Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Inhibition of BMP2 and BMP4 Represses Barrett’s Esophagus While Enhancing the Regeneration of Squamous Epithelium in Preclinical Models

doi: 10.1016/j.jcmgh.2023.01.003

Figure Lengend Snippet: (A) Clustering of RNA expression in biopsies from Barrett’s and squamous tissue and from a validation cohort (Wang 2013, GSE26886).27 Gene-clustered heatmap was drawn for quantitative PCR data using the heatmap package, after log transformation and calculation of z-scores for all samples per gene. (B) Quantitative PCR of SHH/WNT pathway genes in biopsies from Barrett’s and squamous tissue from AMC patients (left) and from a validation cohort (right) (Wang 2013, GSE26886).27 Genes significantly differentially expressed in Barrett’s compared with squamous in the AMC cohort (P < .05). (C) Western blot analysis of BMP2, BMP4, BMP5, and BMP6 in Barrett patient’s (BE) biopsies (left panel). Levels of BMPs in Barrett’s biopsies were quantified and normalized to squamous tissue (SQ). Data are represented in box and whiskers plot of at least 4 independent experiments. (D) BMP2, BMP4, BMP7, and SHH mRNA levels by quantitative PCR in mouse esophageal keratinocytes, human normal squamous esophageal EPC2-hTERT, and Barrett’s CP-A cells, after treatment with glycine-conjugated bile acid (100 μmol/L) for 8, 24, or 48 hours. Data are represented in box and whiskers plots as mean of at least 3 independent experiments. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001. ns: not significant.

Article Snippet: The membranes were blocked with blocking solution (5% nonfat milk in Tris-buffered saline with 1% Tween-20) and afterwards incubated with the appropriate primary antibody solution anti-BMP2 (Peprotech), anti-BMP4 (R&D), anti-BMP5 (Abcam), and anti-BMP6 (R&D) at a 1:500 dilution in blocking solution overnight at 4°C.

Techniques: RNA Expression, Real-time Polymerase Chain Reaction, Transformation Assay, Western Blot

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Inhibition of BMP2 and BMP4 Represses Barrett’s Esophagus While Enhancing the Regeneration of Squamous Epithelium in Preclinical Models

doi: 10.1016/j.jcmgh.2023.01.003

Figure Lengend Snippet: List of the Primers (Sense and Antisense) Used for Quantitative PCR

Article Snippet: The membranes were blocked with blocking solution (5% nonfat milk in Tris-buffered saline with 1% Tween-20) and afterwards incubated with the appropriate primary antibody solution anti-BMP2 (Peprotech), anti-BMP4 (R&D), anti-BMP5 (Abcam), and anti-BMP6 (R&D) at a 1:500 dilution in blocking solution overnight at 4°C.

Techniques:

Journal: Haematologica

Article Title: Iron overload induces BMP6 expression in the liver but not in the duodenum

doi: 10.3324/haematol.2010.031963

Figure Lengend Snippet: Effect of dietary iron-enrichment or Hfe-deficiency on hepatic iron concentrations and Bmp6 gene expression in the liver and the duodenum of 7-week old C57BL/6, DBA/2, and 129/Sv mice. (A) Increase in hepatic iron concentrations and expression ratios of Bmp6 transcripts normalized to the Hprt reference gene mRNA (and 95% confidence intervals) in wild-type animals fed an iron-enriched diet versus animals fed an iron-balanced diet (5 mice per group). (B) Increase in hepatic iron concentrations and expression ratios of Bmp6 transcripts normalized to the Hprt reference gene mRNA (and 95% confidence intervals) in Hfe-deficient versus wild-type mice (5–6 mice per group).

Article Snippet: Similar results were obtained with the Santa Cruz N-19 anti-BMP6 antibody ( data not shown ).

Techniques: Gene Expression, Expressing

Journal: Haematologica

Article Title: Iron overload induces BMP6 expression in the liver but not in the duodenum

doi: 10.3324/haematol.2010.031963

Figure Lengend Snippet: Cellular localization of iron and BMP6 in the liver and the duodenum of mice with iron overload secondary to an iron-enriched diet or due to Hfe-deficiency. Iron deposits are vizualized by Perls’ staining (1). BMP6 expression was detected by immunohistochemistry in the liver (2) and the duodenum (3). As expected, although Bmp6-deficient mice (A) have the highest iron accumulation, no Bmp6 was detected. C57BL/6 mice with secondary iron overload (B) and DBA/2 Hfe-deficient mice (C) have significant amounts of liver iron and Bmp6 in their liver but not in their duodenum. 129/Sv mice with secondary iron overload (D) have the lowest amount of liver iron and this correlates with Bmp6 staining in the liver. Original magnification x100 (1) or x200 (2 and 3).

Article Snippet: Similar results were obtained with the Santa Cruz N-19 anti-BMP6 antibody ( data not shown ).

Techniques: Staining, Expressing, Immunohistochemistry

Journal: Blood

Article Title: Regulation of TMPRSS6 by BMP6 and iron in human cells and mice

doi: 10.1182/blood-2011-04-348698

Figure Lengend Snippet: TMPRSS6 expression is induced by BMP6. (A) Hep3B cells were treated with 5, 25, and 50 ng/mL of human BMP6 for 16 hours and were analyzed for hepcidin and TMPRSS6 relative to RPL19 mRNA expression by quantitative real-time RT-PCR. The mean of 3 to 8 (depending of the dose) independent experiments is presented. Results are reported as the mean ± SEM for the fold change from mock, and significant changes represent the comparisons with mock. (B-C) Hep3B cells were transfected with siRNA control (5nM), siRNA TMPRSS6 (5nM), or TMPRSS6-FLAG (8 μg), and treated with 25 ng/mL of BMP6 for 48 hours. Cells were analyzed for matriptase-2 level relative to pan-cadherin protein by Western blot (B) followed by chemiluminescence quantification (C). (B) *A shorter exposure of lane 5 to better distinguish the 2 bands. (C) The mean of 3 experiments is presented, and results are reported as the mean ± SEM. (D) A total of 15 μg of protein from conditioned media of Hep3B cells transfected with siRNA control (5nM) and siRNA TMPRSS6 (5nM) and treated with BMP6 (25 ng/mL) for 48 hours were incubated with 666μM of N-(tert-butoxycarbonyl)-Gln-Ala-Arg-p-nitroanilide. Activity of matriptase-2 was assessed by measurement of the release of the dye p-nitroaniline during up to 20 minutes at a wavelength of 405 nm at 37°C using a spectrophotometer. The resulting activities (1 U corresponds to a release rate of 1 mmol of p-nitroaniline per minute) were measured in duplicate in 3 independent experiments. Results are reported as the mean ± SEM. (A,C-D) Significant changes are as follows: *P < .05; **P < .01; and ***P < .005.

Article Snippet: For BMP6 injection and BMP6 antibody injection experiments, C57BL/6 mice were purchased from Taconic at 7 weeks of age, then housed in the MGH facility and maintained on a standard rodent diet (Prolab RMH 3000 diet, 380 ppm iron) for 1 week before the start of treatment.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Incubation, Activity Assay, Spectrophotometry

Journal: Blood

Article Title: Regulation of TMPRSS6 by BMP6 and iron in human cells and mice

doi: 10.1182/blood-2011-04-348698

Figure Lengend Snippet: Regulation of TMPRSS6 mRNA expression in response to BMP6. Hep3B cells were treated with 25 ng/mL of BMP6 for several time points between 1 and 48 hours (A), and were analyzed for hepcidin and TMPRSS6 relative to RPL19 mRNA expression by quantitative real-time RT-PCR. (B-C) Hep3B cells received 10 μg/mL of cycloheximide (B) or 60nM of LDN-193189 (C) before BMP6 (25 ng/mL) treatment and were analyzed for gene expression relative to RPL19 mRNA expression by quantitative real-time RT-PCR. The mean of 6 (A-B) and 3 (C) independent experiments is presented. (B-C) Results are reported as the mean ± SEM for the fold change from mock (just before adding BMP6) and significant changes represent the comparisons with mock. Significant changes are as follows: *P < .05; **P < .01; and ***P < .005.

Article Snippet: For BMP6 injection and BMP6 antibody injection experiments, C57BL/6 mice were purchased from Taconic at 7 weeks of age, then housed in the MGH facility and maintained on a standard rodent diet (Prolab RMH 3000 diet, 380 ppm iron) for 1 week before the start of treatment.

Techniques: Expressing, Quantitative RT-PCR

Journal: Blood

Article Title: Regulation of TMPRSS6 by BMP6 and iron in human cells and mice

doi: 10.1182/blood-2011-04-348698

Figure Lengend Snippet: TMPRSS6 expression is controlled by ID1 in response to BMP6. Hep3B cells transfected with 10nM of siRNA control, siRNA SMAD7 (A-B), or siRNA ID1 (C-D), and treated in the absence or presence of 25 ng/mL of BMP6 for 24 hours were analyzed for TMPRSS6 (B,D) SMAD7 (A), and ID1 (C) mRNA expression relative to RPL19 mRNA by quantitative real-time RT-PCR. Results are reported as the mean ± SEM for the fold change from mock, and significant changes represent the comparisons with mock (siRNA control alone). Significant changes are as follows: *P < .05.

Article Snippet: For BMP6 injection and BMP6 antibody injection experiments, C57BL/6 mice were purchased from Taconic at 7 weeks of age, then housed in the MGH facility and maintained on a standard rodent diet (Prolab RMH 3000 diet, 380 ppm iron) for 1 week before the start of treatment.

Techniques: Expressing, Transfection, Quantitative RT-PCR

Journal: Blood

Article Title: Regulation of TMPRSS6 by BMP6 and iron in human cells and mice

doi: 10.1182/blood-2011-04-348698

Figure Lengend Snippet: TMPRSS6 expression is up-regulated in vivo by BMP6 and chronic iron treatment. (A) Eight-week-old male C57BL/6 mice received an intraperitoneal injection of BMP6 750 μg/kg animal weight (BMP6, black bars) or vehicle alone (mock, gray bars; n = 3 per group) for 6 and 12 hours. (B) Eight-week-old male C57BL/6 mice received an intraperitoneal injection of neutralizing BMP6 antibody 15 mg/kg once a day for 1 week (n = 5 per group). (C) Seven-week-old male C57BL/6 mice were killed at time zero (baseline) or after initiation of a 2% carbonyl iron diet for 24 hours, 48 hours, 72 hours, 1 week, or 2 weeks (n = 6 per group). Tissues were analyzed for hepatic hepcidin and Tmprss6 relative to Rpl19 mRNA by quantitative real-time RT-PCR. Results are reported as the mean ± SEM for the fold change from mock and significant changes represent the comparisons with mock. Significant changes are as follows: *P < .05; and ***P < .005.

Article Snippet: For BMP6 injection and BMP6 antibody injection experiments, C57BL/6 mice were purchased from Taconic at 7 weeks of age, then housed in the MGH facility and maintained on a standard rodent diet (Prolab RMH 3000 diet, 380 ppm iron) for 1 week before the start of treatment.

Techniques: Expressing, In Vivo, Injection, Quantitative RT-PCR

Journal: Blood

Article Title: Regulation of TMPRSS6 by BMP6 and iron in human cells and mice

doi: 10.1182/blood-2011-04-348698

Figure Lengend Snippet: Schematic representation showing proposed role of TMPRSS6 regulation by the BMP6-SMAD signaling pathway and iron via ID1. We propose that, in addition to being stimulated by several signals that inhibit hepcidin, such as iron deficiency, erythropoeitic drive, and hypoxia, TMPRSS6 expression is also stimulated indirectly by the hepcidin activators BMP6 and iron. Stimulation by BMP6 and/or iron induces an increase of the BMP6-HJV-SMAD pathway activity, possibly through a mechanism involving HFE and transferrin receptor 2 (TFR2), leading to binding of SMAD complexes to BMP-responsive elements (BMP-REs) on the hepcidin promoter and up-regulation of hepcidin transcription. In parallel, BMP6-SMAD pathway directly up-regulates SMAD7 and ID1 transcription. ID1 induction leads to the up-regulation of TMPRSS6 expression. TMPRSS6 then serves as a negative feedback inhibitor of BMP-SMAD pathway activity and hepcidin expression by cleaving the BMP coreceptor HJV. Inhibitory SMAD7 can also act as a negative feedback inhibitor by blocking SMAD activation. By acting as negative feedback inhibitors, TMPRSS6 and SMAD7 are important to prevent excessive hepcidin increases in response to BMP6 and iron, thereby maintaining tight control of iron homeostasis. Abbreviations: BMPR indicates BMP receptor; TF-Fe, holotransferrin; sHJV, soluble hemojuvelin; and BMP-RE, BMP responsive element.

Article Snippet: For BMP6 injection and BMP6 antibody injection experiments, C57BL/6 mice were purchased from Taconic at 7 weeks of age, then housed in the MGH facility and maintained on a standard rodent diet (Prolab RMH 3000 diet, 380 ppm iron) for 1 week before the start of treatment.

Techniques: Expressing, Activity Assay, Binding Assay, Blocking Assay, Activation Assay

Journal: iScience

Article Title: TNIK drives castration-resistant prostate cancer via phosphorylating EGFR

doi: 10.1016/j.isci.2023.108713

Figure Lengend Snippet: Targeting TNIK suppresses CRPC tumor progression in vivo (A) C4-2 cells were implanted subcutaneously in male BALB/c mice. When tumors became palpable, mice administered daily by oral gavage either with vehicle (10% DMSO in PBS) or NCB0846 (80 mg/kg of body weight) for 10 days (n = 4 mice for each treatment). Tumor volumes were measured with calipers. (B) Tumor size of xenografts of the above represented the growth of tumor over 10 days (n = 4) in athymic nude mice (p < 0.001). Data are shown as mean ± SD. (C) Tumor weight of the control mice tumors and NCB-0846-treated mice tumors (p < 0.001). Data are shown as mean ± SD. (D) Body weight of nude mice after implantation of control or C4-2 xenografts and treatment with vehicle or NCB-0846 for 4 weeks. (E) Quantitation of Ki-67, TNIK, p -EGFR, β-catenin, vimentin, E-cadherin, BMP6, and BMP7 expressions in C4-2 xenograft tumors from each group; specimens were got at 10 days posttreatment. Scale bars: 500 μm. The IHC was scored according to number of cells expressing the indicated proteins, and statistical analysis was performed (non-parametric Kruskal-Wallis test) in order to determine significance. Data are shown as mean ± SD. ∗∗∗p < 0.005.

Article Snippet: Anti- BMP6 antibody , Affinity Biosciences , Cat# AF5196; RRID: AB_2837682.

Techniques: In Vivo, Control, Quantitation Assay, Expressing

Journal: iScience

Article Title: TNIK drives castration-resistant prostate cancer via phosphorylating EGFR

doi: 10.1016/j.isci.2023.108713

Figure Lengend Snippet:

Article Snippet: Anti- BMP6 antibody , Affinity Biosciences , Cat# AF5196; RRID: AB_2837682.

Techniques: Virus, Bacteria, Recombinant, Lysis, Chromatin Immunoprecipitation, Control, Isolation, Labeling, Software