16-Hydroxycleroda-3,13-dien-15,16-olide induces anoikis in human renal cell carcinoma cells: involvement of focal adhesion disassembly and signaling

Figures & data

Table 1 Antibodies

Antibody	Dilution	Brand
Anti-vimentin (2707), anti- pSrc (Tyr418; ab133460), anti-pNF-κB (Ser536; ab76302), anti-glycerol-3-phosphate dehydrogenase (GAPDH; ab9484), p21 (ab109520)	WB: 1:1,000 IF: 1:50	Epitomics (an Abcam company, Burlingame, CA, USA)
Anti-paxillin (610051), anti- pFAK (Tyr397; 611722), anti-FAK (610087), p53 (554293)	WB: 1:1,000 IF: 1:50	BD Biosciences (San Jose, CA, USA)
Anti-cyclin-dependent kinase (CDK) 2, anti-CDK4, anti-cyclin B1, anti-cyclin D1, anti-vascular endothelial growth factor (VEGF, sc-152)	1:500	Santa Cruz Biotechnology, Inc. (Dallas, TX, USA)
Anti-cyclin E1 (GTX103045) anti-MMP-2 (GTX104577), anti-MMP-9 (GTX100458)	1:1,000 1:500 1:500	GeneTex, Inc. (Irvine, CA, USA)
Anti-vinculin (700062)	1:1,000	Thermo Fisher Scientific (Waltham, MA, USA)

Abbreviations: IF, immunofluorescence; MMP, matrix metalloproteinase; pFAK, phosphorylated focal adhesion kinase; WB, Western blotting.

Figure 1 Clonogenic assays were performed to detect the reproductive capacity of 786-O and A-498 cells after CD treatment.

Notes: The figures shown are representative of one experiment; **P<0.01, ***P<0.001 compared with the control (0 µM group).
Abbreviation: CD, 16-hydroxycleroda-3,13-dien-15,16-olide.

Figure 2 Cells (786-O and A-498) were treated with 0.1% DMSO or CD (10–40 µM) for 24 hours.

Notes: Cell cycle distribution was analyzed using flow cytometry. The cell population in the different phases of the cell cycle is demonstrated as a percentage. Representative images of flow cytometry analysis are displayed. Results are shown as mean±SD from 12 independent experiments. *P<0.05, **P<0.01, ***P<0.001 compared with the control (0 µM group).
Abbreviations: CD, 16-hydroxycleroda-3,13-dien-15,16-olide; DMSO, dimethyl sulfoxide.

Figure 3 (A) Immunoblots of cyclins, CDKs, p21, and p53 in CD-treated RCC cells. GAPDH was used as the internal control. (B) The bar graphs show densitometric data (mean±SD) from three to five independent experiments.

Notes: The figures shown are representative of one experiment; *P<0.05, **P<0.01, ***P<0.001 compared with the control (0 µM group).
Abbreviations: CD, 16-hydroxycleroda-3,13-dien-15,16-olide; CDK, cyclin-dependent kinase; RCC, renal cell carcinoma.

Figure 4 (A) Scratch assay of RCC cells treated with the indicated concentration of CD for 8 and 20 hours. (B) Transwell migration assay of RCC cells treated with 0.1% DMSO, 10 or 20 µM, and 40 µM CD for 24 hours. Scale bar =50 µm. 50× magnification. (C) Matrigel invasion assay of RCC cells treated with 0.1% DMSO or 40 µM CD for 24 hours. Scale bar =50 µm. 100× magnification.

Notes: The bar graphs show the quantification of cell numbers that passed through the Transwell membrane from three independent experiments. *P<0.05, **P<0.01, ***P<0.001 compared with the control (0 µM group).
Abbreviations: CD, 16-hydroxycleroda-3,13-dien-15,16-olide; DMSO, dimethyl sulfoxide; RCC, renal cell carcinoma.

Figure 5 RCC cells were treated with 0.1% DMSO (control) or 40 µM CD for 24 hours.

Notes: (A) Immunofluorescence staining was used to detect FA proteins. Stress fibers were determined by staining with FITC-phalloidin. (B) The bar graphs show densitometric data from four to eight different coverslips. Scale bar =50 µm. *P<0.05, **P<0.01, and ***P<0.001.
Abbreviations: CD, 16-hydroxycleroda-3,13-dien-15,16-olide; DMSO, dimethyl sulfoxide; FA, focal adhesion; FITC, fluorescein isothiocyanate; pFAK, phosphorylated focal adhesion kinase; RCC, renal cell carcinoma.

Figure 6 (A) Immunoblots of FAK-related signaling molecules. The figures shown are representative of one experiment. (B) The bar graphs show densitometric data (mean±SD) from three independent experiments.

Note: *P<0.05, ***P<0.001 compared with the control (0 µM group).
Abbreviations: CD, 16-hydroxycleroda-3,13-dien-15,16-olide; FAK, focal adhesion kinase; pFAK, phosphorylated focal adhesion kinase.

Figure 7 (A) Immunoblots of pNF-κB, MMPs, and VEGF in CD-treated RCC cells. The figures shown are representative of one experiment. (B) The bar graphs show densitometric data (mean±SD) from three independent experiments.

Note: *P<0.05, **P<0.01, ***P<0.001 compared with the control (0 µM group).
Abbreviations: CD, 16-hydroxycleroda-3,13-dien-15,16-olide; MMP, matrix metalloproteinase; RCC, renal cell carcinoma; VEGF, vascular endothelial growth factor.