Figure 1 BPV DNA sequences identified by PCR

Figure 2 Cell cycle analysis by flow cytometry: normal skin (A), papilloma 01 (B), 02 (C), 03 (D), esophageal carcinoma (E) and cell cycle distribution (F). Results show a cell percentage increase in S (synthesis) and G2-M in all primary cultures derived from BPV-infected lesions in relation to BPV-free normal skin cells.

Figure 3 Analysis of BPV action on mitochondrial membrane potential (??m) deregulation by immunofluorescence and flow cytometry (FC):

A) control presenting absence of fluorescent signal in cutaneous papilloma cell line not treated with MitoTracker probe, MitoTracker labelling in normal skin, papillomas and esophageal carcinoma cells in second passage. Histograms based on cell percentage labelled with MitoTracker probe (B) and the median of fluorescent intensity (MFI) + standard deviation (C). Results show a reduction in cell percentage with oxidative metabolism in fibropapilloma (FP) and esophageal carcinoma cell lines, suggesting the activation of aerobic glycoslysis. Bonferroni’s multiple comparison analysis based on MFI showed an increase in MFI of papilloma 02 and esophageal carcinoma cells, suggesting an energy compensatory mechanism.Total of 10,000 events analyzed. Scale bar of 100 µm. Flow cytometry graphs showing number of cells (Y axis) and fluorescent intensity in FL4 channel (X axis).

Figure 4 Analysis of BPV E6 oncoprotein on mitochondrial membrane potential (??m) deregulation by immunofluorescence and flow cytometry (FC).

A) negative control (without probe) presenting absence of fluorescent signal in cutaneous papilloma cell line not treated with MitoTracker probe; normal skin cell lines normal skin cell line (uninfected by BPV) and normal skin cell line treated with 1 µg/mL of BPV-1 E6 recombinant oncoprotein. Histogram based on cell percentage labelled with MitoTracker probe (B) and median of fluorescent intensity (MFI) + standard deviation (C). Results show a reduction of mitochondrial membrane potential after the treatment with BPV-1 E6 oncoprotein (B), however, without reduce the MFI (C) in cells expressing oxidative metabolism.

Figure 5 Levels of reactive oxygen species (ROS) observed on five cell lines.

A) flow cytometry showing absence of unspecific labelling in cutaneous papilloma cells not treated with the DCFH-DA probe (negative control) and ROS production in: B) normal skin cells treated with 1 mM of H2O2 and incubated with probe (positive control), C) normal skin, D) cutaneous papilloma 01, E) papilloma 02 (FP – fibropapilloma), F) papilloma 03 (FP – fibropapilloma) and G) esophageal carcinoma cells. Histograms based on cell percentage labelled with DCFH-DA probe (H) and median of fluorescent intensity (MFI) + standard deviation (I). Results show that the normal skin cells presented the highest ROS production, verified by the mean of fluorescence intensity (MFI) (I), but restricted a fewest number of cells (H). Papillomas cells showed intermediate levels of ROS (I), but in a high cell percentage, reinforcing the energy compensatory mechanism. Total of 10,000 events analyzed. Scale bar of 100 µm. Flow cytometry graphs showing number of cells (Y axis) and fluorescent intensity in FL1 channel (X axis). Cells analyzed in second passage (P2).

Figure 6 Levels of reactive oxygen species (ROS) observed after E6 oncoprotein addition.

A) normal skin cell line (uninfected by BPV) not incubated with DCFH-DA probe (negative control), showing absence of labelling, B) normal skin cell line treated with 1 mM of H2O2 and incubated with probe, showing ROS formation (positive control), C) normal skin cell line only incubated with probe, showing reduced ROS production as a consequence of normal oxidative metabolis, D) normal skin cell line treated with 1 µg/mL of BPV-1 E6 recombinant oncoprotein. Histogram based on ROS-producing cell percentage (E) and median of fluorescent intensity (MFI) + standard deviation (F). Results show an increase in ROS production in cells treated with BPV-1 E6 oncoprotein. Total of 10,000 events analyzed. Scale bar of 100 µm. Flow cytometry graphs showing number of cells (Y axis) and fluorescent intensity in FL1 channel (X axis). Cells analyzed in second passage (P2).

Figure 7 Analysis of clastogenic potential of BPV by comet assay.

A) nucleoids showing absence of DNA damage (class 0) observed in normal skin (uninfected by BPV), nucleoids showing maximum level of DNA damage (class 2), observed in cutaneous papilloma 01 (B), 02 (C), 03 (D) and esophageal carcinoma cell lines (F). G) Boxplot showing maximum, minimum and median of comet scores, demonstrating high levels of clastogenesis in esophageal carcinoma cell line.

Figure 8 Linear regression of comet score

Figure 9 Analysis of STAT3 Y705 expression.

A) photomicrograph and flow cytometry showing absence of unspecific labelling in cutaneous papilloma cell line only treated with secondary antibody (Alexa Fluor 633) (control) and normal skin cells not treated with any antibody. Results show the immunodetection of STAT3 activated in nucleus of cutaneous papilloma (papilloma 01), fibropapilloma (papilloma 02 and 03) and esophageal carcinoma, but not in normal skin cells. Histograms based on STAT3 Y705-expressing cell percentage (B) Results show a high STAT3-expressing cell percentage in both papilloma and fibropapilloma (FP) cells. Esophageal carcinoma and normal skin cells show the similar levels of expression of STAT3, however, it is verified the nuclear immunodetection of STAT3 in esophageal carcinoma, indicating the activation of this nuclear transcription factor. Total of 10,000 analyzed events. FP – fibropapilloma. Scale bar of 100 µm. Flow cytometry graphs showing number of cells (Y axis) and fluorescent intensity in FL1 channel (X axis).

Figure 10 Downregulation of SOD2 and GPx antioxidant enzymes

A) Under normal conditions, the singlet oxygen produced by the mitochondrial complex is converted to hydrogen peroxide by the superoxide dismutase 1 (SOD1) or 2 (SOD2) and, the hydrogen peroxide is converted to water molecule by the GPx enzyme. B) However, the E6 oncoprotein expression leads to the SOD2 and GPx downregulation, reducing the catalysis of singlet oxygen and hydrogen peroxide (ROS). However the electron flux is conserved, avoiding the cytochrome c release and, therefore, conferring an anti-apoptotic stimulus.

Normal skin

Passage

Class 0

Class 1

Class 2

Score

P1

90

7

3

13

P2

79

11

10

31

P3

74

4

22

48

P4

61

9

30

69

P5

48

7

45

97

P6

52

6

42

90

Total

404

44

152

348

Cutaneous papilloma 01

Passage

Class 0

Class 1

Class 2

Score

P1

79

1

20

41

P2

65

5

30

65

P3

68

3

29

61

P4

29

12

59

130

P5

35

17

48

113

P6

53

12

41

94

Total

329

50

227

504

Cutaneous papilloma 02

Passage

Class 0

Class 1

Class 2

Score

P1

58

7

35

77

P2

30

12

58

128

P3

47

22

31

84

P4

8

28

64

          156

P5

87

18

69

156

P6

7

20

73

166

Total

237

107

330

767

Cutaneous papilloma 03

Passage

Class 0

Class 1

Class 2

Score

P1

26

11

63

137

P2

24

17

59

135

P3

32

12

56

124

P4

39

16

45

106

P5

35

20

45

110

P6

25

37

38

113

Total

181

113

306

725

Esophageal carcinoma

Passage

Class 0

Class 1

Class 2

Score

P1

2

19

79

177

P2

1

21

78

177

P3

12

27

61

149

P4

5

23

72

167

P5

3

14

83

180

P6

4

18

78

174

Total

27

122

451

1,024

Table1 Comet assay results

Comparition

Post difference

Z calculated

Critical Z

p

Normal skin and Papilloma 01

3.9167

0.7706

2.807

ns

Normal skin and Papilloma 02

11.8333

2.3282

2.807

ns

Normal skin and Papilloma 03

10.7500

2.1150

2.807

ns

Normal skin and esophageal carcinoma

21.0000

4.1317

2.807

< 0.05

Papilloma 01 and Papilloma 02

7.9167

1.5576

2.807

ns

Papilloma 01 and Papilloma 03

6.8333

1.3444

2.807

ns

Papilloma 01 and esophageal carcinoma

17.0833

3.3611

2.807

< 0.05

Papilloma 02 and Papilloma 03

1.0833

0.2131

2.807

ns

Papilloma 02 and esophageal carcinoma

9.1667

1.8035

2.807

ns

Papilloma 03 and esophageal carcinoma

10.2500

2.0167

2.807

ns

Table2 Results of post hoc Dunn test

ns – non significative statiscal difference

Passage

Normal skin

Papilloma 01

Papilloma 02

Papilloma 03

Esophageal carcinoma

P1

13ª*

41ª

77ª,b

137ª,b

177b

P2

31ª

65ª

128ª,b

135ª,b

177b

P3

48ª

61ª

84ª,b

124ª,b

149b

P4

69ª

130ª

156ª,b

106ª,b

167b

P5

97ª

113ª

156ª,b

110ª,b

180b

P6

90ª

94ª

166ª,b

113ª,b

174b

Table3 Statistical results of comet assay

*numbers followed by equal letters indicate the absence of significate statistical differences