Hy b0015

Animal experiments were approved by the Institutional Animal Care and Use Committee at Army Medical University (AMUWEC20201344). For the immunodeficiency mouse model, 4T1 tumors formed by control or Atad3a-knockdown cells (1 × 10⁵ cells in 50 μL PBS mixed with 50 μL Matrigel, Corning) were injected into the fourth left mammary fat pads of female BALB/c nude mice. For the immune-competent mouse model, 4T1 tumors formed by control or Atad3a-knockdown cells (5 × 10⁴ cells in 50 μL PBS mixed with 50 μL Matrigel, Corning) were injected into the fourth left mammary fat pads of female BALB/c mice. For in vivo treatment, mice were treated randomly with paclitaxel (8 mg/kg, HY-B0015, MedChemExpress) or vehicle intraperitoneally on 5, 8, 11, and 14 days after tumor cells inoculation, while mouse anti-PD-L1 antibody (100 μg per mouse, clone 10 F.9G2, BE0101, BioXcell) or rat IgG2b isotype control (100 μg per mouse, clone LTF-2, BE0090, BioXcell) was utilized on 6, 9, 12, 15 days after inoculation. Orthotropic tumor sizes were measured every 4 days with a caliper and tumor volumes = Length × Width²/2 were calculated.

Approximately 5,000 TNBC cells were resuspended in 90 µL culture medium and seeded in 96-well plates. On the next day, PTX reagent (HY-B0015, MedChemExpress, Monmouth Junction, NJ, USA) at an initial concentration of 20 µM was prepared, which was repeatedly diluted at a twofold gradient, with seven varying concentrations obtained. The TNBC cells were treated with PTX reagent at different concentrations for 48 h, followed by addition of 10 µL CCK-8 reagent (ab228554, Abcam) to each well. After 1 h of dark incubation at 37℃, the optical density (OD) value at 460 nm was examined. Cells treated with phosphate-buffered saline (PBS) instead of PTX were set to controls, whose OD460 value was defined as 100%, according to which the viability of PTX-treated cells was evaluated.

Marine sulfated polysaccharide PMGS with a content of sulfate of 36% and a molecular weight (Mw) of 153 kDa was prepared in our lab at the Marine Biomedical Research Institute of Qingdao. It was synthesized from marine alginate, which was purified from brown seaweed as described previously [10 (link),11 (link)]. The chemical structure of PMGS is shown in Figure 1A. It was dissolved in a culture medium and prepared when used.
PTX was obtained from MedChemExpress (HY-B0015, Princeton, NJ, USA). The chemical structure of PMGS is shown in Figure 1B. As a stock solution, PTX was dissolved in DMSO and stored at −20°C. The working solution was prepared by dissolving the stock solution in a culture medium when used.