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. 2019 Jul 18;16(5):842–861. doi: 10.1080/15548627.2019.1643184

Figure 8.

Figure 8.

The PPRV-C and PPRV-N proteins modulate autophagy via IRGM and HSPA1A. (A) Overexpression of PPRV-C, PPRV-N or PPRV-C+ PPRV-N modulates autophagosome formation. GFP-LC3 EECs were transfected with a GST-encoding vector (control) or a vector encoding the PPRV-C and/or PPRV-N proteins. Twenty-four h after transfection, the number of autophagic vesicles was determined by confocal immunofluorescence microscopy. Scale bars, 20 μm. (B) The corresponding graph shows the number of GFP+-LC3 vesicles per cell profile among the transfected EECs. (C) PPRV-C and PPRV-N modulate autophagosome formation partly via IRGM-HSPA1A. GFP-LC3 EECs were treated with si-control, si-IRGM or si-HSPA1A 24 h prior to transfection with a vector encoding PPRV-C or PPRV-N. After an additional 24 h, the cells were fixed, and the number of autophagosomes was determined by confocal immunofluorescence microscopy. Scale bars, 20 μm. (D) The corresponding graph shows the number of GFP+-LC3 vesicles per cell profile among the siRNA-pre-treated and transfected EECs. (E and F) EECs were pre-treated and transfected as described in C. Cell samples were analyzed by immunoblotting with anti-LC3, anti-SQSTM1, and anti-ACTB (loading control) antibodies. The target protein levels relative to the ACTB levels in siRNA-pre-treated and transfected EECs were determined by densitometry. The data represent the mean ± SD of three independent experiments. Two-way ANOVA; **P < 0.01; ***P < 0.001.