Supplementary Material 1

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Appendix
Table of Contents
Appendix Figure legends page 2-4
Appendix Figure S1 page 5
Appendix Figure S2 page 6
Appendix Figure S3 page 7
Appendix Table 1 page 8
Appendix Table 2 page 9

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Appendix Figure S1.
Increased frequency of bone marrow HSPCs in PKCδ-deficient mice.
A. Total BM cell numbers from two femurs and 2 tibias of WT or PKCδ KO mice. Data compiled
from n=14 mice per each genotype.
B. Frequency of Lin-Sca1+ckit+ (LSK) cells, as a percent of live cells, in the fetal liver of embryonic
day 14.5 (e14.5) embryos of mice of the indicated genotype (n=5 embryos analyzed per genotype).
Data represent mean ± SEM., and indicate no statistically significant differences by one-way
ANOVA analysis with subsequent Holm-Sidak’s multiple comparison tests.
C. Quantification of colony formation from total BM cells harvested from WT (n=6) or PKCδ KO
(n=6) mice. Colony forming unit cell (CFUc) frequencies were determined at day 12 after seeding in
methylcellulose medium and presented as number per 20,000 BM cells. Each sample was plated in
duplicate in each experiment. Data are pooled from 3 independent experiments and presented as
mean ± SEM. **p<0.01 by two-tailed Student’s unpaired t-test analysis for comparison of control
and PKCδ KO mice.
D. Schematic of the spleen colony forming unit assay (CFU-s). Bar graph represents the number of
colonies formed per spleen at day 8 (CFU-S8) and day13 (CFU-S13) after transplantation of pooled
total BM (n=2 donor mice for each genotype) into irradiated recipients (n=5 recipients for each
genotype). Data presented as mean ± SEM. **p<0.01 by two-tailed Student’s unpaired t-test
analysis for comparison of control and PKCδ cKO mice. As CFU-S8 reads out primarily oligolineage
progenitor cell activity, whereas CFU-S13 reflects the presence of less differentiated multipotent
progenitors and HSCs (Zhang et al., 2009), these data are consistent with a selective expansion of
the most primitive HSPC subsets in PKCδ KO BM.
E,F!Schematic of competitive transplantation assays using FACS-purified LT-HSCs (E). Percent of
total donor-derived, hematopoietic cells (CD45.2+), B-cells (B220+), myeloid cells (CD11b+Gr1+),
and T cells (CD3+) in the peripheral blood (PB) of recipient mice, as determined by FACS at the

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indicated time points (F). (n=5 recipients per genotype). No statistically significant differences in
reconstitution were detected.
Data Information: All the data presented as presented as mean ± SEM. **p<0.01 by two-tailed
Student’s unpaired t-test analysis (A-D) or two-way ANOVA analysis with Sidak’s multiple
comparison tests (F).
Appendix Figure S2.
Augmented HSPC pool size and contribution to mature peripheral blood cells following loss
of PKCδ in the hematopoietic system.
A. Experimental outline of competitive BM reconstitution assay.
B. Representative FACS plots show the percentage of total donor derived (CD45.2+) cells in PB of
recipient mice at 16-weeks post-pIpC treatment. Bar graph shows the percentages of total donor-
derived cells in the PB of recipient mice at indicated times pre-and post-pIpC treatment. (n=7-8
mice per genotype).
Data information: Statistical significance was determined by repeated measures two-way ANOVA
analysis with Sidak’s multiple comparison tests for comparison of control and PKC
δ
cKO mice at
each time point. *p<0.05 and **p<0.01.
Appendix Figure S3.
PKCδ sets a threshold for metabolic activation of HSPCs during myeloablative regeneration.
A. Experimental design.
B. Mitochondrial OCR rate in bone marrow HSPCs. MitoStress test revealed increased basal
oxygen consumption rates (OCR) (measured before inhibitors treatment, left); the Maximal OCR
capacity after FCCP treatment (middle), and the production of ATP (right) in LSKs from 5-FU
treated PKC
δ
Δ/Δ (cKO) mice as compared to LSKs from treated WT mice.
Data information: All data are presented as mean ± SEM (n=6 mice per genotype), *p<0.05 and

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**p<0.01, by two-tailed Student’s unpaired t-test analysis for comparison of control and PKCδ cKO
mice.

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Appendix Figure S1

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Appendix Figure S2

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Appendix Figure S3

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Appendix Table 1 (related to Materials and Methods and Figure 7). List of genes and primers used
for RT-PCR analysis. All Taqman primers (FAM) were purchased from Applied Biosystems.
Gene
Product ID
Pdk2
Mm00446681_m1
Pdk4
Mm01166879_m1
Ldha1
Mm01612132_g1
Acads
Mm00431617_m1
Hes1
Mm01342805_m1
ATP5a1
Mm00431960_m1
Cox5a1
Mm01176957_m1
Cyc1
Mm00470540_m1
Cdkn2c
Mm00483243_m1
Cdkn1b
Mm00438168_m1
Mcl1
Mm00725832_s1
Bcl2l1
Mm00437783_m1
Egr1
Mm00656724_m1
Gfi1b
Mm00492318_m1
Bad
Mm00432042_m1
Cdkn2a
Mm00494449_m1
Cebp
α
Mm00514283_s1
Nfe2L2 (Nrf2)
Mm00477784_m1
β
-Actin
Mm00607939_s1
TBP
Mm00446971_m1
Gata1
Mm01352636_m1
Gata2
Mm00492301_m1
ID2
Mm00711781_m1
Pkm
Mm00834102_gH
Spi1 (PU.1)
Mm00488142_m1

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Appendix Table 2. List of primary and secondary antibodies used for intracellular flow cytometry.
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Primary antibody
Supplier
Catalogue#
Dilution
Phospho-Akt (Ser473) (D9E) XP®
Rabbit mAb
Cell Signaling Technology
4060
1:200
PhosphoDetect™Anti-PDH-E1α
(pSer293) Rabbit pAb
Millipore
AP1062
1:100
Phospho-FoxO3a (Ser253)
Antibody
Cell Signaling Technology
9466
1:100
Phospho-S6 Ribosomal Protein
(Ser235/236) (D57.2.2E) XP®
Rabbit mAb
Cell Signaling Technology
4858P
1:100
Phospho-Rb (Ser807/811)
(D20B12) XP® Rabbit mAb
Cell Signaling Technology!
8516S
1:400
c-Myc(D84C12) Rabbit mAb
(Alexa Fluor 647 conjugate)
Cell Signaling Technology
13871
1:50
Cyclin D1(92G2) Rabbit mAb
Cell Signaling Technology
2978
1:50
PPARγ (C26H12) Rabbit mAb
Cell Signaling Technology
2435
1:100
Anti-PPARδ (C26H12) Rabbit
polyclonal Ab
Abcam
ab23673
1:100
Phospho-NF-κB p65 (Ser536)
(93H1) Rabbit mAb (Alexa Fluor®
647 Conjugate) #488
Cell Signaling Technology
4887
1:50
Phospho-mTOR (Ser2448) (D9C2)
XP® Rabbit mAb
Cell Signaling Technology
5536
1:50
Phospho-p38 MAPK
(Thr180/Tyr182) (D3F9) XP®
Rabbit mAb
Cell Signaling Technology
4511
1:400
Phospho-p44/42 MAPK (Erk1/2)
(Thr202/Tyr204) (197G2) Rabbit
mAb
Cell Signaling Technology
4377
1:200
Anti-rabbit IgG (H+L), F(ab’)2
fragment (Alexa Fluor 647
conjugate)
Cell Signaling Technology
4414
1:500
Anti-rabbit IgG (H+L), F(ab’)2
fragment (Alexa Fluor 488
conjugate)!
Cell Signaling Technology
4412
1:500