immunofluorescence staining kit p0176/p0179  (Beyotime)

Journal: Research

Article Title: Visomitin Attenuates Pathological Bone Loss by Reprogramming Osteoclast Metabolism via the STAT3/LDHB Axis

doi: 10.34133/research.0784

Figure Lengend Snippet: Visomitin regulates osteoclastogenesis through the LDHB–lactate axis. (A) The heatmap depicting the gene expression profiles of metabolic pathways. (B) Representative immunoblots depicting the protein levels of LDHA and LDHB following Visomitin treatment ( n = 3). (C) Immunofluorescence staining of LDHB in BMMs subjected to osteoclast differentiation, with or without Visomitin treatment; scale bars, 20 μm. (D) Quantification of the relative LDHB MFI in panel (C) ( n = 6). (E) BMMs infected with either the vector or LDHB-overexpressing adenovirus were differentiated into osteoclasts in the presence or absence of Visomitin treatment. Representative images of TRAP staining were shown. Scale bars, 50 μm. (F) Quantification of TRAP + multinuclear cells per well in panel (E) ( n = 3). (G) The classification of metabolites detected through metabolomics. (H and I) The volcano plot and heatmap illustrating the metabolite affinity profiles derived from metabolomics. (J and K) Enrichment analysis of differential metabolites detected by metabolomics using SMPDB and KEGG databases. Data are mean ± SD; * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, not significant.

Article Snippet: The HE Staining Kit (C0105M, Beyotime, China), TRAP Staining Kit (AMK1005, Amizona Scientific, China), and Immunofluorescence Staining Kit (P0176/P0179, Beyotime, China) were used to stain the sections.

Techniques: Gene Expression, Western Blot, Immunofluorescence, Staining, Infection, Plasmid Preparation, Derivative Assay

Journal: Research

Article Title: Visomitin Attenuates Pathological Bone Loss by Reprogramming Osteoclast Metabolism via the STAT3/LDHB Axis

doi: 10.34133/research.0784

Figure Lengend Snippet: STAT3 functions as a direct target of Visomitin to modulate LDHB transcription. (A) The potential transcription factors (TFs) for LDHB were predicted using the KnockTF, ENCODE, and ChIP_Atlas databases. (B) The potential targets of Visomitin were predicted using the SuperPRED database. (C and D) The mRNA and protein expression levels of LDHB under Stattic treatment ( n = 3). (E) The thermal stability of FLAG-STAT3 under Visomitin treatment was detected using WB ( n = 3). (F) The stability of FLAG-STAT3 in the presence of protease following treatment with Visomitin (0, 75, 150, and 300 nmol/l) was detected using WB ( n = 3). (G) Three-dimensional image of molecular docking between Visomitin and STAT3. (H) Representative immunoblots for the indicated nuclear, phosphorylated, or total proteins following treatment with Visomitin (0, 75, 150, and 300 nm) ( n = 3). (I) Representative Immunofluorescence images of STAT3 in BMMs after treatment with RANKL or Visomitin (0, 75, 150, and 300 nm) as indicated. Scale bars, 20 μm. (J) Quantification of Pearson’s correlation coefficient between STAT3 and DAPI in panel (I) ( n = 3). (K) STAT3 ChIP assay of LDHB promoter region. (L) Quantification of the binding affinity between STAT3 and the LDHB promoter ( n = 3). Data are mean ± SD; * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, not significant.

Article Snippet: The HE Staining Kit (C0105M, Beyotime, China), TRAP Staining Kit (AMK1005, Amizona Scientific, China), and Immunofluorescence Staining Kit (P0176/P0179, Beyotime, China) were used to stain the sections.

Techniques: Expressing, Western Blot, Immunofluorescence, Binding Assay