Journal: Advanced Science

Article Title: Burn‐Induced Gut Microbiota Dysbiosis Aggravates Skeletal Muscle Atrophy by Tryptophan‐Kynurenine Mediated AHR Pathway Activation

doi: 10.1002/advs.202409296

Figure Lengend Snippet: Kyn induces L6 myotube proteolysis through the activation of the AHR pathway. A) L6 myotubes were treated with Kyn (50, 100, or 150 µmol) or vehicle for 24 h. Myotube morphology was examined via H&E staining (scale bar, 50 µm). B) Effects of Kyn on the diameter and length of L6 myotubes ( n = 3 per group). C) Changes in SLC38A2 and AHR protein expression in L6 myotubes treated with Kyn (50, 100, or 150 µmol) or vehicle for 24 h ( n = 3 per group). D) L6 myotubes pre‐treated with SLC38A2 siRNA (100 nmol) and incubated with Kyn (100 µmol). H&E staining was used to examine the myotube morphology and effects of different treatments on the diameter and length of L6 myotubes (scale bar, 50 µm. n = 3 per group). E) Bar graph showing RT‐qPCR quantification of AHR gene expression levels in L6 myotubes transfected with SCL38A2 siRNAs or vehicle ( n = 3 per group). F) L6 myotubes were pretreated with DMSO or Kyn supplementation. The expression of AHR, HSP90, Cul4B, and GAPDH was determined by western blot analysis (input). Immunoprecipitation was performed with AHR or IgG antibody. HSP90, Cul4B, HSP90‐bound AHR, and Cul4B‐bound AHR were determined by western blot analysis (IP; n = 3). G) L6 myotubes pre‐treated with AHR siRNA (100 nmol) and incubated with Kyn (100 µmol). H&E staining was used to examine the myotube morphology and effects of different treatments on the diameter and length of L6 myotubes (scale bar, 50 µm. n = 3 per group). H,I) Changes in CYP1A1, CYP1A2, CYP1B1, AHRR, MAFbx, and MuRF‐1 protein expression in L6 myotubes transfected with AHR siRNAs or vehicle ( n = 3 per group). All quantitative data were analyzed using one‐way ANOVA and are shown as mean ± SEM. * p ≤ 0.05; ** p ≤ 0.01; and *** p ≤ 0.001.

Article Snippet: Approximately 100 µL of protein lysates were incubated with 20 µL of Protein A/G Agarose (Millipore, IP05) and the appropriate antibodies (HSP90 (Proteintech, 13171‐1‐AP), Cul4B (Proteintech, 12916‐1‐AP), AHR (Proteintech, 28727‐1‐AP)) at 4 °C overnight with gentle shaking.

Techniques: Activation Assay, Staining, Expressing, Incubation, Quantitative RT-PCR, Gene Expression, Transfection, Western Blot, Immunoprecipitation

Journal: iScience

Article Title: Increased CSN5 expression enhances the sensitivity to lenalidomide in multiple myeloma cells

doi: 10.1016/j.isci.2024.111399

Figure Lengend Snippet: E3 ubiquitin ligase is responsible for CSN5 ubiquitination (A) Western blots representing CSN5, CUL1, CUL2, CUL3, CUL4A, CUL4B, and CUL5 expression levels in MM.1S and RPMI8226 treated with DMSO or 10 μM MLN4924 for 12 h. This experiment was performed in triplicate. β-Actin was used as a loading control. Band densities were quantified using iBright Analysis Software. Data are represented as mean ± standard deviation. Student's t test. (B) Proximity proteomics was performed using RPMI8226 cells overexpressing APEX2-CSN5 or TurboID-CSN5. We identified proteins that were highly enriched (2-fold or more, p < 0.01) in biotin-tyramide- or biotin-treated samples as proximal proteins of CSN5. We selected ubiquitin-related proteins from the enriched proteins and performed an STRING analysis ( https://string-db.org/ ). A protein interaction network generated using STRING is shown. (C) Comparison of the levels of DDB1 and DCN1 expression quantified in proteomics analysis of LEN-sensitive and resistant cell groups among the 15 MM cell lines. Student's t test. (D) Comparison of the protein levels between CSN5 and DDB1 quantified in proteomics analysis of 15 MM cell lines.

Article Snippet: CUL4B Rabbit pAb , Proteintech , Cat#12916-1-AP; RRID: AB_2086699.

Techniques: Western Blot, Expressing, Control, Software, Standard Deviation, Generated, Comparison

Journal: iScience

Article Title: Increased CSN5 expression enhances the sensitivity to lenalidomide in multiple myeloma cells

doi: 10.1016/j.isci.2024.111399

Figure Lengend Snippet:

Article Snippet: CUL4B Rabbit pAb , Proteintech , Cat#12916-1-AP; RRID: AB_2086699.

Techniques: Recombinant, Sequencing, Modification, Bicinchoninic Acid Protein Assay, Mass Spectrometry, Western Blot, Software