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. Author manuscript; available in PMC: 2011 Sep 8.
Published in final edited form as: Cell Metab. 2010 Sep 8;12(3):260–272. doi: 10.1016/j.cmet.2010.08.004

Figure 4. Inhibition of Transcription Does Not Alter Thermotolerance.

Figure 4

(A) Effects of inhibition of transcription by α-amanitin pretreatment prior to inducing HS. Increasing concentrations of α-amanitin blocked HS induced GFP induction. Plotted is the Relative Fluorescence (R.F) versus time during recovery at 25°C. Error bars ±2× S.E.M. (B) Average expression levels of hsp-12.6, hsp-16.1 and hsp-16.2 following a heat shock (HS, 2h at 35°C), with and without a 1h pretreatment with α-amanitin (α-aman). Relative expression levels were derived from Calibrated Normalized Relative Quantities using the geometric mean of 2 ‘house keeping’ genes, gpd-1 and gpd-4 and are plotted as arbitrary units (A.U) ±S.E.M. Data from n=11 individuals. For all genes the heat shock (+, HS) treatment significantly increased relative mRNA levels (p<0.001) compared to controls (-). α-amanitin pretreatment significant reduced stress induced expression for all genes (p<0.001). (C) Immunoblot using anti-HSP-16.2 antibody of untreated (-) and heat shocked (+, HS) (33°C for 2h with a 12h recovery, HS) wild type populations with and without a pretreatment of 100ug/ml α-amanitin (as previously described). (D) Inhibition of transcription does not affect intrinsic thermotolerance in wild type, daf-2(e1370) or daf-16(m26) mutants. Kaplan-Meier survival curves show daf-2(e1370) mutants have significantly increased thermotolerance compared to wild type (p<0.0001) and daf-16(m26) mutants (p<0.0001), respectively. Pretreatment with 100 mg/ml α-amanitin (α-aman) did not alter thermotolerance of any genotype. Median survival were 480 min (wild type, n=50), 480 min (wild type + α-amanitin, n=50), 590 min (daf-2(e1370), n=51), 590 min (daf-2(e1370) + α-amanitin, n=52), 480 min (daf-16(m26), n=52), 590 min (daf-16(m26) + α-amanitin, n=50). (E) Inhibition of transcription significantly reduces resistance to the oxidative stressor juglone. Median survival of wild type was significantly reduced following pretreatment with 100 mg/ml α-amanitin (α-aman), p<0.0001. Median survival were 195 min (wild type, n=46) versus 135 min (wild type + α-amanitin, n=45). Median survival of daf-16(mu86) (185 min, n=47) was also significantly reduced compared to wild type, p<0.05.