Figure 3
Figure 3. HDACi, (MS-275 and vorinostat), target the proteasome. (A) MS-275 decreased mRNA expression of catalytic β5, β2, β1 proteasomal subunits. Real-time PCR analyzed expression levels of catalytic β subunits of the proteasome in Jurkat cells exposed to 5 μM MS-275 for 24 hours. Shown is the relative expression, which was calculated by first normalizing all samples to corresponding actin expression, than to control (cells treated with DMSO). *P < .05 compared with 0 hours. (B) Vorinostat decreases mRNA levels of β5, β2, β1 proteasomal subunits. Jurkat cells were treated with 500 nM vorinostat for 18 hours, and RNA was collected to analyze expression of β subunits by real-time PCR. Shown is the relative expression. *P < .01 compared with 0 hours. (C) MS-275 reduces β5, β2, β1 subunit protein expression. Jurkat cells were exposed to 5 μM MS-275, and protein lysates were collected at indicated time points. Western blot analysis and specific antibodies determined expression of β subunits and actin. Numerical values under each panel are the densitometry ratios of β subunit to actin and are normalized to 0 hours. (D) MS-275 inhibits the chymotrypsin-like and caspase-like activity of the proteasome. Jurkat cells were exposed to 5 μM MS-275 for 24 hours. The chymotrypsin-like and caspase-like activity was determined by measuring the fluorescence (amc) intensity released by the cleavage of fluorogenic substrate suc-LLVY-amc or z-LLE-amc, respectively. Proteasome activity was evaluated in RFU. Treatment with proteasome inhibitor, 10 nM NPI-0052, for 1 hour was used as a positive control. Statistical differences between 0 and 24 hours with MS-275 were determined by Student t test, *P = .002 for chymotrypsin-like and P = .004 for caspase-like. (E) Vorinostat diminishes proteasomal chymotrypsin-like and caspase-like activities. After an 18-hour exposure to 500 nM vorinostat, chymotrypsin-like and caspase-like activities in Jurkat cells were measured using fluorogenic substrates as previously described. Student t test determined statistical differences between 0 and 18 hours. (F) HDACi do not inhibit the rate-limiting activity of the proteasome in isolated proteasomes. Purified 20S proteasomes were combined with diluent, 5 μM MS-275, 500 nM vorinostat, or 10 nM NPI-0052 for 30 minutes at room temperature. The chymotrypsin-like activity was analyzed using fluorogenic substrate suc-LLVY–amc and activity was evaluated in RFU. NPI-0052 was used as a positive control.

HDACi, (MS-275 and vorinostat), target the proteasome. (A) MS-275 decreased mRNA expression of catalytic β5, β2, β1 proteasomal subunits. Real-time PCR analyzed expression levels of catalytic β subunits of the proteasome in Jurkat cells exposed to 5 μM MS-275 for 24 hours. Shown is the relative expression, which was calculated by first normalizing all samples to corresponding actin expression, than to control (cells treated with DMSO). *P < .05 compared with 0 hours. (B) Vorinostat decreases mRNA levels of β5, β2, β1 proteasomal subunits. Jurkat cells were treated with 500 nM vorinostat for 18 hours, and RNA was collected to analyze expression of β subunits by real-time PCR. Shown is the relative expression. *P < .01 compared with 0 hours. (C) MS-275 reduces β5, β2, β1 subunit protein expression. Jurkat cells were exposed to 5 μM MS-275, and protein lysates were collected at indicated time points. Western blot analysis and specific antibodies determined expression of β subunits and actin. Numerical values under each panel are the densitometry ratios of β subunit to actin and are normalized to 0 hours. (D) MS-275 inhibits the chymotrypsin-like and caspase-like activity of the proteasome. Jurkat cells were exposed to 5 μM MS-275 for 24 hours. The chymotrypsin-like and caspase-like activity was determined by measuring the fluorescence (amc) intensity released by the cleavage of fluorogenic substrate suc-LLVY-amc or z-LLE-amc, respectively. Proteasome activity was evaluated in RFU. Treatment with proteasome inhibitor, 10 nM NPI-0052, for 1 hour was used as a positive control. Statistical differences between 0 and 24 hours with MS-275 were determined by Student t test, *P = .002 for chymotrypsin-like and P = .004 for caspase-like. (E) Vorinostat diminishes proteasomal chymotrypsin-like and caspase-like activities. After an 18-hour exposure to 500 nM vorinostat, chymotrypsin-like and caspase-like activities in Jurkat cells were measured using fluorogenic substrates as previously described. Student t test determined statistical differences between 0 and 18 hours. (F) HDACi do not inhibit the rate-limiting activity of the proteasome in isolated proteasomes. Purified 20S proteasomes were combined with diluent, 5 μM MS-275, 500 nM vorinostat, or 10 nM NPI-0052 for 30 minutes at room temperature. The chymotrypsin-like activity was analyzed using fluorogenic substrate suc-LLVY–amc and activity was evaluated in RFU. NPI-0052 was used as a positive control.

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