Fig 1: (a) Alkaline phosphatase staining of absorbable collagen sponges (ACS) at 7 days of ST2 cells seeded on control tissue culture plastic, control ACS, ACS loaded with BMP2 low (10 ng/ml), ACS loaded with BMP2 high (100 ng/ml), ACS loaded with BMP9 low (10 ng/ml), and ACS loaded with BMP9 high (100 ng/ml). (b) bone morphogenetic protein 9 low and high significantly increased alkaline phosphatase staining when compared to control and bone morphogenetic protein 2samples (* denotes significant difference, p < .05; ** denotes significantly higher than all other treatment modalities, p < .05)
Fig 2: Anoikis susceptibility is mediated via ALK3/ALK6. (A) Western blotting of 4T1 cells grown under anoikis conditions either with or without 1 µM dorsomorphin and GDF2 as indicated. Lysates were immunoblotted against CC3. Actin was the loading control (quantification of CC3 levels presented in Supplementary Figure S3E). (B) Kinase mutants ALK3 K-R and ALK6 K-R reduce anoikis. Western blotting of PA1 cells expressing control, ALK3 K-R, or ALK6 K-R was plated for anoikis (Methods) in the absence or presence of GDF2 for indicated times (quantification of CC3 levels presented in Supplementary Figure S3F). (C) MTT absorbance values of PA1 cells expressing shScr, shALK3 or shALK6 after 24 hours of anoikis in the absence and presence of 10 ng/ml of GDF2 are presented. Error bars indicate SEM. (D) Western blotting of same cells as in (C) harvested after 24 hours of anoikis and immunoblotted for CC3. Actin was the loading control. (E) Western blotting as indicated of PA1 cells expressing shSMAD1 or shScr at the indicated times under anoikis conditions (quantification of CC3 presented in Supplementary Figure S3G). SMAD1 transcript levels upon shRNA to SMAD1 relative to controls are presented in the adjacent graph. Actin was the loading control.
Fig 3: GDF2 expression in breast and ovarian cells. (A) Indicated cell lines were lysed and immunoblotted for GDF2 (*lower band). Actin was the loading control. (B) Secreted GDF2 levels as determined using ELISAs in indicated breast and ovarian cell lines. (C) QRT-PCR levels of GDF2 transcript in the presence of increasing AZA for the indicated time points. Error bars indicate SEM. (D) Western blotting for CC3 and pSMAD1/5 in PA1 cells either untreated or treated with AZA under anoikis conditions for the indicated times. Dorsomorphin (5 µM) was added in combination during anoikis where indicated (Dm). Actin was the loading control. (E) Promoter methylation of GDF2. GDF2 promoter methylation status of ovarian cancer patients (N = 47; black line) plotted against normal fallopian tube epithelium (gray line) from the same experiment. Values were plotted using the GraphPad Prism software. Beta values are presented (*P < .05). (F) Percent methylation determined by pyrosequencing (Methods and Supplementary Table) of the CpG1 site contained within the GDF2 promoter in either untransformed P211 or transformed PA1 cells in the presence or absence of 1 µM AZA is presented.
Fig 4: (A) Western blotting for BIM in indicated cell lines plated for anoikis (see Methods) in the absence and presence of GDF2 10 ng/ml (+) or 20 ng/ml (++) as indicated. Immunoblotted lysates are the same as used in Figure 4A. Actin was the loading control. (B) Caspase3/7 activity under anoikis conditions (Methods) in the presence or absence of GDF2 and represented as luminescence units. Data are representative of two independent experiments (**P < .01, **P < .01, and *P < .05 in P76, P211, and PA1 cells, respectively). Error bars indicate SEM. (C and D) Growth curve of viable cells as indicated, treated -/+ GDF2, and harvested 1 and 3 days later. (E-F) Quantification of CC3 levels in Figure 5, A, B, and E, respectively, represented as pixel intensities normalized to actin levels. (H) Anoikis assay at the indicated conditions -/+ 10 µm TAK inhibitor treatment. Immunoblot of CC3 and loading control actin. (I) Anoikis assay at the indicated time points -/+ 160 ng/ml Alk1-ECD. Immunoblot of CC3 and loading control actin.
Fig 5: Analysis of serum BMP9 by different statistical approaches. a, Circulating BMP9 levels in HTN, CHD and HTN + CHD patients; b, The odds ratio of having HTN in different quartile of BMP9; c, The odds ratio of having CHD in different quartile of BMP9; d, The odds ratio of having HTN + CHD in different quartile of BMP9. *P < 0.05, **P < 0.01 vs. controls or quartile 4
Supplier Page from R&D Systems, a Bio-Techne Brand for BMP-9 ELISA