Journal: Antibodies

Article Title: A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody–Drug Conjugates

doi: 10.3390/antib7010012

Figure Lengend Snippet: ( A ) PK profile of brentuximab- 7 ; ( B ) PK profile of Adcetris ® . Open circle = total antibody (by ELISA); solid square = total conjugated antibody (by ELISA); blue diamond = free MMAE (by LC-MS).

Article Snippet: The Eagle Biosciences DM1 Antibody–Drug Conjugate (ADC) ELISA Assay Kit is designed, developed, and produced for the quantitative measurement of antibody DM1 conjugate (trastuzumab emtansine) in serum, tissue, and cell culture samples.

Techniques: Enzyme-linked Immunosorbent Assay, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: AZIN2-dependent polyamine metabolism determines adipocyte progenitor fate and protects against obesity and dysmetabolism

doi: 10.1101/2024.11.19.621837

Figure Lengend Snippet: A-D. RNA-seq in SAT of HFD compared to LFD mice (n=4 mice per group). Volcano plot of differentially expressed genes with log2FC > 0.5 and <-0.5 (A) . Differentially expressed adipogenic genes in SAT of HFD compared to LFD mice (B) . GSEA analysis for inflammatory response-related gene set (C) . Upregulated KEGG signaling pathways (D) and downregulated KEGG metabolic pathways (E) in SAT of HFD mice, as shown by EGSEA analysis (adjp<0.05). F. RNAScope for Azin2 and immunofluorescence for PDGFRα and Caveolin in SAT and GAT of 10 week-old mice fed a chow diet (CD). Scale bar, 50 μm. Representative images from 2 mice, 3 levels per tissue are shown. G. Pdgfra , Ptprc ( Cd45 ) and Pecam1 ( Cd31 ) expression in CD45 + , CD31 + and CD45 - CD31 - SVF populations isolated from SAT and GAT of mice fed for 20 weeks a LFD (n=5 mice per group). H. Pdgfra expression in SAT APs and AF of mice fed for 20 weeks a LFD or a HFD (n=4-5 mice per group). I-J. Azin2 expression in different mouse tissues (n=8 mice) ( I ) and cell types (n=2-6 mice) ( J ). K. Number of APs (PDGFRA + Sca1 + CD31 - CD45 - ) per g of adipose tissue assessed by FACS in SAT and GAT of young (CD-fed), LFD- and HFD-fed wt mice (n=5-8 mice per group). L. Pparg , Cebpa , Acsl1 , Adipoq , Cd36 , Lipe and Plin2 expression in non-differentiated and differentiated SAT APs (n=8 mice per group). Data in G-L are shown as mean±SEM, Kruskal-Wallis ( G,K ) and Mann-Whitney U ( H, L ) tests were used for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001. NES: Normalized enrichment score; FDR: False discovery rate; SAT: subcutaneous adipose tissue; GAT: gonadal adipose tissue; BAT: brown adipose tissue; CD: chow diet; LFD: Low fat diet; HFD: High fat diet; AP: adipocyte progenitors; AF: adipocyte fraction; Macs: Macrophages

Article Snippet: 3T3-L1 cells were transfected with a plasmid overexpressing Azin2 (NM_172875, mouse tagged ORF clone, Origene) or pCMV control plasmid using Lipofectamine LTX Reagent (Thermo Fisher Scientific) according to manufacturer‘s instructions and as previously described .

Techniques: RNA Sequencing, Protein-Protein interactions, RNAscope, Immunofluorescence, Expressing, Isolation, MANN-WHITNEY

Journal: bioRxiv

Article Title: AZIN2-dependent polyamine metabolism determines adipocyte progenitor fate and protects against obesity and dysmetabolism

doi: 10.1101/2024.11.19.621837

Figure Lengend Snippet: A. GSEA analysis of bulk RNA-seq in SAT of mice fed for 20 weeks a HFD or LFD for KEGG_arginine and proline metabolism (n=4 mice per group). B. Arginine metabolism-related genes downregulated in SAT of HFD mice based on RNA-seq data (n=4 mice per group). C. Schematic presentation of polyamine metabolism. D. Azin2 , Odc1 and Sms relative gene expression in SAT of mice fed for 20 weeks a HFD or LFD (n=13 mice per group). E. Immunofluorescence for ARG1 (magenta), ODC1 (magenta) and SAT1 (magenta), PDGFRα (yellow), Perilipin (green) and DAPI staining in SAT of mice fed a CD. Scale bar, 50 μm. Representative images are shown. F. RNAScope for Azin2 and immunofluorescence for PDGFRα and perilipin in SAT and GAT of 8 week-old mice fed a CD and mice fed for 20 weeks a LFD or a HFD. Scale bar, 50 μm. Representative images from 2 mice, 3 levels per tissue are shown. G. Azin2 , Odc1 , Srm , Sms and Sat1 expression in CD45 + , CD31 + and CD45 - CD31 - SVF populations isolated from SAT and GAT of wt mice (n=5 mice per group). H. Arg1 , Azin2 , Odc1 , Srm , Sms and Sat1 expression in differentiated (DIF) or non-differentiated (ND) cultured murine SAT APs (n=4-8 mice per group). I. AZIN2 and SMS expression in differentiated or non-differentiated cultured human preadipocytes (n=4-8). Gene expression in D,G-I was determined by qPCR using 18S as a housekeeping gene. Data in D,G-I are shown as mean±SEM. Mann-Whitney U ( D,H,I ) or Kruskal-Wallis ( G ) tests were used for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001. NES: Normalized enrichment score; FDR: False discovery rate

Article Snippet: 3T3-L1 cells were transfected with a plasmid overexpressing Azin2 (NM_172875, mouse tagged ORF clone, Origene) or pCMV control plasmid using Lipofectamine LTX Reagent (Thermo Fisher Scientific) according to manufacturer‘s instructions and as previously described .

Techniques: RNA Sequencing, Gene Expression, Immunofluorescence, Staining, RNAscope, Expressing, Isolation, Cell Culture, MANN-WHITNEY

Journal: bioRxiv

Article Title: AZIN2-dependent polyamine metabolism determines adipocyte progenitor fate and protects against obesity and dysmetabolism

doi: 10.1101/2024.11.19.621837

Figure Lengend Snippet: A. Number of sites with enriched H3K27ac marks in different DNA regions up- or downregulated in Azin2 -/- compared to wt differentiated SAT APs. B. Genome-wide average log2FC enrichment of H3K27ac marks in regions between transcriptional start sites (TSS) and transcriptional end sites (TES) for representative wt and Azin2 -/- SAT AP samples. C-G. Genes with H3K27ac enrichment in the promoter (1-2 kb and <1kb) ( C ), 5’ UTR region ( D ), exons ( E ), introns ( F ) and distal intergenic regions ( G ) in Azin2 -/- versus wt SAT APs. The Pparg gene is depicted with an arrow. A-G : n=3 pools of APs from 2 mice per group. H. Images of Oil-Red O staining in differentiated wt and Azin2 -/- SAT APs treated during differentiation with spermidine (10 μM). Representative images of cells from 1 out of 8 mice is shown per condition, scale bar: 200 μm. I. Images of immunofluorescence for PPARg in differentiated wt and Azin2 -/- SAT APs. Representative images of cells from 1 out of 8 mice is shown per condition, scale bar: 200 μm. J. Images of Oil-Red O staining in SAT APs transfected 3 times over one week of differentiation with siRNA against Slc7a2 , Arg1 or control siRNA. Representative images of cells from 1 out of 5-8 mice per condition are shown, scale bar: 200 μm.

Article Snippet: 3T3-L1 cells were transfected with a plasmid overexpressing Azin2 (NM_172875, mouse tagged ORF clone, Origene) or pCMV control plasmid using Lipofectamine LTX Reagent (Thermo Fisher Scientific) according to manufacturer‘s instructions and as previously described .

Techniques: Genome Wide, Staining, Immunofluorescence, Transfection, Control

Journal: bioRxiv

Article Title: AZIN2-dependent polyamine metabolism determines adipocyte progenitor fate and protects against obesity and dysmetabolism

doi: 10.1101/2024.11.19.621837

Figure Lengend Snippet: A. Immunofluoeresence image of SAT stained for IL4Rα (magenta), PDGFRα (yellow), Perilipin (green) and DAPI (cyan). Scale bar, 50 μm B. IL4ra expression in the AF and CD11b - SVF cells of SAT of HFD mice (n=6). C. Azin2 and Stat6 expression in non-differentiated SAT APs transfected with si Stat6 or siCtrl for 24 h and treated or not for 4 h with IL4 (20 ng/ml) (n=5). D. Azin2 expression in non-differentiated SAT APs treated for 4 h with IL13 (20 ng/ml) (n=5). E-H . Wt mice were fed for 6 weeks a HFD and treated every 2 days with PBS or IL4 + a-IL4 during the last 2 feeding weeks. The body weight gain ( E ), adipogenic gene expression ( F ) and AP number in SAT ( G ) are shown (n=6-7 mice per group). H. Arg1 , Chil3 , Retnla , Clec10a and Mrc1 expression in SAT SVF of PBS and IL4-treated mice (n=6-7 mice per group). I. Pparg and Cd36 expression in GAT of mice infected with N. brasiliensis or control mice 9 days post-infection (n=4-9 mice per group). J-R. Correlation of AZIN2 expression with IL4, IL13, STAT6, IL5, and IL33 in human subcutaneous ( J-M ) and visceral ( N-R ) adipose tissue based on the GEPIA2 database ( http://gepia2.cancer-pku.cn/#index ). Data in C-I are shown as mean±SEM. Gene expression in B-D , F, H and I was determined by qPCR using 18S as a housekeeping gene. Mann-Whitney U test was used for statistical analysis. *p < 0.05, **p < 0.01

Article Snippet: 3T3-L1 cells were transfected with a plasmid overexpressing Azin2 (NM_172875, mouse tagged ORF clone, Origene) or pCMV control plasmid using Lipofectamine LTX Reagent (Thermo Fisher Scientific) according to manufacturer‘s instructions and as previously described .

Techniques: Staining, Expressing, Transfection, Gene Expression, Infection, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: AZIN2-dependent polyamine metabolism determines adipocyte progenitor fate and protects against obesity and dysmetabolism

doi: 10.1101/2024.11.19.621837

Figure Lengend Snippet: A. IL4ra expression in differentiated and non-differentiated SAT APs assessed by qPCR (n=7 mice per group). B-E. Bulk RNA-seq was performed in mouse SAT APs treated for 4 h with IL4 (20 ng/ml). Volcano plot for differentially expressed genes (DEG) with padj<0.05 log2FC>0.3 and < -0.3 ( B ), positively enriched KEGG metabolic pathways based on EGSEA analysis ( C ), heatmap for arginine metabolism genes ( D ), expression of Slc family 7 cationic amino acid transporters ( E ) (n=4 mice per group for B-E ). F. Azin2, Slc7a2, Arg1 and Srm expression in mouse APs treated for 4 h with IL4 (20 ng/ml) (n=6 mice per group). G. AZIN2 expression in human preadipocytes treated for 24 h with IL4 (12 ng/ml) (n=4). H. Azin2, Slc7a2 and Arg1 expression in GAT explants from Il4ra -/- and wt mice treated for 4 days with 50 ng/ml IL4 or IL13 (n=5-6 mice per group). I. Azin2 expression in SAT SVF, and Slc7a2 and Arg1 in SAT of mice fed for 6 weeks a HFD and treated every 2 days with PBS or IL4 + a-IL4 during the last 2 feeding weeks, as depicted in the scheme (n=3-7 mice per group). J. Azin2, Odc1 and Srm expression in GAT of mice infected with N. brasiliensis or control mice 9 days post-infection (n=4-9 mice per group). Gene expression in A,F-J was determined by qPCR using 18S as a housekeeping gene and shown as mean±SEM. Mann-Whitney U test was used for statistical analysis in A , G,I,J ; Wilcoxon test was used for statistical analysis in F , Kruskal-Wallis test was used for statistical analysis in H *p < 0.05, **p < 0.01.

Article Snippet: 3T3-L1 cells were transfected with a plasmid overexpressing Azin2 (NM_172875, mouse tagged ORF clone, Origene) or pCMV control plasmid using Lipofectamine LTX Reagent (Thermo Fisher Scientific) according to manufacturer‘s instructions and as previously described .

Techniques: Expressing, RNA Sequencing, Infection, Control, Gene Expression, MANN-WHITNEY

Journal: bioRxiv

Article Title: AZIN2-dependent polyamine metabolism determines adipocyte progenitor fate and protects against obesity and dysmetabolism

doi: 10.1101/2024.11.19.621837

Figure Lengend Snippet: A. Azin2 expression in differentiated wt and Azin2 -/- SAT APs (n=4-5 mice per group). B. N1-acetylspermidine concentration in the cell culture supernatant of in vitro differentiated wt and Azin2 -/- SAT APs (n=4-5 mice per group). C. Azin2 expression in SAT APs transfected 3 times over one week of differentiation (every 2-3 days), with si Azin2 or control siRNA (n=5-7 mice per group). D. Putrescine and ornithine levels in cell culture supernatants of SAT APs 24 h after transfection with si Azin2 or control siRNA or (n=7 mice per group). E. Acetyl-CoA levels in differentiated wt and Azin2 -/- SAT APs (n=3-5 mice per group). F. Azin2 mRNA expression (left) and western blot for AZIN2-DDK (right) in 3T3-L1 preadipocytes transfected for 24 h with a plasmid overexpressing AZIN2-DDK or control pCMV plasmid (n=3 for left panel). G-I. Slc7a2 , Arg1 , and Sat1 expression in SAT APs transfected 3 times over one week of differentiation with the respective siRNA (n=5 for G , n=6 for H , n=7-8 for I ). J. Odc1 expression in SAT APs 24 h after transfection with si Odc1 or control siRNA (n=5 mice per group). Gene expression was determined by qPCR using 18S as housekeeping gene. Polyamine levels were determined by LC-MS/MS. Data are shown as mean±SEM. Mann-Whitney U test was used for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: 3T3-L1 cells were transfected with a plasmid overexpressing Azin2 (NM_172875, mouse tagged ORF clone, Origene) or pCMV control plasmid using Lipofectamine LTX Reagent (Thermo Fisher Scientific) according to manufacturer‘s instructions and as previously described .

Techniques: Expressing, Concentration Assay, Cell Culture, In Vitro, Transfection, Control, Western Blot, Plasmid Preparation, Gene Expression, Liquid Chromatography with Mass Spectroscopy, MANN-WHITNEY

Journal: bioRxiv

Article Title: AZIN2-dependent polyamine metabolism determines adipocyte progenitor fate and protects against obesity and dysmetabolism

doi: 10.1101/2024.11.19.621837

Figure Lengend Snippet: A. Intracellular spermidine and N1-acetylspermidine levels in undifferentiated cultured wt and Azin2 -/- GAT APs (n=5 mice per group). B. N1-acetylspermidine and N1-acetylspermine concentrations in supernatants of GAT explants from wt and Azin2 -/- mice kept 18 hours in culture (n=6-8 mice per group). C. N1-acetylspermidine concentration in cell lysates of wt and Azin2 -/- SAT APs treated for 24 h with IL4 (20 ng/ml) (n=3-5 mice per group). D,E. SAT APs were transfected 3 times over one week of differentiation with si Azin2 or siCtrl and N1-acetylspermidine was measured in the cell lysates ( D ) and cell culture supernatants ( E ) (n=7 mice per group). F. Relative abundance of acetyl-CoA in non-differentiated wt and Azin2 -/- SAT APs shown as peak intensity assessed by LC-MS/MS (n=6 mice per group). G. Relative abundance of acetyl-CoA levels in 3T3-L1 preadipocytes transfected with an Azin2 overexpressing plasmid or a control plasmid (pCMV) shown as peak intensity assessed by LC-MS/MS (n=6 biological replicates). H,I. N1-acetylspermidine concentration in cell culture supernatants ( H ) and relative abundance of acetyl-CoA ( I ) in SAT APs treated with spermidine (SPD, 10 μΜ) during 1 week of differentiation (n=4 for H and 6 for I mice per group). J,K. N1-acetylspermidine concentrations in cell lysates ( J ) and cell culture supernatants ( K ) of undifferentiated SAT APs transfected with si Slc7a2 or siCtrl (n=6 mice per group). L,M. N1-acetylspermidine concentrations in cell lysates ( L ) and cell culture supernatants ( M ) of SAT APs transfected 3 times over one week of differentiation with si Arg1 or siCtrl (n=7 mice per group). N. N1-acetylspermidine, putrescine and spermidine concentrations in cell culture supernatants of undifferentiated SAT APs transfected with si Sat1 or siCtrl (n=5 mice per group). O. Relative abundance of acetyl-CoA in non-differentiated APs transfected with si Sat1 or siCtrl (n=3-4 mice per group). Data are shown as mean±SEM. Mann-Whitney U test was used for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001. AU: Arbitary units. Data of ‘siCtrl’ samples are the same in D and L , and E and M .

Article Snippet: 3T3-L1 cells were transfected with a plasmid overexpressing Azin2 (NM_172875, mouse tagged ORF clone, Origene) or pCMV control plasmid using Lipofectamine LTX Reagent (Thermo Fisher Scientific) according to manufacturer‘s instructions and as previously described .

Techniques: Cell Culture, Concentration Assay, Transfection, Liquid Chromatography with Mass Spectroscopy, Plasmid Preparation, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: AZIN2-dependent polyamine metabolism determines adipocyte progenitor fate and protects against obesity and dysmetabolism

doi: 10.1101/2024.11.19.621837

Figure Lengend Snippet: A-E. Bulk ChIP-Seq was performed after IP for H3K27ac in in vitro differentiated wt and Azin2 -/- SAT APs (n=3 pools of APs from 2 mice per group). A. Global DNA distribution of H3K27ac marks upregulated in Azin2 -/- SAT APs. B. Histogram of the H3K27ac signal intensity in wt and Azin2 -/- SAT APs. C. Enriched GO terms and pathways and KEGG pathways in Azin2 -/- compared to wt SAT APs plotted for the –log(adjp) value based on genes with enhanced H3K27ac signal in Azin2 -/- versus wt APs. D. Integrative Genomic Viewer (IGV) screenshots showing H3K27ac marks in the –16 kb region upstream of the Pparg2 ΤSS in representative wt and Azin2 -/- SAT AP samples. E,F. Abundance of H3K27ac ( E ) and H3K9ac ( F ) 251-144 bp upstream of the Pparg2 ΤSS in differentiated wt and Azin2 -/- SAT APs assessed by ChIP-qPCR and shown as % of input (n=8-10 mice per group). G. Pparg and Cepba expression in differentiated wt and Azin2 -/- SAT APs (n=4-5 mice per group). H. Representative images of in vitro differentiated wt and Azin2 -/- SAT APs stained with BODIPY. Scale bar, 200 μm (a representative image of 1 out of 8 mice per group is shown). I. Quantification of Oil Red O staining of in vitro differentiated wt and Azin2 -/- SAT APs (n=3 mice per group). J. Quantification of Oil Red O staining in SAT APs transfected 3 times during one week of differentiation with siRNA against Slc7a2 or Arg1 or non-targeting control siRNA (siCtrl) (n=8 mice per group). K. Pparg expression in SAT APs transfected 3 times during one week of differentiation with siRNA against Arg1 , Sat1 , Odc1 or non-targeting siRNA (siCtrl) (n=5-7 mice per group). L. Quantification of Oil Red O staining in wt and Azin2 -/- SAT APs treated or not with spermidine (SPD, 10 μM) during in vitro differentiation (n=8 mice per group). M. Quantification of Oil Red O staining in wt and Azin2 -/- SAT APs treated with C646 (4 μM) or equal amount of DMSO (Ctrl) during in vitro differentiation (n=8 mice per group). N. Pparg expression in Azin2 -/- SAT APs treated with C646 (4 μM) or DMSO during differentiation (n=5 mice per group). Gene expression in G, K and N was determined by qPCR using 18S as a housekeeping gene. All data are shown as mean±SEM. Mann-Whitney U ( E-G,I,K,L,N ), one-way ANOVA ( J,M ) and Kruskal-Wallis ( K right panel) tests were used for statistical analysis. *p < 0.05, **p < 0.01; ns: not significant

Article Snippet: 3T3-L1 cells were transfected with a plasmid overexpressing Azin2 (NM_172875, mouse tagged ORF clone, Origene) or pCMV control plasmid using Lipofectamine LTX Reagent (Thermo Fisher Scientific) according to manufacturer‘s instructions and as previously described .

Techniques: ChIP-sequencing, In Vitro, ChIP-qPCR, Expressing, Staining, Transfection, Control, Gene Expression, MANN-WHITNEY

Journal: bioRxiv

Article Title: AZIN2-dependent polyamine metabolism determines adipocyte progenitor fate and protects against obesity and dysmetabolism

doi: 10.1101/2024.11.19.621837

Figure Lengend Snippet: A-F. Body weight ( A ), GAT weight ( B ), axillary adipose tissue weight ( C ), mesenteric adipose tissue weight ( D ), liver weight ( E ) and spleen weight ( F ) in 10 weeks-old chow diet-fed wt and Azin2 -/- mice (n=16 mice per group for A-C, E,F and n=6-8 mice per group for C and D ). G,H. Glucose tolerance test (GTT) ( G ) and insulin tolerance test (ITT) ( H ) in 10 weeks-old chow diet-fed wt and Azin2 -/- mice (n=6-8 mice per group). I. Representative H&E images of GAT in 10 weeks-old chow diet-fed wt and Azin2 -/- mice, scale bar: 200 μm (n= 3 mice per group). J. Number of ATMs (CD45 + CD11b + F4/80 + ) per g of GAT assessed by FACS in wt and Azin2 -/- mice (n=6-8 mice per group). K. Percentage of CD11c + ATMs (CD45 + CD11b + ) in GAT of wt and Azin2 -/- mice assessed by FACS (n=6-8 mice per group). L. Percentage of CD9 high APs (PDGFRα + LY6A + CD31 - CD45 - ) in GAT of wt and Azin2 -/- mice assessed by FACS (n=6-8 mice per group). Data in A-H and J-L are shown as mean±SEM. Mann-Whitney U test was used for statistical analysis. *p < 0.05, ns: not significant.

Article Snippet: 3T3-L1 cells were transfected with a plasmid overexpressing Azin2 (NM_172875, mouse tagged ORF clone, Origene) or pCMV control plasmid using Lipofectamine LTX Reagent (Thermo Fisher Scientific) according to manufacturer‘s instructions and as previously described .

Techniques: MANN-WHITNEY

Journal: bioRxiv

Article Title: AZIN2-dependent polyamine metabolism determines adipocyte progenitor fate and protects against obesity and dysmetabolism

doi: 10.1101/2024.11.19.621837

Figure Lengend Snippet: A. Weight of SAT (left: net weight, right: as percentage of body weight) in 10 weeks-old CD-fed wt and Azin2 -/- mice (n=16 mice per group). B. Representative H&E images of SAT in CD-fed wt and Azin2 -/- mice, scale bar: 200 μm (n=3 mice per group). C,D. Number of total APs (PDGFRA + LY6A + CD45 - CD31 - ) ( C ) and Ki67 + APs ( D ) analyzed by FACS in SAT of 10 weeks-old CD-fed wt and Azin2 -/- mice (n=8 mice per group). Data are shown as mean±SEM. Mann-Whitney U test was used for statistical analysis. *p < 0.05, ***p < 0.001, ns: not significant.

Article Snippet: 3T3-L1 cells were transfected with a plasmid overexpressing Azin2 (NM_172875, mouse tagged ORF clone, Origene) or pCMV control plasmid using Lipofectamine LTX Reagent (Thermo Fisher Scientific) according to manufacturer‘s instructions and as previously described .

Techniques: MANN-WHITNEY

Journal: bioRxiv

Article Title: AZIN2-dependent polyamine metabolism determines adipocyte progenitor fate and protects against obesity and dysmetabolism

doi: 10.1101/2024.11.19.621837

Figure Lengend Snippet: A. Experimental setup. B. Body weight gain in wt and Azin2 -/- mice fed for 8 weeks a HFD (n=15 mice per group). C. Insulin tolerance test performed at 7 weeks of HFD feeding in wt and Azin2 -/- mice (n=11 mice per group). Left panel: Blood glucose concentration over time; right panel: area under the curve (AUC). D,E . Energy expenditure (EE) ( D ) and O 2 consumption ( E ) measured in metabolic cages in wt and Azin2 -/- mice fed for 6 weeks a HFD (n=7-9 mice per group). F. Fat versus lean mass in wt and Azin2 -/- mice fed for 8 weeks a HFD in SAT weight (n=3 mice per group). G. SAT weight in wt and Azin2 -/- mice fed for 8 weeks a HFD (n=20-21 mice per group). H. Representative images of H&E staining of SAT from wt and Azin2 -/- mice fed for 8 weeks a HFD (one image is shown from one out of 10-11 mice), scale bar: 200 μm. Adipocyte size quantification in SAT of wt and Azin2 -/- mice fed for 8 weeks a HFD (n=6 mice per group, 9-10 sections per mouse were imaged, each dot represents the size of the sliced area of one adipocyte). I. Relative abundance of acetyl-CoA levels in SAT of wt and Azin2 -/- mice fed for 8 weeks a HFD presented as peak intensity assessed by LC-MS/MS (n=4-6 mice per group). J,K. EGSEA analysis for KEGG signaling pathways upregulated in Azin2 -/- mice ( J ) and GSEA analysis for HALLMARK_Adipogenesis, REACTOME_Fatty Acyl-CoA Biosynthesis and GO_Positive Regulation of Lipid Storage ( K ) based on bulk RNA-seq in SAT of wt and Azin2 -/- mice fed for 8 weeks a HFD (n=4 mice per group). L-N. Relative abundance of TG species ( L ), lipid groups ( M ) and ceramide species ( N ) in SAT of wt and Azin2 -/- mice fed for 8 weeks a HFD presented as peak intensity determined by LC-MS/MS, only differentially regulated TG species are shown (n=5-8 mice per group). O,P. Total and Ki67 + AP (PDGFRα + LY6A + CD31 - CD45 - ) abundance per g of tissue in SAT of wt and Azin2 -/- mice fed for 8 weeks a HFD assessed by flow cytometry (n=7-9 mice per group). Q. GSEA analysis for REACTOME_Cellular Senescence, HALLMARK_Hypoxia, and REACTOME_Oxidative Stress Induced Senescence pathways based on bulk RNA-seq in SAT of wt and Azin2 -/- mice fed for 8 weeks a HFD (n=4 mice per group). R. Percentage of CD9 high SAT APs (PDGFRα + LY6A + CD31 - CD45 - ) in wt and Azin2 -/- mice fed for 8 weeks a HFD (n=6 mice per group). S-U. scRNA-seq was performed in CD45 - CD31 - SAT SVF cells of wt and Azin2 -/- mice fed for 8 weeks a HFD (2 mice pooled per genotype). Down- and up-regulated GO Terms and pathways in Azin2 -/- versus wt mice plotted according to the –log(adjp) value ( S,T ). Violin plots showing gene expression scores of Pparg , Cd36 , Fabp4 , Apoe , and Pltp , in AP in wt and Azin2 -/- HFD mice ( U ). Data in B-F,H,I,L-N,O,P,R are shown as mean±SEM. Mann-Whitney U test ( B,C,F, I, I,O,P,R ), ANCOVA ( D,E ), t test ( H ) and multiple t test ( L-N ) were used for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. NES: Normalized enrichment score; FDR: False discovery rate. V. Schematic presentation of AZIN2-dependent mechanism regulating adipogenesis.

Article Snippet: 3T3-L1 cells were transfected with a plasmid overexpressing Azin2 (NM_172875, mouse tagged ORF clone, Origene) or pCMV control plasmid using Lipofectamine LTX Reagent (Thermo Fisher Scientific) according to manufacturer‘s instructions and as previously described .

Techniques: Concentration Assay, Staining, Liquid Chromatography with Mass Spectroscopy, Protein-Protein interactions, RNA Sequencing, Flow Cytometry, Gene Expression, MANN-WHITNEY

Journal: bioRxiv

Article Title: AZIN2-dependent polyamine metabolism determines adipocyte progenitor fate and protects against obesity and dysmetabolism

doi: 10.1101/2024.11.19.621837

Figure Lengend Snippet: A,B. Food uptake and locomotion measured in metabolic cages in wt and Azin2 -/- mice fed for 6 weeks a HFD (n=5-8 for A and 7-9 for B mice per group). C. GAT weight of wt and Azin2 -/- mice fed for 8 weeks a HFD (n=20-21 mice per group). D. Representative images of H&E staining of GAT from wt and Azin2 -/- mice fed for 8 weeks a HFD (one image is shown from one out of 10-11 mice), scale bar: 200 μm. E. Adipocyte size quantification in GAT of wt and Azin2 -/- mice fed for 8 weeks a HFD (n=6 mice per group, 9-10 sections per mouse were imaged, each dot represents the size of the sliced area of one adipocyte). F. Relative abundance of acetyl-CoA levels in GAT of wt and Azin2 -/- mice fed for 8 weeks a HFD presented as peak intensity assessed by LC-MS/MS (n=4-6 mice per group). G-I, M-O. Bulk RNA-seq in GAT of wt and Azin2 -/- mice fed for 8 weeks a HFD (n=4 mice per group): Volcano plot of differentially expressed genes with log2FC > 0.5, < -0.5 ( G ), EGSEA analysis for upregulated KEGG signaling pathways (padj<0.05) in Azin2 -/- mice ( H ), GSEA analysis for GO_Activation of Immune Response ( I ). EGSEA pathway analysis showing up-(blue) and downregulated (red) KEGG metabolic pathways (padj<0.05) in Azin2 -/- mice ( M ), GSEA analysis for KEGG_Citrate cycle and KEGG Oxidative Phosphorylation ( N ) and GO_Collagen Metabolic Process ( O ). J. Il6 expression in GAT SVF of wt and Azin2 -/- mice fed for 8 weeks a HFD (n=5-6 mice per group). K. Number of CD11c + macrophages (CD45 + CD11b + ) in GAT of wt and Azin2 -/- mice fed for 8 weeks a HFD assessed by FACS (n=5-6 mice per group). L. F4/80 staining in GAT of wt and Azin2 -/- mice fed for 8 weeks a HFD, scale bar: 200 μm (representative images from one out of 3 mice per group). P Col1a1 and Col6a3 expression in GAT of wt and Azin2 -/- mice fed for 8 weeks a HFD (n=11 mice per group). Q. Percentage of CD9 high GAT APs (PDGFRα + LY6A + CD31 - CD45 - ) in wt and Azin2 -/- mice fed for 8 weeks a HFD (n=6 mice per group). R,S. Picrosirius staining ( R ) and quantification (as % of area) ( S ) in GAT of wt and Azin2 -/- mice fed for 8 weeks a HFD ( R : representative images from one out of 10-11 mice per group , S: n=10-11 mice per group, 8-10 images per mouse) T,U. Relative abundance of TG species ( T ) and DG species ( U ) in SAT of wt and Azin2 -/- mice fed for 8 weeks a HFD shown as peak intensity determined by LC-MS/MS, in T only differentially regulated TG species are shown (n=5-8 mice per group). Gene expression in J and P was assessed by qPCR using 18S as a housekeeping gene. Data in A-C, E, F, J, K, P, Q, S, T and U are shown as mean±SEM. Mann-Whitney U test (for A-C,F,J,K,P,Q ), t-test ( E,S ) and multiple t-test ( T,U ) were used for statistical analysis. *p < 0.05, **p < 0.01, ns: not significant. NES: Normalized enrichment score; FDR: False discovery rate

Article Snippet: 3T3-L1 cells were transfected with a plasmid overexpressing Azin2 (NM_172875, mouse tagged ORF clone, Origene) or pCMV control plasmid using Lipofectamine LTX Reagent (Thermo Fisher Scientific) according to manufacturer‘s instructions and as previously described .

Techniques: Staining, Liquid Chromatography with Mass Spectroscopy, RNA Sequencing, Protein-Protein interactions, Activation Assay, Phospho-proteomics, Expressing, Gene Expression, MANN-WHITNEY

Journal: bioRxiv

Article Title: AZIN2-dependent polyamine metabolism determines adipocyte progenitor fate and protects against obesity and dysmetabolism

doi: 10.1101/2024.11.19.621837

Figure Lengend Snippet: Wt and Azin2 -/- SAT APs were differentiated for one week and cultured for another week to induce senescence. A. Senescence was assessed by β-Galactosidase staining (one representative image from 1 out of 5 mice per group is shown). B. Cdkn2a ( p16 ) and Cdkn1a ( p21 ) mRNA expression was assessed by qPCR using 18S as a housekeeping gene (n=8-9 mice per group). C. Immunofluorescence staining against CDKN2A or CDKN1A (magenta) and DAPI staining (cyan); Scale bar, 50 μm; (one representative image from 1 out of 3-4 mice per group is shown). Data in A are shown as mean±SEM. Mann-Whitney U test was performed **p < 0.01, ***p < 0.001.

Article Snippet: 3T3-L1 cells were transfected with a plasmid overexpressing Azin2 (NM_172875, mouse tagged ORF clone, Origene) or pCMV control plasmid using Lipofectamine LTX Reagent (Thermo Fisher Scientific) according to manufacturer‘s instructions and as previously described .

Techniques: Cell Culture, Staining, Expressing, Immunofluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: AZIN2-dependent polyamine metabolism determines adipocyte progenitor fate and protects against obesity and dysmetabolism

doi: 10.1101/2024.11.19.621837

Figure Lengend Snippet: scRNA-seq was performed in CD45 - CD31 - SAT SVF cells of wt and Azin2 -/- mice fed for 8 weeks a HFD (2 mice pooled per genotype). A-C,E. Violin plots showing gene expression of Pdgfra and Ly6a ( A ), Dpp4 , Wnt2 , Bmp7 , Pi16 , Dmkn , and IL33 ( B ) and Lpl , F3 ( Cd142 ) , Bgn , Lum , Mgp , Mmp3 , and Tgfb1 ( C ) in AP in wt HFD mice, and Mt2 , Has1 , Ugdh , Cd44 , Ptx3 , Ier5 , Lmna , and Il4ra in APs of wt and Azin2 -/- HFD mice ( E ). D. Two-dimensional UMAP representation of 10,275 cells colored, according to Azin2 expression (blue) in wt HFD mice.

Article Snippet: 3T3-L1 cells were transfected with a plasmid overexpressing Azin2 (NM_172875, mouse tagged ORF clone, Origene) or pCMV control plasmid using Lipofectamine LTX Reagent (Thermo Fisher Scientific) according to manufacturer‘s instructions and as previously described .

Techniques: Gene Expression, Expressing

Journal: bioRxiv

Article Title: AZIN2-dependent polyamine metabolism determines adipocyte progenitor fate and protects against obesity and dysmetabolism

doi: 10.1101/2024.11.19.621837

Figure Lengend Snippet: Liver weight ( A ), Nile Red staining in liver ( B ), triglyceride content ( C ) and gene expression of Pparg , Cebpa , Cd36 , Acc , Fasn , Col1a1 and Col3a1 in liver ( D ) in wt and Azin2 -/- mice were fed for 8 weeks a HFD (n=6 mice per group). Data in A,C and D are shown as mean±SEM. Mann-Whitney U test was performed *p < 0.01.

Article Snippet: 3T3-L1 cells were transfected with a plasmid overexpressing Azin2 (NM_172875, mouse tagged ORF clone, Origene) or pCMV control plasmid using Lipofectamine LTX Reagent (Thermo Fisher Scientific) according to manufacturer‘s instructions and as previously described .

Techniques: Staining, Gene Expression, MANN-WHITNEY

Journal: bioRxiv

Article Title: AZIN2-dependent polyamine metabolism determines adipocyte progenitor fate and protects against obesity and dysmetabolism

doi: 10.1101/2024.11.19.621837

Figure Lengend Snippet: A-AD . Correlation of AZIN2 expression with DPP4 , WNT2 , PDGFRA , EPHA3 , CD142 , FABP4 , PPARG , ADIPOQ , PNPLA3 , DGAT2 , SREBF1 , PGAP1 , ELOVL5 , FADS3 , EDNRA , LIN7A , and AGMO in visceral (A-Q) and DPP4 , WNT2 , EPHA3 , CD142 , FABP4 , PPARG , ADIPOQ , DGAT2 , ELOVL5 , ELOVL3 , EDNRA , and FHOD3 in subcutaneous adipose tissue (R-AD) based on the GEPIA2 database ( http://gepia2.cancer-pku.cn/#index ).

Article Snippet: 3T3-L1 cells were transfected with a plasmid overexpressing Azin2 (NM_172875, mouse tagged ORF clone, Origene) or pCMV control plasmid using Lipofectamine LTX Reagent (Thermo Fisher Scientific) according to manufacturer‘s instructions and as previously described .

Techniques: Expressing

Journal: Acta Pharmacologica Sinica

Article Title: Humanized dual-targeting antibody–drug conjugates specific to MET and RON receptors as a pharmaceutical strategy for the treatment of cancers exhibiting phenotypic heterogeneity

doi: 10.1038/s41401-024-01458-7

Figure Lengend Snippet: a Schematic structure of PCMbs-MR generated through cDNA recombination from anti-MET mAb PCM-MET01 and anti-RON mAb PCM5B14. A single heavy chain and light chain, representing both anti-MET mAb and anti-RON mAb, respectively, is presented. The knob-into-hole was created in the CH3 domains of both anti-MET and anti-RON mAbs . The crossover was conducted between the CH1 domain of the heavy chain and the CL domain of the light chain from the anti-MET mAb accordingly . b PCMbs-MR is conjugated with MMAE through a cleavable dipeptide linker to form the dual-targeting ADC PCMdt-MMAE [ – ]. The drug-to-antibody ratio (DAR) was calculated as 4.13:1. c Structural 3D analysis of individual complementary-determining regions (black) grafted into the variable regions of humanized heavy chain and light chain (top panel). Antigen-binding surfaces in the variable regions of the humanized anti-MET and anti-RON mAbs are depicted as dark blue (bottom panel). d Cell surface immunofluorescent analysis of PCMbs-MR binding to MET and RON. A panel of cancer cell lines expressing variable levels of MET, RON, or both receptors were used. Anti-MET mAb PCM-MET01 and anti-RON mAb PCM5B14 were included for comparison. Briefly, cells at 1 × 10 6 cells per sample were incubated with 2 µg/mL of PCMbs-MR or other antibodies (red line), followed by rabbit anti-human or mouse IgG coupled with FITC. Regular human IgG was used as the control (black line). Immunofluorescence intensities from individual samples were analyzed using a flow cytometer . Cellular levels of receptor expressions are indicated as follows: HCC1806 (MET-/RON-) ; Hs746T (MET3+/RON-) ; SUM52PE (MET-/RON3+) ; MDA-MB468 (MET1+/RON2+) ; HT29 (MET3+/RON3+) ; BxPC-3 (MET3+/RON2+) ; T-47D (MET3+/RON3+) ; HCC1937 (MET3+/RON3+) ; HCC2185 (MET3+/RON-) ; and MDA-MB-231 (MET3+/RON2+) . e Species specificity of PCMbs-MR. Human HT29, monkey 4MBr-5, canine MDCK, and mouse MS-1 cell lines, known to express both MET and RON, were used. Incubation of cells with PCMbs-MR and specific immunofluorescence analysis were performed as detailed previously . The binding affinity of PCMbs-MR was calculated using the GraphPad Prism 6 software. f PCMbs-MR induced cell-surface MET and RON internalization by individual cancer cells. Seven cancer cell lines expressing variable levels of MET, RON, or both receptors were used. PCMbs-MR at 5 µg/mL was used for incubation. Procedures used to determine cell surface receptor internalization were performed as previously described [ – ]. Internalization efficacy (IE 50 ) among cell lines was calculated using a previously described method [ – ]. g PCMbs-MR-induced internalization efficacy in comparison with anti-MET or anti-RON mAbs. BxPC-3 and HCT116 cell lines expressing high levels of both MET and RON were used. Treatment of cells with mAbs and the methods used to determine IE 50 were carried out to obtain the individual IE 50 values . ADC antibody–drug conjugate, FITC fluorescein isothiocyanate, mAb monoclonal antibody, MET mesenchymal-epithelial transition, MMAE monomethyl auristatin E, PCMbs–MR humanized bispecific monoclonal antibody specific to both MET and RON, RON recepteur d’Origine nantais.

Article Snippet: The amount of MMAE conjugated to PCMbs–MR in plasma was determined by using a MMAE ADC ELISA kit (Eagle Biosciences Inc., Nashua, NH, USA; www.eaglebio.com/product/mmae-adc-elisa-kit ) [ – ].

Techniques: Generated, Binding Assay, Expressing, Comparison, Incubation, Control, Immunofluorescence, Flow Cytometry, Software, Cell Surface Receptor Assay

Journal: Acta Pharmacologica Sinica

Article Title: Humanized dual-targeting antibody–drug conjugates specific to MET and RON receptors as a pharmaceutical strategy for the treatment of cancers exhibiting phenotypic heterogeneity

doi: 10.1038/s41401-024-01458-7

Figure Lengend Snippet: Analysis of PCMdt-MMAE PK profiles: Athymic nude mice (8-week-old) were divided into tumor-bearing ( a ) and tumor-nonbearing ( b ) groups (three animals per group). Mice from the tumor-bearing group were subcutaneously injected with 1 × 10 6 BxPC-3 cells. When tumor volumes reached ~500 mm 3 , both groups of mice were administered once through the tail vein with 3 or 10 mg/kg of PCMdt-MMAE. Blood samples were collected from individual mice at different time intervals. The amount of MMAE-conjugated PCMbs-MR in plasma was determined by using a MMAE ADC ELISA kit (Eagle Biosciences Inc., Nashua, NH, USA). The PK parameters were calculated using the WinNonlin software package (Certara, Princeton, NJ, USA) [ – ]. c Toxic effects of PCMdt-MMAE in vivo: Effect s of multiple doses of PCMdt-MMAE on mouse bodyweight were determined by a single administration of PCMdt-MMAE at 10, 30, and 60 mg/kg, respectively. The mice were weighed and monitored for 12 days. The average bodyweight before PCMdt-MMAE injection was 19.8 ± 3.6 g (five mice per group) and set as 100%. ADC, antibody–drug conjugate; MMAE, monomethyl auristatin E; PCMdt-MMAE, monomethyl auristatin E was conjugated to PCMbs–MR to generate the dual-targeting ADC; PCMbs-MR humanized bispecific monoclonal antibody specific to both MET and RON, PK pharmacokinetic

Article Snippet: The amount of MMAE conjugated to PCMbs–MR in plasma was determined by using a MMAE ADC ELISA kit (Eagle Biosciences Inc., Nashua, NH, USA; www.eaglebio.com/product/mmae-adc-elisa-kit ) [ – ].

Techniques: Injection, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Software, In Vivo

Journal: Acta Pharmacologica Sinica

Article Title: Humanized dual-targeting antibody–drug conjugates specific to MET and RON receptors as a pharmaceutical strategy for the treatment of cancers exhibiting phenotypic heterogeneity

doi: 10.1038/s41401-024-01458-7

Figure Lengend Snippet: a Changes in cell cycle: BxPC-3 and control HCC1806 (1 × 10 6 cells per dish) cells were treated at 37 °C with 5 µg/mL of PCMdt-MMAE for various times. They were then collected, stained with propidium iodide, and analyzed using a flow cytometer [ – ]. Changes in cell cycle were marked with arrows. b Reduction of cell viability: A panel of seven cancer cell lines expressing variable levels of MET, RON, or both receptors (5000 or 8000 cells per well in a 96-well plate in triplicate) were treated with different amounts of PCMdt-MMAE for 96 h. HCC1806 cells without MET or RON expression served as the control. Cell viability was determined by the MTS assay . c Dose-dependent cell death: Three cancer cell lines expressing MET, RON, or both receptors were treated with different amounts of PCMdt-MMAE for 96 h as described in ( b ). HCC1806 cells were used as the control. At the end of the study, the dead cells were counted using the Trypan blue exclusion assay to determine the percentage of cell death . d Morphological evidence of cell death. Treatment of cells with PCMdt-MMAE was performed as described in ( c ). Cellular morphological changes from individual cell lines were observed at 96 h under the Olympus BK-41 inverted microscope and photographed. For all studies described above, the percentages of cell viability and/or cell death and the individual IC 50 values from individual groups were calculated using the GraphPad Prism 6 software. Results shown here are from one of three experiments with similar results. ADC antibody–drug conjugate, CRC colorectal cancer, MET mesenchymal-epithelial transition, PCMdt-MMAE monomethyl auristatin E was conjugated to PCMbs–MR to generate the dual-targeting ADC, RON recepteur d’Origine nantais

Article Snippet: The amount of MMAE conjugated to PCMbs–MR in plasma was determined by using a MMAE ADC ELISA kit (Eagle Biosciences Inc., Nashua, NH, USA; www.eaglebio.com/product/mmae-adc-elisa-kit ) [ – ].

Techniques: Control, Staining, Flow Cytometry, Expressing, MTS Assay, Trypan Blue Exclusion Assay, Inverted Microscopy, Software

Journal: Acta Pharmacologica Sinica

Article Title: Humanized dual-targeting antibody–drug conjugates specific to MET and RON receptors as a pharmaceutical strategy for the treatment of cancers exhibiting phenotypic heterogeneity

doi: 10.1038/s41401-024-01458-7

Figure Lengend Snippet: Mice bearing xenograft tumors were divided into experimental and control groups (five animals per group). ADCs were injected through the tail vein. RhIgG-MMAE was used as the control. Tumor volumes were measured every four days. Individual tumors were collected at the end of the study, photographed, weighed to reach an average value per group, and analyzed for statistical differences using GraphPad Prism 6 software. The tumoristatic concentration (TSC), a minimal dose required to maintain a balance between the growth and inhibition of xenograft tumors, was calculated [ – ]. a Therapeutic efficacy of PCMdt-MMAE in comparison with anti-MET or anti-RON ADCs. HT29 cell-mediated xenograft tumors were used. PCMdt-MMAE, PCM-MET01-MMAE, and PCM5B14-MMAE at 10 mg/kg were injected once into mice. Mice treated with RhIgG-MMAE served as the control. Tumor growth was monitored up to day 36 or 40. b , c Dose-dependent effects of PCMdt-MMAE on xenograft tumor growth. FG and T-47D cell-derived xenograft tumors were used. PCMdt-MMAE from 1, 3, 7, 10, and 15 mg/kg in a Q12×2 schedule was injected once into mice. d Duration of PCMdt-MMAE-mediated anticancer activity. Xenograft tumors derived from H358, BxPC-3, and HCT116 cell lines were used. HCC1806 tumors served as the control. PCMdt-MMAE at 10 mg/kg was used for treatment. Tumor growth from each group was monitored until tumor regrowth was visible. ADC antibody–drug conjugate, MET mesenchymal-epithelial transition, PCMdt-MMAE monomethyl auristatin E was conjugated to PCMbs–MR to generate the dual-targeting ADC, RON recepteur d’Origine nantais.

Article Snippet: The amount of MMAE conjugated to PCMbs–MR in plasma was determined by using a MMAE ADC ELISA kit (Eagle Biosciences Inc., Nashua, NH, USA; www.eaglebio.com/product/mmae-adc-elisa-kit ) [ – ].

Techniques: Control, Injection, Software, Concentration Assay, Inhibition, Drug discovery, Comparison, Derivative Assay, Activity Assay

Journal: Acta Pharmacologica Sinica

Article Title: Humanized dual-targeting antibody–drug conjugates specific to MET and RON receptors as a pharmaceutical strategy for the treatment of cancers exhibiting phenotypic heterogeneity

doi: 10.1038/s41401-024-01458-7

Figure Lengend Snippet: A Therapeutic efficacy of PCMdt-MMAE at a single dose of 10 mg/kg in comparison with that of anti-MET ADC PCM-MET-1-MMAE or anti-RON ADC PCM5B14-MMAE against tumor xenografts mediated by colorectal cancer HT29 cells a . B Dose-dependent efficacy of PCMdt-MMAE against tumor xenografts mediated by PDAC FG and breast cancer T-47D cells in mouse models b . C Therapeutic effect of PCMdt-MMAE on multiple tumor xenografts mediated by T-47D, FG, HT29, and BxPC-3 cell lines in mouse models c .

Article Snippet: The amount of MMAE conjugated to PCMbs–MR in plasma was determined by using a MMAE ADC ELISA kit (Eagle Biosciences Inc., Nashua, NH, USA; www.eaglebio.com/product/mmae-adc-elisa-kit ) [ – ].

Techniques: Drug discovery, Comparison, Control