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: Molecular Cancer

Article Title: Tumor cell invasion of collagen matrices requires coordinate lipid agonist-induced G-protein and membrane-type matrix metalloproteinase-1-dependent signaling

doi: 10.1186/1476-4598-5-69

Figure Lengend Snippet: HEK293 cells transfected with MT1-MMP cDNA invade 3D collagen gels in response to LPA . (A) Tumor cell lysates were prepared for Western blot analysis. Lysates were probed for MT1-MMP to assess protein expression in the four tumor cell lines. Lysates were probed for Actin as a loading control. (B) HEK293 cells were transfected with the pAdTrack-CMV plasmid as a control, or plasmids encoding MT1-MMP, MT2-MMP, or MT3-MMP cDNA 24 hours prior to placement in invasion assays. Cells were allowed to invade 2.0 mg/ml collagen gels in the presence or absence of 1 μM LPA. Data are expressed as mean numbers of invading cells per HPF (20×) (± S.D.) from a minimum of 20 fields. (C) Lysates from HEK293 cells transfected with cDNAs encoding the designated genes were prepared for Western blot analysis and probed for GFP, MT1-MMP, MT2-MMP, MT3-MMP, or Actin as a loading control. TRK = pAdTrack-CMV, MT1 = MT1-MMP, MT2 = MT2-MMP, MT3 = MT3-MMP.

Article Snippet: Human MT1-MMP (AF918, R&D Systems, Minneapolis, MN), MT3-MMP (RP1-MMP-16, Triple Point Biologics, Forest Grove, OR), and Rac1 (ARC01, Cytoskeleton, Denver, CO), and monoclonal antibodies directed against human MT2-MMP (MAB916, R&D Systems), Lamin A/C (MAB3211, Chemicon Corp), Actin (JLA-20, Calbiochem, San Diego, CA), RhoA (ARH01, Cytoskeleton), and Cdc42 (610929, BD Tansduction, San Jose, CA) were used for Western blot analysis as described previously [ ]. si GENOME SMART pool ® reagents were products of Dharmacon (Lafayette, CO).

Techniques: Transfection, Western Blot, Expressing, Control, Plasmid Preparation

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: MT2-MMP associates with ZO-1 in polarized MDCK cells. (A) Western blot analysis of HA, E-cadherin and Rho-GDI in biotinylated cell lysates from Mock, MT2-MMP (MT2FL) and MT2-MMPWK (MT2WK) stable MDCK transfectants pulled down with streptavidin beads; input, unbound and bound fractions are shown (Inp, Unb, Biot). (B) Western blot analysis of ZO-1 and HA in cell lysates from Mock, MT2-MMP and MT2-MMPWK stable MDCK transfectants pulled down with anti-HA antibody; IgG immunoprecipitates and whole lysates (Input) are also shown as controls. A blot of the input lanes after a longer exposure is also shown. (C) Representative maximal projections from apical and basolateral stacks of confocal sections from polarized MDCK transfectants stained for HA (MT2-MMP, green), ZO-1 (red) and nuclei (Hoechst, blue). (D) Orthogonal x–z views of 3D confocal image stacks from C. (E) Representative peak intensity profiles from x–z views of 3D confocal image stacks from C. Graph to the right shows the quantification of MT2-MMP/ZO-1 Pearson correlation coefficient in polarized MT2-FL and MT2-WK MDCK transfectants. Values are mean±s.e.m. n=40 cells per condition in two independent experiments; *P<0.05.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Western Blot, Staining

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: MT2-MMP overexpression induces aberrant apical epithelial cell accumulation in polarized MDCK monolayers. (A) Orthogonal x–z projections of 3D confocal image stacks of MDCK transfectants stained for F-actin (Phalloidin, gray), HA (MT2-MMP, green) and Hoechst (nuclei, blue). Scale bar: 10 μm. (B) Quantification of apical epithelial foci per field (left) and the percentage of foci having more than 8 nuclei (right). 10 fields were counted per condition in n=4 independent experiments. (C) Representative maximal projections are shown from subapical and complete stacks of confocal sections from polarized MDCK transfectants stained for E-cadherin (gray). The dotted yellow line marks apical foci. Orthogonal x–z views are shown to the right. (D) Line and bar graphs show E-cadherin peak and average mean fluorescence intensity (MFI), respectively, around the junctions formed by MDCK transfectants. Data are represented as mean±s.e.m. and were tested by one-way ANOVA versus mock 1 followed by Dunnett's post-test in B and C. **P<0.01, ***P<0.001, ****P<0.001; ns, not significant.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Over Expression, Staining, Fluorescence

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: Apical epithelial cell accumulation depends on MT2-MMP catalytic activity. (A) Representative maximal projections from confocal sections of polarized MDCK transfectants stained for E-cadherin (gray) in the presence of GM6001 (50 μM) or vehicle (DMSO). Orthogonal x–z views are shown below. (B) Line and bar graphs show E-cadherin peak and average intensity, respectively, around the junctions formed by MDCK transfectants treated as in A. Bar graph at the bottom shows the number of apical events on the polarized MDCK monolayer in the presence or absence of DMSO. In the middle and bottom graphs, the difference between mock DMSO and MT2 FL were significant with P<0.01 and P<0.0001, respectively. (C) Representative maximal projections are shown from confocal sections of polarized MDCK transfectants (mock, MT2 and MT2EA) stained for E-cadherin (gray). Orthogonal x–z views are shown to the right. (D) Line and bar graphs show E-cadherin peak and average mean fluorescence intensity (MFI), respectively, around the junctions formed by MDCK transfectants shown in C. Bar graph on the right shows the number of apical events occurring in polarized MDCK monolayers. Data are represented as mean± s.e.m. and were tested by one-way ANOVA followed by Sidak post-test in B. Dunnett's post-test was used in D. *P<0.05, **P<0.01, ****P<0.001; ns, not significant.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Activity Assay, Staining, Fluorescence

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: E-cadherin is cleaved by MT2-MMP after N445 in the EC5 loop. (A) In silico model of canine E-cadherin (green)/human MT2-MMP (blue) interactions in cis association at the plasma membrane; the catalytic MT2-MMP center and the E-cadherin peptide, GPIPEPRNMDFCQKNPQP, are shown in orange and red, respectively. (B) Scheme of E-cadherin structure with the peptide containing the predicted cleavage sites after N445 and N459 in the EC5 loop. (C) Representative extracted ion chromatograms of 3 independent experiments corresponding to the peptides detected following in in vitro digestion of the GPIPEPRNMDFCQKNPQP peptide in the absence or presence of the human MT2-MMP recombinant catalytic domain (rhMT2). (D) Western blot analysis of lysates recovered from MDCK transfectants cultured with different calcium concentrations. Results are representative of two independent experiments. (E) Representative orthogonal x–z views of confocal images for polarized MDCK transfectants co-immunostained for HA (MT2-MMP, green), E-cadherin (red) and nuclei (Hoechst, blue).

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: In Silico, Clinical Proteomics, Membrane, In Vitro, Recombinant, Western Blot, Cell Culture

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: MT2-MMP disrupts apical E-cadherin-mediated signals. (A) Orthogonal x–z projections of 3D confocal image stacks are shown of polarized MDCK transfectants stained for F-actin (Phalloidin, green), myosin IIB (red), and nuclei (Hoechst, blue). (B) Orthogonal x–z projections of 3D confocal image stacks are shown of polarized MDCK transfectants (mock and MT2-MMP) in the presence of 4-HAP (500 nM) or vehicle (DMSO) for 72 h and stained for F-actin (phalloidin, green), myosin IIB (red), and nuclei (Hoechst, blue). (C) Quantification of apical/total MFI of myosin IIB in polarized MDCK transfectants treated with 4-HAP (500 nM) or vehicle (DMSO); n=5 independent experiments. (D) Quantification of cell circularity in MDCK cells presented in C. 25 cells per field were counted in 2 images per condition in 6 independent experiments. (E) Quantification of the number of apical events on polarized MDCK cells presented in panel B. 10 fields were counted per condition in n=4 independent experiments. Difference between mock DMSO1 and MT2 FL1, and mock DMSO2 and MT2 FL2 were significant with P<0.0001 and P<0.05, respectively. Data are represented as mean±s.e.m. and were tested by one-way ANOVA followed by Sidak post-test. *P<0.05, **P<0.01, ****P<0.0001; ns, not significant.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Staining

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: Mislocalization of pSrc in polarized MT2-MMP-MDCK cells contributes to apical cell accumulation. (A) Percentage of cells in G0/G1, S, and G2/M phases of the cell cycle analyzed by flow cytometry in propidium-iodide-stained MDCK transfectants after 72 h of serum deprivation. Means±s.e.m. are shown for 3 independent experiments. (B) Orthogonal x–z projections of 3D confocal image stacks of polarized MDCK transfectants (mock and MT2-MMP) stained for F-actin (Phalloidin, green), pSrc (red), and nuclei (Hoechst, blue). Representative peak intensity profiles are shown on the right for pSrc (red) and F-actin (green). (C) Bar graphs show the apical (left) and junctional (right) pSrc intensity, relative to total mean fluorescence intensity (MFI) in 6 independent experiments. (D) Number of apical events occurring in polarized MDCK cells treated with PP2 or vehicle (DMSO). 10 fields were counted per condition in 3 independent experiments. Differences between mock DMSO1 and MT2 FL1, and mock DMSO2 and MT2 FL2 were significant with P<0.001 and P<0.01, respectively. (E) Representative confocal images of 3D cysts formed by MDCK transfectants in Matrigel and stained for pSrc (green), F-actin (white), E-cadherin (red), and nuclei (Hoechst, blue). (F) Quantification of the percentage of lumenized cysts. Data are represented as the mean±s.e.m. and were tested by two-way ANOVA followed by Dunnett's post-test in A, two-tailed Welch-test comparison was used in B, and one-way ANOVA followed by Sidak post-test was used in C. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; ns, not significant.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Flow Cytometry, Staining, Fluorescence, Two Tailed Test, Comparison

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: MT2-MMP deficiency alters junctional E-cadherin and leads to decreased 3D colon organoid formation ex vivo and smaller crypts in vivo. (A) Representative confocal images of MT2-MMP-silenced colon organoids stained for MT2-MMP (green; top). Graph shows the normalized MT2-MMP mean fluorescence intensity (MFI) in stained organoids 72 h after siRNA transfection (bottom), n=6 images per condition from 3 independent experiments; Mmp15 mRNA levels decreased ∼20% in silenced organoids. (B) Bright-field microscopy images of MT2-MMP-silenced colon organoids. Bar graph shows the percentage of organoid generation efficiency 48 h after siRNA transfection (right) in 3 independent experiments. (C) Representative confocal images of MT2-MMP-silenced colon organoids stained for nuclei (Hoechst/Ho, blue), F-actin (red), E-cadherin (green) and β-catenin (white); magnified views of E-cadherin and β-catenin staining are shown in insets. (D) Representative confocal images of colonic tissues recovered from wild-type or MT2-MMP-null mice, and stained for Ki67 (red) and nuclei (Hoechst, blue). On the right, graph shows the percentage of Ki67-positive cells per crypt. 9–15 crypts were quantified per condition in 3 images taken from 2 mice per genotype (top). (E) Quantification of the cumulative frequency of crypt length (left) and width (right). Data are represented as mean±s.e.m. and were tested by unpaired Student's t-test in A and B and by two-tailed Welch-test comparison in D and E. *P<0.05, **P<0.01, ***P<0.001; ns, not significant.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Ex Vivo, In Vivo, Staining, Fluorescence, Transfection, Microscopy, Two Tailed Test, Comparison

Journal: bioRxiv

Article Title: Kallikrein-related peptidase 14 activates zymogens of membrane type matrix metalloproteinases (MT-MMPs) - a CleavEx library-based analysis

doi: 10.1101/2020.04.23.057109

Figure Lengend Snippet: Activation of proMMPs by KLK14 results in a fully functional mature enzyme. Each proMMP was incubated with the indicated concentrations of KLK14 for an hour at 37°C. The reaction was stopped by the addition of KLK14-specific inhibitors and the reaction mixture was analyzed by SDS-PAGE, followed by a zymogram with gelatin as a substrate. The proMMP2 (A) negative control was not activated. ProMMP14 (B) , proMMP15 (C) and proMMP16 (D) were activated whereas proMMP17 (E) did not show hydrolysis of gelatin, yet a shift corresponding to the loss of the profragment was observed (note that an amino acid substitution was introduced in proMMP17 by the manufacturer (R&D Systems)).

Article Snippet: A total of 0.5 μg native proMMP2 (catalog no. 902-MP-010, R&D systems), 0.5 μg proMMP14 (catalog no. 918-MP-010, R&D systems), 1 μg proMMP15 (catalog no. 916-MP-010, R&D systems), 0.5 μg ProMMP16 (catalog no. 1785-MP-010, R&D systems) and 1 μg ProMMP17 (catalog no. 7796-MP-010, R&D systems) were separately incubated in 10 μl with a range of KLK14 concentrations (25-250 nM, with molar ratios from around 1:65 to 1:10 KLK14:MMP) in the presence of 5 μM batimastat (Sigma) for 1 hour at 37°C in PBS.

Techniques: Activation Assay, Functional Assay, Incubation, SDS Page, Negative Control

Journal: International journal of molecular sciences

Article Title: Silencing PROK2 Inhibits Invasion of Human Cervical Cancer Cells by Targeting MMP15 Expression.

doi: 10.3390/ijms21176391

Figure Lengend Snippet: Figure 3. Effect of knockdown PROK2 on MMP15 expression and cell invasion in human cervical cancer HeLa cells. (A) Human HeLa cells were transfected with or without PROK2 shRNA, then followed by measuring the capacity of cell migration and invasion. (B,C) The protein and mRNA expression of MMP15 were inhibited by shPROK2-HeLa cells were measured by western blotting and R-qPCR assay. (D) Validation of MMP15 gene expression in matched cervical cancer tissues and adjacent noncancerous cervical tissues from the GEPIA databases. T: cervical tumour tissue (n = 306); N: normal cervical tissue (n = 13), * p < 0.05 versus normal cervical tissue. (E) Overall survival rate (OS) in patients with high or low MMP15 expression. The red line indicates high expression, and black line indicates low expression. (F) MMP15 expression was correlated with PROK2 expression in human cervical cancer patients. ** p < 0.01 versus shLuc cells.

Article Snippet: Antibodies against MMP15 (130522, 1:500) was obtained from Novus Biologicals (Briarwood, CO, USA) and horseradish peroxidase (HRP)-conjugated anti-mouse (AP124P, 1:10,000) were purchased from Merck Millipore (Burlington, MA, USA).

Techniques: Knockdown, Expressing, Transfection, shRNA, Migration, Western Blot, Biomarker Discovery, Gene Expression

Journal: International journal of molecular sciences

Article Title: Silencing PROK2 Inhibits Invasion of Human Cervical Cancer Cells by Targeting MMP15 Expression.

doi: 10.3390/ijms21176391

Figure Lengend Snippet: Figure 4. MMP15 involved in PROK2 regulates cell migration and invasion in human cervical cancer HeLa cells. Using transfected with Neo or PROK2 overexpression plasmid in shLuc- or shPROK2-HeLa cells for 48 h. (A) The protein expression of MMP15 and PROK2 were measured by the western blotting. β-actin as a protein loading control. (B) The MMP15 and PROK2 mRNA expression were detected by RT-qPCR assay. GAPDH as a mRNA loading control. (C) In vitro migration and invasion assay was conducted to measures the cell migration and invasion numbers. ** p < 0.01 versus shLuc cells; # p < 0.05 verus shPROK2 cells.

Article Snippet: Antibodies against MMP15 (130522, 1:500) was obtained from Novus Biologicals (Briarwood, CO, USA) and horseradish peroxidase (HRP)-conjugated anti-mouse (AP124P, 1:10,000) were purchased from Merck Millipore (Burlington, MA, USA).

Techniques: Migration, Transfection, Over Expression, Plasmid Preparation, Expressing, Western Blot, Control, Quantitative RT-PCR, In Vitro, Invasion Assay

Journal: Heliyon

Article Title: Melatonin alleviates aging-related heart failure through melatonin receptor 1A/B knockout in mice.

doi: 10.1016/j.heliyon.2024.e38098

Figure Lengend Snippet: Fig. 1. Deletion of MT1 and MT2 inhibits the protective effect of melatonin on cardiac function in aging mice. (A) Survival curves of mice in MT1 group and aging group; (B) Survival curves of mice in MT2 group and aging group; (C) the ratios of heart weight to tibia length (HW/TL); (D) the ratios of heart weight to body weight (HW/BW); (E) M-mode echocardiography; (F) quantification of EF%; (G) quantification of FS%; (H) Heart cross-sections were stained with WGA; (I) The mRNA expression of Nppa and Nppb in each group; (J) The expression of p-ERK 1/2, ERK 1/2, p-AKT, AKT and β-actin in each group.

Article Snippet: MT1 and MT2 KO C57BL/6 male mice were purchased from the Cyagen Biosciences (Suzhou, China).

Techniques: Staining, Expressing

Journal: Heliyon

Article Title: Melatonin alleviates aging-related heart failure through melatonin receptor 1A/B knockout in mice.

doi: 10.1016/j.heliyon.2024.e38098

Figure Lengend Snippet: Fig. 3. Knockout of MT1 and MT2 promotes oxidative stress in the hearts of aging mice (A) Representative DHE staining; (B) Representative γ-H2AX and DAPI staining; (C) The mRNA expression of Nox2 and Nox4 in each group; (D) The protein expression of NOX2, NOX4 and β-actin in each group.

Article Snippet: MT1 and MT2 KO C57BL/6 male mice were purchased from the Cyagen Biosciences (Suzhou, China).

Techniques: Knock-Out, Staining, Expressing

Journal: Heliyon

Article Title: Melatonin alleviates aging-related heart failure through melatonin receptor 1A/B knockout in mice.

doi: 10.1016/j.heliyon.2024.e38098

Figure Lengend Snippet: Fig. 4. Knockout of MT1 and MT2 promoted cardiac senescence and myocardial apoptosis in aging mice. (A) Representative TUNEL; (B) Representative p16 immunohistochemistry in each group; (C) The expression of p53 and β-actin were detected by western Blot.

Article Snippet: MT1 and MT2 KO C57BL/6 male mice were purchased from the Cyagen Biosciences (Suzhou, China).

Techniques: Knock-Out, TUNEL Assay, Immunohistochemistry, Expressing, Western Blot

Journal: Environmental pollution (Barking, Essex : 1987)

Article Title: Biotransformation of graphene oxide within lung fluids could intensify its synergistic biotoxicity effect with cadmium by inhibiting cellular efflux of cadmium.

doi: 10.1016/j.envpol.2022.119421

Figure Lengend Snippet: Fig. 3. The cytotoxicity of Cd and intracellular Cd accumulation with or without GOs pre-treatment. (a) The cellular viability of BEAS-2B cells detected by CCK8 assay after being treated to Cd (1, 5, 10, 20, 25, 30 and 50 μM) for 24 h (n = 5, * indicate P < 0.05, compared to the untreated group). (b) The heat map of the cell viability inspected by CCK8 assay for BEAS- 2B cells after being treated to Cd (1, 5, 10, 20, 25, 30 and 50 μM) for 24 h with or without pre-treatment of P-GO, A-GO, or G-GO at the dose of 10 μg/mL, respectively (n = 5). (c) Intracellular Cd mass quan tification by ICP-MS. BEAS-2B cells were pre-treated with P-GO, A-GO, G-GO at 10 μg/mL for 24 h, and then exposed to CdCl2 at 10 μM for 24 h (n = 5), *P < 0.05. (d) Western blot analysis of MT1M and MT2A protein expression levels in BEAS-2B cells exposed to either GOs (10 μg/mL) or Cd (10 μM) or a combina tion of both (GOs + Cd) for 24 h.

Article Snippet: Antibodies (Abs) used were as follows: anti-NRF2 Ab (1:2000 dilution, Proteintech, USA), anti-SOD1 Ab (1:5000 dilution, Proteintech, USA), anti-SOD2 Ab (1:3000 dilution, Proteintech, USA), anti-MT1M (1:500 dilution, Proteintech, USA), anti-MT2A Ab (1:2000 dilution, Affinity, USA), anti-Caspase-8 Ab (1:5000 dilution, Proteintech, USA), anti-Bax Ab (1:5000 dilution, Proteintech, USA), anti-Bcl-2 Ab (1:5000 dilution, Proteintech, USA), anti-RIP1 Ab (1:2500 dilution, Proteintech, USA), anti-RIP3 Ab (1:2500 dilution, Proteintech, USA), anti-ABCB1 Ab (1:5000 dilution, Proteintech, USA), anti-ABCC1 Ab (1:2500 dilution, Proteintech, USA), anti-ABCG2 Ab (1:1000 dilution, Proteintech, USA).

Techniques: CCK-8 Assay, Western Blot, 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: