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. Author manuscript; available in PMC: 2015 Jul 15.
Published in final edited form as: J Immunol. 2014 Jun 11;193(2):879–888. doi: 10.4049/jimmunol.1303396

Figure 5. Stress enhances TLR4-induced CXCL1 gene transcription.

Figure 5

A. BMDM were treated with DMSO or Tm for 6 hrs followed by LPS for the indicated times and total RNA was used to determine levels of CXCL1 primary transcripts by real-time PCR as described in Materials and Methods. Values presented are the fold induction relative to cultures treated with DMSO alone for 12 hrs and are the mean of duplicate determinations +/- ½ range. The schematic above the graph shows the position of primers amplifying CXCL1 transcripts containing both intronic and exonic sequence. B. BMDM transfected with control or RIPK1 siRNAs were treated with DMSO, Tm or Tg for 6 hrs followed by 6 hrs of LPS treatment. CXCL1 primary transcripts were determined as in A. C. BMDM were treated with DMSO or Tm for 6 hrs and LPS for 5 hrs prior to addition of actinomycin D. RNA was prepared at the indicated time and used to determine the remaining CXCL1 mRNA by northern hybridization. Autoradiographs were quantified as in Fig 1A and results are representative of 2 separate experiments. D. BMDM from TTP+/+ or TTP-/- mice were subjected to DMSO or Tm treatment for 6 hrs followed by LPS treatment for 6 hrs. CXCL1 mRNA was measured by real-time PCR using primers to detect mature mRNA and quantified as in A. E. BMDM were treated with DMSO or Tm for 6 hrs followed by LPS for the indicated times. Nuclear extracts were prepared and used to analyze NFκB p65 DNA binding activity as described in Materials and Methods. Results are presented as the mean of duplicate determinations +/- ½ range. F. BMDM were treated with DMSO or Tm for 6 hrs, then stimulated with LPS for indicated times. Nuclear extracts were prepared and used to determine levels of NFκB p65 by western blot. Results are representative of 3 separate experiments. G. RAW 264.7 cells transiently transfected with the indicated luciferase reporter plasmids were treated with DMSO or Tm for 6 hrs followed by LPS for 6 hrs. Total RNA was prepared and used to determine luciferase mRNA by northern hybridization as in Fig 1. Results are representative of 3 separate experiments.