JCS042861 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Reduction in chorion gene amplification by Cul4KR. The control hs-GAL4 (A) or the dominant-negative Cul4KR driven by hs-GAL4 (B) were treated at 37°C for 30 minutes. Following a 2-hour recovery, ovaries were dissected, incubated with BrdU for one hour and stained for Hoechst (blue) and BrdU antibody (red). BrdU incorporation was greatly reduced at stage 10B egg chambers by acute expression of Cul4KR, as compared to wild type. Cul4KR (K767R) represents a Nedd8 modification defective form of Cul4. Scale bars: 10µm. (C) Quantitative PCR was performed to measure the levels of chorion gene amplification. Stage10B-11 egg chambers were dissected 3 hours after heat treatment. About 10-30 egg chambers were collected for preparation of genomic DNA that serves as DNA templates for quantitative PCR. Data were analyzed by Applied Biosystem sequence detection software v1.4 and shown as mean ± s.e.m. for ten replicas in two experiments. Three genomic regions ACE3, ACE1 and 6C were measured for their DNA contents and the numbers of ACE3 and ACE1 were divided by the non-amplified 6C. The ratios to 6C in non-heat shock sets were normalized to 1 for both ACE3 and ACE1. Primers used were: 5′-CCCATAAGCCATTCACAATTTGT-3′ and 5′- AAACTGCCGCACTAAGCTTTTTATA-3′ for ACE3; 5′- GAAACGCCAAATTCATATTAGAGTTTC-3′ and 5′-GATCTTTAGCTATTCCAATTAGGAATTCA-3′ for ACE1; 5′-TTGTGATGGGCCTCGTATTG-3′ and 5′-CGTGTGCGAGGAGCTCAAG-3′ for 6C.

• Supplemental Figure S2 -

  Fig. S2. Follicle cells in stage 10B carrying homozygous mutant clones for Cul4G1-3 showing GFP signal (A1, B1), Orc2 staining (A2, B2), BrdU incorporation (A3, B3) and nuclear Hoechst 33342 signals (A4, B4) and merge for Orc2 and BrdU signals in green and red, respectively (A5, B5). Most Cul4 mutant cells with ectopic genomic replication (arrows in A3, B3) represent diminished Orc2 focal signals (arrows in A2, B2). In some cases, ectopic genomic replication or reduced BrdU incorporation is present in mutant follicle cells with normal Orc2 focal signals (hollow arrows in A and arrowheads in B, respectively), uncoupling both replication defects with Pre-RC formation.