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Supporting Information
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Supplementary Figure 1. TIM-4 co-localizes with CK-18 in serial lung cancer
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tissue sections. IHC staining was performed to detect TIM-4 and CK-18 expression
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in lung cancer tissues. Photos of IHC staining are representative of at least 10 similar
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observations.
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Supplementary Figure 2. LPS and cytokines promote the expression of TIM-4 in
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lung cancer cells. Cells were set up in 96 or 6-well plates. After grown to 80–90%
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confluence, A549 or NCI-H1975 cells were stimulated with LPS or cytokines. 3 h
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after stimulation, cells were harvested and TIM-4 mRNA was detected by real time
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PCR. (A) TIM-4 transcripts were detected in lung cancer cell lines or THP-1 cells by
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RT-PCR. TIM-4 mRNA in A549 cells (B) or NCI-H1973 cells (C) was examined
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after stimulation with LPS (100 ng/ml), IL-6 (5 ng/ml), TGF-β (50 ng/ml) or TNF-α
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(100 ng/ml).
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Supplementary Figure 3. Knockdown of TIM-4 suppresses lung cancer cell
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growth. A549 cells were set up in 96-well plates. After grown to 80–90% confluence,
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cells were transfected with siRNA targeting TIM-4 or control siRNA, and the cell
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growth was monitored by CCK-8 assay for 5 days (*P<0.05).
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Supplementary Figure 4. Overexpression of TIM-4 inhibits apoptosis of lung
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cancer cells. A549 cells were seeded in 24-well plates. After grown to 80–90%
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confluence, the cells were transfected with pc3 or pc3-hT4 plasmid DNA. 48 h After
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transfection, apoptosis or protein analysis was performed. (A) Cells were collected
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after transfection, stained with Annexin V and PI according to standard protocols
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using Annexin V Cell Apoptosis Analysis Kit (AO2003-H, Sungene Biotech Co.,Ltd,
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Tianjin, China) and further assayed using Beckman Coulter Flow Cytometer (FC500,
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Fullerton). (B) Western blot was performed to detect apoptotic related proteins using
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anti-Caspase-3（#9668，cell signaling）, anti-Caspase-9（#9508，cell signaling）,
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anti-PARP (46D11) (#9532, cell signaling ), anti-Bcl-2（#2870，cell signaling）,
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anti-Cytochrome-C(#4280，cell signaling ), and anti-β-Actin (TA-09,
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Zhongshanjinqiao).
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