Journal: Nature Communications

Article Title: The mitochondrial type IB topoisomerase drives mitochondrial translation and carcinogenesis

doi: 10.1038/s41467-018-07922-3

Figure Lengend Snippet: TOP1MT promotes tumor growth. a Tumor growth of isogenic WT and TOP1MT knockout HCT116 xenografts as determined by caliper measurement. Cells (10,000) from two independent TOP1MT- deficient clones (KO1, n = 5; KO2, n = 9), a TOP1MT -expressing clone (WT ‡ , n = 3), which went through a mock CRISPR/Cas9 process, and the parental cell line (WT, n = 8) were injected subcutaneously in the flanks of female Ncr-nu/nu mice. b Weights of excised tumors were determined after 35 days (WT, KO2, n = 20; WT ‡ , n = 3; KO1, n = 8). c Representative bioluminescence imaging 35 days after transplantation of 10,000 cells of each type. d Quantification of the bioluminescence imaging. The total flux is plotted as photons per second (WT, n = 8; WT ‡ , n = 3; KO1, n = 9; KO2, n = 5). All data are means ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, unpaired, two-tailed Student’s t -test

Article Snippet: Antibodies were obtained from the following sources (Supplementary Table ): abcam: OXPHOS Rodent (ab110413), OXPHOS (ab110411), Ki67 (ab16667); Cell Signaling Technologies: Akt (#4691S), p-Akt (#9271S), cleaved caspase 3 (#9661), GAPDH (#5174); Thermo Fisher Scientific: MRPS22 (#PA5-52249); MT-CO2 (#MS-1372-P1); DSHB: TOP1MT (#CPTC-TOP1-MT-3), A6 (#A6 BCM-s); Sigma-Aldrich: β-actin (#A5441), Santa Cruz: IgG (#sc-2027).

Techniques: Knock-Out, Clone Assay, Expressing, CRISPR, Injection, Imaging, Transplantation Assay, Two Tailed Test

Journal: Nature Communications

Article Title: The mitochondrial type IB topoisomerase drives mitochondrial translation and carcinogenesis

doi: 10.1038/s41467-018-07922-3

Figure Lengend Snippet: Limiting dilution analyses

Article Snippet: Antibodies were obtained from the following sources (Supplementary Table ): abcam: OXPHOS Rodent (ab110413), OXPHOS (ab110411), Ki67 (ab16667); Cell Signaling Technologies: Akt (#4691S), p-Akt (#9271S), cleaved caspase 3 (#9661), GAPDH (#5174); Thermo Fisher Scientific: MRPS22 (#PA5-52249); MT-CO2 (#MS-1372-P1); DSHB: TOP1MT (#CPTC-TOP1-MT-3), A6 (#A6 BCM-s); Sigma-Aldrich: β-actin (#A5441), Santa Cruz: IgG (#sc-2027).

Techniques:

Journal: Nature Communications

Article Title: The mitochondrial type IB topoisomerase drives mitochondrial translation and carcinogenesis

doi: 10.1038/s41467-018-07922-3

Figure Lengend Snippet: Knocking out TOP1MT restrains cell proliferation and sensitizes cells to glucose starvation. a Representative Ki67 immunofluorescence staining of WT and TOP1MT -KO xenograft tumors. Scale bar, 50 μm. b , c Quantification of the fraction of Ki67-positive cells ( b ) and nuclei count per field ( c ) measured by ZEN software (6 images per animal, 5 animals per genotype). d Heat map showing significant changes in gene expression profiles analyzed with the nCounter PanCancer Progression panel in four TOP1MT -deficient vs. four WT tumors (89 genes, p < 0.05). e Transcript levels of selected genes of the PI3K/AKT pathway determined by RT-qPCR ( n = 4, each performed in triplicates). f Kinetics of cell growth under standard culture conditions and under glucose withdrawal (1 g L −1 glucose, dashed lines) (four independent experiments performed in quadruplets). g Growth of WT and TOP1MT -KO multicellular tumor spheroids (MCTS) formed by 10,000 HCT116 cells in six independent experiments, each performed in quintuplets. Day 0 corresponds to 48 h after cell seeding (spheroid maturation). Dashed and solid lines represent growth in the absence and presence of glucose, respectively. Data represent the means ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, Student’s t -test

Article Snippet: Antibodies were obtained from the following sources (Supplementary Table ): abcam: OXPHOS Rodent (ab110413), OXPHOS (ab110411), Ki67 (ab16667); Cell Signaling Technologies: Akt (#4691S), p-Akt (#9271S), cleaved caspase 3 (#9661), GAPDH (#5174); Thermo Fisher Scientific: MRPS22 (#PA5-52249); MT-CO2 (#MS-1372-P1); DSHB: TOP1MT (#CPTC-TOP1-MT-3), A6 (#A6 BCM-s); Sigma-Aldrich: β-actin (#A5441), Santa Cruz: IgG (#sc-2027).

Techniques: Immunofluorescence, Staining, Software, Gene Expression, Quantitative RT-PCR

Journal: Nature Communications

Article Title: The mitochondrial type IB topoisomerase drives mitochondrial translation and carcinogenesis

doi: 10.1038/s41467-018-07922-3

Figure Lengend Snippet: Mitochondria are defective in TOP1MT -KO HCT116 tumor xenografts. a Representative electron micrographs. The insets depict higher magnification of the boxed areas. Scale bar, 2 μm. b Quantification of swollen mitochondria in WT and TOP1MT -KO tumors. At least 48 images were analyzed from 12 independent areas at ×5000 magnification. c Mitochondrial mass determined by MitoTracker Deep Red FM staining of tumor cells isolated from WT and TOP1MT -KO tumor xenografts. The median ± SEM is plotted ( n = 5 for each genotype). d Differential oxygen consumption rate measured by Seahorse XF96 Extracellular Flux Analyzer in WT and TOP1MT -KO cells isolated from tumor xenografts ( n = 5 for each genotype, each performed in quintuplets). e Scheme of mitochondrial functions for cellular biosynthesis, bioenergetics and redox signaling. f Cellular energy levels measured by ATPlite ( n = 7). g Redox state determined by glutathione levels ( n = 5). h Steady-state level of the TCA cycle metabolite α-ketoglutarate, n = 10-12. i Reduced aspartate levels in TOP1MT -KO tumor xenografts ( n = 10). Data represent mean ± SEM except in c , * p < 0.05, ** p < 0.01, Student’s t -test

Article Snippet: Antibodies were obtained from the following sources (Supplementary Table ): abcam: OXPHOS Rodent (ab110413), OXPHOS (ab110411), Ki67 (ab16667); Cell Signaling Technologies: Akt (#4691S), p-Akt (#9271S), cleaved caspase 3 (#9661), GAPDH (#5174); Thermo Fisher Scientific: MRPS22 (#PA5-52249); MT-CO2 (#MS-1372-P1); DSHB: TOP1MT (#CPTC-TOP1-MT-3), A6 (#A6 BCM-s); Sigma-Aldrich: β-actin (#A5441), Santa Cruz: IgG (#sc-2027).

Techniques: Staining, Isolation

Journal: Nature Communications

Article Title: The mitochondrial type IB topoisomerase drives mitochondrial translation and carcinogenesis

doi: 10.1038/s41467-018-07922-3

Figure Lengend Snippet: Lack of TOP1MT impairs mitochondrial translation. a Reduced mtDNA copy number was determined by RT-qPCR in TOP1MT -KO and WT HCT116 tumor xenografts ( n = 9, each genotype, each performed in triplicates). b Conserved mitochondrial transcription profiles of TOP1MT -KO vs. WT tumor xenografts determined by tiling array ( n = 4, each genotype). c Representative western blots showing reduced levels of mitochondrial OXPHOS proteins in TOP1MT -KO tumor xenografts (lane 5–8). d Quantification of mitochondrial OXPHOS proteins ( n = 7, each genotype). e Gene ontology analysis of TOP1MT binding partners identified by TOP1MT immunoprecipitation followed by mass spectrometry. f , g Co-immunoprecipitation of TOP1MT-GFP ( f ) and MRPS22 ( g ) followed by western blotting. h Reduced growth of TOP1MT -KO HCT116 multicellular tumor spheroids. Day 0 corresponds to 48 h after cell seeding (spheroid maturation); n = 5, each performed in quintuplets. Dashed and solid lines represent spheroids treated with and without (CTRL) 5 μM tigecycline ( n = 5, each performed in quintuplets), respectively. i Reduced mitochondrial protein synthesis measured by [ 35 S]-methionine labeling of WT and Top1mt -KO MEFs. A representative gel shows the autoradiography of newly synthesized mitochondrial proteins (left). Equal protein loading was ensured by Coomassie staining (right). Data are mean ± SEM; * p < 0.05, ** p < 0.01, Student’s t -test

Article Snippet: Antibodies were obtained from the following sources (Supplementary Table ): abcam: OXPHOS Rodent (ab110413), OXPHOS (ab110411), Ki67 (ab16667); Cell Signaling Technologies: Akt (#4691S), p-Akt (#9271S), cleaved caspase 3 (#9661), GAPDH (#5174); Thermo Fisher Scientific: MRPS22 (#PA5-52249); MT-CO2 (#MS-1372-P1); DSHB: TOP1MT (#CPTC-TOP1-MT-3), A6 (#A6 BCM-s); Sigma-Aldrich: β-actin (#A5441), Santa Cruz: IgG (#sc-2027).

Techniques: Quantitative RT-PCR, Western Blot, Binding Assay, Immunoprecipitation, Mass Spectrometry, Labeling, Autoradiography, Synthesized, Staining

Journal: Nature Communications

Article Title: The mitochondrial type IB topoisomerase drives mitochondrial translation and carcinogenesis

doi: 10.1038/s41467-018-07922-3

Figure Lengend Snippet: TOP1MT promotes tumor growth in a mouse model of liver carcinogenesis. a Experimental design. Male mice received a single intraperitoneal (i.p.) administration of diethylnitrosamine (DEN, 25 mg kg −1 body weight) 14 days after birth. Beginning at 8 weeks, mice received biweekly injections of carbon tetrachloride (CCl 4 , 0.2 mL kg −1 ) for 14 weeks. b Representative images of liver tumors in WT and Top1mt−/− livers at the end of treatment. Tumors are encircled with white dashed circles. Scale bar, 1 cm. c Hematoxylin and eosin (H&E) staining of representative paraffin-embedded liver sections. Tumor areas are encircled with black dashed lines. Scale bar, 1 mm. d Quantification of tumor burden expressed as proportion of hepatic parenchyma occupied by tumor tissue on H&E sections, n = 10-11. e , f Maximum tumor size ( e ) and tumor number ( f ) determined by analysis of H&E sections, n = 10-11. g Transcript levels of Top1mt in surrounding liver versus tumor tissue ( n = 3, each performed in duplicates). h Quantification of Ki67-positive cells in the liver tumors ( n = 5). i Transcript levels of selected mitochondrial-encoded genes in Top1mt−/− liver tumors relative to WT tumors ( n = 3) determined by mitochondrial tiling array. j Representative western blots depicting protein levels of mitochondrial OXPHOS proteins in WT (lane 1–3) and Top1mt-/- (lane 4-6) tumors. GAPDH was used as loading control. k Quantification of mitochondrial OXHOS protein levels in WT and Top1mt-/- liver tumors relative to GAPDH ( n = 3). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, Student’s t -test

Article Snippet: Antibodies were obtained from the following sources (Supplementary Table ): abcam: OXPHOS Rodent (ab110413), OXPHOS (ab110411), Ki67 (ab16667); Cell Signaling Technologies: Akt (#4691S), p-Akt (#9271S), cleaved caspase 3 (#9661), GAPDH (#5174); Thermo Fisher Scientific: MRPS22 (#PA5-52249); MT-CO2 (#MS-1372-P1); DSHB: TOP1MT (#CPTC-TOP1-MT-3), A6 (#A6 BCM-s); Sigma-Aldrich: β-actin (#A5441), Santa Cruz: IgG (#sc-2027).

Techniques: Staining, Western Blot, Control

Journal: Nature Communications

Article Title: The mitochondrial type IB topoisomerase drives mitochondrial translation and carcinogenesis

doi: 10.1038/s41467-018-07922-3

Figure Lengend Snippet: TOP1MT -KO gene expression signature and expression predict survival of HCC patients. a Supervised hierarchical clustering of gene expression profiles from 3 independent Top1mt -KO and WT mouse HCCs. b Integrative cluster analysis of murine HCCs applied to 53 human HCC patient samples using orthologous genes. Light blue bars indicate HCC patients with good survival prognosis, dark blue bars, HCC patients with poor survival, dark red bars, murine Top1mt -KO HCC, and black bars, murine HCC expressing Top1mt . c Overall survival of HCC patients based on the TOP1MT gene expression signature. d High expression of TOP1MT is associated with poor survival of patients with HCC (370 total cases from the TCGA database including 138 patients with high TOP1MT expression and 232 patients with low TOP1MT expression)

Article Snippet: Antibodies were obtained from the following sources (Supplementary Table ): abcam: OXPHOS Rodent (ab110413), OXPHOS (ab110411), Ki67 (ab16667); Cell Signaling Technologies: Akt (#4691S), p-Akt (#9271S), cleaved caspase 3 (#9661), GAPDH (#5174); Thermo Fisher Scientific: MRPS22 (#PA5-52249); MT-CO2 (#MS-1372-P1); DSHB: TOP1MT (#CPTC-TOP1-MT-3), A6 (#A6 BCM-s); Sigma-Aldrich: β-actin (#A5441), Santa Cruz: IgG (#sc-2027).

Techniques: Gene Expression, Expressing

Journal: Neural Development

Article Title: Meningeal cells and glia establish a permissive environment for axon regeneration after spinal cord injury in newts

doi: 10.1186/1749-8104-6-1

Figure Lengend Snippet: Table of antibodies

Article Snippet: Collagen XII (newt) , Mouse IgG1 mAb , DSHB, MT2 , 1/50, concentrate.

Techniques:

Journal:

Article Title: MT2-MMP-dependent release of collagen IV NC1 domains regulates submandibular gland branching morphogenesis

doi: 10.1016/j.devcel.2009.07.016

Figure Lengend Snippet: (A) qPCR analysis of SMG gene expression at distinct developmental stages shows that MT1, MT2, and MT3 increase at E13 when branching morphogenesis begins.

Article Snippet: Primary antibodies included rabbit anti-mouse MT1, MT2, and MT3 (Triple Point Biologics, OR), rat anti-mouse perlecan, goat or rabbit anti-mouse collagen IV (both Chemicon, CA), and rat anti-mouse syndecan 1 (BD Pharmingen, CA).

Techniques: Gene Expression

Journal:

Article Title: MT2-MMP-dependent release of collagen IV NC1 domains regulates submandibular gland branching morphogenesis

doi: 10.1016/j.devcel.2009.07.016

Figure Lengend Snippet: (A) SMGs were cultured for 36 h with non-silencing (NS) and MT1-, MT2-, and MT3-siRNAs. MT2-siRNA has the most significant effect on epithelial morphogenesis.

Article Snippet: Primary antibodies included rabbit anti-mouse MT1, MT2, and MT3 (Triple Point Biologics, OR), rat anti-mouse perlecan, goat or rabbit anti-mouse collagen IV (both Chemicon, CA), and rat anti-mouse syndecan 1 (BD Pharmingen, CA).

Techniques: Cell Culture