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Basic, Underlying Data for Figures 3-5. Supplemental Figure 1a. GC-Rich Regions of Altered Methylation: Comparison of DMBA-Initiated, 3mg CSC-promoted.

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Presentation on theme: "Basic, Underlying Data for Figures 3-5. Supplemental Figure 1a. GC-Rich Regions of Altered Methylation: Comparison of DMBA-Initiated, 3mg CSC-promoted."— Presentation transcript:

1 Basic, Underlying Data for Figures 3-5

2 Supplemental Figure 1a. GC-Rich Regions of Altered Methylation: Comparison of DMBA-Initiated, 3mg CSC-promoted (8wks) Tissue to Control (DMBA/Acetone) DigestHypomethylated RAMHypermethylated RAMTOTAL RAM HpaII 505 MspI 909 PCR Product Size (bp) % DMBA/Acetone Control

3 Supplemental Figure 1b. GC-Rich Regions of Altered Methylation (New Methylations Only): Comparison of DMBA-Initiated, 3mg CSC-promoted (8wks) Tissue to Control (DMBA/Acetone) PCR Product Size (bp) Peak Area (Peak Area Units) DigestNew Methylations HpaII 3 MspI 3

4 Legend - Supplemental Figure 1. GC-Rich Regions of Altered Methylation: Comparison of DMBA-Initiated, 3mg CSC-promoted (8wks) Tissue to Control (DMBA/Acetone). RsaI/HpaII (blue symbols) and RsaI/MspI (red symbols) digestion and subsequent AP-PCR was performed on DNA isolated from SENCAR mouse skin treated with 3mg CSC for 8wks. Regions of hypomethylation were prevalent (a) Regions of new methylation resulting from treatment are shown in terms of the peak area for each PCR product size (b). A table tallying the regions of altered methylation for each treatment are shown as an inset in each figure. Regions of hypo-, hyper-, and new methylation are expressed in terms of the treated mean for each PCR product size as a percent of the control mean for each PCR product size. All changes projecting below the x- axis represent decreases in methylation (hypomethylation) while all those above the x-axis represent increases in methylation (hypermethylation). All 100% hypomethylations are considered to be significant, and only the hypermethylations and partial hypomethylations that were statistically significantly different from control values (Student’s t-test, p<0.05) are depicted.

5 Supplemental Figure 2a. GC-Rich Regions of Altered Methylation: Comparison of DMBA-Initiated, 9mg CSC-promoted (8wks) Tissue to Control (DMBA/Acetone) DigestHypomethylated RAMHypermethylated RAMTOTAL RAM HpaII 268 MspI 022 PCR Product Size (bp) % DMBA/Acetone Control

6 Supplemental Figure 2b. GC-Rich Regions of Altered Methylation (New Methylations Only): Comparison of DMBA-Initiated, 9mg CSC-promoted (8wks) Tissue to Control (DMBA/Acetone) PCR Product Size (bp) Peak Area (Peak Area Units) DigestNew Methylations HpaII 6 MspI 5

7 Legend - Supplemental Figure 2. GC-Rich Regions of Altered Methylation: Comparison of DMBA-Initiated, 9mg CSC-promoted (8wks) Tissue to Control (DMBA/Acetone). RsaI/HpaII (blue symbols) and RsaI/MspI (red symbols) digestion and subsequent AP-PCR was performed on DNA isolated from SENCAR mouse skin treated with 9mg CSC for 8wks. Regions of hypomethylation and hypermethylation are shown (a). Regions of new methylation resulting from treatment are shown in terms of the peak area for each PCR product size (b). A table tallying the regions of altered methylation for each treatment are shown as an inset in each figure. Regions of hypo-, hyper-, and new methylation are expressed in terms of the treated mean for each PCR product size as a percent of the control mean for each PCR product size. All changes projecting below the x- axis represent decreases in methylation (hypomethylation) while all those above the x-axis represent increases in methylation (hypermethylation). All 100% hypomethylations are considered to be significant, and only the hypermethylations and partial hypomethylations that were statistically significantly different from control values (Student’s t-test, p<0.05) are depicted.

8 Supplemental Figure 3a. GC-Rich Regions of Altered Methylation: Comparison of DMBA-Initiated, 18mg CSC-promoted (8wks) Tissue to Control (DMBA/Acetone) DigestHypomethylated RAMHypermethylated RAMTOTAL RAM HpaII 61016 MspI 101 PCR Product Size (bp) % DMBA/Acetone Control

9 Supplemental Figure 3b. GC-Rich Regions of Altered Methylation (New Methylations Only): Comparison of DMBA-Initiated, 18mg CSC-promoted (8wks) Tissue to Control (DMBA/Acetone) PCR Product Size (bp) Peak Area (Peak Area Units) DigestNew Methylations HpaII 19 MspI 2

10 Supplemental Figure 3c. GC-Rich Regions of Altered Methylation (New Methylations Only): Comparison of DMBA-Initiated, 18mg CSC-promoted (8wks) Tissue to Control (DMBA/Acetone) PCR Product Size (bp) Peak Area (Peak Area Units) DigestNew Methylations HpaII 19 MspI 2 285828 278758 151236 143137

11 Legend - Supplemental Figure 3. GC-Rich Regions of Altered Methylation: Comparison of DMBA-Initiated, 18mg CSC-promoted (8wks) Tissue to Control (DMBA/Acetone). RsaI/HpaII (blue symbols) and RsaI/MspI (red symbols) digestion and subsequent AP-PCR was performed on DNA isolated from SENCAR mouse skin treated with 18mg CSC for 8wks. Regions of hypomethylation and hypermethylation are shown (a). Regions of new methylation resulting from treatment are shown in terms of the peak area for each PCR product size (b). The y- axis scale was expanded in order to facilitate visualization of all data points. In doing this four regions of new methylation whose peak areas exceeded the scale of the chart were labeled above the chart with their corresponding peak area values (c). A table tallying the regions of altered methylation for each treatment are shown as an inset in each figure. Regions of hypo-, hyper-, and new methylation are expressed in terms of the treated mean for each PCR product size as a percent of the control mean for each PCR product size. All changes projecting below the x- axis represent decreases in methylation (hypomethylation) while all those above the x-axis represent increases in methylation (hypermethylation). All 100% hypomethylations are considered to be significant, and only the hypermethylations and partial hypomethylations that were statistically significantly different from control values (Student’s t-test, p<0.05) are depicted.

12 Supplemental Figure 4a. GC-Rich Regions of Altered Methylation: Comparison of DMBA-Initiated, 27mg CSC-promoted (8wks) Tissue to Control (DMBA/Acetone) DigestHypomethylated RAMHypermethylated RAMTOTAL RAM HpaII 202 MspI 010 PCR Product Size (bp) % DMBA/Acetone Control

13 Supplemental Figure 4b. GC-Rich Regions of Altered Methylation (New Methylations Only): Comparison of DMBA-Initiated, 27mg CSC-promoted (8wks) Tissue to Control (DMBA/Acetone) PCR Product Size (bp) Peak Area (Peak Area Units) DigestNew Methylations HpaII 1 MspI 27

14 Legend - Supplemental Figure 4. GC-Rich Regions of Altered Methylation: Comparison of DMBA-Initiated, 27mg CSC-promoted (8wks) Tissue to Control (DMBA/Acetone). RsaI/HpaII (blue symbols) and RsaI/MspI (red symbols) digestion and subsequent AP-PCR was performed on DNA isolated from SENCAR mouse skin treated with 27mg CSC for 8wks. Regions of hypomethylation and hypermethylation are shown (a). Regions of new methylation resulting from treatment are shown in terms of the peak area for each PCR product size (b). A table tallying the regions of altered methylation for each treatment are shown as an inset in each figure. Regions of hypo-, hyper-, and new methylation are expressed in terms of the treated mean for each PCR product size as a percent of the control mean for each PCR product size. All changes projecting below the x- axis represent decreases in methylation (hypomethylation) while all those above the x-axis represent increases in methylation (hypermethylation). All 100% hypomethylations are considered to be significant, and only the hypermethylations and partial hypomethylations that were statistically significantly different from control values (Student’s t-test, p<0.05) are depicted.

15 Supplemental Figure 5a. GC-Rich Regions of Altered Methylation: Comparison of DMBA-Initiated, 27mg CSC-promoted (4wks) Tissue to Control (DMBA/Acetone) DigestHypomethylated RAMHypermethylated RAMTOTAL RAM HpaII 123 MspI 033 PCR Product Size (bp) % DMBA/Acetone Control

16 Supplemental Figure 5b. GC-Rich Regions of Altered Methylation (New Methylations Only): Comparison of DMBA-Initiated, 27mg CSC-promoted (4wks) Tissue to Control (DMBA/Acetone) PCR Product Size (bp) Peak Area (Peak Area Units) DigestNew Methylations HpaII 2 MspI 13

17 Legend - Supplemental Figure 5. GC-Rich Regions of Altered Methylation: Comparison of DMBA-Initiated, 27mg CSC-promoted (4wks) Tissue to Control (DMBA/Acetone). RsaI/HpaII (blue symbols) and RsaI/MspI (red symbols) digestion and subsequent AP-PCR was performed on DNA isolated from SENCAR mouse skin treated with 27mg CSC for 4wks. Regions of hypomethylation and hypermethylation are shown (a). Regions of new methylation resulting from treatment are shown in terms of the peak area for each PCR product size (b). A table tallying the regions of altered methylation for each treatment are shown as an inset in each figure. Regions of hypo-, hyper-, and new methylation are expressed in terms of the treated mean for each PCR product size as a percent of the control mean for each PCR product size. All changes projecting below the x- axis represent decreases in methylation (hypomethylation) while all those above the x-axis represent increases in methylation (hypermethylation). All 100% hypomethylations are considered to be significant, and only the hypermethylations and partial hypomethylations that were statistically significantly different from control values (Student’s t-test, p<0.05) are depicted.

18 Supplemental Figure 6a. GC-Rich Regions of Altered Methylation: Comparison of Tumor Tissue to Control (DMBA/Acetone) DigestHypomethylated RAMHypermethylated RAMTOTAL RAM HpaII 190 MspI 314 PCR Product Size (bp) % DMBA/Acetone Control

19 Supplemental Figure 6b. GC-Rich Regions of Altered Methylation (New Methylations Only): Comparison of Tumor Tissue to Control (DMBA/Acetone) PCR Product Size (bp) Peak Area (Peak Area Units) DigestNew Methylations HpaII 3 MspI 11

20 Legend - Supplemental Figure 6. GC-Rich Regions of Altered Methylation: Comparison of Tumor Tissue to Control (DMBA/Acetone). RsaI/HpaII (blue symbols) and RsaI/MspI (red symbols) digestion and subsequent AP-PCR was performed on DNA isolated from tumors resulting from treatment with CSC. Regions of hypomethylation and hypermethylation are shown (a). Regions of new methylation identified are shown in terms of the peak area for each PCR product size (b). A table tallying the regions of altered methylation for each treatment are shown as an inset in each figure. Regions of hypo-, hyper-, and new methylation are expressed in terms of the treated mean for each PCR product size as a percent of the control mean for each PCR product size. All changes projecting below the x- axis represent decreases in methylation (hypomethylation) while all those above the x-axis represent increases in methylation (hypermethylation). All 100% hypomethylations are considered to be significant, and only the hypermethylations and partial hypomethylations that were statistically significantly different from control values (Student’s t-test, p<0.05) are depicted.

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