Anti foxm1 antibody

The human 293T cell line and pancreatic cancer tissue microarrays (Cat#: HPan-Ade180Sur-02) which contain 73 pairs of PDAC samples and adjacent normal tissues were obtained from Shanghai Outdo Biotech, China. Immunohistochemical staining for FOXM1 and in situ hybridization for miR-552 were respectively performed according to the commercial protocol (Outdo Biotech, Shanghai, China) using anti-FOXM1 antibody (Santa Cruz Biotechnology, USA) or 3' and 5' DIG labeled LNA miR-552 probes (Ambion, USA). All the samples were identified by two experienced pathologists independently blinded to the clinical data. The following expression levels were based on the score obtained by the intensity and percentage of the positive staining. The intensity was recorded as 0, 1, 2, and 3 referring to negative, weak, moderate, and strong staining, respectively. Grade 0, 1, 2 group were classified as 'low expression' while grade 3 group were classified as 'high expression'. The research on the use of human tissue samples was approved by the Medical Ethics Committees of Shanghai East Hospital, Tongji University.

PANC1 cells (1×10⁷) were prepared for ChIP assay. ChIP experiments were performed with chromatin immunoprecipitation assay kit (Millipore, CA, USA) according to the manufacturer's instructions. Briefly, cell sediments were crosslinked with 1% formaldehyde for 15 mins. Crosslinked chromatin was sonicated and incubated with 5 μg anti-FOXM1 antibody (Santa Cruz Biotechnology, USA) at 4 °C overnight. Co-precipitated DNA was analyzed using PCR to amplify a 133-bp region of pri-miR-552 promoter and PCR products were resolved electrophoretically on a 2% agarose gel. Also, the immunoprecipitated DNA was quantitated by real-time PCR to measure FOXM1 binding levels, which were normalized to 2% input. Isotype-matched IgG (5 μg) was used as a negative control. The 133-bp promoter sequence was amplified with the following primer pairs: Forward, 5'-TGCCTGTTGGTTGAAGATG-3'; Reverse, 5'-CCAAAGATTCATCCAAGTTG-3'.

Western blot analysis was performed using standard procedures. The following primary antibodies were used in the experiments: anti-FOXM1 antibody (Santa Cruz), anti-pAKT antibody (Peprotech, USA), anti-AKT antibody (Cell Signaling Technology, Beverly, MA, USA), anti-pGSK3β antibody (Proteintech) at 1:1000, anti-Snai1 antibody (Cell Signaling Technology), anti-pIGF1R antibody (Abcam, Cambridge, UK), anti-IGF1R antibody (Abcam, Cambridge, UK), anti-E-cadherin antibody (BD Biosciences, USA), anti-Vimentin antibody (Cell Signaling Technology), anti-N-cadherin antibody (Cell Signaling Technology), anti-pERK1/2 antibody (Abcam), anti-ERK1/2 antibody (Abcam), anti-HIF1α antibody (Novus Biologicals, USA), anti-ETS1 antibody (Abcam) and anti-β-actin antibody (Sigma, St. Louis, MO,USA).

The cells were fixed in 4% paraformaldehyde (#30525–89–4; FUJIFILM Wako Chemical, Wako, Japan) for 15 min and blocked with 1% bovine serum albumin in PBS‐00.1% Tween‐20 (PBS‐T) to reduce non‐specific antibody binding. The cells were then incubated overnight with anti‐PD‐L1 antibody (#14598382; eBioscience, San Diego, CA, USA) diluted 1:200, and anti‐FOXM1 antibody (#sc‐271746; Santa Cruz Biotechnology) diluted 1:50 at 4 °C, followed by incubation for 1 h with Alexa Fluor 488‐labeled secondary antibody (#A11029; Thermo Fisher Scientific). Nuclei were stained with Hoechst 33342. After incubation, the cells were washed with PBS and mounted on glass slides using Prolong Glass Antifade mounting medium (#P36982; Thermo Fisher Scientific). Confocal microscopy was performed using a Zeiss 730 Meta microscope (Carl Zeiss, Oberkochen, Germany) and analyzed using Axiovision software (Carl Zeiss).

Cells were lysed with IP lysis buffer (Thermo Scientific, Waltham, MA) containing protease inhibitor cocktail III (EMD Millipore, Billerica MA). The proteins were separated by electrophoresis using 4–20% SDS-PAGE gel (Bio-Rad, Hercules, CA) and transferred onto PVDF membranes. The membranes were incubated with the first antibody, respectively: anti-TOPK antibody (BD Biosciences, Franklin Lakes, NJ), anti-FOXM1 antibody (Santa Cruz Biotechnology, Dallas, TX), anti-pan-Akt (Cell Signaling, Danvers, MA), anti-β-actin (Sigma-Aldrich), or anti-GAPDH antibody (Sigma-Aldrich). A mouse anti-MELK monoclonal antibody against partial MELK protein (264–601 amino acids) was used to detect MELK protein, as described previously [9 (link)]. β-actin or GAPDH was used as a loading control.