G-H) Bioenergetic Plot for Basal, FCCP, and Monensin J ATP fluxes (G) and Bioenergetic Capacity (H) derived from (AAG)6, (AAA)4, and (AAA)6 NT2197 cells. Data are presented as means +/- SD (n=4), one-way ANOVA, Dunnett’s post-hoc test.

I) Proliferation of (AAG)6, (AAA)4, and (AAA)6 NT2197 cells. ETFDH rescue measurements are the same as in Supplementary Fig.1F. Data are presented as cell count means +/- SD (n=4), **p<0.01, one-way ANOVA, Dunnett’s post-hoc test.

J-K) Tumor growth of (AAG)6 and (AAA)6 NT2197 cells following mammary fat-pad injection

(J) and endpoint tumor volumes (K). Growth was assessed using calipers. Data are presented as means +/- SEM ((AAG)6 n=6-12, (AAA)6 n=11-13), **p<0.01, unpaired Student’s t test.

L) Schematic representation of the model whereby reduction in ETFDH levels accelerates tumor growth. Reduction of ETFDH triggers accumulation of intracellular amino acids thus increasing mTORC1 signaling. This is orchestrated with BCL-6-dependent reduction in 4E-BP1 levels to drive mitochondrial biogenesis and increase bioenergetic capacity of cancer cells ultimately leading to more aggressive tumor growth.

ETFDH expression is reduced across a broad array of cancer subtypes, whereby ETFDH is a non-essential gene in cancer cells.

A) Schematic of electron transfer from fatty and amino acids towards the electron transport chain (ETC) via electron transfer flavoprotein (ETF) and electron transport flavoprotein dehydrogenase (ETFDH).

B) Gene essentiality in NALM6 cells. 19084 genes are presented and ranked based on essentiality (1 being most essential and 19084 being the least essential). Orange bar depicts essential genes and grey bar depicts non-essential genes. The rank of ETFDH is compared to those of MYC, NRAS, RB1, and TP53.

C) ETFDH mRNA abundance profiles across tumor samples (red) compared to normal tissues (blue). Data are presented as log2CPM (counts per million). Data were derived from The Cancer Genome Atlas (TCGA) repository and accessed on the GEPIA2 server (http://gepia2.cancer-pku.cn) (Colon Adenocarcinoma: Normal n=349, Tumor n=275; Liver Hepatocellular Carcinoma: Normal n=160, Tumor n=369; Rectal Adenocarcinoma: Normal n=318, Tumor n=92; Ovarian Serous Cystadenocarcinoma: Normal n=88, Tumor n=426; Skin Cutaneous Melanoma: Normal n=558, Tumor n=461; Testicular Germ Cell Tumors: Normal n=165, Tumor n=137; Uterine Carcinoma: Normal n=78, Tumor n=57), p<2.2×10-16.

D) ETFDH mRNA levels across healthy (labelled as normal) and breast tumor (labelled as tumor) samples. RNAseq data from both TCGA and GTex databases were processed and normalized as described. TPM values were downloaded from GEO [GSE86354 (healthy) and [GSM1536837 (tumour)]. Expression is presented as log2 standardised mRNA levels. Data were acquired from the ‘Breast cancer gene-expression miner’ (bc-GenExMiner) server (http://bcgenex.ico.unicancer.fr) using ETFDH expression according to nature of the tissue. (Normal n=92, Tumor n=743). p<0.0001, Dunnett-Tukey-Kramer’s test.

E-F) Proliferation of WT EV, ETFDH KO, and ETFDH Rescue HCT-116 (E) and NT2197 (F) cells. ETFDH rescue measurements are the same as in Fig.6I. Data are presented as cell count means +/- SD over multiple time-points. Representative western blot of indicated proteins is provided in the inlets (n=3), *p<0.05, **p<0.01, one-way ANOVA, Tukey’s post hoc test.

G-H) Proliferation of WT and ETFDH KO 4T1 (G) and NALM6 (H) cells. Data are presented as cell count means +/- SD over indicated time-points. Representative western blot of indicated proteins is provided in the inlets (n=3), *p<0.05, paired Student’s t test.

I) Proliferation of WT and ETFDH KO NMuMG cells. Representative western blot of indicated proteins is provided in the inlets (n=3), one-way ANOVA, Dunnett’s post hoc test.

J) Soft agar/colony formation assays in WT EV, ETFDH KO, and ETFDH Rescue HCT-116 cells.

Data are presented as fold change relative to WT EV cells (n=3), *p<0.05, **p<0.01, one-way ANOVA, Tukey’s post-hoc test.

ETFDH loss reprograms mitochondrial bioenergetics

A) Schematic depicting fatty acid and leucine catabolism linked to ETFDH electron transfer.

B) Palmitate oxidation at basal level in WT EV, ETFDH KO, and ETFDH Rescue HCT-116 cells.

Data are presented as means +/- SD (n=6), ****p<0.0001, one-way ANOVA, Tukey’s post-hoc test.

C) Fractional enrichment (%) of citrate m+2 in WT EV, ETFDH KO, and ETFDH Rescue HCT-116 cells. Data are presented as means +/- SD (n=6), ****p<0.0001, one-way ANOVA, Tukey’s post-hoc test.

D-E) Oxygen consumption (D) and extracellular acidification (E) for WT and ETFDH KO NT2197 cells was determined using a Seahorse bioanalyzer. Data are normalized to cell count and presented as means +/- SD (n=5), *p<0.05, **p<0.01, paired Student’s t test

F) Basal J ATP calculations from WT and ETFDH KO NT2197 cells. J ATP ox represents ATP production from oxidative phosphorylation, while J ATP glyc is ATP production from glycolysis. Comparison between J ATP ox (grey bars; top) and J ATP glyc (white bars; bottom) is shown. Data are presented as means +/- SD (n=5), ***p<0.001, paired Student’s t test.

G-H) Bioenergetic Plot for Basal, FCCP, and Monensin J ATP fluxes (G) and Bioenergetic Capacity (H) for WT and ETFDH KO NT2197 cells. Data are presented as means +/- SD (n=5), *p<0.05, paired Student’s t test.

I) Glutamine uptake and glutamate production in WT and ETFDH KO NT2197 cells. Data are presented as mean fold changes of ETFDH KO cells relative to WT cells +/- SD (n=3), *p<0.05, paired Student’s t test.

J) Fractional abundance of 13C-labelled metabolites over indicated timepoints from WT (blue) and ETFDH KO (red) NT2197 cells. Isotopomers labelled in green depict 13C-glutamine tracing in the forward direction of the CAC, while those in purple represent reverse tracing. Data are presented as means +/- SD (n=3), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, two-way ANOVA, Tukey’s post hoc test.

ETFDH loss increases mTORC1 signaling and dependency

A-B) Levels and phosphorylation status of indicated proteins in WT and ETFDH KO HCT-116

(A) and NALM6 (B) cells were determined by western blotting. β-Actin was used as a loading control (representative blots of n=3).

C) Proliferation of HCT116 ETFDH KO and ETFDH Rescue cells treated with indicated concentrations of Torin1 for 72h. Data are presented as fold changes relative to vehicle (DMSO) treatment (n=3), *p<0.05, **p<0.01, ***p<0.01, two-way ANOVA, Tukey’s post hoc test.

D) Indicated protein levels in WT and ETFDH KO HCT-116 cells transfected with shRNA targeting RAPTOR (shRAPTOR), RICTOR (shRICTOR), or scrambled control (SCR) were determined by western blotting. β-Actin was used as a loading control (representative blots of n=3).

E-F) Proliferation of HCT-116 WT and ETFDH KO cells expressing SCR and shRAPTOR (E) or SCR and shRICTOR (F). Data are presented as cell count means +/- SD (n=4), **p<0.01, ***p<0.01 two-way ANOVA, Tukey’s post hoc test.

G) Proliferation of HCT116 ETFDH KO and ETFDH Rescue cells treated with indicated concentrations of BiS-35x for 72 hours. Data are presented as fold changes relative to vehicle (DMSO) treatment (n=3), *p<0.05, **p<0.01, ***p<0.01, two-way ANOVA, Tukey’s post hoc test.

H) Total and phosphoprotein levels in WT or ETFDH KO HCT-116 cells were determined by western blotting using indicated proteins. Cells were serum starved overnight, then depleted of amino acids for indicated timepoints (0, 10, 30, 60 minutes), or stimulated with FBS for 4 hours (Stim). β-Actin was used as loading control (representative blots of n=3).

ETFDH loss induces EIF4EBP1 transcription

A) Protein levels in WT and ETFDH KO NALM6 cells were monitored by western blotting using indicated antibodies. β-Actin was used as a loading control (representative blots of n=3).

B) Indicated protein levels in WT and ETFDH KO 4T1 cells were determined by western blotting using indicated antibodies. β-Actin was used as a loading control (representative blots of n=3).

C) Protein levels in WT and ETFDH KO NMuMG cells were monitored by western blotting using indicated antibodies. β-Actin was used as a loading control (representative blots of n=3).

D) Proliferation of ETFDH KO and ETFDH KO overexpressing 4E-BP1 (ETFDH KO 4E-BP1) HCT-116 cells. Data are presented as cell count means +/- SD over indicated time-points (n=3), **p<0.01, paired Student’s t test.

E) Soft agar/colony formation assay in ETFDH KO and ETFDH KO 4E-BP1 HCT-116 cells. Data are presented as fold change relative to ETFDH KO cells (n=3), *p<0.05, paired Student’s t test.

F) Mitochondrial mass in WT and ETFDH KO NT2197 cells was monitored by flow cytometry.

Data are presented as fold change relative to WT cells (n=3), **p<0.01, paired Student’s t test.

G) The levels of indicated proteins in WT and ETFDH KO HCT-116 cells treated with cycloheximide (CHX) (50μg/ml), MG-132 (10μM), or combination thereoff for 4 hours total were monitored by western blotting. β-Actin was used as loading control, whereas p21 was used as a short-lived protein to control for the effects of the CHX and MG-132 treatments (representative blots of n=2).

H) Polysome profiling of WT and ETFDH HCT-116 KO cells. Absorbance at 254nm (AU 254nm) was used to monitor the distribution of 40S-, 60S-ribosomal subunits, monosomes (80S) and polysomes across the gradient. Indicated mRNA abundances from polysome gradient fractions was obtained using RT-qPCR. Data are shown as a mean percentage of mRNA in each fraction relative to cumulative corresponding mRNA amount across the whole gradient +/-SD (n=3).

I) Correlation between ETFDH and EIF4EBP1 mRNA expression in colon adenocarcinoma samples. Data were derived from TCGA repository.

J) WT and ETFDH KO HCT-116 cells were treated with Actinomycin D (10µg/ml) for indicated timepoints upon which EIF4EBP1 and GAPDH mRNA levels were determined by RT-qPCR. Data are presented as a mean percentage of mRNA relative to DMSO-treated cells +/-SD (n=3).

The effects of ETFDH loss on EIF4EBP1 transcription are mediated by BCL-6 but not STAT1, Snail or Slug

A) Protein levels in WT and ETFDH KO HCT-116 cells were determined by western blotting using indicated antibodies. β-Actin was used as a loading control (representative blots of n=2).

B) MT4788 WT and STAT1 KO cells were treated with IFNγ (1ng/µl) to induce STAT1 or vehicle (water) for 24 hours. Levels and phosphorylation status of indicated proteins were determined by western blotting. β-Actin was used as a loading control (representative blots of n=2).

C) Protein levels in WT and ETFDH KO HCT-116 cells were assessed by western blotting using annotated antibodies. β-Actin served as a loading control (representative blots of n=3).

D-E) Binding of STAT1 to the promoters of EIF4EBP1 (D) and EIF4EBP2 (E) was determined by ChIP-qPCR. IgG was used a negative control. Data are presented as a percentage of the input +/- SD (n=3), one-way ANOVA, Tukey’s post-hoc test.

F) Fluorescence intensity of WT and ETFDH KO HCT-116 cells stained with DCFDA (2μM) and measured by flow cytometry. Median values are depicted in the inlets.

G) Levels of 4E-BP1 in WT and ETFDH KO HCT-116 cells were determined by western blotting using the indicated antibody. β-Actin was used as a loading control (representative blots of n=3).

Poly(A) Track design

A) Insertion of poly(A) tracks within the coding sequence of murine ETFDH. DNA sequences for constructs (AAG)6, (AAA)4, (AAA)6, and WT Etfdh are shown.

B) Protein structure of (AAG)6 / (AAA)6 constructs generated using AlphaFold.