Tryptophanyl-Transfer RNA Synthetase Is Involved in a Negative Feedback Loop Mitigating Interferon-γ-Induced Gene Expression

Figures

• 

  Figure 1. Label-free quantitative proteomics workflow to identify modulation in the proteome of A375 cells following interferon-γ treatment. (A) A375 cells treated with 100 ng/mL of IFN-γ or treated with the vehicle solution (control) (n = 4 for each condition) for 48 h were lysed, and proteins were extracted, precipitated and digested with trypsin. Peptides were run in data-dependent acquisition mode on a Q Exactive HF-X mass spectrometer. The resulting raw data were analyzed with MaxQuant. (B) Volcano plot representing the log2 ratio (+IFN-γ/control) for each protein quantified and the corresponding −Log10 p-value. The blue, red and gray dots represent the proteins more abundant in control cells, more abundant in IFN-γ-treated cells or not differentially expressed, respectively. The grey dashed lines represent the fold change and p-value thresholds. Some of the names of the most differentially regulated proteins are displayed on the volcano plot. (C,D) Results from Gene Ontology pathway analysis for the proteins more abundant in IFN-γ-treated cells (C) and more abundant in control cells (D).

• 

  Figure 2. WARS is highly up-regulated in A375 cells treated with IFN-γ. (A) Histogram displaying the measured protein intensity for WARS in control or IFN-γ-treated cells (errors bars represent standard deviations). (B) Table showing the top-20 most abundant proteins (based on their iBAQ values) in A375 cells treated with IFN-γ and their corresponding ranking in control A375 cells.

• 

  Figure 3. WARS is highly expressed in skin cutaneous melanoma and patients with high expression of WARS display better survival. (A) WARS expression in different cancers and corresponding normal tissues (based on TCGA and Gtex data). The abbreviations are detailed for each tumor type in Supplementary Figure S5A. SKCM is highlighted in red. (B) Expression levels of WARS in SKCM tumor versus normal tissue. The asterisk represents a significant difference at a p-value < 0.05. (C) Survival analysis of patients expressing high and low levels of WARS.

• 

  Figure 4. WARS correlates with genes involved in the immune response. (A) STRING network of the top-50 genes (excluding WARS) correlating with WARS from an analysis using GEPIA [40] (TCGA and Gtex data). (B) Gene expression correlation between WARS and STAT1. The R corresponds to the Spearman ranked correlation coefficient.

• 

  Figure 5. The interactome of WARS reveals an interaction with the transcription factor STAT1. (A) A375 cells were transfected with pLKO or a plasmid coding for WARS tagged with a Flag and treated with 100 ng/mL of IFN-γ or treated with the vehicle solution (control) (n = for each condition) for 48 h. The cells were lysed and WARS Flag was immunoprecipitated using an anti-Flag antibody. Beads were incubated with Flag peptides, eluates were digested with trypsin and peptides were run in data-dependent acquisition mode on a Q Exactive HF-X mass spectrometer. The resulting raw data were analyzed with MaxQuant. (B) STRING network of the proteins identified as interacting with WARS. The different colors represent proteins from different biological processes. (C) Venn diagram displaying the overlap between the genes correlating with WARS from the GEPIA analysis, the proteins up-regulated by IFN-γ in the whole proteomics experiment and WARS-associated proteins from the interactome. (D) Immunoprecipitation of endogenous WARS in IFN-γ-treated A375 cells followed by WARS and STAT1 Western blots. (E) Immunoprecipitation of endogenous WARS and STAT1 in IFN-γ-treated A375 cells and WARS and STAT1…

• 

  Figure 6. Knocking down WARS further increases the expression of a set of interferon-γ-induced genes. (A,B) A375 cells were transfected with pLKO or a plasmid coding for a shRNA-targeting WARS and treated with 100 ng/mL of IFN-γ or treated with the vehicle solution (control) for 4 or 48 h. RNAs were extracted and a reverse transcription-quantitative polymerase chain reaction was performed for several genes known to be induced by IFN-γ. Histograms showing the changes in expression of these genes (A), and IDO1 (B), are displayed (errors bars represent standard deviations). The asterisks represent a significant difference at a p-value < 0.05. (C) Time course experiment of A375 cells transfected with pLKO or a plasmid coding for a shRNA-targeting WARS and treated with 100 ng/mL of IFN-γ or treated with the vehicle solution. Proteins were extracted and a Western blot analysis was performed to monitor Phospho-STAT1, STAT1, WARS, IDO1 and GAPDH (loading control). (D) The same as in (C) but with a dose–response (0 to 100 ng/mL of IFN-γ for 48 h) performed instead of a time course.