A1 microscope

TIRF and Laser Scanning Confocal microscopy imaging were conducted using the Nikon A1 Microscope equipped with 405, 488, and 561 nm LASERs, Apo TIRF 100×/1.45 NA objective, and an Evolve EMCCD camera for the TIRF modality (Photometrics Technology). Images were denoised using Nikon Elements software for images shown in Figures 2 and 3, and Fig. S2. During imaging acquisition, the gain was matched between samples for comparison.

MDA-MB-231 cells were seeded at a density of 1x10⁴ cells/well in 24-well plates containing 12 mm coverslips. After 24 hours, cultures were changed to quiescent medium (serum-free RPMI). After an additional 24 hours, cells were treated with 20 ng/mL IP-10, 50 nM AMG-487, 1 µg/mL LPS (Millipore Sigma) + 20 ng/mL EGF (Corning) in serum-free RPMI or HepM plus 10 µM EdU for 4 hours and then fixed with 2% paraformaldehyde at 4°C for 1 hour. Active proliferation was determined using the Click-iT PLUS EdU Alexa Fluor 488 Imaging Kit (Life Technologies) and detected according to the manufacturer’s instructions. Coverslips were mounted onto slides and imaged using the Nikon A1 microscope fitted with a 20x objective (Center for Biological Imaging, University of Pittsburgh).

CLSM observations were conducted as previously reported (Kang et al., 2018 (link)). After incubation, biofilms were illuminated at 25, 10, and 4°C for 2 h. Then sterile water was used to remove planktonic cells. SYTO 9 was used to stain the biofilm for 15 min at room temperature, protected from the light. After staining, the sheets were rinsed three times with sterile deionized water to remove excess stain. A Nikon A1 microscope (Nikon, Tokyo, Japan) was then used to obtain CLSM images using a 10× objective lens. CLSM images of non-LED-treated biofilms were also obtained as a control to observe the damage caused by LED exposure.

Cells were fixed using 4% paraformaldehyde (PFA) for 20 minutes and washed thoroughly with PBS. To distinguish between cell attachment and invasion, extracellular bacteria were stained with rabbit anti-Rickettsia I1789 antibody (provided by Ted Hackstadt) followed by the secondary antibody, Alexa 594 goat anti-rabbit (Invitrogen, A11005; 1:1000 dilution). To subsequently stain all bacteria (intracellular and extracellular), cells were permeabilized and again stained with rabbit anti-Rickettsia I1789 antibody followed by the secondary antibody, Alexa 488 goat anti-rabbit (Invitrogen, A11008; 1:1000 dilution). Coverslips were mounted with VectaShield HardSet antifade mounting medium with DAPI (Vector Laboratories Inc.) for nuclear staining. Samples were visualized and quantified using a Nikon A1 microscope (S10RR027535).
For growth curves, cells were permeabilized using 0.5% Triton-X100 for 15 minutes and blocked with 3% BSA for 1 hour. Coverslips were then probed for rickettsiae using rabbit anti-Rickettsia I1789 antibody diluted 1:1000, followed by the secondary antibody, Alexa 488 goat anti-rabbit (Invitrogen, A11008; 1:1000 dilution). For all immunofluorescence assays, samples with secondary antibody only served as a control for non-specific binding of the Alexa fluor antibodies.

RNA FISH assays were performed as previously described [17 (link)]. Briefly, complementary probes targeting the “head-to-tail” sequence of circPTEN1 were synthesized by Sangon Biotech (Shanghai, China) (Table S2). The cells were cultured on gelatin-coated glass cover slips, fixed with 4% formaldehyde in phosphate-buffered saline (PBS), and permeabilized for 5 min with 0.2% Triton X-100 in PBS. The cover slips were incubated overnight at 37 °C with a hybridization mix containing 1 ng/μL probe mix. The unbound probes were washed off the next day with hybridization buffer, and cells were mounted after staining with DAPI. Images were taken with an immunofluorescence microscope (Nikon A1).
For protein IF assays, cells seeded on cover glass were fixed with 4% PFA for 15 min and washed twice with PBS. Washed cells were permeabilized with 0.2% Triton X-100 for 5 min and blocked for 1 h using 2% BSA dissolved in PBS. The primary antibody was then applied for 3 h at room temperature, followed by three washes in PBS, and fluorophore-conjugated secondary antibody was applied for 1 h. After being washed twice with PBS, cells were stained with DAPI (Sigma). Coverslips were mounted, and cells were evaluated with fluorescence microscopy. A Nikon A1 microscope was used for confocal microscopy.