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Wang et al. Cancer Drug Resist. 2026;9:8 Page 5 of 18

at 37 °C. PTTP-DCns were then added to each dish at a final concentration of 1 μM, followed by 4 h of
continued incubation. The cells were subsequently analyzed directly by CLSM imaging.

Cellular distribution
Following overnight adhesion in glass-bottom dishes (1 × 10 cells/dish), the MCF-7/ADR cells were
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incubated with PTTP-DCns (1 μM) for 24 h after medium removal, and subsequently treated with
Lyso-Tracker Green DND-26 (5 µM) for 1 h. After being washed twice with PBS buffer containing 1% FBS,
the cell samples were imaged with a high-speed CLSM. Lyso-Tracker Green DND-26 (excited at 488 nm,
emission 500-540 nm) was displayed in green, and PTTP-DCns (excited at 552 nm, emission 660-740 nm) in
red.

Cell viability assay
For the cytotoxicity of PTTP-DCns and chloroquine (CQ), MCF-7/ADR or HEK293 cells were seeded in
96-well plates and incubated for 24 h at 37 °C. The medium was then replaced with 0-25 µM of PTTP-DC4,
PTTP-DC6, PTTP-DC8 or 0-10 µM of CQ. After another 24 h, light-treated groups were irradiated with
525 nm light emitting diode (LED) light (0.2 mW·cm , 30 min); the rest remained in the dark. All Cells were
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cultured in the incubator for an additional 24 h.

To evaluate the effect of PTTP-DCns on drug sensitivity, MCF-7/ADR and HCC827/IR cells were treated
with a fixed concentration (1 µM) of each compound for 24 h, followed by the same light/dark protocol
described above. After irradiation, the medium was changed to a series of DOX solutions (0-100 µg·mL ) or
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icotinib solution (0-25 µM), and cells were cultured for an additional 48 h.

To evaluate the effect of CQ on drug sensitivity, MCF-7/ADR cells were treated with 1 µM of CQ for 24 h,
followed by change of culture medium with 100 μg·mL DOX and an additional incubation of 48 h.
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Viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Absorbance was measured at 570/600 nm. All experiments were performed in triplicate.

Cellular ROS level
Following a 24-hour treatment with or without 1 μM PTTP-DC6, MCF-7/ADR cells were incubated with
10 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) for 1 h at 37 °C. After two washes with PBS
containing 1% FBS, the dishes were filled with serum- and phenol red-free medium for subsequent analysis.
The dishes of light groups were irradiated with a 525 nm LED light at an intensity of 0.2 mW·cm for 30 min,
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while the rest were kept in the dark at room temperature. Thereafter, the cells were directly analyzed via
CLSM imaging. ROS generation was monitored by measuring the fluorescence of the oxidation product
(2,7-dichlorofluorescein, DCF), which was excited at 488 nm with emission collected in the 500-550 nm
range.

AO redistribution assay
Following overnight adhesion in glass-bottom dishes (1 × 10 cells/dish), the MCF-7/ADR cells were
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incubated with blank medium or PTTP-DCns (1 μM) for 24 h, and the dishes of light groups were irradiated
with a 525 nm LED light (0.2 mW·cm , 30 min), while the rest were kept in the dark at room temperature.
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Then the cells were incubated with acridine orange hydrochloride hydrate (AO, 5 μg/mL) for 15 min at 37 °C
and washed twice with PBS buffer. Fluorescence images were captured with CLSM (λ = 488 nm), with
ex
emission collected in the green channel (λ m,green = 500-550 nm) and red channel (λ m,red = 650-710 nm).

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