TAL1/SCL is down-regulated upon histone deacetylase
inhibition in T-cell acute lymphoblastic leukemia cells
Bruno A. Cardoso, Sérgio F. de Almeida, Angelo B. A. Laranjeira, M. CarmoFonseca, J. Andrés Yunes, Paul J. Coffer and João T. Barata
Supplementary figure legends S1-S8
Figure S1. HDACi-mediated down-regulation of TAL1 mRNA is not due to
increased apoptosis in TAL1wt T-ALL cell lines. Jurkat cells were treated for 24h with
5mM SB, 20µM QVD-OPH or both. RNA was extracted and TAL1 transcripts analyzed
by quantitative-RT-PCR. TAL1 mRNA levels were normalized to the levels of the
GAPDH housekeeping gene and normalized again to medium control condition. Values
indicate mean ± standard deviation of duplicates. The data is representative of two
independent experiments.
Figure S2. HDACi up-regulates CDKN1A/p21 mRNA expression. Jurkat cells were
treated with 5mM SB for 24h. The mRNA levels of CDKN1A/p21 were analyzed by
quantitative-RT-PCR, and normalized to GAPDH and to medium control condition.
Values indicate mean ± standard deviation of duplicates (* p<0.05). The data is
representative of two independent experiments.
Figure S3. Schematic representation of TAL1 locus and the primers used to detect
total and processed TAL1 mRNA. (A) TAL1 locus is schematic summarized. TAL1
exons are represented as boxes. White boxes depict untranslated exons or regions, while
the black boxes show the translated exons and regions (adapted from 10). The SIL locus
is located at the 5’ and the MAP17 locus is located at the 3’ end of the TAL1 locus. (B)
Primers that detect total TAL1 mRNA were designed to bind exclusively within exon 5
in order to detect both the processed and unprocessed transcripts. Primers that detect
processed TAL1 mRNA bind within exon 5 and in the exon-exon boundary between
exons 5 and 6, allowing the detection of TAL1 mRNA only after splicing – when exon 5
and 6 are juxtaposed.
Figure S4. TAL1d-expressing T-ALL cells up-regulated TAL1 mRNA upon HDACi
treatment. (A) Structural comparison of TAL1wt (left panel) and TAL1d (right panel)
locus (Adapted from
10
). The SIL locus is located at the 5’ and the MAP17 locus is
located at the 3’ end of the TAL1 locus. Exons are represented as boxes. The white
boxes represent exons or regions that are not translated, while the black boxes show
exons or regions that are translated. The grey box indicates the SIL exon that is placed
in the TAL1 locus upon the Tal1d deletion. The arrows represent the TAL1 promoters
(Pr.) (B) Jurkat and PF382 cells were treated with 5mM SB or 10µM SAHA, RNA was
extracted and TAL1 and SIL-TAL1 transcripts analyzed by quantitative-RT-PCR. TAL1
and SIL-TAL1 mRNA levels were normalized to the levels of the GAPDH housekeeping
gene and depicted as relative values to the medium control condition. Values represent
mean ± standard deviation of duplicates (** p<0.01). The data is representative of two
independent experiments.
Figure S5. HDACi induce T-ALL cell death. T-ALL cells were treated with HDACi
and cellular viability was determined as described in “Materials and Methods. Viability
index represents the viability values normalized to the viability in medium control
condition. (A) Primary T-ALL cells (n=3) were treated for 24h in the indicated
concentrations of SB and SAHA. (B) Jurkat, PF382 and SupT1 cells were treated for
24h with the indicated concentrations of SB or TSA. Values indicate the mean ±
standard deviation of duplicates. The data is representative of at least two independent
experiments (* p<0.05; ** p<0.01; *** p<0.001).
Figure S6. Enforced TAL1 expression partially rescues HDACi-mediated apoptosis
of Jurkat cells. Jurkat cells were transduced with empty or TAL1 expressing
lentiviruses and incubated for 24h with the indicated concentrations of SB. Viability
was determined by flow cytometry analysis of FSC/SSC distribution. Viability index
represents the viability values normalized to the viability in medium control condition.
Values indicate the mean ± standard deviation of triplicates (*** p<0.001).
Figure S7. Model for HDACi-mediated TAL1 down-regulation in T-ALL cells. In
T-ALL cells that contain an intact TAL1 locus (TAL1wt), treatment with HDACi impairs
TAL1 gene transcription by displacing RNA polymerase II from TAL1 native promoters.
In T-ALL cells that contain the TAL1d allele and consequently express TAL1 under the
control of the SIL promoter, HDACi do not negatively affect TAL1 gene transcription,
(on the contrary, they appear to up-regulate it) but shut down TAL1 protein translation.
Both mechanisms lead to the same outcome: down-regulation of TAL1 protein
expression in HDACi-treated T-ALL cells.
Figure S8. TAL1 expression is not affect by inhibition of PI3K and mTOR. PF382
cells were cultured for 24h with 5mM SB, 50µM LY294002 (LY), 200nM Rapamycin
(Rap.) or the indicated combinations. Cells were lysed and the phosphorylation levels of
S6 and Akt/PKB, as well as TAL1 protein expression were analyzed by immunoblot.
Actin was used as loading control.