Journal: Molecular Oncology
Article Title: Subcellular localization of PD‐L1 and cell‐cycle‐dependent expression of nuclear PD‐L1 variants: implications for head and neck cancer cell functions and therapeutic efficacy
doi: 10.1002/1878-0261.13567
Figure Lengend Snippet: Cell cycle dependent PD‐L1 expression and cellular localization. (A) Flow cytometric analysis of HNC (head and neck cancer) cell line FaDu, as representative, after cell cycle inhibition. Cell cycle progression was blocked at the G1, S or G2/M phase with 50 n m palbociclib for 12 h, 1000 n m aphidicolin for 24 h or 50 n m nocodazole for 12 h, respectively. As a control, cells were treated with 0.03% DMSO for 24 h. The efficiency of cell cycle blockade was demonstrated by S phase analysis using DAPI to quantify the DNA content in each cell. Inhibitor toxicity was determined using DAPI/Annexin V double staining to identify the proportion of vital cells in the cell population in percentage [%], illustrated as dot plots. Per sample, 5 × 10 4 cells were analyzed. Tested cell lines ( n = 6). To achieve optimal conditions, these experiments were performed separately for each cell line studied. (B) Exemplary WB (Western blot) analysis of PD‐L1 banding patterns in cellular fractions in HNC cell line PCI 52 blocked with 125 n m palbociclib for 12 h in G1 phase, with 1200 n m aphidicolin for 24 h in S phase and with 480 n m nocodazole for 12 h in G2/M phase. Total protein expression with Ponceau S staining solution was used as loading control. 20 μg of total protein was loaded. For original blots including analysis of HNC cell line FaDu, see Fig. . Tested cell lines n = 2. (C) Signal intensity of PD‐L1 expression in the respective cellular fraction of the representative HNC cell line PCI 52 during cell cycle progression. For original blots, including analysis of HNC cell line FaDu, see Fig. . Tested cell lines n = 2. (D) Immunofluorescence staining of PD‐L1 in the HNC cell line PCI 52, blocked in G1, S, and G2/M phase. Magnification = 40x, scale bar = 50 μm. Assay performed in duplicates. On each slide, n = 10 pictures were taken for documentation. Figure provides the displayed images with a highlighted nuclear border.
Article Snippet: Palbociclib (PD 0332991 isethionate), a potent cyclin‐dependent CDK4/6 inhibitor inducing G1 cell cycle arrest, was obtained from Tocris Bioscience (Bristol, UK).
Techniques: Expressing, Inhibition, Control, Double Staining, Western Blot, Staining, Immunofluorescence
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![Cell cycle dependent PD‐L1 expression and cellular localization. (A) Flow cytometric analysis of HNC (head and neck cancer) cell line FaDu, as representative, after cell cycle inhibition. Cell cycle progression was blocked at the G1, S or G2/M phase with 50 n m <t>palbociclib</t> for 12 h, 1000 n m aphidicolin for 24 h or 50 n m nocodazole for 12 h, respectively. As a control, cells were treated with 0.03% DMSO for 24 h. The efficiency of cell cycle blockade was demonstrated by S phase analysis using DAPI to quantify the DNA content in each cell. Inhibitor toxicity was determined using DAPI/Annexin V double staining to identify the proportion of vital cells in the cell population in percentage [%], illustrated as dot plots. Per sample, 5 × 10 4 cells were analyzed. Tested cell lines ( n = 6). To achieve optimal conditions, these experiments were performed separately for each cell line studied. (B) Exemplary WB (Western blot) analysis of PD‐L1 banding patterns in cellular fractions in HNC cell line PCI 52 blocked with 125 n m palbociclib for 12 h in G1 phase, with 1200 n m aphidicolin for 24 h in S phase and with 480 n m nocodazole for 12 h in G2/M phase. Total protein expression with Ponceau S staining solution was used as loading control. 20 μg of total protein was loaded. For original blots including analysis of HNC cell line FaDu, see Fig. . Tested cell lines n = 2. (C) Signal intensity of PD‐L1 expression in the respective cellular fraction of the representative HNC cell line PCI 52 during cell cycle progression. For original blots, including analysis of HNC cell line FaDu, see Fig. . Tested cell lines n = 2. (D) Immunofluorescence staining of PD‐L1 in the HNC cell line PCI 52, blocked in G1, S, and G2/M phase. Magnification = 40x, scale bar = 50 μm. Assay performed in duplicates. On each slide, n = 10 pictures were taken for documentation. Figure provides the displayed images with a highlighted nuclear border.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0815/pmc10850815/pmc10850815__MOL2-18-431-g008.jpg)