Mechano-signalling, induced by fullerene C₆₀ nanofilms, arrests the cell cycle in the G2/M phase and decreases proliferation of liver cancer cells

Figures & data

Figure 1 Preparation and characterization of C₆₀ nanofilms.

Notes: (A) Nanofilms pattern of dots: 1=0 dots (control); 2=10 dots, C₆₀-20%; 3=17 dots; 4=28 dots; 5=37 dots; 6= covering the entire surface, C₆₀-100%. (B) Characterization of fullerenes by transmission electron microscopy (TEM) (1, 2) and scanning electron microscopy (SEM) (3, 4). Scale bars: 500 nm (1), 100 nm (2), 10 µm (3) and 5 µm (4). Agglomerates were observed in the colloid of nanoparticles (black arrowheads). (C) Infrared spectrum of C₆₀ registered in the middle region (3500–500 cm⁻¹). Characteristic transmission bands were assigned to the appropriate vibrations of groups and bands present in the studied samples. (D) Comparison between the Petri dish uncoated and coated with C₆₀ made by means of AFM. The side width of the images is 10 μm for the upper row and 2 μm for the lower. The left micrographs are of the uncoated plate and the right micrographs are of the C₆₀ coated plate.

Abbreviations: C₆₀, fullerenes; Ra, roughness (nm).

Figure 2 (A) Elasticity modulus (kPa) of HS-5, HepG2 and C3A cells on C₆₀ nanofilms. (B) Viability of HS-5, HepG2 and C3A cells after growth on 20 µg of C₆₀ nanofilms. (C) Proliferation of HS-5, HepG2 and C3A cells on C₆₀ nanofilms. (D) Three-dimensional AFM images of HepG2 (D1, D2) and C3A (D3, D4) cells.

Notes: The differences between the cell lines were significant (P≤0.05). Different letters indicate significant differences between the groups (ANOVA, Tukey’s post-test). The differences between the carbon-grown groups (20 µg of C₆₀ nanofilm) and the control group were significant (P≤0.05) (ANOVA, Tukey’s post-test) for HS-5 cells. Different letters indicate significant differences between the groups. Controls are shown in the pictures D1 and D3. The cell nuclei (black arrowheads) were observed using AFM.

Abbreviations: ANOVA, analysis of variance; C, control; C₆₀, fullerenes; XTT, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide; BrdU, 5-bromo-2ʹ-deoxyuridine. AFM, atomic force microscopy.

Figure 3 Propidium iodide (PI) 488 assay analysis.

Notes: Effect of C₆₀ on the number (percentage) of (A) HS-5, (B) HepG2 or (C) C3A cells in the cycle phases. (D) Relative HS-5, HepG2 and C3A cell population in G2/M cycle (%). (E) Western blot analysis of β-catenin, N-cadherin, vinculin and PCNA. GAPDH was used as a loading control.

Abbreviations: C₆₀, fullerenes; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PCNA, proliferating cell nuclear antigen.

Figure 4 Morphology changes and affinity of HS-5 cells to the C₆₀ nanofilms.

Notes: Hematoxylin-eosin (H+E) staining of HS-5 cells on C₆₀ nanofilms visualized using light optical microscopy. (A1) control group; (A2) C₆₀-20%; and (A3): C₆₀-100%. Black arrows indicate the direction of cell migration. Scale bars: left pictures 100 μm; right pictures 20 μm.

Abbreviation: C₆₀, fullerenes.

Figure 5 Morphology changes and affinity of HepG2 cells to the C₆₀ nanofilms.

Notes: Hematoxylin-eosin staining of HepG2 cells on C₆₀ nanofilms visualized using light optical microscopy. (A1) Control group; (A2) C₆₀-20%; and (A3) C₆₀-100%. Black arrows indicate the direction of cell migration. Scale bars: left pictures 100 μm, right pictures 20 μm.

Abbreviation: C₆₀, fullerenes.

Figure 6 Morphology changes and affinity of C3A cells to the C₆₀ nanofilms.

Notes: Hematoxylin-eosin staining of C3A cells on C₆₀ nanofilms visualized using light optical microscopy. (A1) Control group; (A2) C₆₀-20%; and (A3) C₆₀-100%. Black arrows indicate the direction of cell migration. Scale bars: left pictures 100 μm, right pictures 20 μm.

Abbreviation: C₆₀, fullerenes.

Figure 7 Visualization of the interaction of HS-5 cells with nanofilms using scanning electron microscopy.

Notes: (A1) Control group (A2 and A3) C₆₀-20%. Red stars and yellow arrows indicate lamellipodia and filopodia, respectively. The dotted line indicates edges of the dots. Scale bars: A1 and A2 =300 μm and 20 μm, A3 =2.0 mm.

Abbreviation: C₆₀, fullerenes.

Figure 8 Visualization of the interaction of HepG2 cells with nanofilms using scanning electron microscopy.

Notes: (A1) Control group; (A2 and A3) C₆₀-20%. The dotted line indicates edges of the dots. Scale bars: A1 and A2 =300 μm and 20 μm, A3 =2.0 mm.

Abbreviation: C₆₀, fullerenes.

Figure 9 Visualization of the interaction of C3A cells with nanofilms using scanning electron microscopy.

Notes: (A1) Control group; (A2 and A3) C₆₀-20%. Red and blue points indicate lamellipodia and the 3-D ECM structure, respectively. The dotted line indicates edges of the dots. Scale bars: A1 =300 μm and 20 μm, A2 =300 μm, 50 μm and 20 μm, A3 =2.0 mm.

Abbreviation: C₆₀, fullerenes.

Figure 10 Expression level of integrin α5β1 and changes in cell morphology on fullerenes nanofilms.

Notes: HS-5 cells were stained with DAPI (nuclei, blue), phalloidin-Atto 633 (cytoskeleton, red) and fluorescent secondary antibody 488 FITC (integrin, green) and visualized using confocal microscopy and Nomarski interference contrast. (A1) Control group; (A2) C₆₀-20%; and (A3) C₆₀-100%. Scale bar: 20 μm.

Abbreviations: C₆₀, fullerenes; DAPI, 4′,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate.

Figure 11 Expression level of integrin α5β1 and changes in cell morphology on fullerenes nanofilms.

Notes: HepG2 cells were stained with DAPI (nuclei, blue), phalloidin-Atto 633 (cytoskeleton, red) and fluorescent secondary antibody 488 FITC (integrin, green) and visualized using confocal microscopy and Nomarski interference contrast. (A1) Control group; (A2) C₆₀-20%; and (A3) C₆₀-100%. Scale bar: 20 μm.

Abbreviations: C₆₀, fullerenes; DAPI, 4′,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate.

Figure 12 Expression level of integrin α5β1 and changes in cell morphology on fullerenes nanofilms.

Notes: C3A cells were stained with DAPI (nuclei, blue), phalloidin-Atto 633 (cytoskeleton, red) and fluorescent secondary antibody 488 FITC (integrin, green) and visualized using confocal microscopy and Nomarski interference contrast. (A1) Control group; (A2) C₆₀-20%; and (A3) C₆₀-100%. Scale bar: 20 μm.

Abbreviations: C₆₀, fullerenes; DAPI, 4′,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate.

Table 1 Relative values of β-catenin, N-cadherin, vinculin and PCNA protein levels on C₆₀ compared to the control for HepG2 cells

Adjusted density values of bands for C60 samples relative to the control and loading-control			ANOVA
Cell line Protein	HepG2		SE
	C	C60
β-catenin	1	0.90	0.045
N-cadherin	1	0.73*	0.066
Vinculin	1	5.55***	0.999
PCNA	1	0.94	0.075

Note: Statistically significant differences in comparison to untreated cells (P<0.05): *P-value <0.05, **P-value <0.01, and ***P-value <0.001 (ANOVA; Dunnett’s post-test). GAPDH was used as a loading control. The table presents a quantitative analysis of the pixels using ImageJ^® 1.48v. Adjusted density values of bands were expressed as the relative values to the control (Area C60/Area Control), where the control value is 1. Next, bands were expressed as the relative values to the loading control (Area C60/Area Control)EP/(Area C60/Area Control)GAPDH, where EP is the examined protein (β-catenin, N-cadherin, vinculin or PCNA).

Abbreviations: ANOVA, analysis of variance; C, control group; C60, fullerenes; PCNA, Proliferating cell nuclear antigen; SE, standard error.

Table 2 Relative values of β-catenin, N-cadherin, vinculin, and PCNA protein levels on C₆₀ compared to the control for C3A cells

Adjusted density values of bands for C60 samples relative to the control and loading-control			ANOVA
Cell line Protein	C3A		SE
	C	C60
β-catenin	1	0.79	0.032
N-cadherin	1	0.77	0.046
Vinculin	1	0.89	0.080
PCNA	1	1.02	0.143

Note: Statistically significant differences in comparison to untreated cells (P<0.05): *P-value <0.05, **P-value <0.01, and ***P-value <0.001. (ANOVA; Dunnett’s post-test). GAPDH was used as a loading control. The table presents a quantitative analysis of the pixels using ImageJ^® 1.48v. Adjusted density values of bands were expressed as the relative values to the control (Area C60/Area Control), where the control value is 1. Next, bands were expressed as the relative values to the loading control (Area C60/Area Control)EP/(Area C60/Area Control)GAPDH, where EP is the examined protein (β-catenin, N-cadherin, vinculin or PCNA).

Abbreviations: ANOVA, analysis of variance; C, control group; C60, fullerenes; PCNA, Proliferating cell nuclear antigen; SE, standard error.

Table 3 Relative values of β-catenin, N-cadherin, vinculin and PCNA protein levels on C₆₀ compared to the control for HS-5 cells

Adjusted density values of bands for C60 samples relative to the control and loading-control			ANOVA
Cell line Protein	HS-5		SE
	C	C60
β-catenin	1	0.31***	0.016
N-cadherin	1	0.62**	0.050
Vinculin	1	0.85	0.085
PCNA	1	0.62**	0.003

Notes: Statistically significant difference sin comparison to untreated cells (P<0.05): *P-value <0.05, **P-value <0.01, and ***P-value <0.001. (ANOVA; Dunnett's post-test). GAPDH was used as a loading control. The table presents a quantitative analysis of the pixels using ImageJ^® 1.48v. Adjusted density values of bands were expressed as the relative values to the control (Area C₆₀/Area Control), where the control value is 1. Next, bands were expressed as the relative values to the loading control (Area C₆₀/Area Control)EP/(Area C₆₀/Area Control)GAPDH, where EP is the examined protein (β-catenin, N-cadherin, vinculin or PCNA).

Abbreviations: ANOVA, analysis of variance; C, control group; C₆₀, fullerenes; PCNA, Proliferating cell nuclear antigen; SE, standard error.

Figure S1 Results of trypan blue analysis for HS-5, HepG2 and C3A cells. Total cell count (red color) and numbers of live cells (green color) on C₆₀ nanofilms