NT219 : hsp-70

"We also tested whether NT219 affects the induction of the HSF-1 target gene, hsp-70 by treating worms that express GFP under the control of the hsp-70 promoter (hsp-70p::GFP) with the vehicle (Ve), 150, 300 or 600 lM NT219, exposing them to heat (33C, 3 h) and visualizing them. An increase in GFP signal was observed in worms after treatment with 600 lM NT219, but not after treatment with lower concentrations (Fig. 2C, 600 lM NT219 arrowheads)."

NT219 : hsp-16.2

"worms that express the green fluorescent protein (GFP) under the regulation of the hsp-16.2 promoter (strain CL2070). CL2070 worms were grown on control bacteria harboring the empty vector (EV). The animals were then divided to identical groups and treated at days 1 and 2 of adulthood for 3 h with the chemical vehicle of NT219 [Fig. 2A, lane 1 (Ve)], or with 150, 300, 600 or 900 lM NT219 (lanes 3-6, respectively). An identical worm group was grown from hatching on daf-2 RNAi bacteria (lane 2). At day 2 of adulthood, all worm groups were exposed to 33C for 3 h and harvested, and GFP levels were analyzed by WB. We found that NT219 increased the hsp-16.2 induction levels in heat-stressed animals in a dose-dependent manner. While treatment with 150 lM NT219 had no effect on the level of GFP, 300 lM had a moderate effect and 600 lM NT219 remarkably increased the level of GFP to be similar to that of daf-2 RNAi-treated worms. Treatment with 900 lM NT219 had lower effect than that of 600 lM of the drug. Three independent repeats confirmed these observations (Fig. 2B and S2)."

hsp-16.2 : juglone : juglone, a generator of reactive oxyger species (ROS)

Juglone in all concentrations tested increased activation of the hsp- 16.2 promoter with a maximum response of GFP- fluorescence at 40 and 100 lM of juglone.

daf-2 : hsp-16.2

"worms that express the green fluorescent protein (GFP) under the regulation of the hsp-16.2 promoter (strain CL2070). CL2070 worms were grown on control bacteria harboring the empty vector (EV). The animals were then divided to identical groups and treated at days 1 and 2 of adulthood for 3 h with the chemical vehicle of NT219 [Fig. 2A, lane 1 (Ve)], or with 150, 300, 600 or 900 lM NT219 (lanes 3-6, respectively). An identical worm group was grown from hatching on daf-2 RNAi bacteria (lane 2). At day 2 of adulthood, all worm groups were exposed to 33C for 3 h and harvested, and GFP levels were analyzed by WB. We found that NT219 increased the hsp-16.2 induction levels in heat-stressed animals in a dose-dependent manner. While treatment with 150 lM NT219 had no effect on the level of GFP, 300 lM had a moderate effect and 600 lM NT219 remarkably increased the level of GFP to be similar to that of daf-2 RNAi-treated worms. Treatment with 900 lM NT219 had lower effect than that of 600 lM of the drug. Three independent repeats confirmed these observations (Fig. 2B and S2)."

daf-16 : daf-2 : gst-10 : gst-4 : hsf-1 : skn-1 : sod-3

"Using quantitative real-time PCR (qPCR) and specific primer sets, we tested the effect of NT219 on the expression of DAF-16, HSF-1, and SKN- 1 target genes in temperature sensitive sterile worms (strain CF512)... The worms were grown on EV bacteria and treated for 3 h with 600 lM NT219 at days 1 and 2 of adulthood. An identical group of worms was treated with the vehicle (Ve), and a third group was grown on daf-2 RNAi bacteria... Three independent experiments showed that NT219 (Fig. 2D-F, spotted bars) elevates the expression levels of all genes compared with the levels seen in control worms (black bars). Yet, while DAF-16 (D) and SKN-1 (F) target genes exhibited average increase levels of 19-37%, the expression level of hsp-70 was elevated by approximately 170% (E)."

NT219 : daf-16 : gst-10 : gst-4 : hsf-1 : skn-1 : sod-3

"Using quantitative real-time PCR (qPCR) and specific primer sets, we tested the effect of NT219 on the expression of DAF-16, HSF-1, and SKN- 1 target genes in temperature sensitive sterile worms (strain CF512)... The worms were grown on EV bacteria and treated for 3 h with 600 lM NT219 at days 1 and 2 of adulthood. An identical group of worms was treated with the vehicle (Ve), and a third group was grown on daf-2 RNAi bacteria... Three independent experiments showed that NT219 (Fig. 2D-F, spotted bars) elevates the expression levels of all genes compared with the levels seen in control worms (black bars). Yet, while DAF-16 (D) and SKN-1 (F) target genes exhibited average increase levels of 19-37%, the expression level of hsp-70 was elevated by approximately 170% (E)."