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Tubeimoside-1 induces TFEB-dependent lysosomal degradation of PD-L1 and promotes antitumor immunity by targeting mTOR
182 人阅读发布时间：2022-05-18 10:35
To measure the PD-1 and PD-L1 protein interaction in vitro, cells
were seeded on 6-well plates and then incubated with human recombinant PD-1 Fc protein and anti-human Alexa Fluor 488 dye
conjugated22. DAPI was used to stain the cell nuclei. The green
fluorescence signal was captured by a Zeiss Axio Vert A1 microscope (Carl Zeiss, Thornwood, NY, USA) or quantified by
Novocyte flow cytometer with Novo Express software (Agilent).
Human peripheral blood mononuclear cells (PBMC) were obtained from StemEry Biotech (Fuzhou, China). PBMC-mediated
tumor cell-killing assay was performed by the xCELLigence
system (Agilent). Briefly, each well of E-plate 16 was added
50 mL of full medium for 30 min firstly. Additional 50 mL medium
containing of 5 103 H157 or A375 cells was added in plate and
the final volume reached 100 mL. Each treatment was conducted
two times. After 24 h treatment with the indicated conditions,
PBMC cells (activated by 100 ng/mL anti-CD3, 100 ng/mL antiCD28 and 10 ng/mL IL-2) were incubated with tumor cells at the
ratio of 10:1. The measurements for cell index values were performed by continuous impedance recordings every 10 min.
were seeded on 6-well plates and then incubated with human recombinant PD-1 Fc protein and anti-human Alexa Fluor 488 dye
conjugated22. DAPI was used to stain the cell nuclei. The green
fluorescence signal was captured by a Zeiss Axio Vert A1 microscope (Carl Zeiss, Thornwood, NY, USA) or quantified by
Novocyte flow cytometer with Novo Express software (Agilent).
Human peripheral blood mononuclear cells (PBMC) were obtained from StemEry Biotech (Fuzhou, China). PBMC-mediated
tumor cell-killing assay was performed by the xCELLigence
system (Agilent). Briefly, each well of E-plate 16 was added
50 mL of full medium for 30 min firstly. Additional 50 mL medium
containing of 5 103 H157 or A375 cells was added in plate and
the final volume reached 100 mL. Each treatment was conducted
two times. After 24 h treatment with the indicated conditions,
PBMC cells (activated by 100 ng/mL anti-CD3, 100 ng/mL antiCD28 and 10 ng/mL IL-2) were incubated with tumor cells at the
ratio of 10:1. The measurements for cell index values were performed by continuous impedance recordings every 10 min.