Journal: Limnology and Oceanography

Article Title: Carbon content, carbon fixation yield and dissolved organic carbon release from diverse marine nitrifiers

doi: 10.1002/lno.12252

Figure Lengend Snippet: Elemental stoichiometry of phylogenetically diverse cultured marine nitrifiers during different growth phases (early exponential, late exponential, stationary) including previously published values. C : N ratios were obtained during exponential growth phase. Cellular C content values are derived from DIC incorporation measurements if not stated otherwise.

Article Snippet: Nitrosopumilus adriaticus NF5 (= JCM 32270 T = NCIMB 15114 T ) and Nitrosopumilus piranensis D3C (= JCM 32271 T = DSM 106147 T = NCIMB 15115 T ) were isolated from the Northern Adriatic Sea and have been described in detail (Bayer et al. , 2019 c ).

Techniques: Cell Culture, Derivative Assay, Bacteria

Journal: Limnology and Oceanography

Article Title: Carbon content, carbon fixation yield and dissolved organic carbon release from diverse marine nitrifiers

doi: 10.1002/lno.12252

Figure Lengend Snippet: DIC fixation yields of Nitrosopumilus sp. CCS1 and Nitrospina sp. Nb‐3 under different culture conditions (substrate concentrations: 1 μ mol L −1 , 250 μ mol L −1 , 1 mmol L −1 ; temperature: 15°C, 25°C) and culture media (natural seawater, artificial seawater, HEPES‐buffered artificial seawater). Plotted values include both, the fraction of C incorporated into biomass and the fraction of C released as DOC. Ammonium (50 μ mol L −1 ) or tryptone (150 mg L −1 ) served as additional, reduced nitrogen source for Nitrospina sp. Nb‐3. Statistical significance (adj. p < 0.01) of within‐condition comparisons are indicated by an asterisk (*). Statistical results of all pairwise comparisons are reported in Table .

Article Snippet: Nitrosopumilus adriaticus NF5 (= JCM 32270 T = NCIMB 15114 T ) and Nitrosopumilus piranensis D3C (= JCM 32271 T = DSM 106147 T = NCIMB 15115 T ) were isolated from the Northern Adriatic Sea and have been described in detail (Bayer et al. , 2019 c ).

Techniques:

Journal: Limnology and Oceanography

Article Title: Carbon content, carbon fixation yield and dissolved organic carbon release from diverse marine nitrifiers

doi: 10.1002/lno.12252

Figure Lengend Snippet: Thermodynamic considerations and comparison of DIC fixation efficiencies and biomass yields of marine ammonia‐oxidizing archaea and nitrite‐oxidizing bacteria grown under environmentally relevant conditions (substrate concentration: 1 μ mol L −1 ; temperature: 15°C) in artificial and natural seawater medium. Gibbs free energy calculations for NH 3 oxidation and NO 2 − oxidation can be found in Table S1 .

Article Snippet: Nitrosopumilus adriaticus NF5 (= JCM 32270 T = NCIMB 15114 T ) and Nitrosopumilus piranensis D3C (= JCM 32271 T = DSM 106147 T = NCIMB 15115 T ) were isolated from the Northern Adriatic Sea and have been described in detail (Bayer et al. , 2019 c ).

Techniques: Comparison, Bacteria, Concentration Assay

Journal: Limnology and Oceanography

Article Title: Carbon content, carbon fixation yield and dissolved organic carbon release from diverse marine nitrifiers

doi: 10.1002/lno.12252

Figure Lengend Snippet: DOC release by marine nitrifiers as a fraction of fixed DIC. ( a ) Comparison of DOC release by 10 different phylogenetically diverse marine nitrifiers. Values obtained from cultures grown under different conditions (see panel b ) are included in this plot. DOC release by Ca . N. brevis might be underestimated due to the presence of heterotrophic bacteria that could take up some of the released DOC. ( b ) Comparison of DOC release by Nitrosopumilus sp. CCS1 and Nitrospina sp. Nb‐3 grown under different culture conditions (substrate concentrations: 1 μ mol L −1 , 250 μ mol L −1 , 1 mmol L −1 ; temperature: 15°C, 25°C) in artificial or natural seawater medium. Statistical results of all pairwise comparisons are reported in Table .

Article Snippet: Nitrosopumilus adriaticus NF5 (= JCM 32270 T = NCIMB 15114 T ) and Nitrosopumilus piranensis D3C (= JCM 32271 T = DSM 106147 T = NCIMB 15115 T ) were isolated from the Northern Adriatic Sea and have been described in detail (Bayer et al. , 2019 c ).

Techniques: Comparison, Bacteria

Journal: Nature Communications

Article Title: SPOP mutation induces DNA methylation via stabilizing GLP/G9a

doi: 10.1038/s41467-021-25951-3

Figure Lengend Snippet: a Western blots with indicated antibodies in WCL and co-IP samples from 293T cells transfected with plasmids of empty vector (EV) or indicated SPOP mutants and treated with 20 µM MG132 for 8 h. b Western blots with indicated antibodies in WCL and co-IP samples of anti-Flag antibody from 293T cells transfected with the indicated plasmids and treated with 20 µM MG132 for 8 h. c Western blots of WCL from 293T cells transfected with the indicated plasmids. d Western blots of WCL from 22Rv1 cells stably infected with lentivirus expressing EV, WT or mutant HA-SPOP. e , f Representative IFC images of Myc-SPOP and endogenous GLP staining in 22Rv1 cells transfected with Myc-SPOP F102C ( e ) and the optical density (OD)/nuclear area (pixel) of GLP staining was quantified using ImageJ ( f ). Data shown means ± SD ( n = 50 cells/group). Scale bar, 10 μm. g , h Representative IHC images of GLP and G9a staining in SPOP-WT and Q165P mutant PDX tumors ( g ) and the quantitative data of GLP and G9a staining ( h ). Scale bar in 200X images, 100 μm; Scale bar in 400X images, 50 μm. Data shown means ± SD ( n = 3 replicates/group). i , j Representative IHC images of GLP and G9a staining in 84 PCa patient specimens ( i ) and the quantitative data of GLP and G9a staining ( j ). Scale bar, 50 μm. Statistical significance was determined by unpaired two-tailed Student’s t test in ( f , h ). Statistical significance was determined by two-tailed Wilcoxon rank-sum test in ( j ). Experiments in ( a – d ) were repeated twice. Source data are provided as a Source Data file.

Article Snippet: The antibodies used: SPOP (dilution 1:1000, 16750-1-AP, Proteintech), GLP (dilution 1:1000, A301-642A, Bethyl), G9a (dilution 1:1000, PP-A8620A-00, R&D), MYC (dilution 1:1000, M192-7, MBL), Flag (dilution 1:1000, F180-4, Sigma), HA (dilution 1:1000, M180-7, MBL), Actin (dilution 1:1000, AC028, Abclonal), DNMT1 (dilution 1:1000, 5032 S, CST), DNMT3A (dilution 1:300, SC-373905, Santa Cruz Biotechnology), DNMT3B (dilution 1:300, SC-376043, Santa Cruz Biotechnology), DEK (dilution 1:1000, 13962 S, CST), BRD4 (dilution 1:1000, 13440 S, CST), CUL3 (dilution 1:1000, 2759 S, CST), RBX1 (dilution 1:1000, PA5-29149, Thermo Scientific), FOXO3A (dilution 1:1000, 12829 S, CST), GATA5 (dilution 1:300, SC-373684, Santa Cruz Biotechnology), Histone H3 (dilution 1:3000, 9715, CST), H3K9 2me (dilution 1:1000, ab1220, Abcam), NDRG1 (dilution 1:300, SC-398291, Santa Cruz Biotechnology), AR (dilution 1:1000, SC-815, Santa Cruz Biotechnology), PD-L1 (dilution 1:1000, 13684S, CST), cleaved-PARP (dilution 1:1000, 5625S, CST).

Techniques: Western Blot, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Stable Transfection, Infection, Expressing, Mutagenesis, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: SPOP mutation induces DNA methylation via stabilizing GLP/G9a

doi: 10.1038/s41467-021-25951-3

Figure Lengend Snippet: a Pearson correlation of 5mC and GLP/G9a IHC scores in 84 PCa patient specimens. b – d Western blots of WCL from 22Rv1 cells infected with lentivirus expressing indicated constructs and shRNAs ( b ). Representative IFC images of 5mC and HA-SPOP F133V staining are shown in ( c ), and ImageJ was used to quantify the optical density (OD)/nuclear area (pixel) of 5mC signals ( d ). Data shown means ± SD ( n = 50 cells/group). Scale bar, 10 μm. e , f 22Rv1 cells infected with lentivirus expressing indicated constructs and shRNAs were harvested for Western blots with indicated antibodies ( e ) or used for MTS assay to measure cell proliferation ( f ). Data shown means ± SD ( n = 5 replicates/group). g - i 22Rv1 cells infected with lentivirus expressing EV and SPOP F133V and treated with 2 µM UNC0642 for 24 h for western blots ( g ). Representative IFC images of 5mC and HA-SPOP F133V staining are shown in ( h ) and 5mC signals in each group were quantified using ImageJ ( i ). Data shown means ± SD ( n = 50 cells/group). Scale bar, 10 μm. Statistical significance was determined by unpaired two-tailed Student’s t test in ( a , d , f , i ). Experiments in ( b , e , g ) were repeated twice. Source data are provided as a Source Data file.

Article Snippet: The antibodies used: SPOP (dilution 1:1000, 16750-1-AP, Proteintech), GLP (dilution 1:1000, A301-642A, Bethyl), G9a (dilution 1:1000, PP-A8620A-00, R&D), MYC (dilution 1:1000, M192-7, MBL), Flag (dilution 1:1000, F180-4, Sigma), HA (dilution 1:1000, M180-7, MBL), Actin (dilution 1:1000, AC028, Abclonal), DNMT1 (dilution 1:1000, 5032 S, CST), DNMT3A (dilution 1:300, SC-373905, Santa Cruz Biotechnology), DNMT3B (dilution 1:300, SC-376043, Santa Cruz Biotechnology), DEK (dilution 1:1000, 13962 S, CST), BRD4 (dilution 1:1000, 13440 S, CST), CUL3 (dilution 1:1000, 2759 S, CST), RBX1 (dilution 1:1000, PA5-29149, Thermo Scientific), FOXO3A (dilution 1:1000, 12829 S, CST), GATA5 (dilution 1:300, SC-373684, Santa Cruz Biotechnology), Histone H3 (dilution 1:3000, 9715, CST), H3K9 2me (dilution 1:1000, ab1220, Abcam), NDRG1 (dilution 1:300, SC-398291, Santa Cruz Biotechnology), AR (dilution 1:1000, SC-815, Santa Cruz Biotechnology), PD-L1 (dilution 1:1000, 13684S, CST), cleaved-PARP (dilution 1:1000, 5625S, CST).

Techniques: Western Blot, Infection, Expressing, Construct, Staining, MTS Assay, Two Tailed Test

Journal: Nature Communications

Article Title: SPOP mutation induces DNA methylation via stabilizing GLP/G9a

doi: 10.1038/s41467-021-25951-3

Figure Lengend Snippet: a Diagram showing wild-type GLP, enzymatic inactive mutant C1201A and ANK & NHHC domain double deletion mutant. b Western blots with indicated antibodies in WCL and co-IP samples from 22Rv1 cells transfected with indicated plasmids. c – f 22Rv1 cells infected with lentivirus expressing indicated plasmids and/or shRNAs and transfected with indicated constructs were used for western blot ( c ), IFC ( d ) and MeDIP-qPCR ( f ). ImageJ was used to quantify the optical density (OD)/nuclear area (pixel) of 5mC staining in each group. Data shown means ± SD ( n = 50 cells/group) ( e ). Scale bar, 10 μm. Data shown means ± SD ( n = 3 replicates/group) ( f ). g A working model based on the current findings. Left , WT SPOP recognizes and promotes proteasomal degradation of GLP, thereby destabilizing the GLP/G9a complex, inhibiting GLP/G9a-mediated interaction with DNMTs and DNA methylation, and inducing expression of tumor suppressors such as FOXO3 and inhibition of cell proliferation. Right , PCa-associated SPOP mutations fail to bind and degrade GLP, thereby inducing elevation of the GLP/G9a complex and their interaction with DNMTs, increasing DNA methylation and suppression of expression of tumor suppressors and promoting cell proliferation. However, this process can be reversed by DNA demethylation reagents such as 5-AzaC in SPOP-mutant cells. Statistical significance was determined by unpaired two-tailed Student’s t test in ( e , f ). Experiments in ( b , c ) were repeated twice. Source data are provided as a Source Data file.

Article Snippet: The antibodies used: SPOP (dilution 1:1000, 16750-1-AP, Proteintech), GLP (dilution 1:1000, A301-642A, Bethyl), G9a (dilution 1:1000, PP-A8620A-00, R&D), MYC (dilution 1:1000, M192-7, MBL), Flag (dilution 1:1000, F180-4, Sigma), HA (dilution 1:1000, M180-7, MBL), Actin (dilution 1:1000, AC028, Abclonal), DNMT1 (dilution 1:1000, 5032 S, CST), DNMT3A (dilution 1:300, SC-373905, Santa Cruz Biotechnology), DNMT3B (dilution 1:300, SC-376043, Santa Cruz Biotechnology), DEK (dilution 1:1000, 13962 S, CST), BRD4 (dilution 1:1000, 13440 S, CST), CUL3 (dilution 1:1000, 2759 S, CST), RBX1 (dilution 1:1000, PA5-29149, Thermo Scientific), FOXO3A (dilution 1:1000, 12829 S, CST), GATA5 (dilution 1:300, SC-373684, Santa Cruz Biotechnology), Histone H3 (dilution 1:3000, 9715, CST), H3K9 2me (dilution 1:1000, ab1220, Abcam), NDRG1 (dilution 1:300, SC-398291, Santa Cruz Biotechnology), AR (dilution 1:1000, SC-815, Santa Cruz Biotechnology), PD-L1 (dilution 1:1000, 13684S, CST), cleaved-PARP (dilution 1:1000, 5625S, CST).

Techniques: Mutagenesis, Western Blot, Co-Immunoprecipitation Assay, Transfection, Infection, Expressing, Construct, Methylated DNA Immunoprecipitation, Staining, DNA Methylation Assay, Inhibition, Two Tailed Test

Journal: Nature Communications

Article Title: SPOP mutation induces DNA methylation via stabilizing GLP/G9a

doi: 10.1038/s41467-021-25951-3

Figure Lengend Snippet: a , b Cell viability was measured by MTS assay in 22Rv1 ( a ) and DU145 cells ( b ) expressing indicated constructs and treated with 5-AzaC (2 μM) for 5 days. Data shown means ± SD ( n = 5 replicates/group). c , d Organoids derived from indicated PDX tumors were cultured with matrigel and treated with DMSO or 5-AzaC (2 or 4 μM) for 7 days followed by photography ( c ) and quantification ( d ). Scale bars, 50 μm. Data shown means ± SD ( n = 6 fields/group). e , f DU145 cells expressing EV or SPOP-F102C were treated with DMSO, DTX (0.05 nM), 5-AzaC (2 μM) or both for 48 h and harvested for Western blots ( e ) or subjected to MTS assay ( f ). Data shown means ± SD ( n = 5 replicates/group). g , h DU145 cells expressing EV or SPOP-F102C and treated with drugs as in ( e ) were subjected to colony formation assay for 12 days followed by photographing ( g ) and quantification ( h ). Data shown means ± SD ( n = 3 replicates/group). i – k DU145 cells expressing EV or SPOP-F102C were injected s.c. into SCID male mice and treated with vehicle, DTX (5 mg/kg), 5-AzaC (2 mg/kg) or the combination of DTX and 5-AzaC. Tumor volume was measured at indicated time points ( i ). Tumors were harvested at day-28 and photographed ( j ), and tumor weight was measured ( k ). Data shown means ± SD ( n = 6 replicates/group). l IHC staining for GLP, G9a, 5mC and Ki-67 was performed. Representative images were taken from each group. Experiments were repeated twice. Scale bar, 50 μm. m – p Quantification of IHC data shown in ( l ). Percentage of the cells with different intensity of staining (weak, intermediate and strong) for GLP ( m ), G9a ( n ) and 5mC ( o ) were determined. Percentage of Ki-67 staining-positive cells was quantified in ( p ). Data shown means ± SD ( n = 3 xenograft tissues/group). Statistical significance was determined by unpaired two-tailed Student’s t test in ( a , b , d , f , h , i , k , p ). Experiments in ( e ) were repeated twice. Source data are provided as a Source Data file.

Article Snippet: The antibodies used: SPOP (dilution 1:1000, 16750-1-AP, Proteintech), GLP (dilution 1:1000, A301-642A, Bethyl), G9a (dilution 1:1000, PP-A8620A-00, R&D), MYC (dilution 1:1000, M192-7, MBL), Flag (dilution 1:1000, F180-4, Sigma), HA (dilution 1:1000, M180-7, MBL), Actin (dilution 1:1000, AC028, Abclonal), DNMT1 (dilution 1:1000, 5032 S, CST), DNMT3A (dilution 1:300, SC-373905, Santa Cruz Biotechnology), DNMT3B (dilution 1:300, SC-376043, Santa Cruz Biotechnology), DEK (dilution 1:1000, 13962 S, CST), BRD4 (dilution 1:1000, 13440 S, CST), CUL3 (dilution 1:1000, 2759 S, CST), RBX1 (dilution 1:1000, PA5-29149, Thermo Scientific), FOXO3A (dilution 1:1000, 12829 S, CST), GATA5 (dilution 1:300, SC-373684, Santa Cruz Biotechnology), Histone H3 (dilution 1:3000, 9715, CST), H3K9 2me (dilution 1:1000, ab1220, Abcam), NDRG1 (dilution 1:300, SC-398291, Santa Cruz Biotechnology), AR (dilution 1:1000, SC-815, Santa Cruz Biotechnology), PD-L1 (dilution 1:1000, 13684S, CST), cleaved-PARP (dilution 1:1000, 5625S, CST).

Techniques: MTS Assay, Expressing, Construct, Derivative Assay, Cell Culture, Western Blot, Colony Assay, Injection, Immunohistochemistry, Staining, Two Tailed Test