1. Online Data Supplement
Wnt3a attenuates acute lung injury by reducing P2X7 receptor–mediated lung type I cell death
Yujie Guo, Amarjit Mishra, Tingting Weng, Narendranath Reddy Chintagari, Yang Wang, Chunling Zhao, Chaoqun Huang, and Lin Liu
Online Data Supplement

2. a b HEK293-P2x7R HEK293 E10 R3/1 MLE15 A549 PRE-TII H441 RLE-6TN RLF-6
β-actin a b
P2X2R
P2X4R
P2X5R
P2X6R
P2X7R
P2X1R
E10
Lung
100 bp
200 bp
GAPDH
P2X3R
Supplementary Figure S1: Expression of P2X receptors in the lung cells. (a) Protein expression of P2X7R in different lung cell lines (20 µg total protein) was analyzed by Western blot using β-actin as an internal control. (b) mRNA expression of P2XRs in E10 cells and normal mouse lung tissues was determined by PCR using GAPDH as an loading control.

3. a b * * * ** * * BzATP ̶ + + ̶ oATP ̶ ̶ + +
Supplementary Figure S2: Activation of P2X7R causes E10 cell death. (a) E10 cells were incubated with different concentrations of BzATP for 12 hours. Cell number was counted. (b) E10 cells were treated with 400 µM BzATP in the absence or presence of 500 µM oATP for 12 hours, and the released LDH were measured. Data shown are means ± s.e.m. of three independent experiments. Statistical significance was determined by one-way ANOVA analysis with posthoc Tukey’s test. *P<0.001 v.s. control without any treatment or 0 time, **P<0.001 v.s. 400 µM BzATP.

4. a * # c b
Supplementary Figure S3: BzATP does not cause cell death in the absence of P2X7R. (a) E10 cells were transduced with adenovirus-based shRNA vectors [virus control (si-control), si-P2X7R (1) and si-P2X7R (2)] for 4 days and then treated with 400 µM BzATP for 12 hours. Released LDH were measured. A549 cells (b) and H441 cells (c) were incubated with different concentrations of BzATP for 12 hours. Cell viability was measured. Data shown are means ± s.e.m. of three independent experiments. *P<0.001 v.s. control, #P<0.005 v.s. si-control.

5. Supplementary Figure S4: P2X7R inhibits Wnt/β-catenin signaling
Supplementary Figure S4:  P2X7R inhibits Wnt/β-catenin signaling. The relative mRNA expression of
Wnt/β-catenin downstream genes in E10 cells treated with 400 µM BzATP for various times were determined
using Real-time PCR. Data were normalized to 18S rRNA and expressed as a percent of 0 time. Data shown are
means ± s.e.m. of three independent experiments.

6. BzATP Con_CM BzATP Wnt3a_CM
Control
BzATP
Con_CM
BzATP Wnt3a_CM
YO-PRO
100 μm
Supplementary Figure S5: Effect of Wnt3a on P2X7R activity. E10 cells were treated with 400 µM BzATP
for 15 min and incubated with YO-PRO dye for 10 min. The fluorescence was observed using a Nikon TE-2000
inverted microscope.

7. LiCl (mM) BzATP Control 0 10 25 50 β-Catenin
β-Catenin
LiCl (mM)
Supplementary Figure S6: Effect of LiCl on BzATP-induced cell death. E10 cells were treated with 10, 25,
and 50 mM LiCl in the presence or absence of 400 µM BzATP for 8 hours, and then immuo-stained with
anti-β-catenin antibodies . Scale bar: 50 µm.

8. * **
BzATP
Supplementary Figure S7: Wnt3a reduces BAL proteins in BzATP-treated rat. The rats were intratracheally instilled
with BzATP with control (Con) or Wnt3a conditioned medium for 24 hours. BAL protein concentration was measured.
Values represent means ± s.e.m. Statistical significance was determined by one-way ANOVA analysis with posthoc
Tukey’s test. *P<0.01 v.s. PBS alone group and **P<0.01 v.s. BzATP + Con_CM group (n=8/group).

9. Supplementary Figure S8: Original scans of western blots

10. Supplementary Table S1. PCR primer sequences foe mouse genes
Accession #
Forward primers
Reverse primers
18S
NR_
ATTGCTCAATCTCGGGTGGCTG
CGTTCTTAGTTGGTGGAGCGATTTG
Bmp4
NM_
TGGGATGCTGCTGAGGTTGAAG
CGAGCCAACACTGTGAGGAGTTTC
Axin2
NM_
GCGAGTGGCCAAAGCAATCTATAAG
GAGCCGATCTGTTGCTTCTTGATG
Cyclin D1
NM_
AGAAGTGCGAAGAGGAGG
TGTTCACCAGAAGCAGTTC
Cyclin E1
NM_
CACAACATCCAGACCCACACCAAC
ACCACACTCGGAGGAGGAGAAATC
P2X1R
NM_
TCAAGGACATTGTGCAGAGAAC
CAGTTGCCTGTGCGAATACCT
P2X2R
NM_
GCGTTCTGGGACTACGAGAC
ACGTACCACACGAAGTAAAGC
P2X3R
NM_
AAAGCTGGACCATTGGGATCA
CGTGTCCCGCACTTGGTAG
P2X4R
NM_
CTCGGGTCCTTCCTGTTCG
GTTTCCTGGTAGCCCTTTTCC
P2X5R
NM_
AGGGGGTGGCCTATACCAAC
GGTTAGGAGTCACGATCAGGTT
P2X6R
NM_
GTTAAGGAGCTGGAGAACCG
AGGATGCTCTGGACATCTGC
P2X7R
NM_
GACAAACAAAGTCACCCGGAT
CGCTCACCAAAGCAAAGCTAAT