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Blood, Vol. 104, Issue 13, 3927-3935, December 15, 2004
Telomere dynamics in Fancg-deficient mouse and human cells
Blood Franco et al.
104: 3927
Supplemental materials for: Franco et al, Vol 104, Issue 13, 3927-3935
Files in this Data Supplement:
- Table S1. Chromosomal aberrations of wild-type and Fancg-/- cells exposed to clastogens (MMC,
 -radiation). (PDF, 256 KB)
- Figure S1. Cytogenetic analysis of clastogen-treated Fancg−/− cells. (PDF, 124 KB) -
DAPI-stained metaphases were prepared from lymphocytes treated with MMC (A-B) or γ-radiation (C-D), BMCs treated with -radiation (E-F), and MEFs treated with MMC (G-H) or -radiation (I-J). For each treatment, the percentage of metaphases with cytogenetic aberrations is shown on the left graph (A,C,E,G,I) and the number of aberrations per metaphase on the right graph (B,D,F,H,J). Twenty-five metaphases per littermate mice were analyzed.
- Figure S2. Telomerase activity and ALT-associated PML bodies in Fancg-deficient cells. (PDF, 52 KB) -
(A) Telomerase activity in Fancg+/+ and Fancg−/− splenocytes measured by the TRAP assay. For each sample, 3 µg, 1 µg, and 3 µg treated with RNAse was analyzed. IC indicates internal PCR control. (B) Indirect immunofluorescence of PML reveals grossly similar number, size, and distribution of PML foci (green dots) in Fancg+/+ (top panels) and Fancg−/− (left panels) B cells. Nuclei are counterstained with DAPI (blue). Pictures are representative samples of cytospin preparations of splenocytes grown in LPS for 72 hours. (C) After PML immunofluorescence (green), cells were hybridized with a Cy3-labeled telomeric PNA probe (red) and no colocalization was observed in either WT or Fancg−/− cells, suggesting that recombination-based telomere maintenance is not activated in Fancg−/− lymphoid cells.
- Figure S3. Telomere dynamics of Fancg−/− germ cells. (PDF, 309 KB) -
(A-E) Q-FISH assay on paraffin-embedded 4 µm testis sections hybridized with a Cy3-labeled telomere specific probe (yellow) and counterstained with DAPI (blue). No differences in telomere fluorescence were observed between Fancg+/− (normal size and meiotic progression [A]) and Fancg−/− (reduced testis size, degenerating tubules [B]) testis. Telomere fluorescence of more than 100 pachytene nuclei per mouse was quantified, including those close to degenerative areas (example shown in panel C; pachytene nuclei, red arrows; degenerative area, white arrow). (D) A hematoxylin and eosin stain of the same testis is shown for comparison. Note hypocellularity and abnormal meiotic progression. (E) Telomere fluorescence intensity of individual nuclei was plotted in frequency histograms for Fancg+/− and Fancg−/− pachytene meiocytes; note no significant differences in either average length or length distribution. (F) Nuclei were obtained from organs with high (testis) or low (liver, kidney) level of expression of Fancg. Telomere restriction fragments showed no significant differences in size between genotypes in any tissue. (G) Telomerase activity of organs with high (testis) or low (liver, kidney) level of expression of Fancg. Fancg−/− testis S-100 extracts contained telomerase activity similar to Fancg+/+.
- Figure S4. Telomere dynamics of Fancg−/− MEFs. (PDF, 299 KB) -
(A) Telomere restriction fragments of 4 Fancg+/+ and 4 Fancg−/− MEF cultures. Cells were harvested at passage 2 (P2) and telomeric DNA resolved by pulse-field electrophoresis and hybridized with a 32P-labeled telomeric probe. Molecular weight markers (kbp) are indicated to the left. (B) Q-FISH analysis of 2 wild-type MEF cultures and 2 Fancg−/− MEF cultures. Fluorescence intensity values of individual telomeres were plotted in frequency histograms (10 metaphases per embryo). Average telomere length and length distribution were similar for both genotypes. (C-J) Representative chromosomal aberrations in Fancg−/− MEF metaphases hybridized with a Cy3-labeled telomeric PNA probe (yellow) and counterstained with DAPI (blue) after treatment with clastogenic agents. (C, D) Representative microphotographs of Fancg−/− chromosomes after treatment with MMC 15 ng/mL for 48 hours. Characteristic aberrations include fusions between broken chromatids (C) and quatriradials (D), which are formed by fusions between the 4 broken chromatids of 2 nonhomologous chromosomes. Note the complete absence of telomeric signals at fusion points in all examples, suggesting that telomeres are properly capped in Fancg-deficient cells. (E-J) Representative microphotographs of Fancg−/− chromosomes after treatment with -radiation (4 Gy). Aberrations involving both chromatids ("chromosome-type") predominated (chromosome fusions with [E] and without [F] centromeres, minichromosome [G], and ring [H] are shown as examples and were not different from those observed in Fancg+/+ cells). A small number of chromatid breaks (I) and fusions (J) were also induced by -radiation in Fancg−/− MEFs but not in control Fancg+/− MEFs. (K) Telomerase activity is readily detected in both Fancg+/+ and Fancg−/− MEFs with the TRAP assay. -Irradiation (2 or 4 Gy) elicited significant cytogenetic instability in both genotypes but no measurable concomitant change in telomerase activity in either.
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