Page 139 - Read Online

Page 6 of 18 Wang et al. Cancer Drug Resist. 2026;9:8

Detection of cathepsin B release
The pretreatment of MCF-7/ADR cells was consistent with that of AO redistribution assay. Cells were
incubated in blank medium or PTTP-DC6 (1 μM) solution for 24 h, then irradiated at 525 nm (0.2 mW·cm )
-2
for 30 min or darkened, washed twice with PBS buffer, incubated with cathepsin B substrate (Green
Cathepsin B Assay Kit) for 1 h, washed twice with PBS, as observed by CLSM. λ /λ : cathepsin B (green) was
ex
em
488/500-540 nm, PTTP-DC6 (red) was 552/600-740 nm.

Pro DIA quantitative proteomics assay
MCF-7/ADR cells were cultured in T175 tissue culture flasks (culture area: 175 cm ) and divided into three
2
experimental groups (n = 3): a blank control, a DC6-treated group, and a DC6-treated group with light
exposure (DC6-L). When cell density reached approximately 60%, cells were treated with either blank
medium or PTTP-DC6 (1 μM) solution for 24 h, then irradiated at 525 nm (0.2 mW·cm ) for 30 min or
-2
darkened. Following a subsequent 1-hour incubation, cells were harvested and subjected to quantitative
proteomics analysis at Shanghai OE Biotech Co., Ltd. (China).

Data-independent acquisition mass spectrometry (DIA-MS) raw data were processed using DIA-NN (an
automated software suite for DIA proteomics data processing, version V2.3.2) for protein identification and
quantification. Proteins with excessive missing values across samples and those lacking unique peptides were
excluded. Missing values were imputed using the group mean or half of the minimum value in the
expression matrix. The data were then normalized by median centering and log2-transformed. Differential
expression analysis was performed using a t-test; proteins with a fold change ≥ 1.5 and a P-value < 0.05 were
considered significant. Functional enrichment and gene set analysis of these differentially expressed proteins
were conducted using clusterProfiler (version 4.9.1).

3D cytotoxicity assay
MCF-7/ADR spheroids were formed in the Akura™ PLUS Hanging Drop Plate and then transferred to the
Akura™ 96 Spheroid Microplate. Following a recovery day (day 0), they were treated with 5 μM PTTP-DC6
or control medium for 24 h. One group was then irradiated with 525 nm light (1 mW·cm , 30 min), while
-2
another remained in the dark. All spheroids were subsequently exposed to 100 μg·mL DOX for 48 h. Cell
-1
viability was assessed using the CellTiter-Glo® 3D assay. The cell viability rate of 3D microtissues (VR ) was
3D
calculated as: VR = L/L 0 × 100%, where L and L 0 represent luminescence of treated and untreated control
3D
spheroids, respectively. All data were from four independent experiments. Meanwhile, the morphology of
cell spheroids was recorded by a high-content imaging system on day 0 and day 3.

Statistical analysis
Data are presented as mean ± standard deviation (SD). Student’s t-test was used to evaluate differences
between two groups, whereas one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was
applied for comparisons among three or more groups. Differences were deemed statistically significant when
P < 0.05. All statistical tests were two-tailed. P < 0.05; P < 0.01; P < 0.001.
*
***
**
RESULTS
Preparation of PTTP-DCns and PTTP-DCn@Ls
As shown in Figure 1A, three PTTP-DCns with a shared conjugated backbone (highlighted in purple) and
varying side chains of different lengths (indicated in orange) were designed and synthesized. The synthetic
route of PTTP-DCns is depicted in Supplementary Scheme 1. Initially, the reaction between
1-bromo-3,5-dihydroxybenzene and alkyl dibromide with different carbon numbers generated end groups
with varying side chains. Subsequent stannylation of end groups and two-step Stille coupling reactions with
pyridinethiadiazole (PT) dibromide and bisstannyl thieno[3,2-b]thiophene, followed by a quaternization

132