Figure 7.

PCV2 increased cytosolic Ca²⁺ likely from the endoplasmic reticulum (ER) via inositol 1,4,5-trisphosphate receptor (IP3R) to activate CaMKKβ and its substrates. (A,B) PK-15 cells were infected with PCV2 (MOI ≈ 1) for indicated time points or treated with 10 mM inositol 1,4,5-trisphosphate (IP3) or 100 μM 2-APB at 12 hpi, and then incubated for additional 24 h before being subjected to cytosolic Ca²⁺ measurement based on chemical Ca²⁺ indicator Fluo 3-AM by flow cytometry. (A) Cytosolic Ca²⁺ levels relative to mock at 12, 24 and 36 hpi. (B) Cytosolic Ca²⁺ levels of PCV2-infected cells treated with IP3 or 2-APB at 36 hpi relative to mock-infected cells without IP3 and 2-APB treatments. (C,D) Genetically encoded Ca²⁺ indicator TN-XXL expressing plasmid (pcDNA3-TN-XXL) was transiently transfected into PK-15 cells pre-infected with PCV2 at 6 hpi (MOI ≈ 1). Mock-infected cells (Ctrl) or cells treated with IP3 (10 mM) or 2-APB (100 μM) were included for comparative purposes). (C) Cells were subjected to cytosolic Ca²⁺ measurement based on fluorescence resonance energy transfer (FRET) under the confocal microscope (scale bar, 10 μm) at 36 hpi. (D) Average FRET efficiency from at least 50 cells/experiment in three independent experiments as represented in the panel D. (E) The whole cell lysates with different treatments collected at 36 hpi were also subjected to Western blotting for CaMKKβ, p-AMPK and p-CaMKI, t-AMPK, t-CaMKI, WIPI1, Cap and LC3-II. (F,G) Ratios of target molecules to β-actin were normalized to mock infection without IP3 and 2-APB treatments, and set at 1.0. Data are reported as the mean ± SEM of three independent experiments (ns, p > 0.05; * p < 0.05; and ** p < 0.01).