Journal: Scientific Reports

Article Title: Rhein, a novel Histone Deacetylase (HDAC) inhibitor with antifibrotic potency in human myocardial fibrosis

doi: 10.1038/s41598-020-61886-3

Figure Lengend Snippet: Effect of Rhein on HDAC activity and G2/M cell cycle phase regulation, p53 and p21 abundance and proliferation markers. ( A ) Representative blots and quantification showing Rhein-mediated increase of p53 and p21 abundance (n = 4). ( B ) Representative blots and quantification showing Rhein-driven p53 stabilization by increased HDAC inhibition-mediated acetylation at Lys382 (n = 4). 1 mM sodium butyrate (SB) was used as reference HDAC inhibitor. ( C ) Representative blots and quantification showing Rhein-mediated increase of p53 abundance (n = 4) under conditions described in ( B ). ( D ) Representative blots and quantification showing Rhein-mediated increase of p21 abundance (n = 4) under conditions described in ( B ). Antibodies in ( C , D ) were probed to the same membrane. ( E ) Enzymatic HDAC activity is inhibited in the presence of Rhein. Graph showing HDAC activity after 30 min treatment of normal cell lysates with Rhein or SB (n = 4). All data are presented as mean ± SD. One-way-ANOVA with post-hoc Sidak’s multiple comparison, *p < 0.05, **p < 0.01, ***p < 0.001, as indicated.

Article Snippet: Cell lysates were prepared using 1% NP‐40 (in 1x PBS) and 5 μg of protein was incubated with HDAC class I/II substrate (HDAC-Glo I/II Assay and Screening System, Promega, Madison, IA, USA) for 45 min (RT), before luminescence was determined.

Techniques: Activity Assay, Inhibition, Membrane, Comparison

Journal: Scientific Reports

Article Title: Rhein, a novel Histone Deacetylase (HDAC) inhibitor with antifibrotic potency in human myocardial fibrosis

doi: 10.1038/s41598-020-61886-3

Figure Lengend Snippet: Rhein functionally inhibits TGFβ1-stimulated FMT. ( A ) Representative Western Blot and quantification of αSMA relative protein abundance (n = 4). ( B ) Rhein inhibition of TGFβ1/SMAD signaling. Representative blot and quantification of phospho(Ser465/467)-SMAD2 abundance in Rhein and TGFβ1 treated cells. ( C ) Expression analysis of SMAD7 showing TGFβ1-mediated increase of transcription and absence of Rhein-mediated effects (n = 4). ( D ) Rhein increases SMAD7 abundance independently from TGFβ1. Representative blot and quantification showing increased basal SMAD7 abundance after Rhein treatment and TGFβ1-dependent SMAD7 expression (n = 4). ( E ) Effect of HDAC inhibition on SMAD7 stabilization. Representative blot and quantification showing increased protein abundance after Rhein and SB treatment (N = 4). ( F ) FPCL contraction assay. Quantitative analysis on the contraction of experimental FPCLs (n = 4). Representative overhead pictures of FPCLs before (d0) and after treatment (d0) with TGFβ1 alone or in combination with Rhein. Dashed line indicates baseline (start point at d0). All data are presented as mean ± SD. One-way-ANOVA with post-hoc Sidak’s multiple comparison, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as indicated.

Article Snippet: Cell lysates were prepared using 1% NP‐40 (in 1x PBS) and 5 μg of protein was incubated with HDAC class I/II substrate (HDAC-Glo I/II Assay and Screening System, Promega, Madison, IA, USA) for 45 min (RT), before luminescence was determined.

Techniques: Western Blot, Quantitative Proteomics, Inhibition, Expressing, Contraction Assay, Comparison