Additional file 2: of Inhibition of glutamate oxaloacetate transaminase 1 in cancer cell lines results in altered metabolism with increased dependency of glucose

Figure S2. Rescue of GOT1 down-regulated and GOT1-null cells by oxaloacetate. Relative cell viabilities after 8 h (a) and 24 h (b) in wild type 143B cells and 8 h (c) and 24 h (d) in wild type A549 cells. Relative cell viabilities after 8 h (e) and 24 h (f) in GOT1 siRNA knock-down A549 cells. Rescue of GOT1-null 143B cells with OAA at different concentrations upon glucose deprivation (g). Mean ± s.d. from 3 independent experiments. One-way ANOVA test was performed. *** p < 0.001; ** p < 0.01;* p < 0.05. NS: not significant. (TIF 230 kb

Additional file 3: of Inhibition of glutamate oxaloacetate transaminase 1 in cancer cell lines results in altered metabolism with increased dependency of glucose

Figure S3. Partial prevention of ischemic-like-cell-death morphological changes by NAD+. Bars indicate 25 Οm. (TIF 1353 kb

S1 Raw images -

Deficiency in thymidine kinase 2 (TK2) causes mitochondrial DNA depletion. Liver mitochondria are severely affected in Tk2 complete knockout models and have been suggested to play a role in the pathogenesis of the Tk2 knockout phenotype, characterized by loss of hypodermal fat tissue, growth retardation and reduced life span. Here we report a liver specific Tk2 knockout (KO) model to further study mechanisms contributing to the phenotypic changes associated with Tk2 deficiency. Interestingly, the liver specific Tk2 KO mice had a normal life span despite a much lower mtDNA level in liver tissue. Mitochondrial DNA encoded peptide COXI did not differ between the Tk2 KO and control mice. However, the relative liver weight was significantly increased in the male Tk2 KO mouse model. Histology analysis indicated an increased lipid accumulation. We conclude that other enzyme activities can partly compensate Tk2 deficiency to maintain mtDNA at a low but stable level throughout the life span of the liver specific Tk2 KO mice. The lower level of mtDNA was sufficient for survival but led to an abnormal lipid accumulation in liver tissue.</div

Histopathology and electron microscopy results.

Histopathology analysis of liver tissues from control (A) and livTk2 KO mice (B). Electron microscopy analysis of heart tissues in control (C) and in livTk2 KO mice (D). Electron microscopy analysis of liver tissues in control (E) and livTk2 KO mice (F). Blue arrows indicate lipid droplets in liver and red arrows indicate mitochondria in heart and liver tissues.</p

Primer sequences for real-time PCR analysis.

Deficiency in thymidine kinase 2 (TK2) causes mitochondrial DNA depletion. Liver mitochondria are severely affected in Tk2 complete knockout models and have been suggested to play a role in the pathogenesis of the Tk2 knockout phenotype, characterized by loss of hypodermal fat tissue, growth retardation and reduced life span. Here we report a liver specific Tk2 knockout (KO) model to further study mechanisms contributing to the phenotypic changes associated with Tk2 deficiency. Interestingly, the liver specific Tk2 KO mice had a normal life span despite a much lower mtDNA level in liver tissue. Mitochondrial DNA encoded peptide COXI did not differ between the Tk2 KO and control mice. However, the relative liver weight was significantly increased in the male Tk2 KO mouse model. Histology analysis indicated an increased lipid accumulation. We conclude that other enzyme activities can partly compensate Tk2 deficiency to maintain mtDNA at a low but stable level throughout the life span of the liver specific Tk2 KO mice. The lower level of mtDNA was sufficient for survival but led to an abnormal lipid accumulation in liver tissue.</div

Expression levels of genes involved in mtDNA maintenance and lipid metabolism.

(A-J) Tk2, dCKs, Rmr1, Rmr2, Rmr2b, Fasn, Serbp1, Cpt1gene, Tfam and Ppargc1α, respectively. Data presented as mean ± SD. Statistically significant difference (two-tailed unpaired Student’s t-test), *p<0.05. FC: Fold change (livTk2 KO vs wild type).</p

Generation of liver specific Tk2 knockout mouse model.

(A) Generation of targeting construct for deleting exon V in TK2 through homologous recombination. (B) Breeding strategy of generating livTk2 KO mice. (C and D) Screening of livTk2 KO mice through combining Alb-cre-specific primers (F1 and R1) and mutated-allele-specific primers (F2 and R2). Lanes 2 and 5 indicate homozygous livTk KO mice.</p

Body weights.

(A) Male mice and (B) female mice body weights were measured from week 3 to week 55. 20 mice in control group (n = 10 for each gender) and 8 mice in knockout group (n = 4 for each gender). The error bars indicate standard deviation. The Mann-Whitney test was used to compare the control to the knockout groups. Significant levels were set to p < 0.05(*).</p

1.5-year-old mice body weight and mtDNA copy number.

Male mice body weights (A), organ weights (B), and relative organ weights (C). Female mice body weights (D), organ weights (E), and relative organ weights (F). Mitochondrial DNA copy number from liver (G) and heart (H). SDHA and COX I protein levels in liver (I) and heart (J). n = 4 for each group, and error bars indicate standard deviation. The Mann-Whitney test was used to compare the control to the knockout groups. Significant levels were set to p < 0.05(*).</p

Heterophilic antibody (HA) interference in the different capture ELISA setups.

<p>Impact of HA on the detection of Trx1 in two human plasma samples. Two plasmas were diluted 5-fold in incubation buffer or HA blocking buffer and analyzed by Trx1 ELISA. A: analysis with pAb/pAb system where the capture pAb also was replaced by an irrelevant goat pAb. B: analysis with mAb/pAb system where the capture mAb also was replaced by an irrelevant mouse mAb. The experiment was repeated with similar results.</p