Visualization of the PP2A trimeric holoenzyme complexes Aα/B56γ3/Cα and Aα/B55β2/Cα in cells by BiFC-FRET.

<p>(<b>A</b>) The design of BiFC-FRET analysis of Aα/B56γ3/Cα and Aα/B55β2/Cα is shown. YFP, which serves as a FRET acceptor, is reconstituted via BiFC of YC-Aα/YN-B56γ3 or PP2Acα-YC/YN-Aα, in ternary complex Aα/B56γ3/Cα or Aα/B55β2/Cα, respectively. FRET occurs when CFP-PP2Acα or B55β2-CFP, which serves as a FRET donor, associates with YC-Aα/YN-B56γ3 or YN-Aα/PP2Acα-YC BiFC complex, respectively. (<b>B</b>) NIH3T3 cells were transiently transfected with constructs encoding YC-Aα, CFP-PP2Acα and YN-vector or YN-B56γ3, in the presence of pCMV5-SMT_WT, pCMV5-SMT_MUT, or empty vector. Images of expression of CFP-PP2Acα, YFP (due to BiFC of YC-Aα and YN-B56γ3), and FRET between CFP and YFP were acquired by indicated filters. Corrected FRET images were generated and FRET intensity was assessed by AxioVision (Zeiss), as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116074#s2" target="_blank">Methods and Materials</a>”. Representative images are shown. Scale bars, 20 µm. Quantified data from one of two independent experiments with similar results are shown. Mean (±s.d.) of corrected FRET maximum intensity values of individual cells from at least 15 cells were measured for each group. (<b>C</b>) NIH3T3 cells were transiently transfected with constructs encoding PP2Acα-YC and YN-vector or YN-Aα, and B55β2-CFP in the presence of pCMV5-SMT_WT, pCMV5-SMT_MUT, or empty vector, or transfected with equal amounts of constructs harboring PP2Acα-YC, YN-Aα, and B55β2_MUT-CFP. Representative images of expression of B55β2-CFP and B55β2_MUT-CFP, YFP (due to BiFC of association of PP2Acα-YC and YN-Aα), and FRET between CFP and YFP were acquired by indicated filters as described above. Scale bars: 20 µm. Corrected FRET images were generated and FRET intensity was assessed as described earlier. Quantified data from one of two independent experiments with similar results are shown. At least 10 cells were measured for each group.</p

Subcellular distribution of PP2A subunits.

<p>(<b>A</b>) NIH3T3 cells were transiently transfected with pCA2-6myc-PP2A/Aα or pCMV-HA-PP2Acα-YC, and expression of the exogenous Aα and Cα subunits was assessed by indirect immunofluorescence using anti-Myc tag and anti-HA antibodies, respectively, in conjunction with Cy3-conjugated secondary antibody. (<b>B</b>) Diagrams of B55α, B55β, and the B55βαβ chimera mutant are shown. (<b>C</b>) NIH3T3 cells were transiently transfected with pcDNA3.1/Zeo(+)-B55α-HA, pcDNA3.1/Zeo(+)-B55β-HA, pcDNA3.1/Zeo(+)-B55βαβ-HA, pcDNA3.1/Zeo(+)-B55β2-HA, pcDNA3.1/Zeo(+)- B55δ-HA, or pcDNA3.1/Zeo(+)-B56γ3-HA. Expression of various exogenous B isoforms was assessed by indirect immunofluorescence using the anti-HA antibody and Cy3-conjugated secondary antibody. DAPI was applied for staining of nuclei. Scale bars: 20 µm. Cells with different distribution patterns were scored as follows: predominantly nuclear (N>C), homogenously distributed in both nucleus and cytoplasm (N∼C), and predominantly cytoplasmic (N</p

BiFC analysis enables visualization of association between two subunits of PP2A in cells.

<p>(<b>A</b>) Design of BiFC analysis of dimeric interactions between PP2A subunits is shown. Fluorescence is regained when reconstitution of YFP from two fragments of YFP takes place due to an interaction between PP2A subunits fused to the fragments. (<b>B</b>) Equal amounts of BiFC expression constructs encoding YN-Aα and PP2Acα-YC were co-transfected into NIH3T3 cells. YFP signals due to BiFC of YN-Aα and PP2Acα-YC were measured by fluorescence microscopy. (<b>C</b>) Equal amounts of BiFC expression constructs encoding Aα-YC and YN-B55α, YN-B55β1, YN-B55β2, YN-B55βαβ, or YN-B55δ, or constructs encoding YC-Aα and YN-B56γ3 were transfected into NIH3T3 cells. YFP signals due to BiFC of Aα-YC and YN-B were measured by fluorescence microscopy. (<b>D</b>) Equal amounts of BiFC expression constructs encoding PP2Acα-YC and YN-B55β1 or YN-B56γ3 with or without equal amounts of pCA2-6myc-PP2A/Aα were co-transfected into NIH3T3 cells, and 24 h after transfection, YFP signals due to BiFC of PP2Acα-YC and YN-B55β1 or YN-B56γ3 were measured by direct fluorescence microscopy and expression of 6myc-PP2A/Aα was confirmed by indirect immunofluorescence using anti-Myc tag antibody and Cy3-conjugated secondary antibody. DAPI was applied for staining of nuclei. Scale bars: 20 µm. Graphs show quantitative analysis of distribution of BiFC signals in cells from one of at least two independent experiments with similar results, and at least 100 cells were assessed from several random fields. Cells with different distribution patterns of BiFC signals were scored as described earlier.</p

BiFC analysis confirms that B56γ3, but not B55α, promotes PP2A/Aα accumulation in the nucleus in early S phase.

<p>(<b>A</b>) NIH3T3 cells stably expressing HA-tagged B56γ3 or B55α were synchronized at the early S phase by double thymidine block treatment <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116074#pone.0116074-Harper1" target="_blank">[48]</a> followed by released in the regular medium for 3 h as described before <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116074#pone.0116074-Lee1" target="_blank">[29]</a>. Expression of B56γ3 or B55α was assessed by indirect immunofluorescence using anti-HA antibody in conjunction with Cy3-conjugated secondary antibody. (<b>B</b>) Equal amounts of BiFC expression constructs encoding YC-Aα and YN-B56γ3 or equal amounts of BiFC expression constructs encoding Aα-YC and YN-B55α were co-transfected into NIH3T3 cells. Twenty-four hour after transfection, cells were either treated with 10 µg/ml aphidicolin or left untreated for 18 h and subsequently grown in fresh medium without aphidicolin treatment for 3 h, followed by direct fluorescence microscopy for imaging YFP signals due to BiFC of YC-Aα and YN-B56γ3 or BiFC of Aα-YC and YN-B55α. DAPI was applied for staining of nuclei. Scale bars, 20 µm. Graphs show quantitative analysis of distribution of BiFC signals in cells from one of at least two independent experiments with similar results, and at least 100 cells were assessed from several random fields. Cells with different distribution patterns of BiFC signals were scored as described earlier.</p

Visualization of the α4/PP2Ac complex by BiFC.

<p>(<b>A</b>) BiFC expression constructs encoding YN-α4_WT or YN-α4_MUT and YC-PP2Acα were transfected into NIH3T3 cells. YFP signals due to BiFC of YN-α4_WT or BiFC of YN-α4_MUT and YC-PP2Acα were measured by fluorescence microscopy. Expression of Myc-tagged YN-α4_WT or YN-α4_MUT was confirmed using anti-Myc antibody in conjunction with Cy3-conjugated secondary antibody by indirect immunofluorescence microscopy. DAPI was applied for staining of nuclei. Scale bars: 20 µm. Cells with different distribution patterns were scored as described earlier. Quantified data from one of at least two independent experiments with similar results are shown. At least 150 cells were counted for each group. (<b>B</b>) BiFC expression constructs encoding YN-α4_WT or YN-α4_MUT and YC-PP2Acα were transfected into NIH3T3 cells. Cell lysates were collected 24 h post-transfection and immunoprecipitations were performed using anti-Myc tag antibody. The cell lysates and anti-Myc tag immunocomplexes were then analyzed by SDS-PAGE and Western blotting using the indicated antibodies.</p

α4 facilitates formation of PP2A AC core enzyme.

<p>(<b>A</b>) Equal amounts of BiFC expression constructs encoding YN-Aα and PP2Acα-YC in the presence of equal amounts of pcDNA5/To-Flag-α4_WT, pcDNA5/To-Flag-α4_MUT, or empty vector were transfected into NIH3T3 cells. YFP signals due to BiFC of YN-Aα and PP2Acα-YC were measured by fluorescence microscopy. Expression of Flag-α4_WT or Flag-α4_MUT was verified using anti-FLAG antibody and Cy3-conjugated secondary antibody. DAPI was applied for staining of nuclei. Scale bars, 20 µm. (<b>B</b>) BiFC expression constructs encoding YN-Aα and HA-PP2Acα-YC and pcDNA5/To-Flag-α4_WT, pcDNA5/To-Flag-α4_MUT, or empty vector were transfected into NIH3T3 cells. Cell lysates were collected 24 h post-transfection, and immunoprecipitations were performed using anti-HA Sepharose. The cell lysates and anti-HA immunocomplexes were then analyzed by SDS-PAGE and Western blotting using the indicated antibodies.</p

BiFC between YC-Aα and YN-B56γ3 is disrupted by SV40 SMT.

<p>(<b>A</b>) BiFC expression constructs encoding YC-Aα and YN-B56γ3 in the presence of pCMV5-SMT_WT, -SMT_MUT, or empty vector were transfected into NIH3T3 cells, and 24 h after transfection, YFP signals due to BiFC of YC-Aα and YN-B56γ3 were measured by fluorescence microscopy. Expression of HA-tagged YN-B56γ3 was confirmed using anti-HA antibody as described earlier. (<b>B</b>) BiFC expression constructs encoding YC-Aα and YN-B56γ3 in the presence of pCMV5 SMT_WT, -SMT_MUT, or empty vector were transfected into NIH3T3 cells. Cell lysates were collected 24 h post-transfection, and immunoprecipitation was performed using anti-HA-Sepharose. The cell lysates and anti-HA immunocomplexes were then analyzed by SDS-PAGE and Western blotting using indicated antibodies.</p

Increased expression levels of pIKKβ(S181), pS6K1(T389), and VEGF-A in liver tissues of HBx transgenic mice.

<p>(A). A gross view of representative liver tumors (T1, T2, T3) developed in HBx transgenic mice in several months of breeding. The ALT values are shown. (B). The H&E staining of non-tumor and tumor parts in HBx transgenic mice. (C). The expression levels of pIKKβ (S181), IKKβ, pS6K1 (T389), S6K1, VEGF-A, HBx, and β-actin detected by Western blotting in non-tumor and tumor parts of liver tissues of three HBx transgenic mice (#824, #825, and #826) were compared to the normal liver tissues of the wild-type age-matched mouse. The HBx mRNA levels were also measured by RT-PCR and the GAPDH mRNA levels were used as an internal control. The relative levels of pIKKβ (S181), pS6K1 (T389), and VEGF-A were quantified by densitometry and normalized with total IKKβ, total S6K1, and actin. Results are shown as ratios of average levels of pIKKβ (S181) pS6K1 (T389), and VEGF-A in non-tumor and tumor parts of liver tissues of three HBx transgenic mice (#824, #825, and #826) relative to that in the normal liver tissues of the wild-type age-matched mouse (set as 1). Data are shown as means ± S.D. of measurements of three mice. (D). Immunohistochemistry analyses show expression levels of pIKKβ (S181), pS6K1 (T389), VEGF-A, and CD31 in normal liver tissues of the wild-type mouse, and non-tumor and tumor parts of liver tissues of HBx transgenic mice. One representative data are shown. N = 3.</p

Positive association between pIKKβ(S181), pTSC1(S511), and pS6K1(T389) in HBV-associated human HCC specimens.

<p>(A). Immunohistochemistry analysis of pIKKβ (S181), pTSC1(S511) and pS6K1(T389) in tumor tissues of 95 human HBV-associated HCC specimens. Results of one representative specimens stained by specific antibodies are shown. (B). Upper graph shows percentages of specimens with low or high pIKKβ (S181) expression in which pS6K1 (T389) expression was high or was not observed (low). Lower graph shows percentages of specimens with low or high pIKKβ (S181) expression in which pTSC1 (S511) expression was high or was not observed (low). Positive correlations was noted between pIKKβ (S181) and pS6K1 (T389) (*<i>P</i><0.01) and between pIKKβ (S181) and pTSC1 (S511) (*<i>P</i><0.01)<b>.</b> (C) The Kaplan-Meier disease-free survival curves show that expression of pIKKβ (S181) (<i>p</i> = 0.003), pTSC1 (S511) (<i>p</i> = 0.048), or pS6K1 (T389) (<i>p</i> = 0.0027) is associated with early tumor recurrence. Co-expression of pIKKβ (S181) and pS6K1 (T389) (<i>p</i> = 0.0013) was a better predictor of patients’ recurrence-free time survival in HCC patients who received curative surgery for up to 48-month investigation.</p

The IKKβ/TSC1/mTOR signaling pathway is activated by HBx.

<p>(A). Expression of HBx mRNA in Hep3Bx, HepG2x, and parental Hep3B and HepG2 cells was detected using semi-quantitative RT-PCR. Levels of GAPDH mRNA were used as an internal control. RNAs of a HBV-positive patient’s serum (P) and RNAs of a control HBV-negative serum (N) were used as controls. (B). Levels of HBx protein were detected in lysates of Hep3Bx, HepG2x, and parental Hep3B and HepG2 cells using Western blotting by antibody specific against HBx protein and β-actin. (C). Levels of pIKKβ(S181), pTSC1 (S511), pS6K1 (T389), total IKKβ, total TSC1, total S6K1, and β-actin were assessed in lysates of Hep3Bx, HepG2x, and parental Hep3B and HepG2 cells using Western blotting by specific antibody as indicated. (D). Data shown are ratios of viable cells in Hep3Bx and HepG2x cells relative to that in Hep3B and HepG2 cells (set as 1), respectively, at 24 h after seeding using MTT assay. (E). Levels of pIKKβ (S181), pS6K1 (T389), total IKKβ, total S6K1, HBx, and β-actin were assessed in lysates of Huh7 cells transfected with empty vector alone, payw1.2WT, or payw*7. (F). Levels of pIKKβ (S181), pTSC1 (S511), pS6K1 (T389), total IKKβ, total TSC1, total S6K1, and β-actin were assessed in lysates of Hep3B and Hep3Bx with or without TNF-α treatment using Western blotting as described earlier.</p