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stm.sciencemag.org/cgi/content/full/12/537/eaax1798/DC1
Supplementary Materials for
PBX1 expression in uterine natural killer cells drives fetal growth
Yonggang Zhou, Binqing Fu*, Xiuxiu Xu, Jinghe Zhang, Xianhong Tong, Yanshi Wang, Zhongjun Dong, Xiaoren Zhang,
Nan Shen, Yiwen Zhai, Xiangdong Kong, Rui Sun, Zhigang Tian*, Haiming Wei*
*Corresponding author. Email: [email protected] (H.W.); [email protected] (Z.T.); [email protected] (B.F.)
Published 1 April 2020, Sci. Transl. Med. 12, eaax1798 (2020)
DOI: 10.1126/scitranslmed.aax1798
The PDF file includes:
Materials and methods Fig. S1. Validation of rat anti-human/mouse PBX1 monoclonal antibody. Fig. S2. The transcription factor PBX1 is more conserved than other related NK cell transcription factors. Fig. S3. Expression of the GPFs, PTN and OGN, is up-regulated after overexpression of PBX1 in dNK-like cells. Fig. S4. PBX1G21S retains DNA binding activity. Fig. S5. dNK cells are the dominant subset expressing growth factors in early pregnancy. Fig. S6. PBX1 is highly expressed in uterine NK cells from pregnant Pbx1HA-tag knock-in mice. Fig. S7. Knockout of Pbx1 alters the gene expression profile in uterine NK cells, and the bone development of embryos is limited in pregnant Pbx1f/f;Ncr1Cre mice. Fig. S8. Fetal growth is normal in pregnant EomesNK-KO mice. Fig. S9. Working model of PBX1+ NK cells promoting early fetal development. Table S1. Clinical data for patients with RSA and healthy controls. Table S2. Commercial kits for cell preparation used in this study. Table S3. Recombinant proteins used in this study. Table S4. Antibodies used in this study. Table S5. Human-specific primer sequences. Table S6. Construction of siRNAs of the target gene. Table S7. Primer sequences for the ChIP assay. Table S8. Construction of oligonucleotides of Pbx1HA tag knock-in mice.
Other Supplementary Material for this manuscript includes the following: (available at stm.sciencemag.org/cgi/content/full/12/537/eaax1798/DC1)
Data file S1 (Microsoft Excel format). Original data.
Materials and methods
Preparation of rat anti-human/mouse PBX1 monoclonal antibody
The intact human PBX1 homolog, PBX1b, expressed in and purified from prokaryotic cells,
was used to immunize rats to prepare antibodies. Immunization of rats and preparation of
monoclonal antibody hybridoma cells were entrusted to Absea Biotechnology Ltd (Beijing,
China). Resulting monoclonal antibodies from different clones were used as primary antibodies
in flow cytometry to screen for those suitable for use in flow cytometry detection, and clone
2D11 was selected. Antibody specificity was verified by flow cytometry and western blotting to
detect PBX1b protein in cells where it was knocked down (siRNA-PBX1) or knocked down and
recovered by overexpression from a eukaryotic vector. The anti-PBX1 (2D11) monoclonal
antibody was labeled using an Alexa Fluor 488 Antibody Labeling Kit (Invitrogen, Cat. No.
A10235). After collection of the labeled eluate, the antibody concentration (M) and labeling
efficiency (C) were calculated by measuring absorbance values at 280 and 494 nm (A280 and
A494); M = 1 mg/ml, C = 5.8 moles of Alexa Fluor 488 dye per mole of antibody.
Analysis of the conservation of NK cell-associated genes
Phylogenetic homology analysis of NK cell-associated genes, including transcription
factors, functional factors, and characteristic surface molecules, was conducted by querying
protein sequences using the NCBI HomoloGene system
(https://www.ncbi.nlm.nih.gov/homolo-gene) to analyze the distinguishing features of human
NK cells compared with those from other species. The results of cluster analysis are presented as
heat maps. Gene orthologs of NK cell-associated transcription factors in different species were
queried using the HCOP database, supported by the National Human Genome Research Institute
(https://www.genenames.org/cgi-bin/hcop) and the OrthoDB database, which was developed by
Zdobnov’s Computational Evolutionary Genomics group (http://cegg.unige.ch/).
Generation of Pbx1HA-tag knock-in mice
For PBX1 monitoring and for ChIP assays of murine NK cells, HA-tagged knock-in Pbx1
mice were constructed by CRISPR-Cas9-mediated homology-directed repair technology, as
previously reported (33). Briefly, annealed sgRNA oligos targeting the start codon of the Pbx1
gene were inserted into an expression plasmid (pUC57-sgRNA, Addgene plasmid #51132).
Linearized expression plasmid was used as a template to transcribe sgRNA in vitro with T7 RNA
polymerase. Human codon-optimized SpCas9 linearized vector (Addgene plasmid #44758) was
used as a template to transcribe Cas9 mRNA in vitro with a T7 ULTRA Transcription Kit
(Ambion, AM1345), according to the manufacturer’s instructions. Single-stranded
oligodeoxynucleotides were synthesized for homology-driven repair (Integrated DNA
Technologies). Purified sgRNA, Cas9 mRNA, and ssODNs were diluted to 15 ng/μl, 20 ng/μl,
and 40 ng/μl, respectively, with DEPC-treated water in a final volume of 50 μl. The mixture was
injected into the cytoplasm and male pronucleus of mouse zygotes using standardized
microinjection and embryo transfer procedures. The genotype of genetically ablated mice was
assessed by sequence analysis of the Pbx1 gene (fig. S6A). Oligonucleotide sequences are listed
in table S8.
Immunofluorescence
Purified human dNK and pNK cells were fixed with 4% paraformaldehyde for 15 min and
incubated in blocking solution (5% normal goat serum, 0.5% Triton-X in PBS) for 1 h at room
temperature. Cells were then incubated in primary antibody overnight at 4 ℃ and then with
fluorescent secondary antibody at room temperature for 1 h in the dark, followed by staining
with DAPI. Antibodies were diluted as follows: anti-CD45 (1:200), anti-CD56 (1:200),
anti-PBX1 (1:400), and fluorescent secondary antibodies (1:200). A LSM880+ Airyscan system
(Zeiss) was used to capture images, and ZEN software (ZEN 2012, Carl Zeiss Microimaging)
was applied to process the data.
Multiplex immunohistochemistry
Decidual and villus tissues from healthy controls and patients with URSA were fixed in
neutral formalin, and then embedded in paraffin for sectioning. Paraffin sections were dewaxed
in xylene solution and rehydrated with ultrapure water. Antigen was retrieved using Tris-EDTA
antigen retrieval solution (pH 9.0) in a boiling water bath for 20 min, then naturally cooled to
room temperature. Multiplex immunohistochemistry of paraffin sections was conducted using
Tyramide SuperBoost Kits with Alexa Fluor Tyramides (Thermo Fisher, cat# B40922 and
B40913), according to the kit instructions. Sections were incubated with primary antibody
overnight at 4 ℃; antibodies were diluted as follows: anti-CD56 (1:200), anti-EpCAM (1:200),
anti-PTN (1:50), and anti-OGN (1:50). Fluorescent dye staining was conducted at room
temperature for 3 min and terminated using a stop solution for 20 min at room temperature.
Nuclei were stained with DAPI. Antifade Mounting Medium (Thermo Fisher, cat# P36934) was
added to tissue sections dropwise, followed by a sealing treatment, and an LSM880+ Airyscan
system (Zeiss) was used to capture images.
Immunohistochemistry
Paraffin sections of villus tissue from healthy controls and patients with URSA were
prepared. After dewaxing and rehydration, sections were boiled in 1x citrate unmasking solution
in a water bath for 20 min for antigen retrieval, and naturally cooled to room temperature.
Staining of Ki67 and PCNA was then conducted at 4 ℃ overnight. Antibody dilutions were as
follows: anti-Ki67 (1:400) and anti-PCNA (1:4000). After staining with secondary antibody for
30 min at room temperature, samples were developed using DBA substrate for 30 sec, followed
by counterstaining with hematoxylin.
Fig. S1. Validation of rat anti-human/mouse PBX1 monoclonal antibody.
(A) Schematic representation of vector for prokaryotic expression of PBX1. (B) Schematic
representation of the vector for eukaryotic overexpression of PBX1. (C) Coomassie blue staining
of rat anti-human/mouse PBX1 antibody. (D) Western blot analysis of PBX1 in 293T cells
transfected with siRNA-PBX1, or a mixture of siRNA-PBX1 and eukaryotic PBX1 overexpression
vector with rat anti-human/mouse PBX1 antibody. Lamin B was used as an internal reference.
NC, negative control. (E) Flow cytometry analysis of PBX1 in 293T cells with transfected with
siRNA-PBX1, or a mixture of siRNA-PBX1 and eukaryotic PBX1 overexpression vector, with rat
anti-human/mouse PBX1 antibody and secondary AF647-anti-rat IgG antibody. NC, negative
control.
Experiments (D and E) were independently performed twice with similar results.
Fig. S2. The transcription factor PBX1 is more conserved than other related NK cell
transcription factors.
(A) Strategy for gating dNK and pNK cells from mononuclear cells. Numbers are the percentage
in each indicated subset. (B) Confocal microscopy of the expression of PBX1 in purified dNK
and pNK cells. Scale bar, 5 μm. (C) Phylogenic homology analysis of transcription factors,
functional factors, and characteristic surface molecules associated with NK cells. Protein
sequences were identified using the NCBI HomoloGene system to analyze the distinguishing
features between proteins in human NK cells and those from other species. The results of
homology cluster analysis are presented as a heat map. Numbers and colors indicate the degree
of homology of ortholog sequences from other species with human sequences. (D) Phylogeny of
orthologous signature NK cell transcription factor genes from different species identified by
querying the HCOP and OrthoDB databases. Numbers and colors in the plot indicate the
probability of the existence of an orthologous gene in other species.
Fig. S3. Expression of the GPFs, PTN and OGN, is up-regulated after overexpression of
PBX1 in dNK-like cells.
(A) Schematic representation of the in vitro cell system for induction of dNK-like cells using
cytokine cocktail stimulation of human umbilical cord blood stem cells. (B) Gating strategy for
dNK-like cells during the process of cell induction. (C) Quantitative RT-PCR of PBX1, TBX21,
EOMES, PTN, and OGN in cells during different weeks of the process of inducing dNK-like
cells from hematopoietic stem cells (n = 8), normalized to their expression in week 2 cells (ns >
0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by ANOVA). w, week. (D-I) Intracellular flow
cytometry analysis (D, F, and H) and statistical analysis of the MFI (E, G, and I) of expression of
PBX1 (D and E), PTN (F and G), OGN (F and G), and other transcription factors (H and I) in
week 4 dNK-like cells overexpressing PBX1 from a lentiviral vector (ns > 0.05, **P < 0.01,
****P < 0.0001 by ANOVA). iNK-PBX1, dNK-like cells transfected with PBX1-overexpressing
lentiviral vector. iNK-Mock, dNK-like cells transfected with control lentiviral vector. Data from
three independent experiments. (J) Western blot for PBX1 to assess the effects of siRNA-PBX1
vectors transfected into 293T cells. GAPDH was used as an internal control. (K) Quantitative
RT-PCR analysis of PBX1, PTN, and OGN expression in purified human dNK cells transfected
with siRNA-PBX1 or siRNA-NC (negative control) vectors and co-cultured with EVT cells (n =
6); expression was normalized to that in cells transfected with a combination of siPBX1-1 and
siPBX1-2(siPBX1-1 &2) (****P < 0.0001 by ANOVA). (L and M) CD3-CD45+CD56+ NK cells
were purified from fresh decidual tissues from two first trimester pregnancies. Differential
analysis of enriched gene promoter region sequences by scatter plot (L) and heat map (M) of data
from a ChIP assay from decidual NK cells using anti-PBX1 antibody. No. 1 and No. 2 indicate
data of two experimental replicates generated using anti-PBX1 antibodies. IgG antibody,
negative control. The numbers in (B) are the percentage in each indicated subset. The numbers in
(D, F, and H) represent the MFI for the indicated molecules.
Fig. S4. PBX1G21S retains DNA binding activity.
(A) Representative flow cytometry showing the percentages of CD3-CD45+CD56+ dNK cells in
samples purified from first trimester pregnancies of patients with URSA (representative images
from n = 5) and healthy controls (representative images from n = 3). Numbers show the
percentage of each indicated subset. (B) Representative chromatograms generated by Sanger
sequencing of PBX1 exon 1 PCR products amplified from decidual tissue from patients with
URSA (upper panel). Amino acid sequence, functional domains, and mutation site in PBX1
(lower panel). (C) Exome sequencing results for dNK cells from healthy controls and five
patients with URSA. SNP counts per site (left). Overview of dNK cell mutation status for
transcription factors and functional molecule-related genes (right). (D) Western blot analysis to
detect the presence of PBX1 in samples obtained by DNA pull-down assay using PTN or
OGN-binding site probes in 293T cells with knockdown of PBX1WT (wild-type) and
overexpressing PBX1G21S. PBX1G31S is a natural variant of PBX1. 293T cells with PBX1WT
knocked down served as negative controls. BS, binding site.
Fig. S5. dNK cells are the dominant subset expressing growth factors in early pregnancy.
(A) Gating strategy for the cell subset expressing growth factors PTN and OGN from human
decidual tissue in the first trimester of pregnancy. Red, CD3-CD45+CD56+ NK cells. Blue,
CD45+ immune cells. Black, CD45- non-immune cells. (B) Statistical analysis of the MFI for
PTN and OGN in CD3-CD45+CD56+ NK cells, CD45+ immune cells, and CD45- non-immune
cells from decidual tissue of pregnant women about 40 days (n = 4) and 60 days (n = 3) of
pregnancy (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by ANOVA). (C) Western
blot for PTN and OGN in human dNK cells of healthy controls (n = 3) and URSA patients with
PBX1G21S mutant (n=5). β-actin was used as an internal control. (D and E) Confocal microscopy
of the expression of PTN (top, red) and OGN (bottom, red) in CD56+ dNK cells (green) in
human decidual tissue of healthy controls (D) and URSA patients with PBX1G21S mutant (E) in
the first trimester of pregnancy. Scale bar, 50 μm. (F and G) Confocal microscopy of the
expression of PTN (top, red) and OGN (bottom, red) in extravillous trophoblast (EVT) cells in
human villous tissue of healthy controls (F) and URSA patients with PBX1G21S mutant (G) in the
first trimester of pregnancy. EpCAM+ villous cytotrophoblast (green) proliferation that give rise
to EVT cells. Scale bar, 50 μm. (H and I) Immunohistochemical staining of Ki67 (H) and PCNA
(I) in human villous tissue of healthy controls and URSA patients with PBX1G21S mutant in the
first trimester of pregnancy. Scale bar, 50 μm. (J) Statistical analysis of the volume of gestation
sac from the color B-ultrasound of healthy pregnant women (n = 18) and women with URSA and
impaired PBX1 at 7 weeks ± 5 days of gestation (n = 13) (**P < 0.01 by two-tailed t test).
Fig. S6. PBX1 is highly expressed in uterine NK cells from pregnant Pbx1HA-tag knock-in
mice.
(A) Schematic of the process of construction of Pbx1HA-tag knock-in mice using CRISPR-Cas9
technology. (B) Gating strategy and intracellular flow cytometric analysis of PBX1 in NK cells
from spleen of Pbx1HA-tag knock-in mice at gestational day (gd) 11.5 (n = 4) and virgin mice (n =
4) as controls. (C) Statistical analysis of the MFI of PBX1 in (B) (ns > 0.05 by two-tailed t test).
(D) Gating strategy and intracellular flow cytometric analysis of PBX1 in NK cells from uterine
tissue of Pbx1HA-tag knock-in mice at gestational day (gd) 11.5 (n = 4) and virgin mice (n = 4) as
controls. (E) Statistical analysis of the MFI of PBX1 in (D) (****P < 0.0001 by two-tailed t
test). (F) Flow cytometry and frequencies of eGFP+ cells in Rosa26f-stop-f-eGFP;Vav1Cre and
Rosa26f-stop-f-eGFP;Ncr1Cre mice to assess the efficiency of Cre expression in Cre-transgenic mice.
(G) Intracellular flow cytometric analysis of PBX1 in uterine NK cells from Pbx1f/f;Vav1Cre and
Pbx1f/f;Ncr1Cre mice to assess the effect of the Pbx1 gene knockout. The numbers in (B, D, F,
and G) represent the percentages of indicated subsets or the MFI of indicated molecules.
Fig. S7. Knockout of Pbx1 alters the gene expression profile in uterine NK cells, and the
bone development of embryos is limited in pregnant Pbx1f/f;Ncr1Cre mice.
(A, B, and C) RNA sequencing analysis of uterine NK cells from Pbx1f/f and Pbx1f/f;Ncr1Cre
mice at gestational day (gd) 11.5. KEGG pathway analysis of differentially expressed genes (A).
Heat map of differentially expressed cytokine genes (B) and receptor and chemokine genes (C)
in uterine NK cells. (D) Von Kossa staining of skulls of embryos from Pbx1f/f and Pbx1f/f;Ncr1Cre
pregnant female mice mated with B6 male mice at gd16.5. Scale bar: 1 mm. (E) SafraninO-fast
green staining of skulls of embryos from Pbx1f/f and Pbx1f/f;Ncr1Cre pregnant female mice mated
with B6 male mice at gd16.5. Scale bar, 1 mm. Experiments (D and E) were independently
performed three times.
Fig. S8. Fetal growth is normal in pregnant EomesNK-KO mice.
(A) Representative picture of fetuses from Eomesf/f and Eomesf/f;Ncr1Cre mice at gd16.5 and
gd19.5. Scale bar, 5 mm. (B) Statistical analysis of the number of live fetuses from Eomesf/f and
Eomesf/f;Ncr1Cre pregnant female mice (n = 6) mated with B6 male mice. (C and D) Statistical
analyses of the weight (C) and body length (D) of fetuses from Eomesf/f and Eomesf/f;Ncr1Cre
mice (n = 6) mated with B6 male mice at gd16.5 and gd19.5. (E) Statistical analysis of the
relative MFI for PTN and OGN in uterine NK cells from Eomesf/f and Eomesf/f;Ncr1Cre mice (n =
6) at gd11.5, normalized to MFI in IgG antibody negative control. Statistical analyses (B, C, D,
and E) were conducted by two-tailed t test (ns > 0.05, *P < 0.05).
Fig. S9. Working model of PBX1+ NK cells promoting early fetal development. The embryo-derived HLA-G signal activates the AKT signal of NK cells in the decidual tissue,
drives the expression of the transcription factor PBX1, and enhances the transcriptional expression
of growth-promoting factors, ultimately promoting early fetal development.
Table S1. Clinical data for patients with RSA and healthy controls.
Age (year)
(Mean ± SD)
Number of
pregnancies
(Mean ± SD)
Number of
spontaneous
abortions
(Mean ± SD)
Pregnancy
(weeks)
(Mean ± SD)
Control
(Fresh, n= 45) 26.76 ± 4.72 1.47 ± 0.50 0.0 ± 0.0 8.24 ± 1.25
URSA
(Fresh, n= 20) 30.25 ± 4.25 2.70 ± 0.80 2.45 ± 0.60 8.98 ± 1.36
URSA
(Cryopreserved,
n= 45)
30.15 ± 4.67 2.65 ± 0.74 2.42 ± 0.61 9.11 ± 1.65
RSA-ECA
(Cryopreserved,
n= 30)
27.97 ± 4.60 2.34 ± 0.61 2.24 ± 0.51 9.72 ± 1.40
Table S2. Commercial kits for cell preparation used in this study.
Recombinant Proteins Sources Identifier
Anti-PE Microbeads Miltenyi Biotec Cat# 130-048-801,RRID:AB_244373
NK cell Isolation Kit, human Miltenyi Biotec Cat# 130-092-657
CD34 MicroBead Kit, human Miltenyi Biotec Cat# 130-046-702
Lineage Cell Depletion Kit, mouse Miltenyi Biotec Cat# 130-090-858
Nucleofector Kits for Human
Natural Killer Cells Lonza Cat# VPA-1005
Table S3. Recombinant proteins used in this study.
Recombinant Proteins Sources Identifier
Recombinant Human SCF PeproTech Cat# 300-07
Recombinant Human Flt3L PeproTech Cat# 300-19
Recombinant Human IL-15 PeproTech Cat# 200-15
Recombinant Murine SCF PeproTech Cat# 250-03
Recombinant Murine Flt3L PeproTech Cat# 250-31L
Recombinant Murine IL-2 PeproTech Cat# 212-12
Recombinant Murine IL-3 PeproTech Cat# 213-13
Recombinant Murine IL-6 PeproTech Cat# 216-16
Recombinant Murine IL-7 PeproTech Cat# 217-17
Recombinant Murine IL-15 PeproTech Cat# 210-15
Table S4. Antibodies used in this study.
Reagent or Resource Sources Identifier
Human
Anti-human CD11b PE-CY7 BD Cat# 557743, RRID:AB_396849
Anti-human CD103 BV 605 BioLegend Cat# 350217, RRID:AB_2564282
Anti-human CD27 PE BD Cat# 555441, RRID:AB_395834
Anti-human CD3 PerCP-Cy5.5 BioLegend Cat# 300328, RRID:AB_1575008
Anti-human CD3 APC-Cy7 BD Cat# 557832, RRID:AB_396890
Anti-human CD34 FITC BD Cat# 555821, RRID:AB_396150
Anti-human CD39 PE-CY7 BioLegend Cat# 328211, RRID:AB_2293623
Anti-human CD45 BV 510 BD Cat# 555483, RRID:AB_395875
Anti-human CD45 Purified CST Cat# 13917, RRID:AB_2750898
Anti-human CD49a Alexa Fluor 647 BioLegend Cat# 328310, RRID:AB_2129242
Anti-human CD56 BV 421 BioLegend Cat# 362552, RRID:AB_2566061
Anti-human CD56 Alexa Fluor 647 BD Cat# 557711, RRID:AB_396820
Anti-human CD56 BV 510 BioLegend Cat# 318340, RRID:AB_2561944
Anti-human CD56 Purified CST Cat# 3576, RRID:AB_2149540
Anti-human CD9 PE BioLegend Cat# 312105, RRID:AB_2075893
Anti-human ITGB2 BV 421 BD Cat# 743370
Anti-human EOMES PerCP-eFluor 710 Thermo Cat# 46-4877-41, RRID:AB_2573758
Anti-human EOMES Purified Biolegend Cat# 662001, RRID:AB_2564181
Anti-human T-bet PE Thermo Cat# 12-5825-82, RRID:AB_925761
Anti-human T-bet Purified Thermo Cat# 14-5825-80, RRID:AB_763635
Anti-Human/Mouse phospho-S6 Ribosomal
(S235/S236) PE
Thermo Cat# 12-9007-41, RRID:AB_2572666
Anti-human phospho-S6 Ribosomal Purified Thermo Cat# 14-9007-80, RRID:AB_2572910
Rat-Anti-human/mouse PBX1 Alexa Fluor 488 This paper N/A
Anti-human PBX1 Purified CST Cat# 4342S, RRID:AB_2160295
Anti-human PTN Purified LifeSpan Cat# LS-C162291
Anti-human OGN Purified LifeSpan Cat# LS-B10948
Anti-human PDK1 Purified CST Cat# 13037; RRID: AB_2798095
Anti-human PDK2 Purified Abcam Cat# ab68164, RRID:AB_11156499
Anti-human AKT1 Purified CST Cat# 2938, RRID:AB_915788
Anti-human Phospho-Akt1 (Ser473) Purified CST Cat# 9018, RRID:AB_2629283
Anti-human Phospho-Akt (Thr308) Purified CST Cat# 13038, RRID:AB_2629447
Anti-human HLA-G Purified Biolegend Cat# 335904; RRID: AB_10641840
Anti-human ILT2 Purified Biolegend Cat# 333704; RRID: AB_1089088
Anti-human EpCAM Purified Abcam Cat# ab7504, RRID:AB_ 305949
Anti-human Ki-67 Purified CST Cat# 9449, RRID:AB_ 2797703
Anti-human PCNA Purified CST Cat# 2586, RRID:AB_ 2160343
Anti-human GAPDH Purified CST Cat# 5174, RRID: AB_10622025
Anti-human ACTIN Purified CST Cat# 3700, RRID:AB_2242334
Anti-human Lamin B Purified BOSTER Cat# PB9611
Mouse
Anti-mouse CD3ε PerCP-Cy5.5 BioLegend Cat# 100328, RRID:AB_893318
Anti-mouse CD3 BV 605 BD Cat# 563004, RRID:AB_2737945
Anti-mouse CD45.2 APC-Cy7 BioLegend Cat# 109824, RRID:AB_830789
Anti-mouse CD49a BV 786 BD Cat# 740919, RRID:AB_2740560
Anti-mouse CD49b BV 421 BD Cat# 563063, RRID:AB_2737983
Anti-mouse NK-1.1 PE-Cy7 BioLegend Cat# 108714, RRID:AB_389364
Anti-mouse NK-1.1 BV 605 BioLegend Cat# 108740, RRID:AB_2562274
Anti-mouse Eomes eFluor 660 Thermo Cat# 50-4875-82, RRID:AB_2574227
Anti-mouse T-bet PE BioLegend Cat# 644810, RRID:AB_2200542
Anti-mouse PTN Purified LifeSpan Cat# LS-C295980
Anti-mouse OGN Purified LifeSpan Cat# LS-C387062
Anti-Ki67 BV 510 BD Cat# 563462, RRID:AB_2738221
Anti-HA Purified Transgen Cat# HT301-01
Isotype Control
Mouse IgG1, κ FITC BD Cat# 555909, RRID:AB_396216
Mouse IgG1, κ Alexa Fluor 488 BD Cat# 557702, RRID:AB_396811
Mouse IgG1, κ PE BD Cat# 555749, RRID:AB_396091
Mouse IgG1, κ PerCP-Cy5.5 BD Cat# 552834, RRID:AB_394484
Mouse IgG1, κ PerCP-eFluor 710 Thermo Cat# 46-4714, RRID:AB_1834453
Mouse IgG1, κ PE-Cy7 BD Cat# 557872, RRID:AB_396914
Mouse IgG1, κ Alexa Fluor 647 BD Cat# 557714, RRID:AB_396823
Mouse IgG1, κ APC-Cy7 BD Cat# 557873, RRID:AB_396915
Mouse IgG1, κ BV 421 BD Cat# 562438, RRID:AB_11207319
Mouse IgG1, κ BV 510 BioLegend Cat# 400172, RRID:AB_2714004
Mouse IgG1, κ BV 605 BioLegend Cat# 400161, RRID:AB_11125373
Hamster IgG PerCP/Cy5.5 BioLegend Cat# 400931
Hamster IgG1,λ BV 605 BioLegend Cat# 400943
Hamster IgG2,λ1 BV 786 BD Cat# 565864
Mouse IgG2a, κ PE-Cy7 BD Cat# 552868, RRID:AB_394501
Mouse IgG2a, κ APC-Cy7 BD Cat# 557751,
Mouse IgG2a, κ BV 605 BD Cat# 563144
Rat IgG2a, κ Alexa Fluor 660 Thermo Cat# 50-4321, RRID:AB_10598640
Rat IgM, κ BV421 BD Cat# 562595
Rabbit IgG Purified CST Cat# 2729S, RRID:AB_1031062
Rat IgG Purified Thermo Cat# 02-9602, RRID:AB_2532969
Secondary antibody
Goat Anti-Rabbit IgG FITC BD Cat# 554020, RRID:AB_395212
Goat Anti-Rat IgG Alexa Fluor 647 BioLegend Cat# 405416, RRID:AB_2562967
Goat Anti-Mouse IgG APC BioLegend Cat# 405308, RRID:AB_315011
Goat Anti-Rat IgG BV421 BioLegend Cat# 405414, RRID:AB_10900808
Goat anti-Rabbit IgG Alexa Fluor 488 Thermo Cat# A-11008, RRID:AB_143165
Goat anti-Rabbit IgG Alexa Fluor 647 Thermo Cat# A-32733, RRID:AB_2633282
Goat anti-Mouse IgG Alexa Fluor 546 Thermo Cat# A-11030, RRID:AB_144695
Goat anti-Rat IgG Alexa Fluor 647 Thermo Cat# A-21247, RRID:AB_141778
Goat Anti-Rabbit IgG HRP BOSTER Cat# BA1054
Goat Anti-Mouse IgG HRP BOSTER Cat# BA1050
Rabbit Anti-Rat IgG HRP BOSTER Cat# BA1058
Table S5. Human-specific primer sequences.
Genes Oligonucleotides
Primers for human ACTB
(NM_001101.3)
Forward: TGACGTGGACATCCGCAAAGACC
Reverse : CTCAGGAGGAGCAATGATCTTGA
Primers for human PBX1
(NM_002585)
Forward: CCATCTCAGCAACCCTTACCC
Reverse: GAACCAGCCGAGTTGGGAGT
Primers for human TBX21
(NM_013351.2)
Forward: CACGTCCACAAACATCCTGT
Reverse: GATCATCACCAAGCAGGGAC
Primers for human EOMES
(NM_005442.3)
Forward: CTGGCTTCCGTGCCCACGTC
Reverse: CATGCGCCTGCCCTGTTTCG
Primers for human PTN
(NM_002825)
Forward: CCTCCCTGTCAGGGCGTAAT
Reverse: GACGGATGACTCACTGGTCTCTTT
Primers for human OGN
(NM_024416.4)
Forward: GAGGATAAATACCTGGATGGA
Reverse: GTGCGTAAAGATAGGCTGATT
Table S6. Construction of siRNAs of the target gene.
Oligonucleotides Sources Identifier
siRNA targeting sequence: Negative Control
UUCUCCGAACGUGUCACGUTT GenePharma Cat# A06001
siRNA targeting sequence: siPBX1-1,
GCUUUAAACUGCCACAGAATT GenePharma Lot# 20160808662
siRNA targeting sequence: siPBX1-2,
CCAUCCAGAUGCAGCUCAATT GenePharma Lot# 201608081092
siRNA targeting sequence: siPBX1-3,
CCAAAGAGGAGUUAGCCAATT GenePharma Lot# 201608081257
shRNA targeting sequence: pLKO.1-shPbx1
CAGAAATTCTGAATGAATAT Sangon Lot# 9401112278
siRNA targeting sequence: siAKT1-1,
GCACCUUCAUUGGCUACAATT GenePharma Lot# 20190725463
siRNA targeting sequence: siAKT1-2,
GGAGACUGACACCAGGUAUTT GenePharma Lot# 20190725465
siRNA targeting sequence: siPDK2-1,
CCAAGUACAUAGAGCACUUTT GenePharma Lot# 20190725466
siRNA targeting sequence: siPDK2-2,
GCUGUCCAUGAAGCAGUUUTT GenePharma Lot# 20190725467
Table S7. Primer sequences for the ChIP assay.
Target sequences Oligonucleotides
Primers for ChIP human PTN
(-1000/700)
Forward: GCCTTAGCGTCTTTCCTGTA
Reverse: GTGCCTTTAGCAACCTATTTAGT
Primers for ChIP human PTN
(-650/350)
Forward: AAGGCACCAAGGAAACCAAA
Reverse: CAATTGATGGCATTTTAGGTTACA
Primers for ChIP human OGN
(-2100/1800)
Forward: TGGACTATGTTGTCATTTGGTG
Reverse: ATCTTTGCCAGCATTCTTTC
Primers for ChIP human OGN
(-1350/1050)
Forward: CATTCAGCAAACAAAAGTATC
Reverse: TGAAATAGGACACCAAATGA
Primers for ChIP human OGN
(-950/650)
Forward: TGCCTAGCACAATAGTGAGT
Reverse: CAATGTTCTTTCTCCCTTAT
Primers for ChIP human OGN
(-350/50)
Forward: CAGTTATCTCCCCATTTGTC
Reverse: AGTGAGGTTTAAGTCAGGGA
Primers for ChIP mouse Ptn
(-1500/1201)
Forward: CAGCAATGGGAAGGGAGGC
Reverse: CTTTGCTCTTCCTAATGCTGGAT
Primers for ChIP mouse Ptn
(-1200/901)
Forward: AGGAAGAGCAAAGCTACCAGTAC
Reverse: TTTCCAAGACTGGCAATAGACT
Primers for ChIP mouse Ptn
(-900/601)
Forward: AACTAGTCTATTGCCAGTCTTGG
Reverse: AGACCCATTAATTCTCCAGATC
Primers for ChIP mouse Ptn
(-600/301)
Forward: AATGGGTCTTGATTAAAGTCAGTT
Reverse: TCTTGTTGTTTGCATCACTTGGT
Primers for ChIP mouse Ptn
(-300/1)
Forward: ACAACAAGATTGGGTTTGGGCT
Reverse: CTGGAGTAAAGAGAAAGGGGGAA
Primers for ChIP mouse Ogn
(-2100/1800)
Forward: TACTACAAAATTTTGGAATCAAAC
Reverse: GTTGTTGTTGTTGTTGTTATTGTTA
Primers for ChIP mouse Ogn
(-1300/1000)
Forward: ATATCCTCAGCACATTCTCTCTT
Reverse: GAATCATACTTTCACACACAGTAT
Primers for ChIP mouse Ogn
(-900/600)
Forward: CAGATGTAGGTTAGAACTAGAAG
Reverse: TCACTAGTTAGCTAGTAATGTTGC
Primers for ChIP mouse Ogn
(-750/450)
Forward: CCTGTATTCAGCAGTTAAAATTC
Reverse: TGTAAAAGAAGCAGTTCTGAGAA
Primers for ChIP mouse Ogn
(-350/50)
Forward: ATTTATACTGCAGGTACCCCGAG
Reverse: TGATGACTGTGGAACGAAACTCT
Table S8. Construction of oligonucleotides of Pbx1HA tag knock-in mice.
Oligonucleotides Sources
sgRNA targeting sequence: sgPBX1-ATG,
GCTGCCGGAGCCTTCAGAGA
Sangon
PAGE Ultramer DNA Oligo: Pbx1 Donor
GGATTTGAAGACAGCTTGAAGGATAAAAAGCCTCGGTGCTTCC
CAGGCGCCGATCCGAGGAGCCGAAGAGGAAGAGCCGGGGCTG
CCGGAGCCTTCAGAGATGTACCCATACGATGTTCCAGATTACG
CTGACGAGCAGCCGAGGCTGATGCATTCCCACGCTGGGGTCGG
GATGGCCGGACACCCCGGCCTGTCCCAG
IDT