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. 2014 May 15;7(8):963–976. doi: 10.1242/dmm.014449

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© 2014. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 3. — Fibroblasts show reduced proliferation, a senescence-like phenotype and reduced hypusine modification upon complete loss of DOHH expression. (A) Genotypes of 4-OHT-induced and uninduced cells, as well as a Cre-negative control cell line revealed the genomic deletion of both Dohh alleles in Dohh^flox/flox; Cre^pos. 3T3 cells. Primers are indicated in supplementary material Table S2. (B) By using real-time RT-PCR, a decline of the Dohh transcript was observed in Dohh^flox/flox; Cre^pos. cells relative to Gapdh and a Cre-negative control cell line (Dohh^flox/flox; Cre^neg). In parallel, expression of eIF5A was upregulated at the RNA level, most likely to compensate for the loss of DOHH. (C) Western blot analysis of DOHH in 4-OHT-induced and uninduced cells, as well as a Cre-negative control cell line revealed the reduction of DOHH protein expression in Dohh^flox/flox; Cre^pos. 3T3 cells after deletion of Dohh compared with Dohh^flox/flox; Cre^neg cells. Tubulin served as an internal standard. (D) Growth inhibition of Dohh^flox/flox; Cre^pos. fibroblasts after 4-OHT induction, relative to a Dohh^flox/flox; Cre^neg. control cell line. Level of significance was analysed using one-way ANOVA with Dunnett’s multiple comparison tests. (E) SA-β-Gal-positive cells after 1 week of 4-OHT induction, compared with a CreEsr1-negative control cell line. Level of significance was analysed using two-way ANOVA with Bonferroni post-tests. (F) Representative light microscopic images of cells of the experiments analysed in E, showing enlarged SA-β-Gal-positive cells after knock out of Dohh. (G) 2D western blots against eIF5A and GAPDH of fibroblast lysates treated as indicated on the panel depict a shift of a proportion of eIF5A from pH 5.2 (green arrow) to 5.1 (red arrow) after 4-OHT induction and a shift from pH 5.2 to 5.1 and 5.0 (yellow arrow), indicating a reduced hypusine modification. (H) Network function and gene ontology analysis of differentially expressed genes in Affymetrix microarrays after deletion of Dohh by 4-OHT in 3T3 Dohh^flox/flox; Cre^pos. cells versus 3T3 Dohh^flox/flox; Cre^neg. control cells. (I) Quantititive RT-PCR for some differentially expressed genes identified in the microarray analyses. qRT-PCR was performed in triplicates with Gapdh as a housekeeping control gene. ***P<0.001, **P<0.01.