Journal: Cell Stem Cell

Article Title: Phospho-regulation of ATOH1 Is Required for Plasticity of Secretory Progenitors and Tissue Regeneration

doi: 10.1016/j.stem.2018.07.002

Figure Lengend Snippet: Lineage Tracing of Atoh1 + Cells in Homeostasis and after Injury (A–D) The tdTom reporter is detected in Muc2 + goblet cells in the SI (A), colon (B), and Lyz + Paneth cells (C) but not in ChgA + enteroendocrine cells 24 hr post-tamoxifen (D). Muc2, Mucin 2; Lyz, Lysozyme; ChgA, Chromogranin A. (E) ChgA + cells labeled with tdTom on day 4 after induction. (F) Dclk1 + tuft cells are not labeled with tdTom at 24 hr. (G and H) Reporter-positive clone in the SI (G) and colon (H) 30 days following tamoxifen. (I–L) tdTom + clones at 30 days are composed of alkaline phosphatase (Alpi + ) enterocytes (I), Paneth cells (J), goblet cells (K), and enteroendocrine cells (L). (M, P, and S) Schematic of induction and injury protocol: irradiation (M), azoxymethane (AOM) (P), and dextran sodium sulfate (DSS) (S). (N) Representative pictures of SI whole-mounts containing labeled crypts (arrowheads) 30 days post-induction. (O) Quantification of tdTom + crypts in the SI (n = 4 for 0 Gy, n = 6 for 6 Gy [day 1], n = 4 for 6 Gy [day 5]). (Q and T) Representative images of colonic crypts on day 30 post-tamoxifen and AOM (Q) or DSS treatment (T). Note the large tdTom + regenerative multicrypt patches (MCPs) associated with 2% DSS treatment (T). (R) Quantification of reporter-positive crypts in the colon (n = 6 for untreated, n = 5 for AOM-treated). (U) Quantification of tdTom + MCPs in untreated and DSS-treated colons (n = 3 for both groups). Welch’s t test was used in (O) (mean ± SEM, ∗∗∗∗ p < 0.0001) and Mann-Whitney test in (R) (mean ± SEM, ∗∗ p = 0.0087). Scale bars, 50 μm (A–L) and 100 μm (N, Q, and T). See also Figure S1 .

Article Snippet: Mouse Monoclonal anti-Atoh1 , Developmental Studies Hybridoma Bank , Cat# Math1 (Atoh1): RRID: AB_10805299.

Techniques: Labeling, Clone Assay, Irradiation, MANN-WHITNEY

Journal: Cell Stem Cell

Article Title: Phospho-regulation of ATOH1 Is Required for Plasticity of Secretory Progenitors and Tissue Regeneration

doi: 10.1016/j.stem.2018.07.002

Figure Lengend Snippet: Identification of a Hyperactive Phosphomutant ATOH1 (A) Diagram depicting the location of proline-directed kinase motifs (serine-proline [SP] or threonine-proline [TP]) in Atoh1 protein and mutations of these sites into alanine in ATOH1 phosphomutants. (B) In vitro -translated Atoh1 protein band-shift following incubation with different cyclin-dependent kinases (CDKs). Ngn3 was used as a positive control. (C) WT ATOH1 bands (arrows) collapse following λ phosphatase treatment, demonstrating phosphorylation. (D) DLD-1 cell proliferation following doxycycline (Dox)-induced expression of WT or phosphomutant Atoh1 (n = 3 biological replicates, 2 technical replicates, mean ± SEM). (E) Cell cycle profile of uninduced and Dox-treated cells showing increased G1 and decreased S/M populations upon induction of 9S/T-A Atoh1 . (F) Gene expression of Atoh1 and its target and secretory differentiation genes 72 hr after Dox induction of DLD-1 cells (n = 3 biological replicates, 2 technical replicates; Gapdh-normalized, mean ± SEM). Two-way ANOVA was used for statistical analysis; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Mouse Monoclonal anti-Atoh1 , Developmental Studies Hybridoma Bank , Cat# Math1 (Atoh1): RRID: AB_10805299.

Techniques: In Vitro, Electrophoretic Mobility Shift Assay, Incubation, Positive Control, Expressing

Journal: Cell Stem Cell

Article Title: Phospho-regulation of ATOH1 Is Required for Plasticity of Secretory Progenitors and Tissue Regeneration

doi: 10.1016/j.stem.2018.07.002

Figure Lengend Snippet: 9S/T-A ATOH1 Promotes Secretory Maturation and Reduces Proliferation and the Number of Clonogenic Atoh1 + Cells (A and B) Gene set enrichment analysis (GSEA) of the Atoh1 + SI secretory signature (A) and colon (B) shows enrichment of secretory genes in 9S/T-A Atoh1 tdTom + cells. (C and D) GSEA utilizing a published secretory transcriptome reveals an increase in the secretory gene signature in phosphomutant-expressing tdTom + cells in the SI (C) and colon (D). (E) GSEA using a published intestinal stem cell (ISC) gene signature ( Muñoz et al., 2012 ) shows a de-enrichment of ISC genes in the mutant SI progenitors. (F and G) Bromodeoxyuridine (BrdU) labeling index for a range of cell positions in SI crypts (F) shows a reduction in proliferation above the crypt base (n = 100 crypts, 4 mice per genotype; mean ± SEM; ∗∗ p = 0.0061 and 0.0015). Shown in (G) is the frequency of crypt-villus units containing at least one BrdU + goblet cell after a 24-hr BrdU pulse (n = 5 for both groups, ∗ p = 0.0317). (H) Representative image of a crypt-villus unit with a BrdU + Alcian blue (AB) and periodic acid-Schiff (PAS) + cell. (I) Quantification of tdTom + clonal events in the SI (n = 6 [WT], n = 7 [ 9S/T-A ], mean ± SEM, ∗∗ p = 0.0012). (J) Partly populated tdTom + crypts (PPCs) in the colon (n = 6 [WT], n = 7 [ 9S/T-A ], mean ± SEM, ∗∗ p = 0.0023). (K) Wholly populated tdTom + crypts (WPCs) in WT and 9S/T-A colons (n = 6 [WT], n = 7 [ 9S/T-A ], mean ± SEM, ∗∗ p = 0.0012; the same WT data are shown in Figure 1 R because the experiment was done in parallel). All samples were collected 30 days after tamoxifen. Mann-Whitney test was used for all comparisons. (L and M) Representative images of WT (L) and 9S/T-A (M) colons scored in (J) and (K). (N–Q) Inference of the proportion of the clonogenic fraction of labeled Atoh1 + cells in the proximal SI (N), distal SI (O), colon PPCs (P), and colon WPCs (Q). The numbers next to the dotted lines indicate the inferred proportion of crypts that had one labeled stem cell. Scale bars, 75 μm.

Article Snippet: Mouse Monoclonal anti-Atoh1 , Developmental Studies Hybridoma Bank , Cat# Math1 (Atoh1): RRID: AB_10805299.

Techniques: Expressing, Mutagenesis, Labeling, MANN-WHITNEY

Journal: Cell Stem Cell

Article Title: Phospho-regulation of ATOH1 Is Required for Plasticity of Secretory Progenitors and Tissue Regeneration

doi: 10.1016/j.stem.2018.07.002

Figure Lengend Snippet: Atoh1 (9S/T-A)CreERT2 Mice Are Sensitive to Chemical Colitis (A) Change in mouse body weight during and after DSS treatment (n = 5 [WT], n = 6 [ 9S/T-A ]; two 9S/T-A mice were euthanized on day 9 for health reasons, and one WT animal was taken for comparison [arrowhead]). (B) Representative pictures and schematics of the colon on day 9, showing extensive loss of crypts in 9S/T-A but not in the WT. Scale bars, 1 mm. (C) Total length of colon ulceration on day 9 (n = 6 [WT], n = 4 [ 9S/T-A ], mean ± SEM, ∗∗ p = 0.0095). (D and E) Representative images of the distal colon on day 5 of DSS treatment, showing apoptosis (D) and AB and PAS staining (E) in WT and 9S/T-A animals. (F) FACS analysis of the number of tdTom + cells during DSS-induced colitis. (G and H) Analysis of the number (G) and total area (H) of tdTom + MCPs following 1.5% DSS (n = 4 [WT], n = 7 [ 9S/T-A ], mean ± SEM, ∗∗ p = 0.0061, ∗ p = 0.0424). See also Figure S4 .

Article Snippet: Mouse Monoclonal anti-Atoh1 , Developmental Studies Hybridoma Bank , Cat# Math1 (Atoh1): RRID: AB_10805299.

Techniques: Staining

Journal: Cell Stem Cell

Article Title: Phospho-regulation of ATOH1 Is Required for Plasticity of Secretory Progenitors and Tissue Regeneration

doi: 10.1016/j.stem.2018.07.002

Figure Lengend Snippet:

Article Snippet: Mouse Monoclonal anti-Atoh1 , Developmental Studies Hybridoma Bank , Cat# Math1 (Atoh1): RRID: AB_10805299.

Techniques: Recombinant, Clone Assay, RNA Sequencing Assay, Software

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Entamoeba histolytica Cyclooxygenase-Like Protein Regulates Cysteine Protease Expression and Virulence

doi: 10.3389/fcimb.2018.00447

Figure Lengend Snippet: Differential Math1 transcriptional activity in Math1 GFP mice exposed to EhCoxgs due to increased cysteine proteases activity. (A) Math1 expression heatmap in Math1 GFP mice colons, dotted line indicates the colonic loop ligation. (B) GFP signal quantification in the proximal colon. GFP, green fluorescent protein; AU, arbitrary units. * P < 0.05.

Article Snippet: Math1 GFP mice were purchased from Jackson laboratory and bred in-house.

Techniques: Activity Assay, Expressing, Ligation

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Entamoeba histolytica Cyclooxygenase-Like Protein Regulates Cysteine Protease Expression and Virulence

doi: 10.3389/fcimb.2018.00447

Figure Lengend Snippet: Pro-inflammatory responses are exacerbated in Math1 mice exposed to EhCoxgs compared control Eh . Math1 GFP mice were infected with control Eh or EhCoxgs for 3 h. Gene expression of IL-1β, KC, TNF-α, and IFN-γ was examined from excised tissues from the proximal colon. Myeloperoxidase activity in the proximal colons of mice inoculated with control Eh or EhCoxgs . The bars indicate the means and the error bars indicate the standard errors of the means for different experiments. * P < 0.05.

Article Snippet: Math1 GFP mice were purchased from Jackson laboratory and bred in-house.

Techniques: Control, Infection, Gene Expression, Activity Assay

Journal: STAR Protocols

Article Title: Protocol to photoactivate adipose-derived stem cell differentiation using a tightly-focused femtosecond laser

doi: 10.1016/j.xpro.2022.101574

Figure Lengend Snippet:

Article Snippet: Mouse: Math1-GFP (male mice over 8 weeks old, female mice aged 4–6 weeks) , Novartis , N/A.

Techniques: Recombinant, In Vivo, Isolation, Plasmid Preparation, Software, Cell Culture, Microscopy

Journal: Scientific Reports

Article Title: Combinatorial Atoh1 and Gfi1 induction enhances hair cell regeneration in the adult cochlea

doi: 10.1038/s41598-020-78167-8

Figure Lengend Snippet: Efficient HC ablation after DT injection and adenovirus scala media inoculation. Images show cochleae of adult mice 10 days after adenoviral inoculation of scala media with or without HC ablation. ( a , a ′, b , b ′) Ad. Atoh1 adenovirus inoculation of wild-type (WT) mouse without DT-induced HC ablation. ( c , c ′, d , d ′) Ad. Atoh1 adenovirus inoculation of Pou4F3 DTR mouse with simultaneous DT injection. ( e , e ′, f , f ′) adenovirus Ad. Gfi1.Atoh1 inoculation of Pou4F3 DTR mouse with simultaneous DT injection. Dotted lines in a, b indicate where OHCs would be found in normal mice. In animals receiving only the injection surgery, most outer HCs are damaged and only a few OHCs remain in the most apical region, but inner HCs are largely intact. In contrast, all HCs both outer HCs and inner HCs, are ablated by simultaneous DT injection in both the Ad. Atoh1 group ( c , d ) and the Ad. Gfi1.Atoh1 group ( e , f ). All groups robustly expressed the tdTomato reporter ( a ′– f ′). Myosin VIIa, Myosin VIIa. tdTom, tdTomato. Representative figures from 5 biological replicates for each group. Scale bar = 100 μm.

Article Snippet: Math1 .11D (GenVec, Inc., Gaithersburg, MD, USA), used at a final concentration of 4 × 10 9 PU/ml.

Techniques: Injection