Lsm confocal laser scanning microscope

For Co-IP assay, cells were collected and lysed on ice with lysis buffer containing 0.5% NP40. The lysates were pre-cleared by incubation with protein A beads. The protein complex was then precipitated by a specific antibody together with protein A beads followed by extensive washing. The resulting materials were analyzed by western blotting as previously described^{81 (link)}. For IF analysis, PDLSCs were fixed using 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 and blocked with 4% BSA/PBS. Then the cells were treated with primary antibodies followed by incubation with FITC or TRITC–conjugated secondary antibodies. Coverslips were mounted in a glycerol/PBS solution containing DAPI. Confocal images were obtained using a Zeiss LSM confocal laser-scanning microscope. Antibodies against EZH2 (#5246), E2F1 (#3742), lamin A/C (#4777), H3K27me3 (#9733), H3K9me2 (#4685), H3K9me3 (#13969), RB and phospho-RB (S780, S795, and S807/811) (RB Antibody Sampler Kit #9969) were purchased from Cell Signaling Technology (CST). Antibodies against GAPDH (AC002) and α-Tubulin (AC007) were purchased from Abclonal. Antibody against CEMP-1 (ab134231) was from Abcam. Antibody against CAP (sc-53947) was from Santa Cruz. Antibodies used in IF were: EZH2 (612667, BD Pharmingen), H3K27ac (A7253, Abclonal), MED1 (A300-793A, Bethyl) and VAV2 (YT4864, Immunoway).

GFP-tagged P. aeruginosa were grown in ABTG medium and flowed through flow chambers as previously described (Sternberg and Tolker-Nielsen, 2006 (link)). Each flow chamber is consisted of three-channel flow cells that were supplied with a flow of medium and oxygen, while waste medium would be directed into a waste flask. Briefly, overnight cultures were diluted 1,000 times in ABTG medium and injected into each channel for 1 h incubation time without flow. Next, the medium was allowed to flow into the flow cells and the velocity was maintained at 0.2 mm/s using Cole-Palmer peristaltic pump. Biofilms were grown for 72 h before treatment with compounds for further 48 h. To visualize dead cells, 300 μL of propidium iodide (PI) stain was injected into each flow cells. Biofilm images were taken with LSM confocal laser scanning microscope (Carl Zeiss, Germany) at 20x objective lens. Microscopy images were processed with IMARIS software (Bitplane AG, Zurich, Switzerland). Experiments were done in triplicate manner and repeated at least twice to confirm the results.