Figure 1.
MGO decreases the 20S proteasome activity, depletes free ubiquitin and leads to accumulation of ubiquitin conjugates.
(A) ARPE-19 cells were treated with 1 mM or 3 mM of MGO for 3 hours. Cells were then lysed and the intracellular levels of MGO were determined by HPLC after derivatization with DDB. The results represent the mean ± SD of at least three independent experiments. * p<0.05, significantly different from control (one-way ANOVA). (B) ARPE-19 cells were treated with 3 mM MGO for 3 hours and the cell extracts were used to measure the proteolytic activities of the 20S proteasome (chymotrypsin-like, caspase-like and trypsin-like activities) using specific fluorogenic substrates. The results correspond to the measurement at 30 minutes of activity and represent the mean ± SD of at least three independent experiments. ** p<0.01, significantly different from control; paired T test. (C) ARPE-19 cells were treated with 1 mM MGO, 3 mM MGO or 20 µM MG132 for 3 hours and the proteins were separated by SDS-PAGE, transferred to PVDF membranes and probed with monoclonal antibodies against ubiquitin and actin.
Figure 2.
MGO decreases the levels of Hsc70 and Hsp90.
(A, B and C) ARPE-19 cells were treated with 1 mM MGO or 3 mM MGO for 3 hours and the cell lysates were analyzed by western blotting using anti-Hsc70 (A), anti-Hsp90 (B) and anti-actin antibodies. The quantified results represent the mean ± SD of at least three independent experiments. *** p<0.001, ## p<0.01, ### p<0.001, significantly different from control; one-way ANOVA (Dunnet's post hoc test) (C).
Figure 3.
MGO induces formation of large aggregates containing ubiquitin, CHIP and Hsp40.
(A) ARPE-19 cells were treated with 1 mM MGO or 3 mM MGO for 3 hours and the cell lysates were analyzed by western blotting using specific antibodies against CHIP and actin. (B) ARPE-19 cells were transfected with myc-CHIP wt and treated with 3 mM MGO for 15, 30, 50, 79 and 90 minutes. Proteins were separated by SDS-PAGE, transferred to PVDF membranes and probed for c-myc. (C) ARPE-19 cells were treated with 1 mM MGO or 3 mM MGO for 3 hours. The cell lysates were used to immunoprecipitate Hsp40 and immunoblot the immunoprecipitates against ubiquitin. (D) ARPE-19 cells were transfected with myc-CHIP wt and treated with 3 mM MGO for 3 hours. c-myc (CHIP) was immunoprecipitated and the immunoprecipitates were probed using a monoclonal antibody against ubiquitin. (E) ARPE-19 cells were transfected with c-myc CHIP wt, c-myc CHIP K30A or c-myc CHIP H260Q. Cells were subsequently treated with 3 mM MGO for 3 hours and the cell lysates were immunoblotted for c-myc and actin.
Figure 4.
MGO induces accumulation of oxidized and argpyrimidine-modified proteins and decreases cell viability.
(A) ARPE-19 cells were treated with 1 mM MGO or 3 mM MGO for 3 hours and the cell lysates were used to derivatize proteins with DNPH. Derivatized proteins were separated by SDS-PAGE, transferred to PVDF membranes and probed with an antibody against dinitrophenylhydrazone derivatives. (B) ARPE-19 cells were treated with 1 mM or 3 mM MGO for 3 hours and the cell lysates were immunoblotted for argpyrimidine and actin. (C) ARPE-19 cell were treated with 1 mM MGO or 3 mM MGO for 3 hours and used to assess cell viability through the MTT colorimetric assay. The quantified results represent the mean ± SD of at least three independent experiments. * p<0.05, significantly different from control; one-way ANOVA (Dunnet's post hoc test).
Figure 5.
MGO-induced stress activates Hsf-1 through increased oligomerization and DNA-binding, eliciting a cell response to stress.
(A) ARPE-19 cells were treated with 50 µM CHX together with 1 mM MGO for 1, 2, 3, 4 or 5 hours and the cell lysates were analyzed by western blot using specific antibodies against Hsf-1 and actin. * Monomeric Hsf-1 (∼80 kDa); ** Dimeric Hsf-1 (∼160 kDa); *** Trimeric Hsf-1 (∼230 kDa). (B) ARPE-19 cells were treated with 1 mM MGO for 1, 3 or 5 hours and the cell lysates were analyzed by western blot using specific antibodies against Hsf-1 and actin. (C) ARPE-19 cells were transiently transfected with the HSE wt (x4)-luciferase or HSE mut (x4)-luciferase vectors and were treated with 1 mM MGO for 3, 5 or 8 hours. Subsequently, the luciferase activity was determined by luminescence. The quantified results represent the mean ± SD of at least three independent experiments. ** p<0.01, significantly different from control; one-way ANOVA (Dunnet's post hoc test). (D) ARPE-19 cells were treated with 1 mM MGO for 3, 5 or 8 hours. Total RNA was used to synthesize cDNA, which, in turn, was used as template to quantify HspB1, HspB2, Hsc70, Hsp70 and Hsp90 mRNA and 18S rRNA through RT-PCR. The quantified results represent the mean ± SD of at least three independent experiments. * p<0.05, ** p<0.01, *** p<0.001, significantly different from the respective control; ## p<0.01 and ### p<0.001, significantly different from the corresponding “MGO 3 h” condition; § p<0.05, §§ p<0.01, significantly different from the corresponding “MGO 5 h” condition; one-way ANOVA (Tukey's post hoc test). (E) ARPE-19 cells were treated with 1 mM MGO for 3, 5 or 8 hours and the cell lysates were analyzed by western blot using anti-Hsc70, -Hsp90 and -actin antibodies. The quantified results represent the mean ± SD of at least three independent experiments. * p<0.05, ** p<0.01 and *** p<0.001, significantly different from the respective control; one-way ANOVA (Tukey's post hoc test); ## p<0.01, significantly different from the corresponding “MGO 5 h” condition; one-way ANOVA (Tukey's post hoc test). (F) ARPE-19 cells were treated with 1 mM MGO for 1, 3, 5, 8 or 10 hours and the cell lysates were analyzed by western blot using anti-Hsp27, -Hsp70, -GAPDH and -actin antibodies. (G) ARPE-19 cells, transfected with c-myc tagged Hsf-1, were treated with 1 mM MGO for 1, 3, 5 or 8 hours. c-myc was immunoprecipitated and immunoprecipitates were probed against c-myc and Hsp90. (H) ARPE-19 cells were treated with 1 mM MGO for 3, 8 or 10 hours and used to assess cell viability through the MTT colorimetric assay. The quantified results represent the mean ± SD of at least three independent experiments. * p<0.05, significantly different from control; # p<0.05, significantly different from “1 mM MGO 8 h” condition; one-way ANOVA (Dunnet's post hoc test).