Supplementary Text
Deregulation and crosstalk among Sonic hedgehog, Wnt, Hox
and Notch signaling in chronic myeloid leukemia progression
Primary Cells, Cell lines, Cell Culture Conditions and Nucleofection
Bone marrow and peripheral blood samples were obtained from CML patients
suffering in various phases of the disease after informed consent and strictly
following institutional ethical guidance. Conventional cytogenetics and BCR-ABL
specific qRT-PCR for both b3a2 and b2a2 transcripts were done for confirmation.
Characteristics of individual CP/BP CML patient samples are provided in the
Supplementary Table. 1. We considered chronic phase (CP) as with < 10% of
blast cells and blast crisis (BP) having > 30% blasts in peripheral blood and >
50% blasts plus promyelocytes in bone marrow. CD34+ progenitor cells were
subsequently enriched by immunoaffinity selection (MACS CD34 Cell Isolation
kit, Miltenyi Biotec). Preparations were > 90% viable BCR-ABL+ leukemic blasts.
In few cases, bone marrow specimens were collected from CP CML patients
after withdrawal from chemotherapy.
All primary cells were cultured in IMDM (Iscove’s Modified Dulbecco
Medium, Stem Cell Technologies, Canada) supplemented with 10% fetal calf
serum (FCS), 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM GlutaMAX
(Invitrogen/Life Technologies, USA). All cytokines (SCF, Flt3 ligand and IL-3) and
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recombinant N-Shh and Wnt3a (All purchased from R&D systems, USA) were
dissolved in 0.2μ filtered 1X Dulbecco PBS, pH 7.4, containing 0.1% Bovine
serum albumin (BSA) and were finally used as mentioned in the text. Freshly
isolated CD34+ primary cells were washed twice with IMDM supplemented with
2% FCS and 5x105 cells were nucleofected (Program; U-008, AMAXA
Biosystems, USA) by taking 5 μg of respective purified plasmid DNA (Qiatip endo
free maxyprep kit, Qiagen Inc, USA) for every 100 μl of cells suspended in the
supplemented nucleofector solution (for detailed protocols please see: Optimized
Protocol Human CD34+ Cell Nucleofection Kit and General Protocol for
Nucleofection of Suspension Cells; Catalog No. VPA-1003 and DLA-1002,
respectively, AMAXA Biosystems, USA).
Real-time Quantitative RT-PCR (qRT-PCR) for self-renewal associated genes
RNA has been isolated from bone marrow samples of each CP CML patients and
from either bone marrow or peripheral blood of individual BP CML patients as
shown in the Supplementary Table 1. However, there are only four cases (CML1,
CML7, CML8 and CML20) where RNA has been simultaneously isolated from
bone marrow and peripheral blood of CML patients in order to compare the
plausible discrepancy arising from differential sampling. Total RNA was extracted
from CD34+ cells using Tripure isolation reagent (TRIZOL, Roche) and
subsequently treated with RNAse free DNAse (DNAfreeTM Ambion, USA) and
quality assayed using RNA gel electrophoresis as well as spectroscopy
(Eppendorf BioPhotometer, Germany). Isolated RNA were then reverse
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transcribed for 30 minutes at 48ºC using random hexamer or oligo (dT) primers
and Multiscribe reverse transcriptase (Taqman reverse transcriptase reagents,
Applied Biosystems, USA). Quantitative PCR was subsequently performed using
SYBR Green core PCR reagents (Applied Biosystems, USA) and HPRT1 was
used as the endogenous control. Amplification was performed with 40 cycles of
two-step PCR (15s at 95ºC and 60s at 60ºC) after initial denaturation (95ºC for
10 min) using an ABI 7500 Sequence Detector System (Applied Biosystems,
USA). All the gene specific primers that we used in the study, was strictly exon
specific and intron-spanning in order to exclude amplification of potential
genomic contaminants in the RNA extracts. Sequences of all primer pairs used in
the study are provided in the Supplementary Table 2.
Relative quantitation (RQ) was calculated from the 2-ΔΔCt values of
respective samples (Applied Biosystems, USA). ΔCt=Ct (Target Gene) -Ct
(HPRT1). In addition, ΔΔCt=ΔCt (Test sample)-ΔCt (Calibrator sample). The
experimental control sample or the sample having the lowest ΔC t value was
considered as the calibrator. Moreover, unit change in ΔCt value represents twofold change in the RNA, which has also been considered for determining
standard deviation (SD) and standard error of the mean (±SEM). Expression
profile of self-renewal associated genes in CD34+ CP and BP CML cells is
described in the Supplementary Table 3. Again for the ∆∆Ct calculation to be
valid, the efficiency of the target amplification and that of the reference must be
approximately equal. This has been validated for all the respective amplicons by
monitoring at how ∆Ct varies with template dilution. Briefly, input amount of total
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RNA was serially diluted (100.0 ng, 50.0 ng, 25.0 ng, 10.0 ng, 5.0 ng, 2.0 ng and
1.0 ng) and subjected for qRT-PCR analysis as described above using all 24
pairs of gene specific primers. Following the dilution series ΔCt values were
independently calculated for 24 amplicons using HPRT1 as the reference gene.
Finally log input amount versus ΔCt was plotted (24 independent plots) and
slopes were calculated from the equation y=mx+c, where m is the slope and c is
the intercept. For all 24 amplicons slopes were ranging from 0.00 to 0.10 (0.000.10). Notably, if the efficiencies of the two amplicons (target and reference) are
approximately equal, the slope has a value of approximately zero. Therefore
∆∆Ct
calculation
was
considered
appropriate
for
subsequent
analysis.
Furthermore extensive standardization of PCR reactions has been initially
performed through melting curve analysis of respective amplicons in order to
minimize the possibility of primer pair formation.
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