Journal: Molecular & Cellular Proteomics : MCP

Article Title: Application of Mass Spectrometry Profiling to Establish Brusatol as an Inhibitor of Global Protein Synthesis

doi: 10.1074/mcp.M115.055509

Figure Lengend Snippet: Effects of brusatol on total protein synthesis. (A) Scatterplot showing the melting temperatures of all proteins detected in both lysates and intact cells under the DMSO condition. The solid line shows the best fit with an R2 concordance of 0.43, while the red dashed line indicates a hypothetical unity response, to demonstrate that most proteins are more thermostable when heated in lysis buffer compared with intact cells. (B) The ratios of protein abundance of 3499 proteins at the 38 °C temperature comparing brusatol-treated to DMSO-treated A549 cells. Protein abundance was quantified from summed TMT reporter ion intensities of peptide-spectrum-matchings. The scatter plot shows data from two replicate measurements indicating that the majority of proteins show reduced abundance following brusatol treatment. cystatin C (red), IGFBP4 (blue), PAF (purple), and NRCAM (brown) are indicated. (C) MS abundance data for cystatin C, IGFBP4, and PAF over the temperature ranges used are shown. X axis: temperature points to heat intact cells in the CETSA assay. Y axis: protein abundance at each temperature point normalized to the average of DMSO and brusatol-treated protein abundance at 38 °C. Green: DMSO treatment; red: brusatol treatment. (D) The same lysates used for MS analysis were used for Western blotting using antibodies specific for cystatin C, IGFBP4, and PAF.

Article Snippet: RNA was harvested via RNEasy Mini kits (Qiagen), cDNA synthesized with High Capacity cDNA reverse transcription kit (Life Technologies, Foster City, CA), and Taqman assays run using the following primer/probe sets (Life Technologies): Nrf2 (Hs00975961_g1), cystatin C (Hs00264679_m1), PAF1 (Hs00219496_m1), NRCAM (Hs01031598_m1), GCN1L (Hs00412445_m1), RPL11 (Hs00831112_s1), and RL22 (Hs01865331_s1).

Techniques: Lysis, Western Blot

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Application of Mass Spectrometry Profiling to Establish Brusatol as an Inhibitor of Global Protein Synthesis

doi: 10.1074/mcp.M115.055509

Figure Lengend Snippet: Brusatol-induced effects on protein abundance are independent of changes in mRNA. (A) A549 cells were treated with 0.1% DMSO, 50 nm or 500 nm brusatol or 5 or 50 μg/ml cycloheximide for the indicated times before harvesting the cells. Lysates were separated by SDS-PAGE and Western blotted using the indicated antibodies. (B) mRNA levels of Nrf2, cystatin C, PAF, NRCAM, GCN1L, RL22, and RL11 were analyzed at the 30 min and 4 h time points of brusatol and cycloheximide treated cells compared with no treatment control. NTC = nontargeted control siRNA. Error bars show standard deviation (n = 2). (C) Protein abundance change quantified after 4-h brusatol treatment or 6-h cycloheximide treatment (data from (24)). To filter out noise and look at more profound changes, a minimum protein level change of 0.6 (2σ) was required for the brusatol dataset. Proteins showing greater than 3σ changes were highlighted. Pearson correlation was calculated. (D) A549 cells were treated with 0.1% DMSO, 50 nm or 500 nm brusatol or 5 or 50 μg/ml cycloheximide for the indicated times before harvesting the cells. Lysates were separated by SDS-PAGE and Western blotted using the indicated antibodies.

Article Snippet: RNA was harvested via RNEasy Mini kits (Qiagen), cDNA synthesized with High Capacity cDNA reverse transcription kit (Life Technologies, Foster City, CA), and Taqman assays run using the following primer/probe sets (Life Technologies): Nrf2 (Hs00975961_g1), cystatin C (Hs00264679_m1), PAF1 (Hs00219496_m1), NRCAM (Hs01031598_m1), GCN1L (Hs00412445_m1), RPL11 (Hs00831112_s1), and RL22 (Hs01865331_s1).

Techniques: SDS Page, Western Blot, Standard Deviation