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. 2022 Jun 17;4(2):zcac019. doi: 10.1093/narcan/zcac019

Figure 1.

Figure 1.

Dynamics of PD-1-mCherry-SMASh fusion protein in EL4 murine T-lymphoma cells and Jurkat human T-lymphoma cells treated with asunaprevir (ASV). (A) Schematic diagram of the SMASh degron system on mCherry fusion protein. (B) Fluorescent mCherry (mC) and bright-field (BF) images of mCherry-SMASh-expressing EL4 cells with (+) or without (–) ASV treatment for 24 h. Scale bars: 10 μm. (C) Western blotting of cell lysate proteins from the mCherry-SMASh vector-transfected EL4 cells using anti-mCherry and anti-β-actin antibodies. The mCherry levels relative to β-actin (ACTB) were quantified as shown in the graph. The 24- and 44-hour samples comprised the polyclonal cells that were treated at the same time; the experiment was performed once. (D) Fluorescent mCherry and BF images in mCherry-SMASh-expressing EL4 cells. (Top left) without ASV (–), (top right) 48 h after addition of ASV (+), (bottom) cultured for 24 h with ASV, and then ASV was removed and cultured for 24 h (+/–). Scale bars: 10 μm. (E) Western blotting of cell lysate proteins from the same cells in (D) using anti-mCherry and anti-β-actin (ACTB) antibodies. The level of mCherry relative to ACTB was quantified and shown in the graph. ASV+, ASV-, and ASV+/- samples comprised the polyclonal cells that were treated at the same time; the experiment was performed once. (F) A schematic diagram of mCherry-SMASh (left) and PD-1-mCherry-SMASh (right) fusion protein in a cell without ASV. (G) Fluorescent mCherry (mC) and bright-field (BF) images of Jurkat cells expressing mCherry-SMASh (mCSh), PD-1-mCherry-SMASh (PD-1-mCSh) without ASV (ASV–), PD-1-mCherry-SMASh (PD-1-mCSh) with ASV (ASV+) and the backbone vector (negative control) 1 day after transfection. The bottom right insert of each image corresponds to a magnified view. White scale bars: 20 μm, light blue scale bars: 10 μm. (H) Flow cytometric analyses of cell-surface PD-1 (top) and mCherry (bottom) in PD-1-mCherry-SMASh-expressing Jurkat cells without ASV (ASV–, grey) or with ASV (ASV+, red) for 3 days and backbone vector (negative control, blue).