Fig 1: Adiponectin enables CLSP to be active in the presence of higher concentrations of apolipoprotein E4.a–c SH-SY5Y cells, transfected with the empty vector or pcDNA3.1/MycHis-V642I-APP, were cultured in media containing 1 nM of GST-MycHis or CLSP-MycHis with 10 nM of ApoE3 or BSA with 10 nM of adiponectin (ADN) (a), annexin 2 (b), annexin 5(c), or BSA. GST-MycHis and BSA were used as negative controls. At 48 h, cells were harvested for trypan blue exclusion assays. d SH-SY5Y cells, transfected with the vector or pcDNA3.1/MycHis-V642I-APP, were cultured in media containing 1 nM of GST-MycHis or CLSP-MycHis with/without 10 nM of ApoE4 with/without indicated concentrations of adiponectin (ADN). At 48 h, the cells were harvested for the trypan blue exclusion and calcein assays. e SH-SY5Y cells, transfected with the vector or pcDNA3.1/MycHis-V642I-APP, were cultured in media containing 1 nM of GST-MycHis or CLSP-MycHis with/without stepwise increasing concentrations of ApoE4 with/without 1 nM of ADN. At 48 h, the cells were harvested for trypan blue exclusion and WST-8 assays. The cell lysates were immunoblotted using the APP antibody.
Fig 2: Antitumor phenotype in ApoE–/- mice is rescued upon T-cell depletion. A, Experimental design schematic for T-cell depletion in WT and ApoE–/- mice. B, Final tumor weight (g) from WT (n = 6), WT anti-CD4/CD8 (n = 3), ApoE–/- (n = 6), and ApoE–/- anti-CD4/CD8 (n = 6). Statistical significance was determined with a nonparametric Mann–Whitney test. C, Representative SPADE analysis of cellular infiltrate in WT tumor. Identified populations include nonimmune cells, CD8 T cells, CD4 T cells, B cells, immature myeloid cells, macrophages, and CD11c+ myeloid cells. The SPADE plot is colored to indicate CD45 expression. Red, high expression; blue, low expression. D, Manual gating quantitation of cell populations in WT (n = 4), WT anti-CD4/CD8 (n = 2), ApoE–/- (n = 4), and ApoE–/- anti-CD4/CD8 (n = 5) tumors. Populations include CD4 T cells (CD3+ CD4+) and CD8 T cells (CD3+ CD8+) E, total myeloid cells (CD45+ CD11b+), macrophages (CD11b+ F4/80+), CD11c+ myeloid cells (CD11b+ CD11c+), and immature myeloid cells (Ly-6C+ Ly-6G+). Statistical significance was determined by two-tailed t tests between groups. F, Representative SPADE analysis colored by Ly-6G expression in WT, WT anti-CD4/CD8, ApoE-/-, and ApoE–/- anti-CD4/CD8 tumors. Red, high expression; blue, low expression.
Fig 3: ApoE gene and protein levels are increased in MWCNT-instilled Mmp12 KO mice at 60 days. (A) ApoE gene expression was increased in MWCNT-instilled C57Bl/6 at 10 days, and in Mmp12 KO mice at both time points from BAL cells. ApoE was not increased in MWCNT-instilled C57Bl/6 mice relative to sham at 60 days. ApoE expression in MWCNT-instilled Mmp12 KO was increased at 60 days relative to 10 days (* p = 0.05; ** p = 0.01; n = 5/group). (B) ApoE BALF protein levels were increased in MWCNT-instilled 60-day Mmp12 KO relative sham (* p = 0.05; ** p = 0.01; n = 9/group).
Fig 4: Schematic representation of the proposed mechanism. In Mmp12 KO mice, M2c macrophages are the predominant immune cell. In the presence of MWCNT, granulomas form at 10 days in the Mmp12 KO mice. An increase in IL-13 leads to the predominance of the M2a macrophage in MWCNT-instilled Mmp12 KO mice at 60 days. This shift in M2 macrophage phenotype correlates with the resolution of granulomas at 60 days in Mmp12 KO mice. We theorize that the role of M2a macrophages in granuloma resolution is through increased MMP14 surface expression which will function to degrade collagen, a defining feature of mature granulomas. These M2a cells also secrete increased ApoE, which can bind to collagen fragments, marking them for uptake. Through this collagen degradation and uptake, the stability of the granuloma will be diminished, leading to granuloma resolution.
Fig 5: Apolipoproteins E suppresses CLSP activity.a ApoE3, E4, adiponectin (ADN), and annexin 2, C-terminally tagged with HA, were overexpressed in F11 neurohybrid cells. Resulting cell lysates were mixed with bacterially produced GST-MycHis- or CLSP-MycHis-conjugated sepharose 4B and incubated at 4 °C overnight. The pulled-down precipitates were immunoblotted using HA and myc antibodies. For reference, inputs including the sepharose 4B beads conjugating GST-MycHis (GST-MH) and CLSP-MycHis (CLSP-MH) and the cell lysates were similarly immunoblotted. b SH-SY5Y cells, transfected with the empty vector or pcDNA3.1/MycHis-V642I-APP, were cultured in the presence of indicated concentrations of CLSP-MycHis. At 48 h, cells were harvested for trypan blue exclusion cell mortality assays (Trypan Blue Test) and cell viability assays using the WST-8 assay kit or staining with calcein AM. Cell lysates were immunoblotted using the APP antibody. c, d SH-SY5Y cells, transfected with the vector or pcDNA3.1/MycHis-V642I-APP, were cultured in media containing 1 nM of GST-MycHis or CLSP-MycHis with/without indicated concentrations of BSA, ApoE3 (c), or ApoE4 (d). At 48 h, cells were harvested for trypan blue exclusion assays. Cell lysates were immunoblotted using the APP antibody.
Supplier Page from Abcam for Mouse Apolipoprotein E ELISA Kit