Ab14389

The human IL‐17A recombinant protein (rhIL‐17A, #8928), rabbit anti‐ZO‐1 polyclonal antibody (#8193), rabbit anti‐MMP‐9 polyclonal antibody (#15561), MMP‐2 antibody (#4022) the rabbit anti‐Slug polyclonal antibody (#9585), the rabbit anti‐AKT polyclonal antibody (#9272), the rabbit anti‐phospho‐AKT(Ser473) polyclonal antibody (#9271), the rabbit anti‐phospho‐PI3K polyclonal antibody (#4249), the rabbit anti‐phospho‐PI3K p85 (Tyr458)/p55 (Tyr199) polyclonal antibody (#4228), the anti‐rabbit IgG HRP‐linked antibody (#7074) and anti‐mouse IgG HRP‐linked antibody (#7076) were purchased from Cell Signaling Technology (Danvers, MA, USA). The mouse anti‐α‐SMA monoclonal antibody (ab7817), the mouse anti‐Twist monoclonal antibody (ab175430), the mouse anti‐Bmi1 monoclonal antibody (ab14389) were obtained from Abcam (Cambridge, UK). The mouse anti‐GAPDH monoclonal antibody was purchased from Beyotime Biotechnology (Shanghai, China). The Alexa Fluor^® 488 fluorescent second antibody and CellTracker CM‐Dil (C7000) were purchased from ThermoFisher Scientific (Waltham, MA, USA).

Cells were trypsinized and Accutase (Millipore) was used to dissociate GSCs. After washing with PBS, cells were centrifuged and stained with primary antibody, followed by incubation with FITC or PE conjugated secondary antibodies. The cells were then subjected to flow cytometry analysis on BDAria FACS machine (BD Biosciences, San Jose, CA, USA) and the data was analyzed using CellQuest software (BD Biosciences). Flow cytometry antibodies included: anti-human Fc-receptor (Catalog #130-095-979, Miltenyi Biotec, Bergisch Gladbach, Germany), anti-human CD133/2-PE (Catalog #130-080-901, Miltenyi Biotec), GFAP (Catalog #60048; STEMCELL Technologies, Vancouver, Canada), Nestin Antibody (S1409, Abgent, San Diego, USA), BMI1 (ab14389; Abcam, Cambridge, UK), E2F3 (LS-C87464; Lifespan-Bioscience Inc., Seattle, WA, USA), FITC or PE-conjugated goat anti-rabbit (ab6717; Abcam) and FITC or PE-conjugated goat anti-mouse mouse (ab6785; Abcam) secondary antibodies.

The cells were fixed using 10% buffered formaldehyde (Biovitrum, Saint Petersburg, Russia). The fixed cells were incubated with primary antibodies in a block solution based on Phosphate Buffered Saline (PBS) (PanEco, Moscow, Russia) with 10% fetal bovine serum (Capricorn Scientific, Ebsdorfergrund, Germany) and 0.3% TRITON X-100 (Sigma-Aldrich, St. Louis, Missouri, MO, USA) at 4 °C overnight and then with secondary antibodies in PBS with 0.3% TRITON X-100 for 2 h at room temperature. Nuclei were stained with DAPI (Biotium, Fremont, CA, USA).
The antibodies used were as follows:
Primary anti-collagen I antibodies (RAH C11, Imtek, Moscow, Russia)
Primary anti-collagen VI antibodies (ab6586, Abcam, Cambridge, UK)
Primary anti-fibronectin antibodies (ab2413, Abcam, Cambridge, UK)
Primary anti-S100A4 antibodies (ab27957, Abcam, Cambridge, UK)
Primary anti-collagen VII antibodies (C6805, Merck, Kenilworth, NJ, USA)
Primary anti-Ki-67 antibodies (ab16667, Abcam, Cambridge, UK)
Primary anti-hTERT antibodies (ab230527, Abcam, Cambridge, UK)
Primary anti-BMI-1 antibodies (ab14389, Abcam, Cambridge, UK)
Secondary anti-mouse Alexa-594 (A21201, Invitrogen, Carlsbad, CA, USA)
Secondary anti-rabbit Alexa-594 (A21442, Invitrogen, Carlsbad, CA, USA)

The chromatin immunoprecipitation (ChIP) DNA samples were prepared by ChIP Assay Kit (17-295; Upstate, Millipore, Billerica, MA, USA). Cross-linked DNA was sheared to 300–1000 bp in length by SONICS Uibra cell (Sonics & Materials, Newtown, CT, USA). Every ChIP assay contains more than 1 × 10⁶ cells. ChIP grade antibodies against BMI1 (ab14389; Abcam, Cambridge, MA, USA), acetyl-H3 (06-599; Millipore), acetyl-H4 (06-598; Millipore), acetyl-H3K27 (ab4729; Abcam), ubiquitin-H2A (05-678; Millipore), trimethyl-H3K9 (ab8898; Abcam), trimethyl-H3K27 (ab6002; Abcam) and normal Mouse IgG(12-371; Millipore) were used at 2 μg per condition. The products of ChIP were sent to Shanghai Biochip Co. for second-generation sequencing by illummina HiSeq 2000.

Cellular protein of CC was obtained with ice-cold RIPA lysis solution, and Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, 23227) was used to detect protein concentration in cell lysates. Then, the protein was dissolved in loading buffer and high-temperature denatured for 10 minutes. After separating by 10% SDS polyacrylamide gel electrophoresis, the protein was transferred to the PVDF membranes (Millipore, Germany). The membrane was blocked for 1 h prior to overnight incubation of primary antibodies (anti-TAB2, Proteintech, 14410-1-AP-50 μl; anti-BMI1, Abcam, ab14389-25 μg; anti-SOX2, Abcam, ab97959-100 μg; and anti-GAPDH, Abcam, ab70699) at 4°C, followed by secondary antibody incubation at room temperature for 1 h on the other day. The protein-antibody complex was detected with enhanced chemiluminescence (ECL) reagent.

Proteins were isolated using RIPA lysis buffer (Beyotime). After the qualification of protein concentration with the bicinchoninic acid assay, approximately 30 μg of extracted protein was added on Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) for separating and then transferred to polyvinylidene difluoride (PVDF) membranes. Subsequently, the membranes were blocked with 5% skim milk powder for 1 h at room temperature and then incubated with primary antibody against Cyclin D1 (1:20000, ab134175, Abcam, Cambridge, MA, USA), p27 (1:5000, ab32034, Abcam), BMI1 (1:3000, ab14389, Abcam), Caspase-3 Cleavage (Cas-3 Cleavage) (1:1000, Cat#:9664, Cell Signaling, Danvers, MA, USA), Caspase-9 Cleavage (Cas-9 Cleavage) (1:1000, Cat#:9505, Cell Signaling) and GAPDH (1:10000, ab181602, Abcam), followed by incubation with HRP-conjugated antibody (1:1000, ab9482, Abcam). Finally, blot bands were visualized and quantitated using the electrochemiluminescence method. GAPDH, came from the same gel as the target protein, was used as a normalization control.