Figure 1.
Expression of A20 in skin cells.
(A) Expression of A20 was detected using Western blot analysis. High expression of A20 is observed in squamous carcinoma cell line SCC12 and SCC13. (B) Expression of A20 was detected in skin tissues by immunohistochemistry. In normal skin, A20 expression is increased in upper layers of epidermis. In SCC, high expression of A20 is detected in cancer lesions. In negative control, primary antibody was omitted.
Figure 2.
Imiquimod-induced apoptosis of SCC12 cells.
(A) Cells were treated with imiquimod at the indicated concentrations for 24 h. Cell viability was determined by MTT assay. Data are expressed as percentage of control (0 µg/ml imiquimod). The mean values ± SD are averages of triplicate measurements. (B) To determine whether imiquimod induces apoptosis of SCC12 cells, cleavage of PARP, a prominent feature of the apoptotic execution phase, was checked by Western blot. Anti-actin antibody was used as a loading control. Imiquimod induces cleavage of PARP in a dose-dependent manner. (C) Cells were treated with imiquimod at the indicated concentrations and/or for the indicated time points. Expression of A20 was detected by Western blot. Imiquimod induces down-regulation of A20 in a dose- and time-dependent manner. (D) Cells were pretreated with MG132 then treated with imiquimod (150 µg/ml) for the indicated time points. MG132 blocks imiquimod-driven A20 down-regulation.
Figure 3.
Effect of A20 overexpression on imiquimod-induced apoptosis of SCC12 cells.
(A) Cells were transduced with 10 multiplicity of infection (MOI) of adenovirus expressing GFP-tagged A20 (Ad/GFP-A20) or control adenovirus (Ad/GFP) for 6 h. Cells were replenished with fresh medium, and incubated for a further 2 d. Then, cells were treated with imiquimod (150 µg/ml) for 16 h. Apoptosis was determined by flow cytometry. Annexin V high and propidium iodide (PI) dim cells (bold box) represent apoptotic cells. Imiquimod-induced apoptosis is markedly reduced in GFP-A20 overexpressed group (Ad/GFP-A20) compared to GFP overexpressed control group (Ad/GFP). (B) Cleavages of PARP and caspase-3 were detected by Western blot. In GFP-A20 overexpressed cells, imiquimod-driven PARP and caspase-3 cleavages are reduced compared to control group. (C) After adenoviral transduction, cells were treated with imiquimod (150 µg/ml) for the indicated time points, and phosphorylation of MAPKs was detected by Western blot. Imiquimod induces phosphorylation of JNK, which is inhibited by overexpression of GFP-A20.
Figure 4.
Effect of A20 knockdown on imiquimod-induced apoptosis of SCC12 cells.
(A) The stable SCC12 cells expressing shRNA were established, and phosphorylation of MAPKs was detected by Western blot. In A20-knockdowned cells (shRNA-A20 (#1), shRNA-A20 (#2)), phosphorylation of JNK is increased compared to control cells (shRNA-Scr). (B) Cells were treated with imiquimod (150 µg/ml) for 16 h and apoptosis was determined by flow cytometry. Bold boxes represent apoptotic cells. Imiquimod-induced apoptosis is markedly potentiated in A20-knockdowned cells (shRNA-A20 (#2)) compared to control cells (shRNA-Scr).
Figure 5.
Effect of TLR7 knockdown on imiquimod-induced apoptosis of SCC12 cells.
(A) Cells were transduced with 10 multiplicity of infection (MOI) of adenovirus expressing miR-TLR7 or control adenovirus (miR-Scr) for 6 h. Cells were replenished with fresh medium, and incubated for a further 2 d. Endogenous expression of TLR7 is markedly decreased by miR-TLR7. (B) After adenoviral transduction, cells were treated with imiquimod (150 µg/ml) for 16 h and apoptosis was determined by flow cytometry. Bold boxes represent apoptotic cells. There is no difference in apoptotic cell populations between TLR7-knockdowned group (miR-TLR7) and control group (miR-Scr). (C) Cleavages of PARP was detected by Western blot. TLR7 knockdown does not affect imiquimod-driven PARP cleavage.