1. Volume 37, Issue 4, Pages 377-386 (May 2016)
The Centrosome Undergoes Plk1-Independent Interphase Maturation during Inflammation and Mediates Cytokine Release 
Anastassiia Vertii, Maria Ivshina, Wendy Zimmerman, Heidi Hehnly, Shashi Kant, Stephen Doxsey 
Developmental Cell 
Volume 37, Issue 4, Pages (May 2016)
DOI: /j.devcel
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2. Developmental Cell 2016 37, 377-386DOI: (10.1016/j.devcel.2016.04.023)
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3. Figure 1
LPS Treatment of Macrophages for 4–6 hr Induces PCM Recruitment to the Centrosome
(A) Left: Maximum projections from confocal images of the centrosome protein pericentrin (PCNT) staining in control (cntrl) or LPS-treated THP1-differentiated human macrophages (scale bar, 10 μm). Right: enlargement of the centrosomes. Graph: semi-quantitative analysis of pericentrin signal intensity at the centrosomes from control and LPS-treated human macrophages (×106 a.u., n = 33–51 cells/sample, mean ± SEM).
(B) Images (as in A) of Cep152 staining in control or LPS-treated human macrophages (scale bar, 10 μm). Graph: semi-quantitative analysis of Cep152 signal intensity at the centrosomes from control and LPS-treated THP1-derived human macrophages (×105 a.u., n = 53–65 cells/sample, mean ± SEM).
(C) Images (as in A) of ninein staining in control or LPS-treated human macrophages showing the increase in ninein signal at the centrosome after LPS challenge (scale bar, 5 μm). Graph: semi-quantitative analysis of ninein signal intensity at the centrosome from control and LPS-treated THP1-derived human macrophages (×105 a.u., n = 25–30 cells/sample, mean ± SEM).
(D) PCM components are recruited to the centrosomes of bone marrow-derived mouse macrophages after LPS challenge. Left: Maximum projections from confocal images of γ-tubulin staining in control or LPS-treated human macrophages showing the increase in γ-tubulin signal at the centrosome after LPS challenge (scale bar, 10 μm). Right: enlargement of the centrosomes. Graph: semi-quantitative analysis of γ-tubulin signal intensity at the centrosomes from control and LPS-treated primary macrophages (×105 a.u., mean ± SEM).
(E) Images (as in D) of pericentrin staining in control or LPS-treated mouse macrophages showing the increase in pericentrin signal at the centrosome after LPS challenge (scale bar, 10 μm). Graph: semi-quantitative analysis of pericentrin signal intensity at the centrosomes from control and LPS-treated primary mouse macrophages (×106 a.u., n = 34–35 cells/sample, mean ± SEM).
(F) Schematic summary of LPS-induced PCM recruitment to the centrosome.
See also Figure S1.
Developmental Cell  , DOI: ( /j.devcel )
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4. Figure 2
Pro-inflammatory Cytokine Exposure Induces Pericentrin and γ-Tubulin Recruitment to the Centrosome in Non-immune Cells
(A) Left: maximum projections from confocal images of pericentrin staining in control or TNF-α-treated primary human fibroblasts (scale bar, 10 μm). Right: enlargement of the centrosomes. Graph: semi-quantitative analysis of pericentrin signal intensity at the centrosomes from control and TNF-α-treated cells (×106 a.u., mean ± SEM).
(B) Images (as in A) show the increase in γ-tubulin recruitment to the centrosomes after TNF-α challenge (scale bar, 10 μm). Graph: semi-quantitative analysis of γ-tubulin signal intensity at the centrosome from control and TNF-α-treated cells (×106 a.u., mean ± SEM).
(C) Maximum projections from confocal images of γ-tubulin staining in control or TNF-α- and IL-1β-treated primary MEFs demonstrate the increase in PCM components to the centrosome after inflammatory stimuli (DNA, DAPI, in blue; γ-tubulin in red) (scale bar, 10 μm). Smaller images: enlargement of the centrosomes. Graph: semi-quantitative analysis of γ-tubulin signal intensity at the centrosome from control, IL-1β-, and TNF-α-treated primary MEFs (×105 a.u., mean ± SEM).
(D) Increase in pericentrin recruitment to centrosomes after TNF-α challenge using semi-quantitative intensity profiles of pericentrin intensity in primary MEFs. Graph: semi-quantitative analysis of pericentrin signal intensity at the centrosome from control and TNF-α-treated primary MEFs (×106 a.u., mean ± SEM).
(E) Maximum projections from confocal images show the increase in γ-tubulin recruitment to the centrosomes after TNF-α challenge in hRPE (scale bar, 10 μm). Graph: semi-quantitative analysis of γ-tubulin signal intensity at the centrosome from control and TNF-α-treated cells (×106 a.u., mean ± SEM).
(F) As in (E), but cells were treated with IL-1β.
(G) Isolation of centrosomes from hRPE cells by sucrose gradient shows the increase in centrosome size after IL-1β-treatment: sucrose gradients of centrosomes from control and IL-1β-treated lysates were separated by SDS-PAGE and immunoblotted for γ-tubulin. Fractions 4 and 5 in control are shifted to 5 and 6 in IL-1β-treated cells (arrows) indicating the increase in centrosomal γ-tubulin after IL-1β treatment, while total input (In) of γ-tubulin is not significantly changed.
Developmental Cell  , DOI: ( /j.devcel )
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5. Figure 3
Inflammatory Stimuli Induce Plk1-Independent Interphase Centrosome Maturation
(A) Maximum projections from confocal microscopy images of MTs from control and LPS-treated THP1-derived macrophages cells (scale bar, 10 μm). Graph: semi-quantitative analysis of centrosomal α-tubulin signal intensity at the centrosome area from control and LPS-treated cells (×107 a.u., mean ± SEM).
(B) Centrosomes from LPS-challenged macrophages have increased ability to nucleate MTs. Left: MT nucleation at 0 and 1 min after nocadazole release (α-tubulin, green; γ-tubulin, red) in control and LPS-treated THP1-derived macrophages (scale bar, 10 μm). Right: enlargement of the centrosomes. Graph: semi-quantitative analysis of centrosomal α-tubulin signal intensity at the centrosome area after 1 min of MT regrowth from control and LPS-treated cells (×107 a.u., mean ± SEM).
(C) Cytokine exposure increases recruitment of the recycling endosome component, FIP3, to centrosomes. Graph: semi-quantitative analysis of FIP3 signal intensity at the centrosomes from control or LPS-treated human macrophages (×106 a.u., mean ± SEM). Images: maximum projections from confocal microscopy images of control and LPS-treated human macrophages (DNA, DAPI in blue, PCNT in red, FIP3 in green) (scale bar, 10 μm). Upper panels: enlargement of the centrosomes.
(D) Maximum projections from confocal microscopy images of control and TNF-α-treated mouse fibroblasts shows that Plk1 is not recruited to the TNF-α-treated interphase centrosomes (I) but to mitotic centrosomes (M) (DNA, DAPI in blue, γ-tubulin in red, Plk1 in green) (scale bar, 10 μm).
(E) Interphase centrosome maturation is MLK dependent. Maximum projections from confocal microscopy images of control and LPS-treated human macrophages also treated with Plk1, MLK, p38, and JNK inhibitors (γ-tubulin, red; DAPI, blue; scale bar, 5 μm). Graph: semi-quantitative analysis of γ-tubulin signal intensity at the centrosome from control or LPS-treated human macrophages (×106 a.u., mean ± SEM) in the presence of Plk1 or MAPK inhibitors p38i, JNKi, MLKi, and LPS.
(F) Mlk2,3-null cells do not accumulate pericentrin in response to TNF-α exposure. Graph: semi-quantitative analysis of pericentrin signal intensity at the centrosome from control or TNF-α-treated wild type (WT) and MLK2/3-deficient MEF cells (×105 a.u., mean ± SEM).
See also Figure S2.
Developmental Cell  , DOI: ( /j.devcel )
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6. Figure 4 Centrosome Integrity Is Critical for Cytokine Production
(A) Compromised centrosome integrity in pericentrin-null MEFs leads to loss of centrosomal FIP3 and attenuated IL-6 production. Maximum projections from confocal microscopy images of FIP3 (in green) and pericentrin (in red) from TNF-α-treated primary WT and pericentrin (Pcnt)-deficient MEFs (scale bar, 5 μm). Upper right panels: enlargement of the centrosomes. Graph: IL-6 secretion from TNF-α-treated primary WT and Pcnt-deficient MEF cells was analyzed by ELISA assay.
(B) Human primary fibroblasts from a healthy individual and a MOPDII patient with C1190fs mutation in pericentrin were treated as in (A). Scale bars, 10 μm; and inset, 5 μm.
(C) Maximum projections from confocal microscope images of RAW264.7 mouse macrophages showing that chemical ablation of the centrosome by treatment with centrinone or centrinone B (Wong et al., 2015) (i.e., loss of Centrin 2, green) reduces localization of recycling endosomes at centrosomes (FIP3, in red) (scale bar, 10 μm). Upper right panels: enlargement of the centrosomes.
(D) Centrosome depletion leads to impaired cytokine production. RAW264.7 macrophage cells were treated with centrinone and centrinone B for 7 days, then challenged with LPS for another 24 hr. IL-6 was analyzed as in (A).
(E) Raw cells were treated as in (D) and analyzed for IL-10 secretion.
(F) Raw cells were treated as in (D) and analyzed for MCP1 secretion.
(G) Raw cells were treated as in (D) and analyzed for TNF-α secretion. ns, not significant.
(H) Model. Exposure to inflammatory stimuli enhances pericentriolar material recruitment to the interphase centrosome in MLK-dependent manner, which mediates cytokine production.
See also Figures S3 and S4.
Developmental Cell  , DOI: ( /j.devcel )
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