Journal: International Journal of Molecular Sciences

Article Title: Expression Patterns and Gonadotropin Regulation of the TGF-β II Receptor (Bmpr2) during Ovarian Development in the Ricefield Eel Monopterus albus

doi: 10.3390/ijms232315349

Figure Lengend Snippet: Nucleotide sequence information and amino acid sequence of the coding region of Bmpr2 in ricefield eel, Monopterus albus . The untranslated regions and translated regions are indicated by lowercase letters and uppercase letters, respectively. The predicted N-glycosylation sites are red boxes. The initiation codon (ATG) and stop codon (TAA) are marked in red color. Asterisks (*) indicate the translation stop codon.

Article Snippet: Before purchasing the primary antibody, the Bmpr2 sequence alignment between M. albus and H. sapiens BMPR2 (LS-C178875) was carried out using the DNAMAN software translation tool (version 6.0, Lynnon Biosoft, San Ramon, CA, USA).

Techniques: Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Expression Patterns and Gonadotropin Regulation of the TGF-β II Receptor (Bmpr2) during Ovarian Development in the Ricefield Eel Monopterus albus

doi: 10.3390/ijms232315349

Figure Lengend Snippet: Phylogenetic analysis and domain characteristics of Bmpr2 in ricefield eel, Monopterus albus . ( A ), The phylogenetic tree was researched by the neighbor-joining algorithm of Mega 11. The phylogeny was tested using the bootstrap method with 1000 replications. The numbers at nodes are bootstrap values (%). M. albus is marked in bold. The Bmpr2 protein sequences of vertebrates were obtained from Entrez (NCBI). ( B ), The characteristic Bmpr2 domains are conserved in ricefield eel orthologues. Bmpr2 is conservative in ligand-binding domain (ActRI/ActRII domain) and kinase domain, while the kinase domain is more conserved in Bmpr2. Numbers represent percentage identity of the predicted protein sequences with other type II BMP receptor orthologues (hBMPR2— Homo sapiens BMPR2, rBMPR2— Rattus BMPR2, gBMPR2— Gallus BMPR2, xBMPR2— Xenopus tropicalis BMPR2, mBmpr2— Micropterus salmoides Bmpr2b, sBmpr2— Scatophagus argus Bmpr2b, dBmpr2a— Danio rerio Bmpr2a, and dBmpr2b— Danio rerio Bmpr2b).

Article Snippet: Before purchasing the primary antibody, the Bmpr2 sequence alignment between M. albus and H. sapiens BMPR2 (LS-C178875) was carried out using the DNAMAN software translation tool (version 6.0, Lynnon Biosoft, San Ramon, CA, USA).

Techniques: Ligand Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Expression Patterns and Gonadotropin Regulation of the TGF-β II Receptor (Bmpr2) during Ovarian Development in the Ricefield Eel Monopterus albus

doi: 10.3390/ijms232315349

Figure Lengend Snippet: Expression of bmpr2 in different tissues of ricefield eel, Monopterus albus . ( A ), Relative mRNA levels of bmpr2 in tissues of ricefield eels were analyzed using RT-qPCR. ( B ), Bmpr2 immunoreactivity in the ovaries was evaluated using Western blot analysis. Anti-Bmpr2, primary antibody; Control, negative control; Bl, blood; Br, brain; Ey, eyes; He, heart; Ig, intersex gonads; In, intestines; Ki, kidneys; Li, liver; Mu, muscle; Ov, ovaries; Pi, pituitary; Sp, spleen; Te, testes. Results are expressed as means ± SEMs ( n = 5). *, **, *** and **** are the significant differences of Duncan’s multiple comparisons, representing * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001, respectively.

Article Snippet: Before purchasing the primary antibody, the Bmpr2 sequence alignment between M. albus and H. sapiens BMPR2 (LS-C178875) was carried out using the DNAMAN software translation tool (version 6.0, Lynnon Biosoft, San Ramon, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Expression Patterns and Gonadotropin Regulation of the TGF-β II Receptor (Bmpr2) during Ovarian Development in the Ricefield Eel Monopterus albus

doi: 10.3390/ijms232315349

Figure Lengend Snippet: Expression of bmpr2 in developing ovaries was analyzed in ricefield eel, Monopterus albus . ( A ), Expression of bmpr2 during ovarian development. ( B ), The localization of Bmpr2 immunoreactive signals (yellow and dot-like) in the ovary. ( i ), Primary growth stage; ( ii ), early vitellogenic stage; ( iii ), negative control. EV, early vitellogenic stage; MLV, middle to late vitellogenic stage; OM, oocyte in mature stage; OG, oogonium; PG, primary growth stage; PV, previtellogenic stage. Results are expressed as means ± SEMs ( n = 5). * and **** are the significant differences in Duncan’s multiple comparisons, representing * p < 0.05 and **** p < 0.0001, respectively.

Article Snippet: Before purchasing the primary antibody, the Bmpr2 sequence alignment between M. albus and H. sapiens BMPR2 (LS-C178875) was carried out using the DNAMAN software translation tool (version 6.0, Lynnon Biosoft, San Ramon, CA, USA).

Techniques: Expressing, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Expression Patterns and Gonadotropin Regulation of the TGF-β II Receptor (Bmpr2) during Ovarian Development in the Ricefield Eel Monopterus albus

doi: 10.3390/ijms232315349

Figure Lengend Snippet: Immunofluorescence analysis of Bmpr2 and Gdf9 in the ovary of ricefield eel, Monopterus albus . Fluorescent images of Bmpr2 in the ovary captured by FITC: ( A , a ), DAPI; ( B , b ), FITC; ( C ), merged fluorescent images of ( A , B ). ( c ), Merged fluorescent images of ( a , b ). Fluorescent images of Gdf9 in the ovary captured by TRITC: ( D , d ), TRITC; ( E ), merged fluorescent images of ( A , D ); ( e ), merged fluorescent images of ( a , d ); ( F ), merged fluorescent images of ( C , E ); ( f ), merged fluorescent images of ( c , e ). PG, primary growth stage; PV, previtellogenic stage; EV, early vitellogenic stage. Immunofluorescence (green) shows Bmpr2 expression in the ovary. Immunofluorescence (orange-red signal) shows Gdf9 expression in the ovary. Nuclei are labeled with DAPI (blue). All photomicrographs were taken by an Olympus inverted research microscope.

Article Snippet: Before purchasing the primary antibody, the Bmpr2 sequence alignment between M. albus and H. sapiens BMPR2 (LS-C178875) was carried out using the DNAMAN software translation tool (version 6.0, Lynnon Biosoft, San Ramon, CA, USA).

Techniques: Immunofluorescence, Expressing, Labeling, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Expression Patterns and Gonadotropin Regulation of the TGF-β II Receptor (Bmpr2) during Ovarian Development in the Ricefield Eel Monopterus albus

doi: 10.3390/ijms232315349

Figure Lengend Snippet: Expression of gdf9 and bmpr2 in ovarian tissue of ricefield eel after FSH and hCG incubation in vitro. Results are expressed as means ± SEM (error bars), which were compared with the control group. ( A , C ) The relative expression level of gdf9 and bmpr2 after FSH incubation; ( B , D ), the relative expression level of gdf9 and bmpr2 after hCG incubation. FSH, follicle-stimulating hormone; hCG, human chorionic gonadotropin; IU, international unit. *, **, *** and **** are the significant differences in Duncan’s multiple comparisons, representing * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001, respectively.

Article Snippet: Before purchasing the primary antibody, the Bmpr2 sequence alignment between M. albus and H. sapiens BMPR2 (LS-C178875) was carried out using the DNAMAN software translation tool (version 6.0, Lynnon Biosoft, San Ramon, CA, USA).

Techniques: Expressing, Incubation, In Vitro

Journal: International Journal of Molecular Sciences

Article Title: Expression Patterns and Gonadotropin Regulation of the TGF-β II Receptor (Bmpr2) during Ovarian Development in the Ricefield Eel Monopterus albus

doi: 10.3390/ijms232315349

Figure Lengend Snippet: Primers used for cloning and RT-qPCR.

Article Snippet: Before purchasing the primary antibody, the Bmpr2 sequence alignment between M. albus and H. sapiens BMPR2 (LS-C178875) was carried out using the DNAMAN software translation tool (version 6.0, Lynnon Biosoft, San Ramon, CA, USA).

Techniques: Clone Assay

Journal: bioRxiv

Article Title: Autocrine TGFβ2 enforces a transcriptionally hybrid cell state in Ewing sarcoma

doi: 10.1101/2025.06.11.653876

Figure Lengend Snippet: ( A ) scSeq data from 6 EwS cell lines with CAF-like subpopulations. Cells scored as EWS::FLI1 “true low” (below median expression of activated/above median expression repressed targets ) or “hybrid” (above median expression of activated and repressed targets), other cells termed “high”. ( B ) Single cell TGFBR2 expression grouped by EWS::FLI1 activity state (n=6 cell lines). ( C ) Flow cytometry of TGFBR2 and CD73. n=3 ( D ) TGFBR2 expression by GeoMx digital spatial profiling of 12 regions of CHLA10 xenografts in NSG mice, previously scored as EWS::FLI1 “low”, “hybrid”, or “high” activity . ( E ) 40 EwS patient tumors from stroma-poor or stroma-rich (ý30% non-tumor stroma) samples . Genes ranked by mean stroma-rich/mean stroma-poor sample values. Dashed lines = top/bottom 5% of genes. ( F ) Ranked enrichment in stroma-rich vs. stroma-poor samples in 40 patient tumors of EWS::FLI1 activated or repressed target genes (0 = not enriched). P-value = Mann-Whitney test. ( G ) Immunoblots of phospho vs. total SMAD2 and EWS::FLI1 in CHLA10 cells transduced with dominant negative TGFBR2 vs. empty vector control -/+ 24 hrs treatment with TGFβ1. Representative of n=2. A549 = positive control for TGFβ induced pSMAD2 and negative control for EWS::FLI1. Loading control = GAPDH. ( H ) RT-qPCR of TNC and COL1A1 in parent, empty vector, or dominant-negative TGFBR2 transduced CHLA10 cells -/+ 24 hrs treatment with TGFβ1. n=3, p-values = unpaired t-tests. ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: The transfer plasmids utilized in this study included pLenti CMV Blast DEST (Addgene #17451), pLenti CMV Blast DN-TGFBR2-HA (Addgene #130888), pLKO.1 shFLI1 (Sigma TRCN0000005322), and pLKO.1 non-silencing control (Sigma SCH002).

Techniques: Expressing, Activity Assay, Flow Cytometry, MANN-WHITNEY, Western Blot, Transduction, Dominant Negative Mutation, Plasmid Preparation, Control, Positive Control, Negative Control, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Autocrine TGFβ2 enforces a transcriptionally hybrid cell state in Ewing sarcoma

doi: 10.1101/2025.06.11.653876

Figure Lengend Snippet: (A ) % GFP+ after transduction with SBE-GFP SMAD reporter-/+ TGFβ1 (48 hours, n=2). ( B ) TGFB1 and TGFB2 expression in CAF-like (top 30% expression CAF-like gene signature) vs. non-CAF-like cells by single cell sequencing (9 EwS lines). ( C ) ELISAs of TGFβ1 in conditioned media (3 days of culture). n=3. Dashed line indicates TGFβ1 level detected in media only controls. ( D ) ELISAs of secreted total TGFβ2 in conditioned media (3 days of culture). n=3. TGFβ2 not detected in media only controls. ( E ) Genes significantly (padj < 0.05) upregulated by TGFβ1 or TGFβ2 in A673, CHLA10, and PDX305 (bulk RNAseq, n=3). ( F ) Gene ontology (GO:BP) of the 365 genes commonly significantly induced (padj<0.05) by TGFβ1 and TGFβ2 in A673, CHLA10, and PDX305 cells. ( G ) ELISAs of secreted total TGFβ2 after 3 days of culture of CHLA10 dominant negative TGFBR2 or empty vector transduced cells. n=3. p-value = unpaired t-test. ( H ) Expression of TNC , TGFB1 , and TGFB2 in PDX305 cells treated with TGFβ1 (10 ng/mL) -/+ vactosertib, TGFβ2 (10 ng/mL) -/+ vactosertib, or vactosertib alone (1 μM), n=2, p-values = unpaired t-tests. ( I ). Expression of TGFβ induced genes in CHLA10 cells treated with TGFβ ligand blocking antibodies (10 ug/mL, 4 days). ( J ) IF of TNC in CHLA10 cells treated with TGβ ligand blocking antibodies vs. IgG control (10 ug/mL, 4 days). ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: The transfer plasmids utilized in this study included pLenti CMV Blast DEST (Addgene #17451), pLenti CMV Blast DN-TGFBR2-HA (Addgene #130888), pLKO.1 shFLI1 (Sigma TRCN0000005322), and pLKO.1 non-silencing control (Sigma SCH002).

Techniques: Transduction, Expressing, Sequencing, Dominant Negative Mutation, Plasmid Preparation, Blocking Assay, Control

Journal: bioRxiv

Article Title: Autocrine TGFβ2 enforces a transcriptionally hybrid cell state in Ewing sarcoma

doi: 10.1101/2025.06.11.653876

Figure Lengend Snippet: (A ) GSEA NES values of EWS::FLI1 target genes in TGFβ2 treated cell lines (bulk RNAseq, n=3, p-values = padj from GSEA). ( B ) TGFBR2 expression (RNA-seq) of cell lines treated with TGFβ1, TGFβ2, or vactosertib (n=3, error bars = standard error, p-values = padj). ( C ) Expression of TGFBR2 , TGFB1 , TGFB2 in shFLI1 vs. shNS CHLA10 cells by RT-qPCR (n=3). ( D ) RT-qPCR of TGFB2 and TGFBR2 in CHLA10 cells transduced with shNS or shFLI1 shRNAs (48hrs) then treated with vactosertib (1μM, 24 hrs). n=3. ( E ) RT-qPCR of TGFB2 and TGFBR2 in CHLA10 cells transduced with shNS or shFLI1 shRNAs (48hrs) then treated with TGFβ2 (10 ng/mL, 24 hrs). n=3. ( F ) RT-qPCR of EWS:FLI1 and its direct activated targets SOX2 and NR0B1 in CHLA10 cells transduced with shNS or shFLI1 shRNAs (48hrs) then treated with TGFβ2 (10 ng/mL, 24 hrs). Normalized to housekeeping genes then to the average of shNS samples. n=3. P-values = unpaired t-tests. ( G ) RT-qPCR of TGFB2 and TGFBR2 in TC71 cells transduced with shNS or shFLI1 shRNAs (48hrs) then treated with TGFβ2 (10 ng/mL, 24 hrs). n=3. ( H ) Model of the positive feedback loop between TGFBR2 activity, TGFβ2 expression, and de-repression of EWS::FLI1 downregulated targets in permissive EWS::FLI1 “low” or EWS::FLI1 “hybrid” states vs. EWS::FLI1 “high” cells. ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: The transfer plasmids utilized in this study included pLenti CMV Blast DEST (Addgene #17451), pLenti CMV Blast DN-TGFBR2-HA (Addgene #130888), pLKO.1 shFLI1 (Sigma TRCN0000005322), and pLKO.1 non-silencing control (Sigma SCH002).

Techniques: Expressing, RNA Sequencing, Quantitative RT-PCR, Transduction, Activity Assay