Journal: Journal of Cellular and Molecular Medicine

Article Title: Metformin attenuates trauma‐induced heterotopic ossification via inhibition of Bone Morphogenetic Protein signalling

doi: 10.1111/jcmm.16076

Figure Lengend Snippet: List of antibodies and protein used in this study

Article Snippet: Smad1 (D59D7) XP® Rabbit mAb , 6944S , Cell Signaling Technology , Danvers, USA.

Techniques: Recombinant

Journal: Journal of Cellular and Molecular Medicine

Article Title: Metformin attenuates trauma‐induced heterotopic ossification via inhibition of Bone Morphogenetic Protein signalling

doi: 10.1111/jcmm.16076

Figure Lengend Snippet: AMPK activators inhibit alkaline phosphatase activity during osteogenic differentiation. MC3T3‐E1 cells were incubated with osteogenic differentiation medium in the presence of different doses of metformin (Met) and Ibuprofen. After one week, the Alkaline phosphatase activity (metformin (Met) (A) and Ibuprofen(C)) was measured. The graph represents averages of three independent experiments (mean ± SD, N = 3). *Significant difference from metformin (A) and Ibuprofen(C) versus control was assessed using Student's t test (* P < .05). Western blot (metformin (B) and Ibuprofen(D)) was also performed to study activation of AMPK and Smad1/5. NM: no differentiation medium, α‐MEM complete medium

Article Snippet: Smad1 (D59D7) XP® Rabbit mAb , 6944S , Cell Signaling Technology , Danvers, USA.

Techniques: Activity Assay, Incubation, Control, Western Blot, Activation Assay

Journal: International Journal of Biological Sciences

Article Title: MicroRNA-196a promotes renal cancer cell migration and invasion by targeting BRAM1 to regulate SMAD and MAPK signaling pathways

doi: 10.7150/ijbs.60805

Figure Lengend Snippet: Bram1 inhibits cell migration and invasion via MAPK and BMP pathways by binding to EGFR and BMPR1A. (A) HEK293T cells were transfected with Bram1 or shBram1 and the expression level of phosphorylated SMAD1/5/8 and tubulin was examined by Western Blot assay. Data is representative of three independent experiments. (B) HEK293T cells were co-transfected with SMAD luciferase plasmids and Bram1 or shBram1 plasmids, and the lysate was used for luciferase assay. *P<0.05. (C, D) cells were transfected with Bram1 or shBram1, and the expression level of indicated proteins was examined by Western Blot assay. (E) Protein level of phosphate p42/44 and total p42/44 in empty vector (-) and Bram1 overexpression (+) RCC4 cells with or without U0126 treatment (10 µM, 4 hours). (F) Wound healing assay analysis the effect of MAPK inhibitor and Bram1 overexpression on cell migration (10 µM U0126 treatment for 4 hours). (G) The physical interaction between Bram1 and BMPR1A or EGFR was investigated by immunoprecipitation experiments. HEK293T cells were transfected with flag-Bram1, and treated with 50 µM BMP4, 48 hr post-transfection the lysates were collected and incubated with normal beads as a negative control or flag-beads. (H) RCC4 cells were co-transfected with miR-196a and Bram1 plasmids, the expression of Bram1 was detected by RT-PCR. *P<0.05. (I) RCC4 cells were co-transfected with miR-196a and Bram1 plasmids and seeded in the upper chamber of transwell plates. Duplicates were performed in three independent experiments. ***P<0.001. (J) RCC4 cells were co-transfected with miR-196a and Bram1 plasmids, followed by wound healing assay measured at 0 hr and 24 hr. Data is representative of three independent experiments.

Article Snippet: The antibodies used in our experiments included: Anti-Rabbit Bram1( Cell Signaling Technology (CST), Inc Danvers, MA, U.S.A); Anti-Rabbit BMPR1A (Gene tex Inc, San Antonio, Texas, U.S.A); Anti-Rabbit p-ERK (CST, Danvers, MA, U.S.A); Anti-Rabbit total-ERK (CST, Danvers, MA, U.S.A); Anti-Rabbit p-p38 (CST, Danvers, MA, U.S.A); Anti-Rabbit total-p38 (CST, Danvers, MA, U.S.A); Anti-Rabbit p-JNK (CST, Danvers, MA, U.S.A); Anti-Rabbit total-JNK (CST, Danvers, MA, U.S.A); Anti-Rabbit p-SMAD1/5 (CST, Danvers, MA, U.S.A); Anti-Rabbit EGFR (CST, Danvers, MA, U.S.A); Anti-Mouse Tubulin (Sigma-Aldrich Co St. Louis, MO, U.S.A.).

Techniques: Migration, Binding Assay, Transfection, Expressing, Western Blot, Luciferase, Plasmid Preparation, Over Expression, Wound Healing Assay, Immunoprecipitation, Incubation, Negative Control, Reverse Transcription Polymerase Chain Reaction

Journal: Developmental biology

Article Title: Multi-modal effects of BMP signaling on Nodal expression in the lateral plate mesoderm during left-right axis formation in the chick embryo.

doi: 10.1016/j.ydbio.2012.11.027

Figure Lengend Snippet: Fig. 6. BMP signaling and NODAL signaling mutually suppress their effects on Nodal expression in the LPM (A) Control MO, Smad1 MO, or Smad2 MO were electroporated into the middle primitive streak region of the epiblast at HH4 and Nodal expression was examined at HH8. (B and B0) No effect on Nodal expression in the LPM was seen in the control MO (black arrowheads). (C and C0) Smad1 MO induced ectopic Nodal expression in the right LPM (red arrowheads). (D and D0) Smad2 MO repressed endogenous Nodal expression in the left LPM (blue arrow- heads). (E) Bar chart summary of the results showing percentages of each phenotype. The extent of Nodal expression was classified as shown in Fig. 1. Asterisks indicate significant differences in ectopic Nodal induction (between control and Smad1 MO) or Nodal inhibition (between control and Smad2 MO): **Po0.01; *Po0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Proteins were detected using antibodies against Smad1 (1:1000, D59D7, #6944, Cell Signaling), Smad2 (1:2000, D43B4, #5339, Cell Signaling), phosphorylated-Smad1/5/8 (1:1000, #AB3848, Millipore), and actin (1:3000, A2066, Sigma), diluted in Signal Enhancer HIKARI (Nacalai Tesque, Japan).

Techniques: Expressing, Control, Inhibition

Journal: Developmental biology

Article Title: Multi-modal effects of BMP signaling on Nodal expression in the lateral plate mesoderm during left-right axis formation in the chick embryo.

doi: 10.1016/j.ydbio.2012.11.027

Figure Lengend Snippet: Fig. 8. Multi-modal effects of BMP signaling on Nodal expression: A model for the multiple roles of BMP signaling in Nodal expression. Variations in BMP signaling strength produce four different states of Nodal expression in the LPM. The thickness of the arrows represents the traffic quantity of each step. (A) High BMP signaling level. Steps 1– 5: In the presence of a high concentration of BMP, Cfc expression is strongly activated. Step 6: Increased CFC production causes an increase in the sensitivity of the NODAL receptor to NODAL. Steps 7 and 8: NODAL at background level activates the NODAL receptor. Step 9: Increase in levels of pSMAD2/3 raises the probability of formation of pSMAD2/3-SMAD4 complexes. Steps 10–12: As a result, Nodal transcription and the NODAL positive-feedback loop are activated. This state is equivalent to an LPM with an experimentally applied high concentration of BMP. (B) High intermediate BMP signaling level. Steps 1 and 2: BMPs bind to and activate BMP receptors. Steps 3 and 4: pSMAD1/5/8 associate with SMAD4 and translocate to the nucleus. Step 5: pSMAD1/5/8-SMAD4 complexes stimulate expression of target genes including the NODAL co-receptor Cfc. Steps 6–10: At this level of BMP signaling, CFC production for NODAL binding to the receptor is maintained, and NODAL present at the basal level activates the NODAL receptor; however, the predominance of binding between pSMAD1/5/8 and SMAD4 causes a shortage of free SMAD4, preventing pSMAD2/3 forming a complex with SMAD4. Steps 11 and 12: As a result, Nodal transcription and the NODAL positive-feedback loop are inactivated. This state is equivalent to the native right LPM, or to the left LPM with an experimentally applied low concentration of BMP. (C) Low intermediate BMP signaling level. Steps 1–6: In the presence of a low concentration of the BMP antagonist, BMP binding to the receptor is weakly inhibited and free SMAD4 is released from pSMAD1/5/8-SMAD4 complexes, although Cfc expression is still maintained. Steps 7 and 8: NODAL from the node or present at the basal level binds to the receptor and activates the NODAL receptor. Steps 9 and 10: Phosphorylated SMAD2/3 associates with free SMAD4 and translocates to the nucleus. Steps 11 and 12: This activates Nodal transcription and the NODAL positive-feedback loop. This state is equivalent to the native left LPM, or to the right LPM with an experimentally applied with low concentration of a BMP antagonist. (D) Low BMP signaling level. Steps 1–5: In the presence of a high concentration of a BMP antagonist, BMP binding to the receptor and downstream events leading to Cfc expression are strongly inhibited. Steps 6–12: As a result of decreased CFC production, NODAL binding to the receptor and downstream events leading to Nodal expression and the NODAL positive-feedback loop are strongly inhibited. This state is equivalent to the LPM with an experimentally applied high concentration of a BMP antagonist such as NOGGIN.

Article Snippet: Proteins were detected using antibodies against Smad1 (1:1000, D59D7, #6944, Cell Signaling), Smad2 (1:2000, D43B4, #5339, Cell Signaling), phosphorylated-Smad1/5/8 (1:1000, #AB3848, Millipore), and actin (1:3000, A2066, Sigma), diluted in Signal Enhancer HIKARI (Nacalai Tesque, Japan).

Techniques: Expressing, Concentration Assay, Binding Assay