Supplemental Figure 1: Additional phenotype of the immune system in BI-1 KO
splenocytes. (a) Total cell number in the thymus, bone marrow (BM), peritoneal cavity (PC)
and spleen are similar in WT and BI-1 KO mice. Bar graphs represent the average cell
number as the mean ± SD from 12 mice. (b) The relative fractions of bone marrow cell types
do not differ between WT and BI-1 KO mice. Bar graphs represent the relative percentage of
segment cells (Seg), erythroblasts (EB), normoblasts (NB), lymphocytes (Lym), myelocyte
promyelocytes (My), promyelocytes (Pro), eosinophils (Eos) and plasma cells (P) as the
mean ± SD from n = 3 mice (c) Percentages of pro/pre, immature as well as mature
recirculating B cells do not differ between WT and BI-1 KO mice. Bone marrow cells were
stained withIgM andB220 and gated for IgM+/B220- (pro/pre B cells), IgM+/B220+ (immature
B cells) and IgM++/B220+ (mature recirculating B cells). Dot plots show representative gates
from one WT and one BI-1 KO mouse. Bar graphs represent the average percentage of the
indicated cell types as the mean ± SEM from two independent experiments. (d) The
percentages of CD4 SP (single positive), CD8 SP and CD4CD8 DP (double positive) cells in
the thymus are similar in WT and BI-1 KO mice. Thymus cells were stained withCD4
andCD8 and gated accordingly with a flow cytometer. The dot plots show representative
gates for one WT and one BI-1 KO mouse. Bar graphs show the average percentage of
CD4+ and CD8+ as the mean ± SEM from 3 versus 3 mice. Data was analyzed by two-tailed
t-tests, *P < 0.05.
Supplemental Figure 2: No changes in the expression levels of class I NF-κB, IKKs
and ubiquitination. (a) No changes in p100/p52 and p105/p50 abundance in cytosolic and
nuclear fractions and (b) IKKα, IKKβ and IKKγ in unfractionated lysates of BI-1 KO
splenocytes compared to WT splenocytes. Immunoblots were incubated with the indicated
antibodies. α/β Tubulin served as loading control for the cytosolic fraction, HDAC for the
nuclear fraction, and Actin for whole cell lysates. Molecular weight is indicated. Bar graphs
show mean intensity values normalized to WT ± SD from six mice. (c) Similar level of
ubiquitination in WT and BI-1 KO splenocytes. Immunoblots were incubated with a primary -
poly-ubiquitin antibody. GAPDH served as loading control on the same blot and size is
indicated. Bar graphs show mean intensity values normalized to WT ± SD from six mice.
Data were analyzed by two-tailed t-tests, *P < 0.05.
Supplemental Figure 3: No evidence of ER stress or altered autophagy in BI-1 KO
splenocytes immediately after preparation. (a) No upregulation of ER chaperones and ER
folding proteins in BI-1 KO splenocytes. ERp57 is significantly downregulated. Immunoblots
were incubated with the indicated antibodies. Actin served as loading control on the same
blot and size is indicated. Bar graphs show mean intensity values normalized to WT ± SD
from six mice. (b) Decreased XBP-1 splicing in BI-1 KO splenocytes analyzed by PCR and
visualized on an agarose gel. Band intensity was normalized to the expression of the
housekeeping gene hprt. Bar graphs show the mean percentage ± SD of spliced XBP-1 from
six mice. (c) The autophagy marker ATG12 is significantly upregulated in kidney tissue from
BI-1 KO mice but not in splenocytes or liver. Immunoblots were incubated with an antibody
against ATG12. Actin served as a loading control on the same blot and size is indicated. Bar
graphs show the relative expression of ATG12 normalized to its expression in WT mice as
mean ± SEM from six mice. Data were analyzed by two-tailed t-tests, *P < 0.05.
Supplemental Figure 4: Ultrastructural changes in BI-1 KO B cells. Representative
electron-microscopic images of magnetically sorted and negatively selected WT and BI-1 KO
B cells shows an altered texture of the cytoplasm (shaded in blue), defective organelles
(shaded in blue) and presence of nuclear inclusions (shaded in red) in BI-1 KO B cells.
Magnification is indicated.