Journal: Development (Cambridge, England)

Article Title: Notch3 signaling gates cell cycle entry and limits neural stem cell amplification in the adult pallium

doi: 10.1242/dev.095018

Figure Lengend Snippet: Notch3 inhibition accounts for the effect of Notch blockade on RG activation. (A-F) Triple immunohistochemistry for the RG marker glutamine synthetase (GS, green), MCM5 (magenta) and HuC/D (blue) on telencephalic cross-sections from adult notch3+/+ siblings and notch3fh332/+ heterozygotes under control conditions (top row) or upon LY treatment (middle and bottom rows). Scale bar: 20 μm. Confocal projection images from four optical planes, each 1 μm thick. (G,H) Total number of MCM5-positive progenitors per section (G) and proportion of RG cells in proliferation (H) in the different genotypes and treatment conditions, as well as in the standard AB wild-type line. P<0.0001 (n=3 brains for AB, notch3+/+ and notch3fh332/+, respectively). (I) Schematic of the notch3-MO knock-down experiment: a BrdU pulse is applied 2 days after electroporation (EP) of fluorescein-labeled notch3-MO or control-MO in pallial ventricular cells. Brains are analyzed immediately. (J,K) Analysis of the proliferation status (anti-BrdU) (blue) of electroporated (fluorescein-positive) (green) RG (anti-S100β) (magenta), assessed by immunocytochemistry. Arrows indicate fluorescein-labeled radial glia BrdU-positive cells. Scale bar: 20 μm. Confocal projection images from four optical planes, each 1 μm thick. (L) Proportion of BrdU-positive cells within the radial glia MO-targeted population. P<0.001 (n=3 brains for each condition).

Article Snippet: Immunohistochemistry For analyses of adult brains, the following primary antibodies were applied onto free-floating 50 μm vibratome sections: GFP (1:500, Aves Laboratories), MCM5 (1:500, kindly provided by Soojin Ryu, Max Planck Institute for Medical Research, Heidelberg, Germany), HuC/D (1:600, mouse, Invitrogen; 1:2000, human, a gift from Dr B. Zalc, Salpêtrière Hospital, Paris), BrdU (1:1000, mouse clone MoBU-1, Invitrogen; 1:250, rat, Abcam), glutamine-synthase (1:500, mouse, Millipore) and S100β (1:1000, rabbit, Dako).

Techniques: Inhibition, Activation Assay, Immunohistochemistry, Marker, Electroporation, Labeling, Immunocytochemistry

Journal: Development (Cambridge, England)

Article Title: Notch3 signaling gates cell cycle entry and limits neural stem cell amplification in the adult pallium

doi: 10.1242/dev.095018

Figure Lengend Snippet: Individual RG cells maintain stem cell properties after Notch blockade. (A) Experimental design. (B-K) Single optical section of the pallial ventricular zone in gfap:gfp transgenic brains, triple labeled for GFP (RG, green), CldU (magenta) and HuC/D (gray). (C-F) High magnification of the area boxed in B; example of a self-renewing, symmetric gliogenic division. (H-K) High magnification of the area boxed in G; example of a self-renewing and asymmetric division. Scale bars: yellow, 20 μm; white, 2 μm.

Article Snippet: Immunohistochemistry For analyses of adult brains, the following primary antibodies were applied onto free-floating 50 μm vibratome sections: GFP (1:500, Aves Laboratories), MCM5 (1:500, kindly provided by Soojin Ryu, Max Planck Institute for Medical Research, Heidelberg, Germany), HuC/D (1:600, mouse, Invitrogen; 1:2000, human, a gift from Dr B. Zalc, Salpêtrière Hospital, Paris), BrdU (1:1000, mouse clone MoBU-1, Invitrogen; 1:250, rat, Abcam), glutamine-synthase (1:500, mouse, Millipore) and S100β (1:1000, rabbit, Dako).

Techniques: Transgenic Assay, Labeling

Journal: Nature

Article Title: Functional genomic landscape of cancer-intrinsic evasion of killing by T cells

doi: 10.1038/s41586-020-2746-2

Figure Lengend Snippet: a, Overlap of core intrinsic CTL-evasion genes and hits from the Renca IFNγ screen. b, Per cent viability of Fitm2 or intergenic gRNA-transduced Renca-HA, B16-Ova or CT26-HA cells treated with escalating doses of antigen-specific (CL4) T cells or IFNγ. Error bars equal s.e.m. of indicated number (n) of independent experiments. For CT26HA panel, * denotes Fitm2–1 P-value <0.05. P-values determined by Two-way ANOVA with Fisher’s LSD comparison. c, Per cent viability of Fitm2 or intergenic gRNA-transduced human A375 cells treated with escalating doses of antigen-specific (WT-1) T cells. Data representative of 3 independent experiments, with line highlighting mean effect. P values determined by two-way ANOVA with Fisher’s LSD comparison. d, Microscopic views of Fitm2 (right panels) or intergenic (left panels) gRNA-transduced Renca cells after 72 h of IFNγ treatment shown ( lower panels). Untreated control cells are shown in the top panels. Data represent a single experiment. e, Expression of surface MHC-I by flow cytometry for B16-Ova cells transduced with Fitm2 or intergenic gRNAs and treated with IFNγ. Data representative of 4 independent experiments, with line highlighting mean effect. P values determined by two-way ANOVA with Fisher’s LSD comparison. f, Expression of surface MHC-I/Ova(SIINFEKL) by flow cytometry for B16-Ova cells transduced with Fitm2 or intergenic gRNAs and treated with IFNγ. Data representative of 4 independent experiments, with line highlighting mean effect. P values determined by two-way ANOVA with Fisher’s LSD comparison. g, Per cent viability of Fitm2 or intergenic gRNA-transduced RencaHA cells treated with increasing doses of TNF for 72 h. Errors bars equal s.e.m. of 5 independent experiments. For 100 ng ml−1 dose, * or ** denote P values <0.05 and <0.01, respectively, for Fitm2-2 and Fitm2-3, respectively. P values determined by two-way ANOVA with Fisher’s LSD comparison. Note: overlap of data for intergenic control with Fig. 3b.

Article Snippet: After washing, cells were stained at 4 °C for 30 min in FACS buffer containing MHC-I (anti mouse MHC-I (H-2 kb) EFluor450 at 1:200, clone AF6–88.5.5.3, Ebiosciences) and MHC-I bound to OVA (anti mouse OVA 257–264 peptide bound to H2kb PE at 1:100, clone 25-D1.16, Ebiosciences) antibodies.

Techniques: Expressing, Flow Cytometry, Transduction