Journal: Oxidative Medicine and Cellular Longevity

Article Title: Acetylshikonin, A Novel CYP2J2 Inhibitor, Induces Apoptosis in RCC Cells via FOXO3 Activation and ROS Elevation

doi: 10.1155/2022/9139338

Figure Lengend Snippet: Analysis of protein expression level in A498 and ACHN cells treated with acetylshikonin by western blot analysis. (a) Cells were treated with different concentrations of acetylshikonin (0, 1.25, 2.5, and 5 μ M) for 24 h, and western blot was performed to measure pro- or antiapoptotic protein expression level using specific antibodies. β -Actin was used for a gel loading control. (b) Nuclear localization of FOXO3 and p27 proteins from the cytoplasm in A498 and ACHN cells treated with acetylshikonin. Nuclear fractional western blotting results of A498 and ACHN cells treated with acetylshikonin (0, 1.25, 2.5, and 5 μ M). The cytoplasmic and nucleic protein contents of FOXO3 and p27 were analyzed by western blotting using specific antibodies. GAPDH and Lamin A/C were used as loading control proteins for the cytoplasm and nucleus, respectively.

Article Snippet: Caspase-3 (Cat #9662), caspase-6 (Cat #9762), caspase-7 (D2Q3L, Cat #12827), caspase-9 (Cat #9504), cleaved-caspase-3 (Cat #9661), cleaved-caspase-7 (D6H1, Cat #8438), cleaved-caspase-6 (Cat #9761), cleaved-caspase-8 (Cat #9429), cleaved-caspase-9 (Cat #9509), Akt (Cat #9272), pAkt (D9E, Cat #4060), ERK (Cat #9102), pERK (Cat #9101), PARP (Cat #9542), cleaved-PARP (D64E10, Cat #5625), Bim (C34C5, Cat #2933), Bcl-2 (D17C4, Cat #3498), Bad (Cat #9292), pBad (Cat #9291), p21 (12D1, Cat #2947), p27 (D37H1, Cat #3688), H2aX (Cat #2595), and γ H2aX (Cat #2577) primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). β -Actin (C4, Cat #sc-47778), CYP2J2 (D-6, Cat #sc-137127), caspase-8 (D-8, Cat #sc-5263), JNK2 (A-7, Cat #sc-271133), pJNK (G-7, Cat #sc-6254), p38 (G-7, Cat #sc-166357), and pp38 (D-8, Cat #sc-7973) primary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Acetylshikonin, A Novel CYP2J2 Inhibitor, Induces Apoptosis in RCC Cells via FOXO3 Activation and ROS Elevation

doi: 10.1155/2022/9139338

Figure Lengend Snippet: Cell viability and antiproliferative effect of acetylshikonin treatment combined with CYP2J2 siRNA transfection against RCC. The A498 and ACHN cells were transfected with control or CYP2J2 siRNA and additionally treated with 1 μ M acetylshikonin. (a) Graphical data from GEPIA displays the overall survival and disease-free survival (DFS) analysis of kidney renal papillary cell carcinoma (KIRP) patients with high (red) or low (blue) level of CYP2J2 expression. (b) Cytotoxic effect was analyzed at 24, 48, and 72 h after treatment. The proliferation rates were determined by using the MTT assay. (c) The cell numbers were analyzed by the cell counting assay after 0, 24, 48, and 72 h. (d) Colony forming assay of A498 and ACHN cells for 14 days. The bar graphs represent a quantitation of the colonies. Data are represented with the mean ± SD of triplicated results. Single and double asterisks indicate significant differences from the control cells ( ∗ p < 0 : 05 and ∗∗ p < 0 : 01, respectively). (e) Western blot analysis of control or CYP2J2 siRNA transfected A498 and ACHN cells followed by 1 μ M acetylshikonin treatment for 24 h. β -Actin is used for a gel loading control.

Article Snippet: Caspase-3 (Cat #9662), caspase-6 (Cat #9762), caspase-7 (D2Q3L, Cat #12827), caspase-9 (Cat #9504), cleaved-caspase-3 (Cat #9661), cleaved-caspase-7 (D6H1, Cat #8438), cleaved-caspase-6 (Cat #9761), cleaved-caspase-8 (Cat #9429), cleaved-caspase-9 (Cat #9509), Akt (Cat #9272), pAkt (D9E, Cat #4060), ERK (Cat #9102), pERK (Cat #9101), PARP (Cat #9542), cleaved-PARP (D64E10, Cat #5625), Bim (C34C5, Cat #2933), Bcl-2 (D17C4, Cat #3498), Bad (Cat #9292), pBad (Cat #9291), p21 (12D1, Cat #2947), p27 (D37H1, Cat #3688), H2aX (Cat #2595), and γ H2aX (Cat #2577) primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). β -Actin (C4, Cat #sc-47778), CYP2J2 (D-6, Cat #sc-137127), caspase-8 (D-8, Cat #sc-5263), JNK2 (A-7, Cat #sc-271133), pJNK (G-7, Cat #sc-6254), p38 (G-7, Cat #sc-166357), and pp38 (D-8, Cat #sc-7973) primary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Control, Expressing, MTT Assay, Cell Counting, Quantitation Assay, Western Blot