ldn 193189  (MedChemExpress)

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: The BMP receptor inhibitor (LDN193189, MCE, HY-12071) was dissolved in sterile ddH 2 O to prepare a 1 mM 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 ) Changes in the peaks of all transcription factors identified by CUT&Tag following enalapril treatment, with red dots indicating transcription factors with increased peak intensity (upregulated TFs). ( B ) Integrative Genomics Viewer (IGV) showing pSmad1/5/9 signals near the ID1 region between the control group (Ctrl) and the enalapril treatment group (Enalapril). The vertical yellow boxes indicate regions with increased signal intensity. ( C ) Western blot analysis showing the changes in the protein levels of ID1 and ID2 following enalapril treatment. ( D ) Changes in the RNA levels of ID1 and ID2 following enalapril treatment. ( E ) Western blot analysis showing the protein levels of ID1 and ID2 following treatment with a BMP receptor inhibitor (LDN193189, LDN). ( F, I ) Western blot showing the changes in the protein levels of p16 and p21 following the knockdown of ID1 or ID2 ( F ) or the inhibition of ID1 ( I ). ( G ) RNA expression of SASP factors after ID knockdown, with pink representing ID1 knockdown and orange representing ID2 knockdown. ( H ) Normalized average RNA expression levels of selected SASP factors and cell cycle arrest factors after enalapril treatment and ID knockdown. Positive values indicate upregulation, while negative values indicate downregulation. The values represent the expression levels relative to those of the Ctrl. ( J ) Western blot analysis showing pSmad1/5/9 levels following ID1 and ID2 knockdown. ( K ) Senescence-associated β-galactosidase (SA-β-Gal) staining (left) and SA-β-Gal ratio quantification (right) in the control group (Ctrl) and ID -knockdown groups (ID1_KD, ID2_KD) with or without enalapril treatment. Scale bars, 200 μm. Enlarged scale bars, 100 μm. ( L ) Ki67 immunofluorescence intensity in the control group (Ctrl) and ID knockdown groups (ID1_KD, ID2_KD), with or without enalapril treatment. A two-tailed t-test was employed, ns indicates no significant difference, * p <0.05, ** p <0.01, *** p <0.001. Figure 3—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 3—source data 2. Original files for western blot analysis displayed in .

Article Snippet: The BMP receptor inhibitor (LDN193189, MCE, HY-12071) was dissolved in sterile ddH 2 O to prepare a 1 mM stock solution.

Techniques: Control, Western Blot, Knockdown, Inhibition, RNA Expression, Expressing, 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 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: In the LDN treatment group, mice received intraperitoneal injections of the BMP receptor inhibitor LDN193189 (3 mg/kg, MCE, HY-12071) every other day.

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 showing the protein levels after the addition of LDN193189. ( B ) Senescence-associated β-galactosidase (SA-β-gal) staining (left) after adding LDN193189 and its corresponding staining proportion statistical chart (right). Scale bars, 200 μm. Enlarged scale bars, 100 μm. ( C ) Immunofluorescence staining for Ki67 (left) after adding LDN193189, along with the corresponding intensity statistical chart (right). Scale bars, 80 μm. Enlarged scale bars, 40 μm. ( D ) Western blot showing the protein levels following BMPR1A knockdown. ( E ) Relative RNA levels of BMPR1A following BMPR1A knockdown. ( F ) SA-β-gal staining (left) and its corresponding staining proportion statistical chart (right) in the control group (Ctrl) and BMPR1A -knockdown groups (BMPR1A_KD) with enalapril treatment. Scale bars, 200 μm. Enlarged scale bars, 100 μm. ( G ) Ki67 immunofluorescence (left) and its corresponding staining proportion statistical chart (right) in the control group (Ctrl) and BMPR1A -knockdown groups (BMPR1A_KD) with enalapril treatment. Scale bars, 80 μm. Enlarged scale bars, 40 μm. A two-tailed t-test was employed, ns indicates no significant difference, ** p <0.01, *** p <0.001, **** p <0.0001. Figure 2—figure supplement 3—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 2—figure supplement 3—source data 2. Original files for western blot analysis displayed in .

Article Snippet: In the LDN treatment group, mice received intraperitoneal injections of the BMP receptor inhibitor LDN193189 (3 mg/kg, MCE, HY-12071) every other day.

Techniques: Western Blot, Staining, Immunofluorescence, Knockdown, Control, 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 ) Changes in the peaks of all transcription factors identified by CUT&Tag following enalapril treatment, with red dots indicating transcription factors with increased peak intensity (upregulated TFs). ( B ) Integrative Genomics Viewer (IGV) showing pSmad1/5/9 signals near the ID1 region between the control group (Ctrl) and the enalapril treatment group (Enalapril). The vertical yellow boxes indicate regions with increased signal intensity. ( C ) Western blot analysis showing the changes in the protein levels of ID1 and ID2 following enalapril treatment. ( D ) Changes in the RNA levels of ID1 and ID2 following enalapril treatment. ( E ) Western blot analysis showing the protein levels of ID1 and ID2 following treatment with a BMP receptor inhibitor (LDN193189, LDN). ( F, I ) Western blot showing the changes in the protein levels of p16 and p21 following the knockdown of ID1 or ID2 ( F ) or the inhibition of ID1 ( I ). ( G ) RNA expression of SASP factors after ID knockdown, with pink representing ID1 knockdown and orange representing ID2 knockdown. ( H ) Normalized average RNA expression levels of selected SASP factors and cell cycle arrest factors after enalapril treatment and ID knockdown. Positive values indicate upregulation, while negative values indicate downregulation. The values represent the expression levels relative to those of the Ctrl. ( J ) Western blot analysis showing pSmad1/5/9 levels following ID1 and ID2 knockdown. ( K ) Senescence-associated β-galactosidase (SA-β-Gal) staining (left) and SA-β-Gal ratio quantification (right) in the control group (Ctrl) and ID -knockdown groups (ID1_KD, ID2_KD) with or without enalapril treatment. Scale bars, 200 μm. Enlarged scale bars, 100 μm. ( L ) Ki67 immunofluorescence intensity in the control group (Ctrl) and ID knockdown groups (ID1_KD, ID2_KD), with or without enalapril treatment. A two-tailed t-test was employed, ns indicates no significant difference, * p <0.05, ** p <0.01, *** p <0.001. Figure 3—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 3—source data 2. Original files for western blot analysis displayed in .

Article Snippet: In the LDN treatment group, mice received intraperitoneal injections of the BMP receptor inhibitor LDN193189 (3 mg/kg, MCE, HY-12071) every other day.

Techniques: Control, Western Blot, Knockdown, Inhibition, RNA Expression, Expressing, 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 ) Bar plot showing the enriched Gene Ontology (GO) terms and pathways associated with the CUT&Tag-upregulated peaks (left) and the upregulated RNA-seq genes (right) following enalapril treatment. ( B ) Heatmap showing the changes in the RNA expression of antioxidative genes. ( C ) Profile plot showing the increase in the pSmad1/5/9 binding signal of antioxidative genes in the transcription start site (TSS) region following enalapril treatment. ( D ) Western blot analysis showing the protein levels of representative antioxidative genes after enalapril treatment. ( E ) Relative RNA levels of TXN , GPX4 , and PRDX5 during cellular senescence. The blue bars represent young cells, and the red bars represent senescent cells. ( F, H ) Integrative Genomics Viewer (IGV) showing pSmad1/5/9 signals near the PRDX5 ( F ) and TXN ( H ) regions between the control group (Ctrl) and the enalapril treatment group (Enalapril). The vertical yellow boxes indicate regions with increased signal intensity. ( G ) ChIP-qPCR results showing pSmad1/5/9 levels at many peaks following enalapril or combination treatment with enalapril and LDN193189. The y-axis represents the normalized pSmad1/5/9 signals relative to 10% input. pSmad1/5/9 enrichment for HPRT1 and HBB served as a negative control, and pSmad1/5/9 enrichment for ID1 and ID2 served as positive controls, as previously described. A two-tailed t-test was employed, * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001. ( I ) Detection of reactive oxygen species (ROS) fluorescence levels via the DCFH-DA probe in young cells, senescent cells (Ctrl), cells treated with enalapril, and cells treated with a combination of enalapril and LDN193189. Scale bars, 200 μm. Figure 4—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 4—source data 2. Original files for western blot analysis displayed in .

Article Snippet: In the LDN treatment group, mice received intraperitoneal injections of the BMP receptor inhibitor LDN193189 (3 mg/kg, MCE, HY-12071) every other day.

Techniques: RNA Sequencing, RNA Expression, Binding Assay, Western Blot, Control, ChIP-qPCR, Negative Control, Two Tailed Test, Fluorescence