JCS03412 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Anti-histone-H4 staining of male gut cells. (Upper panels) A patch of gut tissue showing cells in which heterochromatin reversal has not occurred yet. Note the strict nuclear localization of histone H4. Note also that H4 is enriched between euchomatin and heterochomatin. (Bottom panels) Heterochromatin reversal is accompanied by redistribution of histone H4 throughout the nucleus and cytoplasm.

• Supplemental Figure 2 -

  Fig. S2. A patch of female gut tissue immunostained using anti-Me(3)K20H4. The staining is limited to nuclei with no evidence of cytoplasmic staining.

• Supplemental Figure 3 -

  Fig. S3. PCHET2 levels are decreased in whole-cell extracts taken from pchet2-dsRNA-treated embryos. Total protein extracts from pchet2-dsRNA-treated embryos were separated by SDS-PAGE, transferred to nitrocellulose and then probed with an anti-PCHET2 antibody. This showed that PCHET2 protein levels were reduced in the pchet2-dsRNA-treated embryos compared with mock-treated embryos. The loading control α-tubulin was not affected by the treatment.

• Supplemental Figure 4 -

  Fig. S4. Exposure of embryos to pchet1 dsRNA has no affect on the chromocenter (S4’) or on C1A9 staining (S4’’; DAPI in red, C1A9 immunostaining in green). Bar, 10 μm.

• Supplemental Figure 5 -

  Fig. S5. Effect of RNAi of pchet2 on Me(3)K9H3 and Me(3)K20H4 levels. Following the soaking of P. citri embryos in the pchet2 (HP1-like) dsRNA interfering solution, total proteins were extracted, run on SDS-PAGE and blotted to filters. The filters were probed with either anti-Me(3)K9H3 or the anti-Me(3)K20H4 antibodies. The amount of Me(3)K20H4 but not of Me(3)K9H3 is greatly reduced in dsRNA-treated embryos compared with mock-treated ones. α-tubulin was used as loading control. In the blot probed with anti-Me(3)K20H4 antibody, the 11 kDa canonical histone H4 band and also an upper band is apparent, probably corresponding to an acetylated H4 isoform