Journal: The FEBS journal

Article Title: Nuclear patterns of phosphatidylinositol 4,5- and 3,4-bisphosphate revealed by super-resolution microscopy differ between the consecutive stages of RNA polymerase II transcription.

doi: 10.1111/febs.17136

Figure Lengend Snippet: Fig. 1. Correlation of the consecutive stages of RNAPII transcription and nPIPs. Transcription initiation and elongation marker P-S5 and P-S2, respectively, and nuclear PI(4,5)P2 or PI(3,4)P2 indirectly immunolabeled with AF647 (magenta) and AF555 (green), respectively, with zoom-in to the boxed ROIs in the nucleoplasm (Np) or nuclear speckles (Sp) imaged by dSTORM (A). Normalized control (ctrl) and random (rand.) NND distributions between P-S5 or P-S2 and nPI(4,5)P2 or nPI(3,4)P2 in the Np and Sp ROIs (B). Color-coded pixel maps of the NND between P-S5 or P-S2 and nPI(4,5)P2 or nPI(3,4)P2 (C) with zoom-in to the boxed ROIs. (D) The Mode NNDs and the Fraction of the NNDs in the mode NND in the Np and at Sp ROIs. Scale bars = 5 lm; zoom-in = 1 lm. Measurements P-S5 to PI(4,5)P2 in the Np are from 28 and in Sp from 29 ROIs from n = 12 nuclei from N = 4 independent experiments; P-S2 to PI(4,5)P2 Np and Sp both 32 ROIs n = 14, N = 3; P-S5 to PI(3,4)P2 Np 27 and Sp 30 ROIs n = 11, N = 3; P-S2 to PI(3,4)P2 Np 43 and Sp 36 ROIs n = 8, N = 3. The error bars in the normalized NND distributions in (B) represent the SEM. The data in (D) are plotted as Tukey whisker plots and were evaluated statistically using one-way ANOVA followed by Bonferoni post-test expressing the statistical significance: **P < 0.01; ***P < 0.005.

Article Snippet: The secondary antibodies and concentrations used were: goat anti-mouse IgM (l-chain), AF555 (10 lg mL 1; A24126; Jackson ImmunoResearch), donkey anti-mouse IgG AF555 (10 lg mL 1; A31570 Invitrogen Thermo Fisher Scientific) and goat anti-rabbit IgG AF647 (10 lg mL 1; A21245 Invitrogen Thermo Fisher Scientific).

Techniques: Marker, Immunolabeling, Control, Whisker Assay, Expressing

Journal: Cell Stem Cell

Article Title: Defining the Identity and Dynamics of Adult Gastric Isthmus Stem Cells

doi: 10.1016/j.stem.2019.07.008

Figure Lengend Snippet: Rapidly Cycling Isthmus Progenitors Can Maintain Long-Term Self-Renewal Potential (A) Schematic showing the fluorescence signal associated with each phase of the cell cycle in the Rosa26-Fucci2a mouse. (B) Representative confocal images of stomach corpus gland sections (150 μm) of Rosa26-Fucci2a mice. Red, hCdt1-mCherry(30/120-G1); green, hGem(1/110-S/G2/M); grey, β-catenin (I), Ki67 (II), HKATPase (III). Scale bars: 50 μm. (C) Venn diagram showing the overlap between genes more highly expressed in isolated proliferative isthmus cells and genes downregulated following 5-FU treatment, taken to be prospective candidate markers for proliferating isthmus cells. (D) Immunohistochemical staining for Ki67, counterstained with Mayer’s hematoxylin (leftmost panel). In situ hybridization with Stmn1 anti-sense (middle panel) and Stmn1 sense negative control (rightmost panel) probes in mouse stomach corpus sections are shown. Stmn1 mRNA was restricted to the isthmus region delineated by Ki67 staining. Scale bars: 25 μm. (E) Schematic of the genetic strategy to trace Stmn1+ cell-derived progeny in the corpus gland. (F) Representative confocal images of the stomach corpus of Stmn1-CreERT2 mice. Red, Ki67; green, EGFP. Scale bars: 50 μm. (G) Representative confocal images of 150-μm-thick sections of the stomach corpus of Stmn1-CreERT2;R26R-tdTomato mice at 2 days, 2 weeks, 1 month, 3 months, and 6 months post-injection of 0.5–1 mg tamoxifen/20 g mouse body weight. Labeling of clones in the isthmus region with tdTomato can be detected as early as 2 days post-injection. Red, tdTomato. Scale bars: 50 μm. (H) Representative confocal images of the mouse stomach corpus glands of Stmn1-CreERT2;R26R-tdTomato mice at 5 months post-labeling. Red, tdTomato for labeled cells; grey, Atp4b for parietal cells; green, Muc5ac for pit cells and GS-II for neck cells. Scale bars: 50 μm. See Figure S6 and and .

Article Snippet: Sections were incubated in blocking solution (2% donkey or goat serum, 5% DMSO and 0.5% Triton X-100 in PBS) at room temperature for 2 h. The following primary antibodies were used: chicken anti-EGFP (1:500; Abcam, ab13970), rabbit anti-Ki67 (1:250; A. Menarini, MP-325-CRM1), Alexa 647-conjugated mouse anti-β-catenin (1:200; Cell Signaling Technology, 4627S), rabbit anti-H+/K+ ATPase β Antibody (D-18) (1:500; Santa Cruz, sc-84304) and Alexa 555/647-conjugated rabbit anti-ATP4b (1:200; Bioss, bs-2433R-AF555, bs-2433R-AF647).

Techniques: Fluorescence, Isolation, Immunohistochemical staining, Staining, In Situ Hybridization, Negative Control, Derivative Assay, Injection, Labeling, Clone Assay

Journal: Cell Stem Cell

Article Title: Defining the Identity and Dynamics of Adult Gastric Isthmus Stem Cells

doi: 10.1016/j.stem.2019.07.008

Figure Lengend Snippet:

Article Snippet: Sections were incubated in blocking solution (2% donkey or goat serum, 5% DMSO and 0.5% Triton X-100 in PBS) at room temperature for 2 h. The following primary antibodies were used: chicken anti-EGFP (1:500; Abcam, ab13970), rabbit anti-Ki67 (1:250; A. Menarini, MP-325-CRM1), Alexa 647-conjugated mouse anti-β-catenin (1:200; Cell Signaling Technology, 4627S), rabbit anti-H+/K+ ATPase β Antibody (D-18) (1:500; Santa Cruz, sc-84304) and Alexa 555/647-conjugated rabbit anti-ATP4b (1:200; Bioss, bs-2433R-AF555, bs-2433R-AF647).

Techniques: Recombinant, Blocking Assay, Labeling, Multiplex Assay, Isolation, Software

Journal: Mucosal Immunology

Article Title: Group 2 innate lymphoid cells utilize the IRF4-IL-9 module to coordinate epithelial cell maintenance of lung homeostasis

doi: 10.1038/mi.2015.59

Figure Lengend Snippet: Alveolar type II (ATII) cells are the source of interleukin (IL)-33 and thymic stromal lymphopoietin (TSLP) in response to chitin. ( a ) ELISA of IL-33 (left) and TSLP (right) from whole lung homogenates of mice at indicated times after chitin administration. ( b ) Quantitative reverse transcription-PCR analysis of Il33, Tslp, Il2, and Il7 expression from whole lung RNA of mice 6 h after chitin challenge. Values normalized to 18s expression and graphed as fold increase over the mean of the 0 h mice. ( c ) Immunohistochemistry of naive mouse lungs. Original magnification, × 200. Scale bar, 50 μm. ( d ) Western blot analysis of whole lung homogenates of mice at indicated times after chitin. “rIL-33” is recombinant protein (Ser109-Ile266; R&D Systems). IB, immunoblot. ( e ) Ratio of 20-kDa anti-IL-33-reactive band pixel density to total IL-33 pixel density (sum of 20-kDa band and 35-kDa band densities) in each lane, compiled from three independent experiments. ( f ) Flow cytometry (left) and immunocytochemistry (right) of post-sort ATII cells (DAPI lo CD45 lo CD11b lo CD31 lo EpCAM + ) isolated from lungs of mice 3 h after chitin. Original magnification, × 200. Scale bar, 10 μm. ( g ) ELISA of TSLP in lung populations sorted from mice 3 h after PBS or chitin (left) and in supernatants from MLE12 cells cultured with 40 ng ml −1 phorbol 12-myristate 13-acetate for the indicated times (right), compiled from four independent experiments, respectively. Where applicable: * P <0.05, ** P <0.005, *** P <0.0005 and NS, not significant (two-tailed t -test). Data are representative of at least three independent experiments with 2–4 mice per exposure ( a , f ) or at least two independent experiments with 3 mice per exposure ( b – d ). DAPI, 4',6-diamidine-2'-phenylindole dihydrochloride; SPC, surfactant protein C.

Article Snippet: Briefly, cell homogenates were incubated with goat anti-mouse TSLP detection antibodies (R&D Systems) and subsequently probed with horseradish peroxidase (HRP)-conjugated donkey anti-goat IgG antibodies (SouthernBiotech, Birmingham, AL), followed by application of Substrate Solution (R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Reverse Transcription, Expressing, Immunohistochemistry, Western Blot, Recombinant, Flow Cytometry, Immunocytochemistry, Isolation, Cell Culture, Two Tailed Test

Journal: The FEBS journal

Article Title: Nuclear patterns of phosphatidylinositol 4,5- and 3,4-bisphosphate revealed by super-resolution microscopy differ between the consecutive stages of RNA polymerase II transcription.

doi: 10.1111/febs.17136

Figure Lengend Snippet: Fig. 1. Correlation of the consecutive stages of RNAPII transcription and nPIPs. Transcription initiation and elongation marker P-S5 and P-S2, respectively, and nuclear PI(4,5)P2 or PI(3,4)P2 indirectly immunolabeled with AF647 (magenta) and AF555 (green), respectively, with zoom-in to the boxed ROIs in the nucleoplasm (Np) or nuclear speckles (Sp) imaged by dSTORM (A). Normalized control (ctrl) and random (rand.) NND distributions between P-S5 or P-S2 and nPI(4,5)P2 or nPI(3,4)P2 in the Np and Sp ROIs (B). Color-coded pixel maps of the NND between P-S5 or P-S2 and nPI(4,5)P2 or nPI(3,4)P2 (C) with zoom-in to the boxed ROIs. (D) The Mode NNDs and the Fraction of the NNDs in the mode NND in the Np and at Sp ROIs. Scale bars = 5 lm; zoom-in = 1 lm. Measurements P-S5 to PI(4,5)P2 in the Np are from 28 and in Sp from 29 ROIs from n = 12 nuclei from N = 4 independent experiments; P-S2 to PI(4,5)P2 Np and Sp both 32 ROIs n = 14, N = 3; P-S5 to PI(3,4)P2 Np 27 and Sp 30 ROIs n = 11, N = 3; P-S2 to PI(3,4)P2 Np 43 and Sp 36 ROIs n = 8, N = 3. The error bars in the normalized NND distributions in (B) represent the SEM. The data in (D) are plotted as Tukey whisker plots and were evaluated statistically using one-way ANOVA followed by Bonferoni post-test expressing the statistical significance: **P < 0.01; ***P < 0.005.

Article Snippet: The secondary antibodies and concentrations used were: goat anti-mouse IgM (l-chain) AF555 (10 lg mL 1; A24126; Jackson ImmunoResearch, Ely, UK), donkey anti-mouse IgG AF555 (10 lg mL 1; A31570; Invitrogen Thermo Fisher Scientific, Waltham, MA, USA) and goat anti-rabbit IgG AF647 (10 lg mL 1; A21245; Invitrogen Thermo Fisher Scientific).

Techniques: Marker, Immunolabeling, Control, Whisker Assay, Expressing