Journal: Stem Cell Reviews and Reports

Article Title: Nlrp3 Inflammasome Signaling Regulates the Homing and Engraftment of Hematopoietic Stem Cells (HSPCs) by Enhancing Incorporation of CXCR4 Receptor into Membrane Lipid Rafts

doi: 10.1007/s12015-020-10005-w

Figure Lengend Snippet: Confocal analysis of membrane lipid rafts in purified murine SKL cells . Panel A. Defective lipid raft formation in murine C57Bl/6 Nlrp3-KO BM-purified SKL cells or wild type BM-purified SKL cells exposed to adenosine (10 μM) or CoPP (50 μM). Representative images of SKL cells sorted from WT BM, stimulated with SDF-1 (50 ng/ml) and LL-37 (2.5 μg/ml), stained with cholera toxin subunit B (a lipid raft marker) conjugated with FITC and rat anti-mouse CXCR4 followed by anti-rat Alexa Fluor 594, and evaluated by confocal microscopy for formation of membrane lipid rafts. Lipid rafts were formed in SKL cells (control) but not in SKL cells isolated from Nlrp3-KO animals or SKL cells isolated from WT animals after adenosine and CoPP treatment. Panel B. The chemotactic responsiveness of mBMMNCs untreated or treated with 10 Panx, SC Panx, or apyrase in unsupplemented medium or medium supplemented with SDF-1, S1P, or ATP, as determined by counting the number of CFU-GM clonogenic progenitors. Results are combined from two independent experiments. *p > 0.05, ** p > 0.01, *** p > 0.001

Article Snippet: The cholera toxin B subunit conjugated with FITC (Sigma-Aldrich) was applied to detect the ganglioside GM1, and rat monoclonal anti-CXCR4 IgG antibody (R&D Systems) and Alexa Fluor 594 goat anti-rat IgG antibody (Invitrogen) were applied to detect CXCR4.

Techniques: Membrane, Purification, Staining, Marker, Confocal Microscopy, Isolation

Journal: Stem Cell Reviews and Reports

Article Title: Nlrp3 Inflammasome Signaling Regulates the Homing and Engraftment of Hematopoietic Stem Cells (HSPCs) by Enhancing Incorporation of CXCR4 Receptor into Membrane Lipid Rafts

doi: 10.1007/s12015-020-10005-w

Figure Lengend Snippet: The impact of pannexin 1 channel blockade on short- and long-term engraftment of HSPCs in WT mice. Panel A. Lethally irradiated WT mice (9 per group) were transplanted with bone marrow mononuclear cells (BMMNCs) that had been previously labeled with a PKH67 cell linker and then treated with 10 Panx or SC Panx. Twenty-four hours after transplantation, the femoral BMMNCs were harvested, the number of PKH67 + cells evaluated by FACS, and the CFU-GM clonogenic progenitors enumerated in an in vitro colony assay. Panel B. Lethally irradiated WT mice (9 per group) were transplanted with BMMNCs treated with 10 Panx or SC Panx, and 12 days after transplantation the femoral BMMNCs were harvested and plated to count the number of CFU-GM colonies and the spleens removed for counting the number of CFU-S colonies. No colonies were formed in lethally irradiated, untransplanted mice (irradiation control). * p < 0.05, ** p < 0.01, *** p < 0.001. Panel C. Lethally irradiated WT mice (9 per group) were transplanted with BMMNCs treated with 10 Panx. White blood cells (above) and platelets (below) were counted at intervals (at 0, 3, 7, 14, 21, and 28 days after transplantation). * p < 0.05. Panel D . Defective lipid raft formation in murine BM-purified SKL cells from C57Bl/6 mice exposed to 10 Panx inhibitory peptide (200 μM). Representative images of SKL cells sorted from WT BM, stimulated with SDF-1 (50 ng/ml) and LL-37 (2.5 μg/ml) (positive control) or exposed to 10 Panx inhibitory peptide, stained with cholera toxin subunit B (a lipid raft marker) conjugated with FITC and rat anti-mouse CXCR4 followed by anti-rat Alexa Fluor 594, and evaluated by confocal microscopy for formation of membrane lipid rafts. Lipid rafts were formed in SKL cells (control), but not in SKL cells after 10 Panx inhibitory peptide treatment

Article Snippet: The cholera toxin B subunit conjugated with FITC (Sigma-Aldrich) was applied to detect the ganglioside GM1, and rat monoclonal anti-CXCR4 IgG antibody (R&D Systems) and Alexa Fluor 594 goat anti-rat IgG antibody (Invitrogen) were applied to detect CXCR4.

Techniques: Irradiation, Labeling, Transplantation Assay, In Vitro, Colony Assay, Purification, Positive Control, Staining, Marker, Confocal Microscopy, Membrane

Journal:

Article Title: Phosphoinositide 3-Kinase Activation in Late G 1 Is Required for c-Myc Stabilization and S Phase Entry ▿

doi: 10.1128/MCB.00783-06

Figure Lengend Snippet: Late-G1 PI3K inhibition reduces c-Myc and cyclin A expression as well as Cdk2 activity. (A) Total RNA and protein extracts were prepared from cells entering the cell cycle synchronously. Ly294002 was added at 7 h after serum addition; cell harvesting was at 0 and 9 h. Samples were examined by Northern blotting (see Materials and Methods) or by WB, using a c-myc probe or anti-Myc antibody, respectively. The Northern blot corresponds to two different experiments (Exp.). (B) Cell extracts were prepared from cells entering the cell cycle synchronously. Ly294002 was added at 7 h after serum addition. Western blots show the expression of c-Myc, phospho-c-Myc, cyclin D2, Cdk4, and actin. The percentages of cells with sub-G1, G0/G1, S, or G2/M DNA content are indicated. (C) Synchronized cells were [35S]Met labeled during the first 9 h after serum addition, and then the medium was replaced with nonlabeled Met/Cys-rich medium alone or with Ly294002 (10 μM), and cells were collected at 12 and 16 h. For the sample treated with Ly294002 at 9 h, the inhibitor was added 30 min before harvesting. [35S]c-Myc was examined by autoradiography. Quantitative-analysis data for three experiments are shown below the blots. (D) The cell extracts used for panel B were examined by WB using anti-c-Myc, -cyclin-D3, -E, -A, and -Rb antibodies. The percentages of cells in S and G2/M phases are indicated. (E) Cyclin E/Cdk2 and cyclin A/Cdk2 kinase activities in cyclin E and cyclin A immunoprecipitates, respectively, from cell extracts prepared as described for panel B. Histone H1 (5 μg) was used as a substrate. 32P-histone was quantitated, and the activities are represented (in arbitrary units [a.u.]). Data show means ± standard deviations for three experiments. (F) Cells entering the cell cycle synchronously, treated as described for panel B, were fractionated into cytosolic, nuclear, and chromatin fractions and were examined by WB using anti-MCM2 and anti-histone antibodies. Panels A to D and F show data for one representative experiment of three with similar results.

Article Snippet: Anti-Rb antibody was from Zymed Laboratories, anti-phospho(T58/S62)-c-Myc and anti-c-Myc (9B11) were from Cell Signaling Technologies, and anti-cyclin A and -phosphotyrosine were from Upstate Biotechnology.

Techniques: Inhibition, Expressing, Activity Assay, Cell Harvesting, Northern Blot, Western Blot, Labeling, Autoradiography