Journal: Genetics in medicine : official journal of the American College of Medical Genetics

Article Title: Identification of 4 novel human ocular coloboma genes ANK3 , BMPR1B , PDGFRA , and CDH4 through evolutionary conserved vertebrate gene analysis

doi: 10.1016/j.gim.2021.12.014

Figure Lengend Snippet: Summary of in silico analysis of single nucleotide variants (SNVs) identified

Article Snippet: For rescue, plasmid containing wild-type or variant BMPR1B was linearized by AscI (New England BioLabs) and heat inactivated for 20 minutes at 80 °C.

Techniques: In Silico

Journal: Genetics in medicine : official journal of the American College of Medical Genetics

Article Title: Identification of 4 novel human ocular coloboma genes ANK3 , BMPR1B , PDGFRA , and CDH4 through evolutionary conserved vertebrate gene analysis

doi: 10.1016/j.gim.2021.12.014

Figure Lengend Snippet: A. F1 is a White British nonconsanguineous pedigree with 1 affected male patient (proband 1–3) harboring a de novo sporadic heterozygous missense variant in ANK3 NM_020987.5:c.11650A>T, p.(Thr3884Ser). B. Corresponding widefield color fundus photographs of the right and left eye of the proband aged 17 years showing bilateral inferior chorioretinal coloboma with extensive optic disc involvement (right eye is more severely affected than the left eye as indicated also by the residual best corrected visual acuity). C. F3 is an Indian nonconsanguineous pedigree with autosomal dominant inheritance; the mother and the 2 daughters were affected with a heterozygous missense variant in BMPR1B NM_001203.2:c.272G>T, p.(Arg91Ile). D. Widefield color fundus photograph of the right eye of the proband (3–4) aged 13 showed a large optic disc coloboma extending inferiorly with a region of associated retinal pigment epithelium atrophy below this. The left eye image was not available. Widefield color fundus photographs of the right and left eye of the proband (3–3) aged 18 showing bilateral optic disc coloboma sparing the macula, with irregular asymmetrical peripapillary atrophy in the right eye. E. F5 is a White Northern European nonconsanguineous pedigree with 1 affected male patient (proband 5–3) with a heterozygous missense variant in BMPR1B NM_001203.2:c.671 G>A, p.(Arg224His). Parental samples for segregation were not available. F. Anterior segment color photographs of the right and left eye of the proband aged 4 showing bilateral inferior iris coloboma and corresponding widefield color fundus photographs showing bilateral inferior chorioretinal coloboma with extensive optic disc and macular involvement. F1, family 1; F3, family 3; F5, family 5.

Article Snippet: For rescue, plasmid containing wild-type or variant BMPR1B was linearized by AscI (New England BioLabs) and heat inactivated for 20 minutes at 80 °C.

Techniques: Variant Assay, Northern Blot

Journal: Genetics in medicine : official journal of the American College of Medical Genetics

Article Title: Identification of 4 novel human ocular coloboma genes ANK3 , BMPR1B , PDGFRA , and CDH4 through evolutionary conserved vertebrate gene analysis

doi: 10.1016/j.gim.2021.12.014

Figure Lengend Snippet: Injection of the MOs alone showed reduction in eye size and the presence of a coloboma. A. Coinjection with in vitro synthesized human BMPR1B messenger RNA (mRNA) rescues the phenotypic effects of the MO alone. Human variants identified within the Genomics England cohort were expressed and mRNAs coinjected with each MO. Scale bar 500 μm. Eye diameter was reported for (B) bmpr1ba and (C) bmpr1bb compared with wild-type uninjected; MO; MO + human wild-type BMPR1B ; and variants Arg91Ile, Arg224His, Arg224Leu, and Arg376Glu. ( n for bmpr1ba experiments = 23, 25, 55, 13, 21, 28, 42 and n for bmpr1bb experiments = 23, 56, 55, 32, 34, 33, 24). Unpaired t tests were used to compare data. ** P < .01, *** P < .001, **** P < .0001, ns P > .05. MO, morpholino; ns, nonsignificant.

Article Snippet: For rescue, plasmid containing wild-type or variant BMPR1B was linearized by AscI (New England BioLabs) and heat inactivated for 20 minutes at 80 °C.

Techniques: Injection, In Vitro, Synthesized

Journal: bioRxiv

Article Title: Tunneling nanotubes propagate a BMP-dependent preneoplastic state

doi: 10.1101/2025.11.11.687852

Figure Lengend Snippet: | Actors of the BMP signaling pathway are transferred to acceptor cells through TNTs. a, GOBP subset from a list of 6 protein hits dysregulated between NoTf accept and NoTf no accept cells, generated through STRING from proteins of the BMP and p38 pathways, chosen from the literature. The size of each circle represents the number of genes in the respective category; the intensity of the color represents the P-value, determined by a hypergeometric test. b, Box and whiskers plots of Log 2 LFQ values (Protein Levels) of ID1 ( ID1 ), Nup214 ( NUP214 ), Spartin ( SPART ), ATF2 ( ATF2 ), Rac1 ( RAC1 ) and desmoglein-3 ( DSG3 ), for indicated sorted cell populations. Statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparison test (n = 5 biological replicates; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). For clarity, only comparison between NoTf and NoTf accept cells is shown. c, Immunofluorescence images of actin (red) and BMPR1b (green) in indicated cells. Nuclei (blue). Scale bars, 20 μm. d, Correlative light scanning electron microscopy (SEM) of BMPR1b, immunostained with Alexa Fluor ® 488-Fluoronanogold ™ (green) in M1B26 cells. From left to right: confocal, SEM, zooms, correlative (overlay). Scale bars, 10 μm, unless otherwise indicated. e, Immunofluorescence of actin (green), after transfection with siRNA pools targeting BMPR1b (siBMPR1b) or GPER (siGPER). Non-targeting siRNA: siCTRL. f,g, BMPR1B and GPER transcript (f) and protein expression upon their silencing (f),with siCTRL set at 1 (n=4), and TNT number (> 8 μm) per cell and % of long TNTs (> 50 μm) upon indicated silencing, normalized against siCTRL (g). Error bars depict SD. Statistical significance was calculated using a Student’s two-sided t test with Welch’s correction (n = 4 biological replicates, *** p < 0.001). h, Schematic of experimental design: after sorting, cell populations were either lysed and subjected to RT-qPCR for selected transcript analyses, or grown for several weeks with or without BMP2/IL6, and immunoblot analyses performed at end points. i, RT-qPCR measurement of BMPR1b mRNA in indicated cell populations, the expression in NoTf cells set at 1. Statistical significance was calculated using one-way ANOVA with post-hoc Tukey test (n = 5 biological replicates). For clarity, only comparison between NoTf accept and NoTf cells is shown (**** p < 0.0001).

Article Snippet: After PBS washing, primary antibody against BMPR1b (1:300 #sc-293428 Santa Cruz) and its corresponding isotype were incubated overnight at 4°C.

Techniques: Generated, Comparison, Immunofluorescence, Electron Microscopy, Transfection, Expressing, Quantitative RT-PCR, Western Blot

Journal: bioRxiv

Article Title: Tunneling nanotubes propagate a BMP-dependent preneoplastic state

doi: 10.1101/2025.11.11.687852

Figure Lengend Snippet: | Non-transformed cells acceptor of information via TNTs from transformed cells display preneoplastic features. a, Left, quantification of BMPR1b immunoblots performed on NoTf accept cells grown in the presence of BMP2/IL6, for indicated weeks post sorting. Box and whiskers plot of 6 biological replicates. Statistical significance was calculated using a one-way ANOVA with non-parametric Kruskal-Wallis test, and for clarity, only p-values ≤ 0.05 are indicated. Right, quantification of BMPR1b immunoblots performed on indicated cells, 14 weeks post sorting, with NoTf conditions set at 1 (n = 6 biological replicates). Statistical significance was calculated using a one-way ANOVA with post-hoc Tukey test (*** p < 0.001). b,c, Quantification of p-SMAD1/5/8/SMAD1/5/8 (b) and p-p38/p38 (c) immunoblots performed on NoTf accept cells grown in the presence of BMP2/IL6, for indicated weeks post sorting. Box and whiskers plot of 6 biological replicates. Statistical significance was calculated using a one-way ANOVA with non-parametric Kruskal-Wallis test, and for clarity, only p-values ≤ 0.05 are indicated. d, RNA-sequencing results of transcriptomic expression levels of HOXB6, CCND2, EREG and HRAS in indicated cell lines (n = 3 biological replicates). Data deposited on the Gene Expression Omnibus repository, GSE186734 . Error bars depict SD. Statistical significance was calculated using a one-way ANOVA with post-hoc Tukey test (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). e, RT-qPCR measurements of HOXB6, CCND2, EREG and HRAS mRNA in indicated cell populations, with the expression in NoTf cells set at to 1. Error bars depict SD. Statistical significance was calculated using a one-way ANOVA with post-hoc Tukey test (Biological replicates: n = 8 for HOXB6, n = 5 for CCND2, n = 7 for EREG, n = 8 for HRAS). For clarity, only comparison between NoTf accept and NoTf cells is shown (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). f, Schematic of long-term soft agar colony formation assays. g, Quantification of soft agar clones of indicated sorted cells, after 16 weeks in culture with or without BMP2/IL6 (n = 3 biological replicates). Error bars represent SD. Statistical significance was calculated using a one-way Welch and Brown-Forsythe ANOVA test (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). For clarity, only p-values ≤ 0.05 are indicated, and comparisons with Tf cells are omitted. h, Model depicting how TNTs are involved in the propagation of a BMP-dependent preneoplastic state. Fibrils and fibers depict extracellular matrix components.

Article Snippet: After PBS washing, primary antibody against BMPR1b (1:300 #sc-293428 Santa Cruz) and its corresponding isotype were incubated overnight at 4°C.

Techniques: Transformation Assay, Western Blot, RNA Sequencing, Expressing, Gene Expression, Quantitative RT-PCR, Comparison, Clone Assay