Article Figures & Data
Figures
- FIG. 1.
Curcumin decreases replication of CVB3. (A) HeLa cells were sham infected with PBS or infected with CVB3 in the absence or presence of curcumin (30 μM). Six hours postinfection, viral RNA was detected by in situ hybridization using antisense riboprobes for CVB3 (red). Cell nuclei were counterstained with hematoxylin (blue). The data represent the results from two independent experiments. (B) HeLa cells were infected and treated with various concentrations of curcumin as for panel A. Seven hours postinfection, CVB3 viral-protein synthesis was examined using a monoclonal antibody that recognizes the CVB3 capsid protein VP1. To verify equal loading, the same blots were stripped and reprobed for β-actin. The data represent results from three independent experiments. (C) HeLa cells were infected and treated with curcumin as for panel A. Nine hours postinfection (p.i.), medium was collected and CVB3 progeny were measured by plaque assay. The data are means plus standard deviations (SD); n = 3; #, P < 0.005 compared to DMSO treatment. (D) HeLa cells were infected with 35S-labeled CVB3 for 1 h. Cell lysates were collected and measured for radioactivity using a scintillation counter (mean ± SD; n = 6). CPM, counts per minute. (E) HeLa cells were infected with CVB3, and curcumin (30 μM) was added to the cells at different times as indicated. Seven hours postinfection, cell lysates were collected and expression levels of VP1 and β-actin were examined.
- FIG. 2.
Curcumin reduces CVB3-induced cytopathic effect and apoptosis. HeLa cells were sham infected with PBS or infected with CVB3 in the absence or presence of curcumin (30 μM). (A) Seven hours postinfection, the morphologies of infected cells were examined under a phase-contrast microscope. Representative images from three independent experiments are presented. (B) Cell lysates were prepared 7 h postinfection (p.i.), and cleavage of caspase 3 was examined using a monoclonal anti-capase 3 antibody. β-Actin was examined to show equal protein loading. The data represent the results from two independent experiments.
- FIG. 3.
Curcumin does not inhibit replication of CVB3 through MAPK pathways. HeLa cells were infected with CVB3 in the absence or presence of curcumin (10 and 30 μM). Seven hours postinfection (p.i.), the phosphorylation status of MAPKs was examined using anti-phospho-ERK1/2, anti-phospho-p38, and anti-phospho-JNK1/2 antibodies, respectively. Expression levels of total ERK1/2, p38, and JNK1/2 were examined as the loading controls. The data represent the results from two independent experiments.
- FIG. 4.
Curcumin probably does not inhibit replication of CVB3 through CKII and CSN. HeLa cells were transiently transfected with specific siRNAs to knock down the expression of CKIIα (A), CKIIα/β (B), or Jab1 (C), respectively, for 24 h. Seven hours after CVB3 infection, viral protein expression (VP1) and levels of CKIIα, CKIIβ, and Jab1 were determined by Western blotting. β-Actin was examined to verify equal loading. The data represent the results from three independent experiments.
- FIG. 5.
Curcumin promotes protein polyubiquitination and decreases free ubiquitin levels. (A) HeLa cells were treated with DMSO or curcumin (30 μM) for 5 h. Protein polyubiquitination and free ubiquitin levels were examined by Western blot analysis using a polyclonal anti-ubiquitin antibody. β-Actin was examined to verify equal loading. (B) HeLa cells were incubated with either DMSO, MG132 (5 μM), or curcumin (30 μM) for 5 h. Ubiquitin conjugates were determined as for panel A. Multiple lanes represent the results from independent experiments.
- FIG. 6.
Curcumin inhibits 20S proteasome activities. (A) HeLa cells were treated with DMSO, curcumin (30 μM), or MG132 (5 μM) for 5 h. The activities of 20S proteasome, including trypsin-like, chymotrypsin-like, and PDGH activities, in the cell lysates were measured as described in Materials and Methods. (B) Purified 20S proteasome (100 ng) was used to examine the effects of curcumin (30 μM) and MG132 (5 μM) on proteasome hydrolytic activities. The data are means plus standard deviations; n = 3. *, P < 0.01; #, P < 0.005; δ, P < 0.001 compared to DMSO treatment.
- FIG. 7.
Curcumin reduces cellular deubiquitinating activities. (A) HeLa cells were sham infected with PBS or infected with CVB3 for 6 h. Cell lysates were prepared, and cellular deubiquitinating activities were measured using a fluorogenic substrate, ubiquitin-AMC, as described in Materials and Methods. (B) HeLa cells were treated with DMSO, curcumin (30 μM), or MG132 (5 μM) for 5 h, and cellular deubiquitinating activities were measured. The data are means plus standard deviations; n = 3. #, P < 0.005 compared to DMSO treatment.
