Online Data Supplement
Autophagy Is Essential for Ultrafine Particle-Induced Inflammation and Mucus
Hyperproduction in Airway Epithelium
Zhi-Hua Chen, Yin-Fang Wu, Ping-Li Wang, Yan-Ping Wu, Zhou-Yang Li, Yun Zhao,
Jie-Sen Zhou, Chen Zhu, Chao Cao, Yuan-Yuan Mao, Feng Xu, Bei-Bei Wang,
Stephania A Cormier, Song-Min Ying, Wen Li, Hua-Hao Shen
Supplementary Reagents:
An additional set of siRNAs targeting LAMP2, RELA and JUN were designed and
synthesized by Shanghai GenePharma (Shanghai, China). The sequences of siRNAs
are listed below:
LAMP2-siRNA: Forward: 5’-CAGCAUGUAUUUGGUUAAUTT -3’; Reverse:
5’-AUUAACCAAAUACAUGCUGTT-3’
RELA-siRNA:
Forward:
5’-CCUCCUUUCAGGAGAUGAATT-3’;
Reverse:
5’-UUCAUCUCCUGAAAGGAGGTT-3’
JUN-siRNA:
Forward:
5’-GGAACAGGUGGCACAGCUUTT-3’;
Reverse:
5’-AAGCUGUGCCACCUGUUCCTT -3’
Control-siRNA:
Forward:
5’-UUCUCCGAACGUGUCACGUTT-3’;
5’-ACGUGACACGUUCGGAGAATT -3’
Reverse:
Figure S1. PM-induced mRNA expression of other cytokines in HBE cells. Cells
were treated with the indicated concentrations of PM for 24 h, and the relative levels
of IL1A, IL1B, TNF, and IL18 mRNA transcripts were detected by quantitative PCR.
Figure S2. knockdown effects of the siRNAs used in this study. HBE cells were
transfected with the indicated siRNA for 48 h, and then were harvested for analysis of
target gene or protein by quantitative PCR or western blot.
Figure S3. Expression of CXCL2 and IL6 in PM-exposed WT and lc3b-/- mice. Mice
(n=5 to 7 for each group) were instilled intratracheally with PM at 100 μg/d for 2 days.
Mice were sacrificed after 24 h, and the expression of the mRNA levels of Cxcl2 or
Il6 in lung tissue were analyzed by quantitative PCR, and protein levels of CXCL2 or
IL6 in the BALF were measured by ELISA.
Figure S4. Effects of an additional LAMP2-siRNA on PM-induced expression of IL8
and MUC5AC in HBE cells. Cells were transfected with the indicated siRNA for 24 h,
and then were treated with PM (100 g/ml) for an additional 24 h. Cells were then
harvested for analysis of mRNA expression of IL8 or MUC5AC by quantitative PCR.
Figure S5. Effects of Baf A1 (A), LAMP2- (B), RELA- (C), or JUN- (D) siRNA on
PM-induced expression of IL6 in HBE cells. (A) Cells were treated with 100 g/ml PM
and Baf A1 (10 nM) for 24 h. (B to D) Cells were transfected with the indicated siRNA
for 24 h, and then were treated with PM (100 g/ml) for an additional 24 h. Cells
were then harvested for analysis of mRNA expression of IL6 by quantitative PCR.
Figure S6. Effects of additional RELA- or JUN-siRNA on PM-induced expression of
IL8 and MUC5AC in HBE cells. Cells were transfected with the indicated siRNA for
24 h, and then were treated with PM (100 g/ml) for an additional 24 h. Cells were
then harvested for analysis of mRNA expression of IL8 or MUC5AC by quantitative
PCR.