Fig 1.
(A) Mastomys coucha keratinocytes at passage 6. (B) Kera5 at passage 175, showing a typical cobblestone phenotype and an increased cell size (arrowheads mark ongoing mitoses; magnification: 200x).
Fig 2.
Kera5, MaFi132, 308 and NIH 3T3 cells were grown on cover slips, fixed with acetone, stained with keratin 14 or vimentin specific antibodies and detected with AlexaFluor488 or AlexaFluor594-conjugated secondary antibodies, respectively. Murine keratinocytes (308 cells) and fibroblasts (NIH 3T3) were used as controls. Green fluorescence indicates keratin 14 expression only in Kera5 and 308 cells, whereas red fluorescence shows vimentin expression only in MaFi132 and NIH 3T3 (original magnification: 200x).
Fig 3.
Karyotype analysis of Kera5 cells.
(A) Representative metaphases of freshly isolated Mastomys coucha splenocytes in comparison with Kera5 at passage 8 and passage 163. Splenocytes are diploid (n = 36), whereas polyploidy can already be discerned at p8 (average n = 100; ranging from n = 92 to 111). The chromosome set in Kera5 at p163 is smaller (average n = 83; ranging from n = 78 to 86). (B) Chromosomal instability is further demonstrated by chromatid breaks and fragments (black arrowheads), acentric fragments (white arrowheads), rearrangements leading to marker chromosomes (*) and dicentric chromosomes (**). Metaphasic chromosomes were obtained by subsequent block with colcemid, hypotonic treatment and fixation in methanol-acetic acid. Cell suspensions were spread on microscope slides, stained with DAPI and imaged at 630x magnification.
Fig 4.
p53 cDNA sequence and transcriptional analyses.
(A) The relevant nucleotides of the mutated p53 cDNA are shown. At passage 13, an insertion of 19 nt is detectable in the cDNA of p53 (exon 7: green, exon 8: grey, insertion: orange). (B) Comparison of translated wildtype and mutant sequences derived from the cDNA. The insertion leads to a premature stop codon (*) that results in a truncated form of p53. (C) Semi-quantitative RT-PCR of Trp53 at different passages. GAPDH was used as reference gene. (D) Semi-quantitative RT-PCR to detect the insertion within the p53 cDNA, resulting in a slower-migrating PCR product at higher passages (p27-p105). GAPDH was used as reference gene. (E) Western blotting. Left panel: lack of p53 expression in Kera5. Kera5 (p155) were exposed to UVB (40 or 250 mJ/cm2) or treated with adriamycin (1.0 μg/ml) and harvested as indicated. 50 μg protein/lane were loaded. To control the specificity of the p53 antibody, MaFi132 were transiently transfected with pPK-CMV-E3 containing the cDNA for wildtype (p53wt) or the truncated (p53trunc)) form of p53. Since only 25 μg of protein were loaded for transfected MaFi132, a longer exposure time was chosen for actin. Right panel: NIH 3T3 cells were used as reference for stabilization of p53 after cells damage with UV or Adriamycin, respectively. NIH 3T3 cells were exposed to UVB (20 or 100 mJ/cm2) or treated with Adriamycin (0.75 μg/ml) and harvested as indicated. 50 μg protein/lane were loaded. After low dose UVB, p53 stabilization occurs slowly, whereas after high dose UVB the stabilization is fast, but transient. Adriamycin permanently stabilizes p53 already after 7h. Actin was used as loading control.
Fig 5.
Sequencing of the Mastomys p53 gene (Trp53) and partial alignment with mouse and rat sequences.
(A) Sequencing of Trp53 in Kera5 (passages 8 and 103) shows a G>A transition at the first position of intron 7 (underlined). Freshly isolated primary keratinocytes (Kera5, p0) as well as five individual Mastomys samples do not harbor this mutation and are similar to murine and rat sequences at this position. Numbers refer to the sequences of murine Trp53 and rat Tp53. For intron 7, only 5´-start and 3´-ends are shown. Exon 7: green, exon 8: grey, insertion: orange, frames: splicing signals; “a” indicates the original splicing donor, “b” alternative splicing donor signal in intron 7, “c”: splicing acceptor. (B) Sequencing chromatograms of Trp53 reveal the G>A transition in a subpopulation of Kera5 cells at p8 which is not present at p0. A switch of the major peak from G to A from p8 to p103 occurs, suggesting the outgrowth of a single cell colony. At p146 only the A peak is left, revealing homozygosity. The arrows indicate the position of the mutation.
Fig 6.
Calcium-induced differentiation of Kera5.
Kera5 cells at passage 139 were grown on cover slips. After 24 h, the dKSFM (<0.1 mM Ca2+) was additionally supplemented with 0.35 mM, 0.7 mM or 1.05 mM Ca2+ to induce differentiation. After additional 24 h incubation, the cells were fixed with acetone, stained with an involucrin antibody and detected with an AlexaFluor488 secondary antibody, respectively. Green fluorescence indicates elevated involucrin expression in a dose-dependent manner (original magnification: 200x).