Supplemental Figure Legends
Sup. Fig. 1. Construct maps of Mcl-1 genomic locus and southern blots. (a) Schematic
map of Mcl-1 genomic locus. Sac I restriction sites are indicated by (S). External probe
for Southern blotting is indicated by box. Lengths of subsequent restriction fragments
detected by 5' external probe are noted under bars beneath the Mcl-1 loci. (b) Schematic
map of Mcl-1 genomic locus. EcoR1 restriction sites are indicated by (E). External
probe for Southern blotting is indicated by box.
Lengths of subsequent restriction
fragments detected by 3' external probe are noted under bars beneath the Mcl-1 loci. (c)
Southern blot of genomic mouse DNA digested with Sac I and probed with a 5' external
probe. (d) Southern blot of genomic mouse DNA digested with EcoR1 and probed and
with a 3' external probe..
Sup. Fig. 2. Peripheral lymphoid flow cytometric data. (a) Spleen and lymph node cells
from Mcl-1flox/wt (f/wt), and Mcl-1flox/null (f/null) plus Lck-Cre animals were stained for
expression of CD3+ and B220+ lymphocytes. Numbers denote the percentage of cells
within the quadrant. (b) Spleen and lymph node cells from f/wt and f/null plus Lck-Cre
animals were gated on CD3+ lymphocytes and analyzed for expression of CD4 and CD8
co-receptors to determine the ratios of T lymphocyte lineages. Numbers denote the
percentage of cells within the quadrant. (c) Lymph node cells from f/wt, f/null, LckCref/wt and Lck-Cref/null mice were harvested were analyzed by flow cytometry to
determine the percentage of total T lymphocytes (CD3+). Total cell numbers were
calculated by applying the percentage to the total organ cellularity. Total T lymphocytes
were further subfractionated into helper T (CD4+) and cytotoxic T (CD8+) lineages. Cell
numbers are from a cohort of 3 littermate animals and error bars denote the standard error
of the mean (SEM). (d) Spleen and lymph node cells from f/wt and CD19-Cref/null
animals were stained for expression of CD3 and B220 on lymphocytes. Numbers denote
the percentage of cells within the quadrant.
(e) Lymph node cells from f/wt, f/null,
CD19-Cref/wt and CD19-Cref/null mice were counted and analyzed by flow cytometry to
determine the total numbers of B220+ cells in the peripheral lymphoid organs. Cell
numbers are from a cohort of 3 littermates and error bars denote the SEM.
Sup. Fig 3. (a) Lysates of thymocytes were separated by SDS-PAGE and immunoblots
developed for MCL-1 and actin. (b) Analysis of T cell thymic development. Thymic
profile of CD4 and CD8 expression from f/wt and Lck-Cref/null thymi. Numbers denote
percentage of thymocytes in each quadrant. Plots are representative of more than 10
independent analysis. (c) Subsets of CD4-CD8- double negative (DN) thymocytes were
determined by flow cytometry in which the percentage of DN1 (upper left, CD44+CD25), DN2 (upper right, CD44+CD25+), DN3 (lower right, CD44-CD25+), and DN4 (lower
left, CD44-CD25-) thymocytes from f/wt and Lck-Cref/null, genotypes are presented.
Sup. Fig. 4. Analysis of B cell development using cell surface marker staining of bone
marrow from f/wt and CD19-Cref/null mice.
B cell progenitors are fractionated
accordingly: CD4+B220+ cells are subfraction A (Pre-Pro-B), CD127+B220+ subfraction
B (Pro-B cells), BP-1+B220+ subfraction C (Pro-B), CD25+B220+ subfraction C' (Pro-B),
and CD43-B220+ subfraction D (Pre-B) stages. Box indicates subfraction population and
number denotes the percentage of BM lymphoid cells in each population. Plots are
representative of 5 independent analyses.
Sup. Fig. 5. MCL-1 expression in peripheral lymphocytes. MCL-1 protein levels in
thymocytes (Thy), purified splenic T (CD4+ and CD8+), and B (B220+) lymphocyte
populations as determined by immunoblot. Actin represents a loading control.