rothia mucilaginosa  (ATCC)

Journal: mSphere

Article Title: Antibiotics Drive Expansion of Rare Pathogens in a Chronic Infection Microbiome Model

doi: 10.1128/msphere.00318-22

Figure Lengend Snippet: Experimental model organisms used in synthetic community experiments

Article Snippet: Rothia mucilaginosa , ATCC 49042 , 9.1.

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Cytosolic carboxypeptidase CCP6 is required for megakaryopoiesis by modulating Mad2 polyglutamylation

doi: 10.1084/jem.20141123

Figure Lengend Snippet: Mad2 is polyglutamylated by TTLL4 and TTLL6 in MKs. (A) TTLL4 and TTLL6 expression in BM MKs. mRNAs were analyzed by real time qPCR. n = 6. Relative gene expression folds were normalized to endogenous β-actin. Primer pairs are shown in Materials and methods. Relative gene expression fold change was counted as means ± SD. **, P < 0.01. (B) Representative confocal images of immunofluorescence staining of TTLL6 and VWF in BM. Bars, 20 µm. White boxes in top panels are shown in bottom panels. (C) Assessment of rGST-Mad2 polyglutamylation by both TTLL4 and TTLL6 in GST pulldown assays. (D) Assessment of CCP6-mediated rGST-Mad2 deglutamylation by TTLL4 or TTLL6 in GST pulldown assays (left). Ratios of GT335/Mad2 were calculated and shown as means ± SD. **, P < 0.01 (right graphs). (E) Assessment of Mad2 polyglutamylation by TTLL6 at Glu168. 293T cells were transfected with Flag-TTLL6 and lysates were incubated with GST-Mad2 or GST-Mad2 mutant at 37°C for 2 h for in vitro glutamylation followed by GST pulldown assays. (F) Assessment of Mad2 colocalization with TTLL6 or tubulin in low ploidy MKs. Primary mouse MKPs were isolated and cultured for maturation as described in Materials and methods. Representative confocal images are shown (top). Bars, 5 µm. Mad2, red; nucleus, blue; TTLL6, green (top); tubulin, green (middle). For each staining, >50 typical cells were enumerated. Pearson’s correlation coefficient (PCC) between Mad2 and TTLL6 of each cell was calculated by ImageJ and colocalization was identified as PCC rate ≥ 0.5. Percentages of colocalized cells were counted as means ± SD (bottom). **, P < 0.01. (G) Schematic representation of transplantation. (H) Primary mouse MKP cells were sorted. TTLL6 or Mad2 was silenced (shTTLL6 or shMad2). shMad2 silenced MKPs were then rescued with Mad2-wt (Mad2-wt/shMad2) or Mad2E168A (Mad2E168A/shMad2) by retrovirus infection and transplanted into recipients along with helper cells ( n = 6). After 7 d, GFP + and VWF + MKs derived from donor cells were analyzed by immunofluorescence (top left). AchE staining was also analyzed in separate sections for MK verification (top right). Representative microscopy images are shown. Bars, 20 µm. White and red arrows denote MKs. Percentages of transplanted immature MKs (GFP + VWF − , small in size) and mature MKs (GFP + VWF + , large in size) were calculated and shown as means ± SD (bottom). All data are representative of at least four separate experiments.

Article Snippet: Antibody against TTLL6 was from Novus Biologicals.

Techniques: Expressing, Immunofluorescence, Staining, Transfection, Incubation, Mutagenesis, In Vitro, Isolation, Cell Culture, Transplantation Assay, Infection, Derivative Assay, Microscopy

Journal: The Journal of Experimental Medicine

Article Title: Cytosolic carboxypeptidase CCP6 is required for megakaryopoiesis by modulating Mad2 polyglutamylation

doi: 10.1084/jem.20141123

Figure Lengend Snippet: CCP6 deficiency causes Mad2 hyperglutamylation, promoting Aurora B hyperactivation with generation of dysplastic MKs. (A) Assessment of Mad2 polyglutamylation and association with Aurora B upon CCP6 silencing (shCCP6) in an immunoprecipitation assay (shCtrl as control). K562 lysates were immunoprecipitated with anti-Mad2 antibody followed by immunoblotting. (B) Assessment of Mad2 colocalization with Aurora B (AuroB) in primary CD41 + MKs. Representative confocal images are shown (left: Aurora B, red; Mad2, green; nucleus, blue. Bar, 5 µm). 50 primary CD41 + cells were analyzed for each condition. In left panels, arrows point to cells shown in right panels. White arrows denote colocalization of Mad2 with Aurora B. Pearson’s correlation coefficient (PCC) between Mad2 and Aurora B of each cell was calculated by ImageJ and colocalization was identified as PCC rate ≥ 0.5. Percentages of colocalized cells are shown as means ± SD. **, P < 0.01 (right graph). (C) Assessment of polyglutamylated Mad2 association with Aurora B by GST pulldown assays. rGST-Mad2 was incubated with lysates from 293T cells transfected with Flag-TTLL6 and Myc-CCP6 at 37°C for 2 h, followed by GST pulldown in K562 cell lysates. (D) Assessment of Thr232 phosphorylation of Aurora B and Thr288 phosphorylation of Aurora A upon CCP6 silencing (shCCP6; shCtrl as control). K562 lysates were analyzed by immunoblotting with anti-Aurora B, anti–Thr232-Aurora B, anti–Aurora A, and anti–Thr288-Aurora A antibodies (top). Ratios of pThr/Aurora kinase were calculated and shown as means ± SD. **, P < 0.01 (bottom graph). (E) Assessment of Thr232 phosphorylation of Aurora B in the presence of an E168A Mad2 mutant. Flag-TTLL6, pMY-Mad2-wt and pMY-Mad2-E168A vectors were transfected into 293T cells, followed by immunoblotting (top). Ratios of pAurora B/actin were calculated and shown as means ± SD. **, P < 0.01 (bottom graph). (F) Assessment of Thr232 phosphorylation of Aurora B in CCP6 -deficient MKs. Representative confocal images are shown (left). Phospho-Thr232 of Aurora B (pAuroB), green; nucleus, red. Bar, 5 µm. The percentage of cells with high phospho-Thr232 of Aurora B (pAuroB high ) was quantitated in >50 primary CD41 + cells for each group and shown as means ± SD. **, P < 0.01 (right graph). White boxes in left panels are shown in right panels. White arrows denote pAuroB high MKs. The fluorescence intensity of pAuroB in each cell was calculated by ImageJ and pAuroB high was identified as higher intensity than the mean fluorescence intensity in the CCP6 +/+ group. (G) Schematic representation of transplantation. (H) Mad2 silenced BM MEPs from CCP6 +/+ or CCP6 −/− donor mice were rescued with pMY-Mad2-wt or pMY-Mad2E168A together with BM helper cells, and then transplanted into irradiated recipients. BM MKs of recipients were analyzed 12 d after transplantation. n = 6. Percentages of CD41 + MKs are shown as means ± SD. **, P < 0.01. (I and J) Primary MKPs were treated with the Aurora B inhibitor AZD-1152, and then stimulated with 20 ng/ml TPO for 5 d. Cell morphology was analyzed by Giemsa staining (Bar, 20 µm; red arrows denote mature MKs; n = 6; I). DNA contents were analyzed by flow cytometry. Percentages of DNA ploidy are shown as means ± SD. **, P < 0.01 (J). (K and L) Primary MKPs were infected with the indicated retrovirus then stimulated with 20 ng/ml TPO for 5 d. Cell morphology was analyzed by Giemsa staining (Bar, 20 µm; red arrows denote mature MKs; n = 6; K). DNA contents were analyzed by flow cytometry. Percentages of DNA ploidy are shown as means ± SD. *, P < 0.05; **, P < 0.01 (L). Data represent at least three separate experiments.

Article Snippet: Antibody against TTLL6 was from Novus Biologicals.

Techniques: Immunoprecipitation, Western Blot, Incubation, Transfection, Mutagenesis, Fluorescence, Transplantation Assay, Irradiation, Staining, Flow Cytometry, Infection