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. 2015 Feb;29(2):494-7.
doi: 10.1038/leu.2014.285. Epub 2014 Sep 24.

MARIMO cells harbor a CALR mutation but are not dependent on JAK2/STAT5 signaling

K Kollmann  1 , J Nangalia  2 , W Warsch  1 , H Quentmeier  3 , A Bench  4 , E Boyd  4 , M Scott  4 , H G Drexler  3 , A R Green  5
Affiliations
Free PMC article

MARIMO cells harbor a CALR mutation but are not dependent on JAK2/STAT5 signaling

K Kollmann et al. Leukemia. 2015 Feb.
Free PMC article
No abstract available

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Figures

Figure 1
Figure 1
Identification of a CALR-mutated human cell line. (a) Top panel shows the mutated region in CALR exon 9 (red bases). The commonest CALR mutations are shown above the DNA sequence. Solid gray line shows type 1 (52- bp deletion; c.1099_1150del; L367fs*46) and gray arrow shows type 2 (5- bp insertion; c.1154_1155_ins; K385fs*47 mutations). The CALR mutation in human cell line MARIMO is shown below the DNA sequence. Solid red line and capillary sequencing image show a heterozygous 61-bp deletion (c.1099_1159del; L367fs*43) in MARIMO. Dashed gray and red lines represent the homologous sequence flanking the deleted regions in type 1 and MARIMO mutations, respectively, also highlighted in the capillary sequencing image (pale blue) for MARIMO. Lower panel shows the predicted protein sequence of the commonest CALR mutations and of MARIMO with total protein sizes. Amino acids (AA) in the new reading frame are shaded blue and the common novel peptide sequence shared by the different CALR variants are in bold blue. (b) PCR amplification of CALR exon 9 followed by fragment size analysis, as used for diagnostic testing for CALR mutations. Vertical heights of peaks represent dye signal intensity and horizontal position of peaks reflect the fragment size of the PCR amplicon. Wild type (wt) peak occurs at 132- bp. Left panel shows wt and mutated alleles of MARIMO (61-bp separation in peaks), middle panel shows Type 1/L367fs*46 with peak separation of 52 bp and right panel shows Type 2/K385fs*47 peaks separated by 5  bp. (c) Agarose gel image showing wt (upper band) and mutated-CALR (lower band) in MARIMO DNA and cDNA. (d) Quantitative real-time PCR of total CALR mRNA levels expressed as a fold change relative to house-keeping RPLP0 levels, for the cell lines MARIMO, the BCR-ABL1+ CML cell line K562, and the JAK2V617F+ cell lines HEL, UKE-1 and SET-2. Graph depicts all data points generated in two independent experiments performed in duplicate. ***P<0.001 (e) Western blot showing total CALR protein levels of MARIMO and four other myeloid cell lines.
Figure 2
Figure 2
Characterization of the cell line MARIMO. (a and b) Basal cytoplasmic calcium level (a) and changes in cytoplasmic calcium levels over time upon addition of thapsigargin (b) in MARIMO and five other leukemic cell lines. (c) Dose response curves for the JAK2-inhibitor Ruxolitinib in the cell lines MARIMO, K562, HEL and UKE-1. (d and e) Western blots showing protein levels of the inactive and phosphorylated forms of JAK2 and STAT5 (d) and STAT1 and STAT3 (e). PC, positive control. (f) JAK2 mRNA levels expressed as fold changes relative to RPLP0 in MARIMO and four myeloid cell lines.
See this image and copyright information in PMC

References

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