Journal: Viruses

Article Title: Porcine Circovirus Type 2 Activates CaMMKβ to Initiate Autophagy in PK-15 Cells by Increasing Cytosolic Calcium

doi: 10.3390/v8050135

Figure Lengend Snippet: PCV2 infection upregulated Trp-Asp (WD) repeat domain phosphoinositide-interacting protein 1 (WIPI1). Knock-down of WIPI1 downregulated LC3-II and Cap protein without affecting CaMKI. PK-15 cells were infected with PCV2 (MOI ≈ 1) in the presence of siRNA targeting WIPI1 (siWIPI1) for the indicated hpi and the whole cell lysates were subjected to Western blotting. ( A ) Representative images of Western blotting for target proteins of cells; ( B ) ratios of WIPI1, p-CaMKI to β-actin; ( C ) representative images of Western blotting for the target proteins of PCV2-infected cells treated with siWIPI1 at 36 hpi; and ( D ) ratios of WIPI1, p-CaMKI, Cap, and LC3-II to β-actin. Ratios of targeted proteins to β-actin were normalized to mock infection set at 1.0. Scrambled, negative control RNA (siNC) was included. Data are reported as the mean ± SEM of three independent experiments (ns p > 0.05; * p < 0.05; and ** p < 0.01).

Article Snippet: All small interfering RNAs (siRNAs) against CaMKKβ, AMPK, CaMKI, and WIPI1 were purchased from Genepharma (Shanghai, China) and shown in .

Techniques: Infection, Knockdown, Western Blot, Negative Control

Journal: Viruses

Article Title: Porcine Circovirus Type 2 Activates CaMMKβ to Initiate Autophagy in PK-15 Cells by Increasing Cytosolic Calcium

doi: 10.3390/v8050135

Figure Lengend Snippet: Inhibition of CaMKI by KN93 or RNA interference reduced expression of LC3-II and Cap proteins without affecting WIPI1. PK-15 cells were infected with PCV2 (MOI ≈ 1) in the presence of KN93 (2 μM) or siRNA targeting CaMKI (siCaMKI). Cells were collected at 36 hpi and the lysates were subjected to Western blotting. ( A ) Representative images of Western blotting for target proteins from cells treated with KN93; ( B ) Ratios of WIPI1, p-CaMKI, Cap and LC3-II to β-actin; ( C ) Representative images of Western blotting for target proteins from cells treated with siCaMKI; ( D ) Ratios of WIPI1, t-CaMKI, p-CaMKI, Cap and LC3-II to β-actin. Ratios of targeted proteins to β-actin were normalized to mock infection 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).

Article Snippet: All small interfering RNAs (siRNAs) against CaMKKβ, AMPK, CaMKI, and WIPI1 were purchased from Genepharma (Shanghai, China) and shown in .

Techniques: Inhibition, Expressing, Infection, Western Blot

Journal: Viruses

Article Title: Porcine Circovirus Type 2 Activates CaMMKβ to Initiate Autophagy in PK-15 Cells by Increasing Cytosolic Calcium

doi: 10.3390/v8050135

Figure Lengend Snippet: Formation of WIPI1 puncta and colocalization of WIPI1 and LC3 were increased by PCV2 infection, but decreased by treatment with STO-609 or KN93. ( A ) PCV2-infected cells were transfected with pcDNA3.1-DsRed-WIPI1 at 6 hpi, treated with STO-609 (10 μM) or KN93 (2 μM) at 6 hours post-transfection (hpt), and incubated for additional 24 h. 0.5 μM thapsigargin (TG) or 1 μM wortmannin (WM) was used as positive or negative control, respectively. Cells were examined for formation of DsRed-WIPI1 puncta under the confocal microscope (scale bar, 10 μM) at 36 hpi; ( B ) average number of DsRed-WIPI1 puncta per cell from at least 50 cells/experiment in three independent experiments; ( C ) pcDNA3.1-DsRed-WIPI1 was transiently transfected into PCV2 pre-infected PK-15 cells stably expressing EGFP-LC3 (PK15/EGFP-LC3) at 6 hpi (MOI ≈ 1) and treated with STO-609 or KN93 as above. Representative confocal images of colocalization of DsRed-WIPI1 and EGFP-LC3 in PK15/EGFP-LC3 cells are shown (scale bar, 10 μM); and ( D ) average number of colocalization of DsRed-WIPI1 and EGFP-LC3 puncta per cell from at least 50 cells/experiment in three independent experiments. Data are reported as the mean ± SEM (ns, p > 0.05; * p < 0.05; and ** p < 0.01).

Article Snippet: All small interfering RNAs (siRNAs) against CaMKKβ, AMPK, CaMKI, and WIPI1 were purchased from Genepharma (Shanghai, China) and shown in .

Techniques: Infection, Transfection, Incubation, Negative Control, Microscopy, Stable Transfection, Expressing

Journal: Viruses

Article Title: Porcine Circovirus Type 2 Activates CaMMKβ to Initiate Autophagy in PK-15 Cells by Increasing Cytosolic Calcium

doi: 10.3390/v8050135

Figure Lengend Snippet: Silencing of AMPK downregulated LC3-II and Cap protein expression, but had no effect on CaMKKβ, CaMKI, and WIPI1. PK-15 cells were first transfected with siAMPK, then infected with PCV2 (MOI ≈ 1) and incubated for 36 h. The cell lysates were subjected to Western blotting. ( A ) Representative images of Western blotting for target proteins; ( B , C ) ratios of CaMKKβ, t-AMPK, p-AMPK, and p-CaMKI, WIPI1, LC3-II, and Cap to β-actin. Ratios of targeted proteins to β-actin were normalized to mock infection 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).

Article Snippet: All small interfering RNAs (siRNAs) against CaMKKβ, AMPK, CaMKI, and WIPI1 were purchased from Genepharma (Shanghai, China) and shown in .

Techniques: Expressing, Transfection, Infection, Incubation, Western Blot

Journal: Viruses

Article Title: Porcine Circovirus Type 2 Activates CaMMKβ to Initiate Autophagy in PK-15 Cells by Increasing Cytosolic Calcium

doi: 10.3390/v8050135

Figure Lengend Snippet: PCV2 increased cytosolic Ca 2+ 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 Ca 2+ measurement based on chemical Ca 2+ indicator Fluo 3-AM by flow cytometry. ( A ) Cytosolic Ca 2+ levels relative to mock at 12, 24 and 36 hpi. ( B ) Cytosolic Ca 2+ 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 Ca 2+ 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 Ca 2+ 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).

Article Snippet: All small interfering RNAs (siRNAs) against CaMKKβ, AMPK, CaMKI, and WIPI1 were purchased from Genepharma (Shanghai, China) and shown in .

Techniques: Infection, Incubation, Flow Cytometry, Expressing, Plasmid Preparation, Transfection, Fluorescence, Förster Resonance Energy Transfer, Microscopy, Western Blot

Journal: Viruses

Article Title: Porcine Circovirus Type 2 Activates CaMMKβ to Initiate Autophagy in PK-15 Cells by Increasing Cytosolic Calcium

doi: 10.3390/v8050135

Figure Lengend Snippet: A proposed model of Ca2+ signaling in PCV2-induced autophagy in PK-15 cells. PCV2-nduced autophagy likely results from activation of IP3R and elevation of cytosolic Ca2+, which then upregulates CaMKKβ with subsequent activation of its downstream molecules AMPK and CaMKI. Two branches of the CaMKKβ signaling pathway are involved: CaMKKβ/AMPK/extracellular signal-regulated kinase (ERK)/tuberous sclerosis complex 2 (TSC2)/mammalian target of rapamycin (mTOR) and CaMKKβ/CaMKI/WIPI1 in PCV2-infected cells.

Article Snippet: All small interfering RNAs (siRNAs) against CaMKKβ, AMPK, CaMKI, and WIPI1 were purchased from Genepharma (Shanghai, China) and shown in .

Techniques: Activation Assay, Infection