Journal: Glia

Article Title: Histone‐deacetylase 8 drives the immune response and the growth of glioma

doi: 10.1002/glia.24065

Figure Lengend Snippet: HDAC8 inhibition affects glioma growth and increases mice survival. (a) RT–PCR analyses of hdac8 expression in tumor cells, normal cells, and human tissues from patients ( n = 3). (b and c) mean tumor volume (b) and Kaplan–Meier curve (c) in CT2A‐bearing mice. Mice were treated with PCI‐34051, as indicated in the scheme on top ( n = 7–8 mice per treatment; * P < 0.05 ** P < 0.01, one‐way ANOVA). (d) Data show the Ki67 + cells in brain tumors (expressed as the mean area ± S.E.M. percentage of the tumor) at 17 days after glioma implantation in mice treated with PCI‐34051 40 mg/kg, as indicated ( n = 4 mice per treatment; * P < 0.05, Student's t test). Representative immunofluorescence images of proliferating Ki67 + cells (green) under the two experimental conditions are shown on the right. Scale bars, 50 μm. ( e ) Growth curves of human and murine glioma cells treated with PCI‐34051 for the indicated time points. The results are expressed as percentage of vehicle‐treated cells ± S.E.M. ( n = 4–7; * P < 0.05 ** P < 0.01, one‐way ANOVA). ( f ) Growth curves of CT2A glioma cells treated with PCI‐34051 or iPCI‐34051 10 μM for the indicated time points. The results are expressed as percentage of vehicle‐treated cells ± S.E.M. ( n = 3; ** P < 0.01, one‐way ANOVA). (g) RT‐PCR analysis of hdac8 expression in CT2A cells transfected with c‐ or hdac8‐siRNA for 48 h. one representative western blot analysis of HDAC8 protein in silenced cells is shown on the right. ( n = 4; * P < 0.05, one‐way ANOVA). (h) Growth curves of CT2A glioma cells transfected with c‐ or hdac8‐siRNA. The results are expressed as percentage of c‐siRNA treated cells ± S.E.M. ( n = 4; ** P < 0.01, one‐way ANOVA)

Article Snippet: CT2A cells were infected by HDAC8 siRNA particles (provided by Santa Cruz Biotechnology).

Techniques: Inhibition, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunofluorescence, Transfection, Western Blot

Journal: Glia

Article Title: Histone‐deacetylase 8 drives the immune response and the growth of glioma

doi: 10.1002/glia.24065

Figure Lengend Snippet: HDAC8 inhibition reduces glioma cell migration and invasion, and increases alpha‐tubulin acetylation. (a) Human and murine glioma cell chemotaxis toward vehicle, EGF (100 ng/mL) or CXCL12 (50 ng/mL), preincubated with PCI‐34051 at the indicated doses. Results are expressed as the fold increase in comparison with vehicle ± S.E.M. ( n = 3–6; * P < 0.05 ** P < 0.01, one‐way ANOVA). (b) CT2A glioma cell chemotaxis toward vehicle, EGF (100 ng/mL) preincubated with PCI‐34051 10 μM, iPCI‐34051 10 μM or transfected with c‐ or hdac8‐siRNA. Results are expressed as the fold increase in comparison with vehicle ± S.E.M. ( n = 4; ** P < 0.01, one‐way ANOVA). (c) Mean number (± S.E.M.) of glioma cells invading the brain parenchyma for >150 μm (n = 5 mice per condition; ** P < 0.01, Student's t test). Right, representative coronal brain sections stained with hematoxylin and eosin. Black arrows indicate invading glioma cells beyond the main tumor border (dashed line). Scale bars, 20 μm. (d and e) analysis of acetylated alpha‐tubulin protein by western blot (ac‐lys40) (d) and immunofluorescence (e) in glioma cells incubated with vehicle or PCI‐34051 10 μM, iPCI‐34051 10 μM or transfected with c‐ or hdac8‐siRNA for48 h. Results are expressed as the fold increase in comparison with vehicle‐treated glioma cells ( n = 4–7; * P < 0.05, Student's t test). Right, representative blot and images of alpha‐tubulin acetylation in glioma cells under the two experimental conditions. Scale bars, 5 μm

Article Snippet: CT2A cells were infected by HDAC8 siRNA particles (provided by Santa Cruz Biotechnology).

Techniques: Inhibition, Migration, Chemotaxis Assay, Transfection, Staining, Western Blot, Immunofluorescence, Incubation

Journal: Glia

Article Title: Histone‐deacetylase 8 drives the immune response and the growth of glioma

doi: 10.1002/glia.24065

Figure Lengend Snippet: HDAC8 regulates microglia migration and morphology dynamic change in different activation states. (a) Microglia chemotaxis toward the control medium (vehicle), EGF (100 ng/mL) or CT2A‐glioma conditioned medium (CT2A), pretreated with vehicle or PCI‐34051 10 μM for 48 h. results are expressed as fold increases in comparison with vehicle ( n = 5; * < 0.05, one‐way ANOVA). (b and c) the mean (± S.E.M.) area of Iba1 + cells (b) and TMEM119 + cells (c) (as % of the tumor area) 17 days after CT2A transplantation in mice treated with PCI‐34051 40 mg/kg, as indicated. Right, representative immunofluorescences of Iba1 + and TMEM119 + cells glioma infiltrated (green) ( n = 4 mice per condition, ** P < 0.01, Student's t test). Scale bars, 20 μm. (d) RT‐PCR analysis of hdac8 expression in primary microglia incubated with vehicle, IL‐4 or LPS + IFN‐γ for 48 h ( n = 7–4, ** P < 0.01, Student's t test). For boxplots, the center line, boxes and whiskers represent the median, inner quartiles, and rest of the data distribution, respectively. (e) Primary microglia incubated with vehicle, IL‐4 or LPS + IFN‐γ for 48 h, in presence or absence of PCI‐34051 10 μM. Form factor values (calculated as indicated in methods) are shown as mean ± S.E.M ( n = 5, * P < 0.05, one‐way ANOVA). Right, representative immunofluorescences of microglia stained with Iba1 (green). Scale bars, 10 μm

Article Snippet: CT2A cells were infected by HDAC8 siRNA particles (provided by Santa Cruz Biotechnology).

Techniques: Migration, Activation Assay, Chemotaxis Assay, Transplantation Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Incubation, Staining

Journal: Glia

Article Title: Histone‐deacetylase 8 drives the immune response and the growth of glioma

doi: 10.1002/glia.24065

Figure Lengend Snippet: HDAC8 modulates gene expression in GAMs. (a) rtPCR of anti‐ ( arg‐1, chil3, tgfβ and retnla ) and pro‐inflammatory ( tnfα , il1β , nos2 , and cd86 ) genes in CD11b + cells sorted from ipsi‐ and contralateral hemisphere of CT2A‐bearing mice treated with vehicle or PCI‐34051 40 mg/kg, as indicated. Data are the mean ± S.E.M. versus vehicle contra ( n = 6 mice per condition, * P < 0.05 ** P < 0.05, one‐way ANOVA). (b) rtPCR analysis of anti‐ ( arg‐1, chil3, tgfβ and retnla ) and pro‐inflammatory ( tnfα, il1β, nos2 and cd86 ) genes in primary microglia incubated with vehicle, IL‐4 or LPS + IFN‐γ for 48 h, in presence or not of PCI‐34051 10 μM. Data are the mean ± S.E.M. expressed as fold increased in comparison with vehicle‐treated microglia ( n = 5, ** P < 0.01, one‐way ANOVA). (c) rtPCR of human pro‐ ( cxcl10 , nos2 , and il12a ) and anti‐inflammatory ( cd163, mmp12 and tgfβ ) genes in CD11b + cells sorted from patient‐derived GBM tissue, after tissue treatment with PCI‐34051(10 μM, 48 h) or vehicle. Above: Scheme of human GBM treatment. Data are the mean ± S.E.M., n = 5, * P < 0.05 versus vehicle, Student's t‐test

Article Snippet: CT2A cells were infected by HDAC8 siRNA particles (provided by Santa Cruz Biotechnology).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation, Derivative Assay

Journal: Glia

Article Title: Histone‐deacetylase 8 drives the immune response and the growth of glioma

doi: 10.1002/glia.24065

Figure Lengend Snippet: HDAC8 inhibition increases NKG2D ligands in glioma cells boosting NK cell cytotoxicity. (a) ChIP experiments performed on CT2A cells treated or not with the HDAC8 inhibitor for 24 h using the indicated antibody ( n = 3; * P < 0.05, Student's t ‐test). (b) NK cells were incubated with CT2A cells pretreated with PCI‐34051 or vehicle for 48 h. degranulation of NK cells (having subtracted basal degranulation) in a 1:1, 2.5:1, and 5:1 E:T ratio with CT2A cells was assessed by FACS analysis of CD107a + cells (right panel); cell viability in glioma cells is shown in the left panel ( n = 6, * P < 0.05 ** P < 0.01, one‐way ANOVA). Error bars show mean ± SEM. (c) NK cells, isolated from the spleen of wt or prf1 ko mice, were incubated with CT2A cells pretreated with PCI‐34051 or vehicle for 48 h. Glioma cell viability in a 2.5:1 E:T ratio is shown as mean ± S.E.M. ( n = 6, ** P < 0.01, one‐way ANOVA). (d) Percentage of CD69 + , NKG2D + and IFN‐γ + , cells in the CD3 − /NK1.1 + cell population obtained from the brain of vehicle or PCI‐34051 treated mice ( n = 4; * P < 0.05, Student's t ‐test). For boxplots, the center line, boxes and whiskers represent the median, inner quartiles, and rest of the data distribution, respectively

Article Snippet: CT2A cells were infected by HDAC8 siRNA particles (provided by Santa Cruz Biotechnology).

Techniques: Inhibition, Incubation, Isolation

Journal: Analytical and Bioanalytical Chemistry

Article Title: Comparison of functional and discrete data analysis regimes for Raman spectra

doi: 10.1007/s00216-021-03360-1

Figure Lengend Snippet: The mean spectra per class ( C . basilensis , N . aromaticivorans , R . opacus ) for the pre-processed experimental data (left) and their functional approximation (right). The discrete mean spectra per class are shown on the left. Their functional counter parts are illustrated on the right side. A reduction of noise in the functional version is deduced, where the mean intensity with the standard deviation for specific wavenumbers of both versions is illustrated in ESM Table . Beside this noise reduction, no Raman spectral features are removed

Article Snippet: The naphthalene-degrading soil bacteria Rhodococcus opacus DSM 8531 ( R. opacus ), Novosphingobium aromaticivorans DSM 12444 ( N. aromaticivorans ), and Cupriavidus basilensis DSM 9750 ( C. basilensis ) were included in the study and purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures.

Techniques: Functional Assay, Standard Deviation