Wnt3a attenuates acute lung injury by reducing P2X7 receptor–mediated lung type I cell death

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

P2X3Rb

HEK29

3-P2

x7R

HEK29

3E1

0R3/

1M

LE15

A549

PRE-

TIIH44

1RLE

-6TN

RLF-6

P2X7R

β-actin

a

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.

P2X2R

P2X4R

P2X5R

P2X6R

P2X7R

P2X1R

E10

Lung

100 bp200 bp

E10GAPDH

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.

b c

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.

a

**

# #

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.

Control

BzATPCon_CM

BzATP Wnt3a_CM

YO-P

ROCo

ntro

l

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.

BzA

TP

Cont

rol 0 10 25 50

β-Ca

teni

n

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.

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).

* **

*

BzATP

Supplementary Figure S8: Original scans of western blots

Supplementary Table S1. PCR primer sequences foe mouse genes

Gene Accession # Forward primers Reverse primers

18S NR_003278.1 ATTGCTCAATCTCGGGTGGCTGCGTTCTTAGTTGGTGGAGCGATTT

GBmp4 NM_007554.2 TGGGATGCTGCTGAGGTTGAAG CGAGCCAACACTGTGAGGAGTTTC

Axin2 NM_015732.4GCGAGTGGCCAAAGCAATCTATAA

G GAGCCGATCTGTTGCTTCTTGATGCyclin D1 NM_007631.2 AGAAGTGCGAAGAGGAGG TGTTCACCAGAAGCAGTTCCyclin E1 NM_007633.2 CACAACATCCAGACCCACACCAAC ACCACACTCGGAGGAGGAGAAATC

P2X1R NM_008771.3 TCAAGGACATTGTGCAGAGAAC CAGTTGCCTGTGCGAATACCTP2X2R NM_153400.4 GCGTTCTGGGACTACGAGAC ACGTACCACACGAAGTAAAGCP2X3R NM_145526.2 AAAGCTGGACCATTGGGATCA CGTGTCCCGCACTTGGTAGP2X4R NM_011026.2 CTCGGGTCCTTCCTGTTCG GTTTCCTGGTAGCCCTTTTCCP2X5R NM_033321.3 AGGGGGTGGCCTATACCAAC GGTTAGGAGTCACGATCAGGTTP2X6R NM_011028.2 GTTAAGGAGCTGGAGAACCG AGGATGCTCTGGACATCTGCP2X7R NM_011027.2 GACAAACAAAGTCACCCGGAT CGCTCACCAAAGCAAAGCTAAT