DEV020743 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S4 (Adobe PDF) -

  Table S4. Segregation analysis of wee1 and atm mutant alleles amongst csn mutant progeny of csn/CSN wee1/Wee and csn/CSN atm/ATM parents, respectively.

• Supplemental Table S5 (Adobe PDF) -

  Table S5. Results of the flow cytometric analysis of wee1 and atm mutant alleles amongst csn mutant progeny of csn/CSN wee1/Wee and csn/CSN atm/ATM parents.

• Supplemental Figure S1 -

  Fig. S1. RT-PCR expression analysis of cell cycle regulatory and DNA damage response genes. RT-PCR amplification of two G2 and M phase-specific transcripts (CYCA2;2 and CYCB1;1, upper panel) and three DNA damage response genes (BRCA1, PARP1, RAD51, middle panel). ACTIN served as a loading control (lower panel). PCR cycles as indicated below each panel. For primer sequences, see Table 1.

• Supplemental Figure S2 -

  Fig. S2. Immunostaining with the cytokinesis marker KNOLLE suggests normal cytokinesis and cell divisions in csn mutants. (A-J) Immunostaining of wild-type and csn mutant root tips stained for the cytokinesis marker KN (green). The root tips were counterstained using an anti-TUBULIN antibody (red) and the DNA stain DAPI (blue). (A,C,E,G,I) Root tip; (B,D,F,H,J), magnification of a cytokinetic cell. Two- and 5-day-old wild-type and 7-day-old csn mutant seedlings were used. Scale bars: 500 µm for left panels; 50 µm for right panels.

• Supplemental Figure S3 -

  Fig. S3. Genetic interaction between CSN and the DNA damage response regulatory protein kinases ATM and WEE1. (A-G) Seven-day-old atm-2, wee1-2, atm-2 csn and wee1-2 csn mutant seedlings, with the genotypes as indicated. For the phenotypes of the csn single mutants, see Fig. 1A. The atm-2 and wee1-2 mutants are indistinguishable from the wild type at the seedling stage. Scale bars: 4 mm for A,B; 1 mm for C-G. (H,I) FACS analyses of root tips from atm-2, wee1-2, csn4, atm-2 csn4 and wee1-2 csn4 seedlings as specified. 2C denotes the DNA content of cells in G0/G1 phase, 4C denotes cells that have passed S phase, 8C and 16C denote endoreduplicated cells (see Table S5 for quantitative data).

• Supplemental Figure S4 -

  Fig. S4. DNA damage also occurs in the neddylation-deficient axr1 mutants and in mutants of E3 ligase subunits. (A-F) Confocal images of primary roots of 7-day-old dark-grown wild-type and mutant seedlings, with the genotypes as indicated, after the TUNEL assay (left panel). Roots were counterstained with DAPI (right panel). The experiment was repeated twice and a representative image from one experiment is shown. Scale bars: 1 mm. (G-L) Flow cytometric analysis of wild-type and mutant seedlings. 2C denotes the normal diploid DNA content of cells in G0/G1 phase, 4C denotes cells that have passed S phase, 8C and 16C denote endoreduplicated cells (see Table S3 for quantitative data).

• Supplemental Table S1 -

  Table S1. List of cell cycle genes. (A) List of the 57 cell cycle genes as defined in Menges et al. (Menges et al., 2005). (B) Cell cycle genes that are differentially expressed in the csn mutant experiment. (C) Eight cell cycle genes that are 2-fold induced or repressed in all three csn mutants.

• Supplemental Table S2 -

  Table S2. Expression of DNA repair genes in csn mutants. (A) Annotated list of DNA repair genes as listed in www.uea.ac.uk/∼b270/repair.htm. (B) DNA repair genes represented on the ATH1 GeneChip. (C) DNA repair genes that are differentially expressed in response to DNA damage and their expression in csn mutants.

• Supplemental Table S3 -

  Table S3. Results of the flow cytometric analysis for data presented in Fig. 3 and Fig S4. (A) Raw data of three replicate measurements. (B) Average of the data shown in A.