P erk1 2

Bromocriptine was bought from Gedeon Richter Ltd. (Budapest, Hungary). Hordenine was bought from Chengdu Must Bio-Technology Co. Ltd. (Chengdu, PR China). The antibodies of prolactin (PRL), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) were obtained from Affinity Biosciences (Cincinnati, USA). The antibodies of nuclear factor kappa-B (NF-κB), p38, p-p38, JNK, p-JNK, ERK1/2, and p-ERK1/2 for rats were supported by Proteintech Group, Inc (Wuhan, PR China). The PRL enzyme-linked immunosorbent assay (ELISA) kit was bought from Wuhan Huamei Bioengineering Co., Ltd. (Wuhan, China).

Protein levels of PFKFB3, E-cadherin, N-cadherin, Vimentin, total/phosphorylated-ERK 1/2 (t/p-ERK1/2), and β-actin were tested by western blot as narrated before [29 (link)]. The specific schedule was as follows: transmembrane (90 min, 300 milliampere); blocking with milk (45 min); primary antibody incubation time was 2 h; secondary antibody (1:10,000) incubation time was 1 h. Mouse monoclonal antibody against β-actin (1:50,000, Cat No.: 66009–1-Ig), E-cadherin (1:4000, Cat No.: 60335–1-Ig), N-cadherin (1:4000, Cat No.: 66219–1-Ig), Vimentin (1:50,000, Cat No.: 60330–1-Ig) and rabbit polyclonal antibodies against PFKFB3 (1:1000, Cat No.: 13763–1-AP), total-ERK 1/2 (t-ERK1/2, 1:1000, Cat No.: 11257–1-AP), and phosphorylated-ERK 1/2 (p-ERK1/2, 1:3000, Cat No.: 28733–1-AP) (all from Proteintech Group, USA) were adopted.

The 20 (R)-Rg3 was provided by Dalian Fu Sheng Pharmaceutical Co. (Dalian, China). A solution of Rg3 as freshly prepared in DMEM (500 μg/ml) and filtered by 0.22-μm membranes. It was diluted with cell culture media to final concentration in different treatments. DMEM, fetal bovine serum (FBS), TRIzol and Lipofectamine™ 2000 reagents were purchased from Invitrogen (Camarillo, CA, USA). DMSO and MTT were purchased from Sigma-Aldrich (St. Louis, MO, USA). The enhanced chemiluminescence (ECL) assay kit was purchased from Amersham (Pittsburgh, PA, USA). Anti-rabbit FUT4, PCNA, p-ERK1/2, ERK, β-actin, HRP-conjugated anti-mouse IgM, HRP-conjugated anti-rabbit and anti-mouse IgG antibodies were purchased from Proteintech group (Wuhan, China). EGFR and p-EGFR were purchased from Cell Signaling Technology (Boston, MA, USA). FITC conjugated goat anti-mouse IgG and TRITC conjugated goat anti-rabbit IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HRP-Ulex Europaeus (UEA) lectin was purchased by EY Laboratories (San Mateo, CA, USA), which preferentially recognizes the total fucose. Mouse anti-Giantin Golgi marker antibody and mouse anti-LeY antibody (BG-8) were purchased from Abcam (Cambridge, UK). AG1478 inhibitor was obtained from Sigma (St. Louis, MO, USA). Coomassie protein assay reagent was purchased from Bio-Rad (Hercules, CA, USA).

Western blotting was performed as previously described [17 , 20 (link)]. Briefly, cells were collected, lysed, and then quantified with a BCA kit (Beyotime, China). 20 μg of protein was loaded into a SDS gel, followed by electronic transfer onto PVDF membranes (Millipore, USA), and then incubated with primary antibodies against DHX9 (No.17721–1-AP, Proteintech), STAT1 (No.10144–2-AP, Proteintech), STAT1 phospho Ser727 (p-STAT1, No.39634, Active motif), p38 (No.14064–1-AP, Proteintech), Phospho-P38 MAPK (Thr180/Tyr182) (p-p38, No.28796–1-AP, Proteintech), JNK (No.66210–1-Ig, Proteintech), Phospho-JNK (Tyr185) (p-JNK, No.80024–1-RR, Proteintech), ERK1/2 (No.11257–1-AP, Proteintech), Phospho-ERK1/2 (Thr202/Tyr204) (p-ERK1/2, No.80031–1-RR, Proteintech), p65 (No.66535–1-Ig, Proteintech), p65 (phospho S536) (p-p65, sc-136548, Santa Cruz), GAPDH (ab8245, Abcam), flag (ab205606, Proteintech), or Lamin B1 (No.66095–1-Ig, Proteintech). After incubating with the secondary antibodies, the protein bands were detected using an ECL kit (Pierce, USA). The experiment was repeated three times independently.

Pancreatic cancer cell lines PANC-1 and MIA PaCa-2 were purchased from the Shanghai and Kunming Cell Banks of the Chinese Academy of Sciences. The KIF22 primers used were purchased from Beijing Kinco Biotechnology Co., Ltd., fetal bovine serum was obtained from PEAK, United States, and Dulbecco's modified Eagle medium (DMEM) was purchased from Gibco, United States; MTT cell proliferation kit (colorimetric method) was purchased from BioVision, United States; KIF22 SiRNA and supporting transfection reagents were obtained from Shanghai Jima Pharmaceutical Company, PI staining solution used for cell cycle detection was purchased from Beijing Soleibao Company, and p-MEK1/2 (phosphorylated mitogen-activated protein kinase kinase, p-MAPKK; catalogue number: 9154) was purchased from Cell Signaling Technology in the United States, p-ERK1/2 (phosphorylated extracellular regulated kinase 1/2; catalogue number: 28733-1-AP), and P21 (cyclin-dependent kinase inhibitor 1A, CDKN1A; catalogue number: 10355-1-AP) antibodies were purchased from Proteintech Company in the United States.