Journal: eLife

Article Title: Enalapril mitigates senescence and aging-related phenotypes in human cells and mice via pSmad1/5/9-driven antioxidative genes

doi: 10.7554/eLife.104774

Figure Lengend Snippet: ( A ) Western blot results for the detection of pSmad5, pSmad1/5/9, and other proteins after enalapril treatment. ( B ) GSEA showing the enrichment of the BMP signaling pathway after enalapril treatment. NES, normalized enrichment score. ES, enrichment score. ( C ) Profile of pSmad1/5/9 enrichment at the TSS regions in the control group (Ctrl) and the enalapril-treated group (Enalapril). ( D ) Heatmap of cell cycle (green) and SASP-related genes (purple) in the control group (Ctrl), BMP receptor inhibitor-treated group (LDN193189, LDN), and enalapril and BMP receptor inhibitor-cotreated group (EP+LDN). ( E ) Western blot analysis showing the protein levels of pSmad1/5/9, p16, and p21 in response to different combinations of enalapril, a BMP receptor inhibitor (LDN193189, LDN), and BMP4. ( F ) Senescence-associated β-galactosidase (SA-β-Gal) staining (left) and corresponding ratio statistical chart (right) of IMR90 cells treated with different combinations of enalapril, a BMP receptor inhibitor (LDN193189, LDN), and BMP4. Scale bars, 200 μm. Enlarged scale bars, 100 μm. ( G ) Ki67 immunofluorescence experiment (left) and intensity statistical chart (right) of IMR90 cells treated with different combinations of enalapril, a BMP receptor inhibitor (LDN193189, LDN), and BMP4. Scale bars, 80 μm. Enlarged scale bars, 40 μm. A two-tailed t-test was employed; ns indicates no significant difference, * p <0.05, ** p <0.01, *** p <0.001. Figure 2—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 2—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Recombinant BMP4 protein (MCE, HY-P7007) was dissolved in sterile ddH 2 O to prepare a 100 μg/mL stock solution.

Techniques: Western Blot, Control, Staining, Immunofluorescence, Two Tailed Test

Journal: eLife

Article Title: Enalapril mitigates senescence and aging-related phenotypes in human cells and mice via pSmad1/5/9-driven antioxidative genes

doi: 10.7554/eLife.104774

Figure Lengend Snippet: ( A ) Western blot analysis showing the changes in protein levels following BMP4 treatment. ( B ) Senescence-associated β-galactosidase (SA-β-Gal) staining (left) and corresponding ratio analysis chart (right) following BMP4 treatment. Scale bars, 200 μm. Enlarged scale bars, 100 μm. ( C ) Ki67 immunofluorescence (right) and corresponding ratio analysis chart (left) following BMP4 treatment. Scale bars, 80 μm. Enlarged scale bars, 40 μm. A two-tailed t-test was employed, ** p <0.01, **** p <0.0001. Figure 2—figure supplement 2—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 2—figure supplement 2—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Recombinant BMP4 protein (MCE, HY-P7007) was dissolved in sterile ddH 2 O to prepare a 100 μg/mL stock solution.

Techniques: Western Blot, Staining, Immunofluorescence, Two Tailed Test

Journal: The FASEB Journal

Article Title: Low‐Intensity Pulsed Ultrasound Promotes Osteogenesis in Porous Titanium Alloys Through miR ‐1187/ BMP4 Pathway

doi: 10.1096/fj.202403395RR

Figure Lengend Snippet: miR‐1187 directly targets BMP4. (A) The predicted binding site between miR‐1187 and BMP4 by bioinformatics analysis. (B) The luciferase activity of the BMP4‐WT and BMP4‐MUT in MC3T3‐E1 cells treated with miR‐1187 mimics or NC. (C) mRNA expression of Bmp4 was significantly downregulated in the miR‐1187 mimics‐transfected group. (D) Western blot analysis for the expression of BMP4 protein in miR‐1187 mimics and inhibitor‐transfected MC3T3‐E1 cells on Pti. (E) ELISA assay analysis for the expression of supernatant BMP4 secreted protein in miR‐1187 mimics and inhibitor‐transfected MC3T3‐E1cells on Pti. All values represent means ± SD.( n = 3). * p < 0.05, ** p < 0.01.

Article Snippet: BMP4 protein levels in the supernatant were quantified using a mouse BMP‐4 ELISA kit (EK0316, Boster, China) following the manufacturer's protocol.

Techniques: Binding Assay, Luciferase, Activity Assay, Expressing, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: The FASEB Journal

Article Title: Low‐Intensity Pulsed Ultrasound Promotes Osteogenesis in Porous Titanium Alloys Through miR ‐1187/ BMP4 Pathway

doi: 10.1096/fj.202403395RR

Figure Lengend Snippet: BMP4 positively regulates osteogenic differentiation in MC3T3‐E1 cells. (A) Level of BMP4 decreased after transfected with different siRNA‐BMP4, si‐BMP4‐785 was identified to have the highest transfection efficiency. (B) BMP4 transcript levels was determined by RT‐PCR after transfected with overexpression plasmids of BMP4. (C) qRT‐PCR analysis. Level of Col‐1 , Alp , Runx2 , and Ocn mRNA expression was significantly downregulated in BMP4 silencing. (D) qRT‐PCR analysis. Level of Col‐1 , Alp , Runx2 , and Ocn mRNA expression was significantly upregulated in BMP4 overexpression. (E) Western blotting. Expression of BMP4 protein and osteogenesis‐related proteins COL‐1, ALP, RUNX2 as well as statistical analysis after transfecting si‐BMP4. (F) Western blotting. Expression of BMP4 protein and osteogenesis‐related proteins COL‐1, ALP, RUNX2 as well as statistical analysis after transfecting pCDNA3.1‐BMP4. (G) ALP activity detection on day 7 in si‐BMP4 and pCDNA3.1‐BMP4 transfected MC3T3‐E1cells. (H) ALP staining on day 10 in si‐BMP4 and pCDNA3.1‐BMP4 transfected MC3T3‐E1cells (scale bar = 500 μm). (I) Alizarin red S staining and quantitative analysis of Alizarin Red S accumulation on day 21 in si‐BMP4 and pCDNA3.1‐BMP4 transfected MC3T3‐E1cells (scale bar = 200 μm/100 μm), the black arrow indicate the magnified area, the magnified pictures were marked within the square frame in the image. All values represent means ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: BMP4 protein levels in the supernatant were quantified using a mouse BMP‐4 ELISA kit (EK0316, Boster, China) following the manufacturer's protocol.

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Over Expression, Quantitative RT-PCR, Expressing, Western Blot, Activity Assay, Staining

Journal: The FASEB Journal

Article Title: Low‐Intensity Pulsed Ultrasound Promotes Osteogenesis in Porous Titanium Alloys Through miR ‐1187/ BMP4 Pathway

doi: 10.1096/fj.202403395RR

Figure Lengend Snippet: LIPUS promotes osteogenic differentiation of MC3T3‐E1 through inhibiting miR‐1187 and upregulating BMP4. (A) qRT‐PCR analysis. Effect of daily LIPUS stimulation on mRNA expression of Bmp4. (B) ELISA. Supernatant BMP4 secreted by cultured MC3T3‐E1 cells was determined by ELISA assay. (C) Western blotting. BMP4 level was analyzed by western blot after 7 days LIPUS treatment. (D) Expression of osteogenesis‐related mRNA was analyzed by qRT‐PCR after si‐BMP4 or si‐bmp4 + LIPUS treatment. (E) Expression of osteogenesis‐related protein was analyzed by western blot after si‐BMP4 or si‐BMP4 + LIPUS treatment. (F) ALP activity detection on day 7 in si‐BMP4 and si‐BMP4 + LIPUS‐treated MC3T3‐E1 cells. (G) Bmp4 and osteogenesis‐related genes Col‐1, Alp, Runx2, and Ocn expression in MC3T3‐E1 cells on Pti as indicated treatment by qRT‐PCR. (H) ALP activity in MC3T3‐E1 cells on Pti on day 7 as indicated treatment. (I) Expression of BMP4 and osteogenesis‐related proteins including COL‐1, ALP, RUNX2 in MC3T3‐E1 cells on Pti as indicated treatment by Western blot analysis. All values represent means ± SD ( n = 3). Expression of osteogenesis‐related protein was analyzed by western blot (E1) and quantitative analysis (E2) after si‐BMP4, LIPUS or si‐BMP4+LIPUS treated. Expression of BMP4 and osteogenesis‐ related proteins including COL‐ 1, ALP, RUNX2 in MC3T3‐ E1 cells on Pti as indicated treatment by Western blot analysis(I)andquantitative analysis (J). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: BMP4 protein levels in the supernatant were quantified using a mouse BMP‐4 ELISA kit (EK0316, Boster, China) following the manufacturer's protocol.

Techniques: Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot, Activity Assay

Journal: The FASEB Journal

Article Title: Low‐Intensity Pulsed Ultrasound Promotes Osteogenesis in Porous Titanium Alloys Through miR ‐1187/ BMP4 Pathway

doi: 10.1096/fj.202403395RR

Figure Lengend Snippet: LIPUS regulates bone formation in vivo. (A) Study plan of the vitro study. (B) qRT‐PCR analysis of mRNA expression of Bmp4 as well as other osteogenesis‐related gene in the de novo bone of different groups. B1: MRNA expression of Bmp4; B2, B3: Osteogenesis‐related gene including Col‐1, Alp, Runx2, and Ocn at 4 weeks and 8 weeks respectively. (C) Micro‐CT analysis. C1: 3D reconstruction of the bone defect area at week 4 and 8. More bone ingrowth is observed as time increases in each group. The white part is the scaffold and the red part is the bone tissue. C2: The POF values for the Pti at 4 and 8 weeks. The POF values differ significantly between the LIPUS and control group, between the si‐BMP4 and control group, as well as between the si‐BMP4 and si‐BMP4 + LIPUS group. (D) D1: The representative merged images of fluorescent double labeling of calcein(green color) and xylenol orange(orange color). The red square indicate the magnified area, the magnified pictures were marked within the white square frame in the image. (scale bar = 200 μm/100 μm). D2: The MAR of the four groups at 4 and 8 weeks. All values represent means ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: BMP4 protein levels in the supernatant were quantified using a mouse BMP‐4 ELISA kit (EK0316, Boster, China) following the manufacturer's protocol.

Techniques: In Vivo, Quantitative RT-PCR, Expressing, Micro-CT, Control, Labeling

Journal: The FASEB Journal

Article Title: Low‐Intensity Pulsed Ultrasound Promotes Osteogenesis in Porous Titanium Alloys Through miR ‐1187/ BMP4 Pathway

doi: 10.1096/fj.202403395RR

Figure Lengend Snippet: The schematic of LIPUS promotes osteogenesis of porous titanium alloys through inhibiting miR‐1187 and upregulating BMP4.

Article Snippet: BMP4 protein levels in the supernatant were quantified using a mouse BMP‐4 ELISA kit (EK0316, Boster, China) following the manufacturer's protocol.

Techniques: