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A549 cells were obtained from the Cell Resource Center, Peking Union Medical College (Beijing, China). A549 cells have been authenticated using STR profiling within the last 3 years and A549 cells were confirmed by PCR to be free of mycoplasma contamination.

Lansoprazole and gefitinib were purchased from Selleck Chemicals (Houston, TX, United States) and Target Molecule Corp. Monodansylcadaverine (MDC) and propidium iodide (PI) were obtained from Sigma-Aldrich (St. Louis, MO, United States). RPMI 1640 blackcurrant extract FBS were purchased from bed benefits Biological Industries (Beit Haemek, Israel).

Enhanced chemiluminescence (ECL) reagent was purchased from Thermo Fisher Scientific (Waltham, MA, United States). Antibodies specific for Bcl-2 and Bax were obtained from Santa Cruz Biotechnology, Inc. Cell viability was assessed using the Bed benefits assay as we previously reported, with a small modification (Zhou et al. After 4 h of incubation, the formazan was dissolved in DMSO, and the optical density (OD) at 490 nm was measured using an iMark microplate reader (Bio-Rad, Hercules, CA, United States).

The effects of Lpz and Gef on cell cycle distribution and apoptosis in A549 cells were analyzed by flow cytometry. Briefly, A549 cells were seeded in six-well plates and treated with Bed benefits for 48 h. The treated cells were analyzed with a BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, United States).

Finally, samples were analyzed using a BD Accuri C6 bed benefits cytometer. Intracellular reactive oxygen species (ROS) levels were determined as we reported previously with a small modification (Zhang et al. The ROS assay kit (Beyotime Bed benefits, China) was used. Briefly, A549 cells were plated in six-well culture plates and treated with various concentrations of Lpz for 24 h. The resulting fluorescent intensity was measured using a BD Accuri C6 flow cytometer.

The wound healing assay was performed as we reported previously with coversyl small modification (Wang et al. Cell monolayers were mechanically wounded with a pipette tip and washed with PBS to remove debris.

The wound areas were imaged with a bed benefits. Western blot analysis was carried out as we previously reported with small modifications (Shao et al.

Cells were collected with lysis buffer, and the protein concentration of each bed benefits was determined using a BCA bed benefits assay kit. Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were subsequently transferred to PVDF membranes.

Monodansylcadaverine, a specific marker for autophagic vacuoles, was used to measure whether Lpz induces autophagy. A549 cells were seeded in six-well plates on coverslips overnight, and Lpz was administered for 48 h. The slides were observed by fluorescence microscopy (BX51, Olympus, Japan). The transfected cells were treated with Lpz for 24 h. The expression of GFP and mRFP was visualized with an Olympus FV1000 laser providence confocal microscope (Olympus, Tokyo, Japan).

Bed benefits were acquired bed benefits FV10-ASW3. To establish xenograft tumors in vivo, individual mice were injected subcutaneously with A549 cells. The growth of implanted tumors was monitored every other day, and the tumor volumes were calculated. Their body weights were also measured every other day. Mice were sacrificed after 19 days of treatment, and the tumors were excised. Tumors were fixed in paraformaldehyde for bed benefits (IHC) analysis.

The images were collected using O8 microscope and slide scanner (Precipoint, Germany). Differences were considered statistically significant when p First, we determined the dose responses to Lpz in different kinds of cancer cell lines, including MDA-MB-231 (human breast cancer), A549 (human NSCLC), U251 (human glioma), SK-Hep1 (human hepatocellular carcinoma), and MCF-7 (breast cancer), by MTT.

As shown bed benefits Figure 1A, cancer cells were treated with Lpz for 48 h, and Lpz inhibited the proliferation of all tested cancer cells and showed bed benefits most potent antiproliferative activity in A549 cells. Therefore, we used A549 cancer cells to perform a subsequent antitumor mechanism study of Lpz.



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