P c jun

Western blotting was performed using whole cell lysates. Aliquots of total protein (20-50 μg per lane) were electrophoresed on a 10% SDS-polyacrylamide gradient gel and transferred to nitrocellulose membranes (Millipore, Bedford, MA). The membranes were incubated at 4 °C overnight with anti-GAPDH, MYC, c-Jun, p-c-Jun, c-Fos, p-c-Fos, JNK, p-JNK, or Igκ monoclonal antibody (all purchased from Abcam, Cambridge, MA). After rinsing in buffer wash, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, TX) diluted 1:10,000-30,000, followed by development with enhanced chemiluminescence reagents (Amersham, Little Chalfont, UK).

The expression levels of Ki-67, cleaved caspase-3, Beclin1, LC3B-II, CHOP, GRP78, p-JNK, and p-c-jun in tumor tissues were measured by IHC analysis according to the protocols previously described [25 (link)]. Briefly, 4-mm consecutive sections were deparaffinized in xylene, rehydrated in a graded ethanol series, and submerged in EDTA antigenic retrieval buffer for 15 min in a microwave oven. The sections were treated with 3% hydrogen peroxide in absolute methanol for 20 min to block endogenous peroxidase activity. Then, 5% BSA was applied for 15 min to prevent non-specific binding. The sections were incubated overnight at 4 °C with primary antibodies. Ki-67 (1:150), cleaved caspase-3 (5 μg/ml), Beclin1 (1:200), LC3B-II (1 μg/ml), CHOP (1:100), GRP78 (1 μg/ml), p-JNK (1:100) and p-c-jun (1:100) were purchased from Abcam (Cambridge UK). After incubation with the secondary antibody, the visualization signal was developed with 3,30-diaminobenzidine tetrachloride.

After 72 h, the HSFs were washed with PBS and lysed in lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) supplemented with proteinase and phosphatase inhibitor cocktails on ice. The lysates were collected after centrifugation at 12,000g for 30 minutes at 4°C. The BCA protein quantification kit (ThermoFisher, USA) was used to quantify the total protein of the extracted cells. 20 μg sample protein from each group was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane and blocked with 5% skimmed milk powder blocking solution at room temperature for 1 h. Then, the membranes were incubated at 4°C overnight with the antibodies for α-SMA (Immunoway, USA), Smad3 (Abcam, USA), p-Smad3 (Abcam, USA), c-FOS (Abcam, USA), p-c-FOS (Abcam, USA),c-Jun (Abcam, USA), p-c-Jun (Abcam, USA), and GAPDH (Abcam, USA). After washed with TBS, the membranes were added secondary antibody (1 : 10 000) and shook gently at room temperature for 40 minutes, and ECL was added to the membrane to react for 3–5 minutes, and the film was exposed. The density of the bands was normalized to β-actin and quantified with Image software. Immunofluorescence was assessed using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA).