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HeLa cells were plated on glass-bottomed dishes (#D35-20-0-TOP, Cellvis, Mountain View, CA, USA). Cells were then transfected with the indicated plasmids by using Lipofectamine 3000 (Invitrogen, Waltham, MA, USA). Twenty-four hours post-transfection, the samples were mounted onto a Nikon Ti2 Inverted microscope equipped with a Yokogawa W-1 dual spinning disk scan-head, Micro-Scanner for photo-stimulation, and a stage top incubator for live cell imaging. Blue light stimulation was carried out using the built-in 488-nm laser source with 5% input at an interval of 5 s for 5–20 min. To monitor the recruitment of TBK1-YFP, HeLa cells were transfected with plasmids encoding CRY2-pLxIs or mCh-CRY2 (200 ng) and TBK1-YFP (200 ng). 488 nm and 561 nm laser sources were used to excite TBK1-YFP and mCh-CRY2-pLxIs (or mCh-CRY2), respectively. In the experiment for monitoring the reversible clustering of LiSmore, cells were repeatedly illuminated in two dark-light cycles (5 s ON + 10 min OFF). The captured images were analyzed by the Nikon Elements imaging processing software (Nikon, NIS-element AR version 4.0, Minato City, Tokyo, Japan) or the ImageJ program (NIH). All imaging data shown were representative of at least three biological replicates.

To examine LCN2 and EGFR colocalization, OSCC cells were plated on glass-bottomed dishes (#D35-20–0-TOP, Cellvis). The cells were then transfected with the indicated plasmids by using Lipofectamine 3000 (Invitrogen). After transfection for 24 h, the samples were observed and captured on a Zeiss microscope.
For time-lapse imaging of NP uptake by OSCC cells, GFP-endo14 labeled CAL-27 cells at approximately 30–40% confluency were seeded on glass-bottomed dishes for 16 h. After the medium was replaced with 2 mL of fresh medium, glutathione-responsive NPs (G-NPs) loaded with Cy5-labeled siLCN2 were added at an siRNA concentration of 50 nM, and the cells were incubated for 8 h. Images were captured for at least 8 h.
The captured images were analyzed by the Zen Blue microscope imaging software (Zeiss) or ImageJ program. Dozens of cells were selected to define regions of interest (ROIs) for analysis.

Cells were seeded on glass dishes (#D35-20–0-TOP, Cellvis) overnight. The cells were treated with TGF-α for an additional 2 h. At the time of analysis, the cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min, permeabilised with a solution containing 0.1% Triton X-100 in PBS for 10 min and blocked with 1% BSA in PBS for 1 h at room temperature. Primary antibodies (EGFR and LCN2) were then incubated overnight at 4 °C. The slides were washed and secondary antibodies were applied for 1 h in PBS at a final dilution of 1:1000. After washing, the cell nuclei were labelled with DAPI.