1：Experimental Design and Method Selection:

The study designed an upconversion optogenetic nanosystem consisting of upconversion rods (UCRs) encapsulated in a flexible capsule (UCRs-capsule) to convert NIR light to blue light, and optogenetic plasmids (p53-Cry2 and NLS-Cib1) to induce protein-protein interaction and autophagy up-regulation. Methods included cell culture, transfection, confocal microscopy, western blotting, flow cytometry, and in vivo mouse models.

2：Sample Selection and Data Sources:

HeLa and 293T cell lines were used for in vitro experiments. Female BALB/C nude mice with HeLa cell tumors were used for in vivo studies. Data were collected from fluorescence imaging, protein assays, and RNA sequencing.

3：List of Experimental Equipment and Materials:

Equipment included confocal microscope (PerkinElmer), fluorescence spectrophotometer (Cnilaser), SEM (Nanosem430), flow cytometer (BD FACSCalibur), microplate reader (Thermo Scientific). Materials included UCRs (synthesized with rare-earth elements), PDMS (Sylgard 184), PET film (Chenglida), plasmids (constructed by GENEWIZ), antibodies (e.g., LC3 from proteintech), and reagents (e.g., Lipofectamine2000 from Invitrogen).

4：Experimental Procedures and Operational Workflow:

Cells were transfected with plasmids, exposed to light (LED blue or NIR laser), and analyzed for protein localization and autophagy markers. In vivo, mice were implanted with UCRs-capsule, transfected intratumorally, irradiated with NIR light, and tissues were analyzed post-sacrifice.

5：Data Analysis Methods:

Data were analyzed using ImageJ, AzureSpot software, Excel, OriginPro8.0, FLOWJO for flow cytometry, and DAVID for GO analysis. Statistical significance was assessed with t-tests.