Journal: Bioengineered

Article Title: Small activating RNA activation of ATOH1 promotes regeneration of human inner ear hair cells.

doi: 10.1080/21655979.2022.2045835

Figure Lengend Snippet: Figure 2. Screening of ATOH1 small activating RNAs in 293 T cells and mesenchymal stem cells. Cells were transfected with the indicated small activating RNAs using RNAiMAX at the indicated concentrations for 72 h. Mock samples were transfected in the absence of duplex RNA. Plasmid DNA was transfected at 1 µg/µl using Lipofectamine 3000. The transfected cells were subjected to RT–qPCR after RNA isolation, an RT reaction to generate cDNA and a Western blotting assay after protein isolation. A and B. Relative mRNA expression levels of the ATOH1 gene in 293 T cells (a) and mesenchymal stem cells (b) 72 h after transfection. C and D. ATOH1 protein levels assessed by Western blotting in 293 T cells (c) and mesenchymal stem cells (d) at 72 h after transfection. E and F. Relative ATOH1 mRNA expression levels in response to different small activating RNA concentrations.

Article Snippet: Induction of hair cell-like cells The induced hair cell progenitor cells were maintained in 6-well plates (2 × 105 cells per well) and in a mixed medium consisting of serum-free DMEM/F12 and N2/B27 and were transfected with ATOH1 small activating RNAs using RNAiMAX or an ATOH1 plasmid (ORIGENE, #RC210192) using Lipofectamine 3000 for 10 days.

Techniques: Transfection, Plasmid Preparation, Quantitative RT-PCR, Isolation, Western Blot, 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: 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: 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

Journal: Cell Death and Differentiation

Article Title: Trim32 suppresses cerebellar development and tumorigenesis by degrading Gli1/sonic hedgehog signaling

doi: 10.1038/s41418-019-0415-5

Figure Lengend Snippet: Trim32 is selectively expressed in inner EGL and displays an uneven cytoplasmic distribution during GNP differentiation. a RT-qPCR analysis of Trim32 in P0, P7, P14, and adult mouse cerebella. Data are expressed as means ± SD (n = 3). An asterisk indicates P < 0.05 and triple asterisks indicate P < 0.001. b Immunoblotting analysis of Trim32, Gli1, and MycN in P0, P7, P14, and adult mouse cerebellums. c Immunofluorescence staining of Trim32 (red) in the EGL of P7 Math1-GFP transgenic mouse cerebellum. Nuclei were counterstained with DAPI (blue). EGL external granule layer, ML molecular layer, PCL Purkinje cells layer, and IGL internal granule layer. The scale bars represent 100 µm in the first panel and 25 µm in the second panel. d Confocal analysis (left) and quantification of immunofluorescence intensities (right) of distribution of Trim32 (green) in the dividing GNPs in different phases of the cell cycle. The dashed line highlights the cells that are in the indicated phase of cell cycle. The cell-cycle phases were identified by PH3 staining (red) for DNA. Nuclei were counterstained with DAPI (blue). The scale bars represent 10 µm. Data are expressed as means ± SD (n = 3). ns indicates P > 0.05 and triple asterisks indicate P < 0.001

Article Snippet: For protein detection, the following antibodies were used: Trim32 (1:1000, GeneTex, #GTX113,937), Gli1 (1:1000, CST, #2643), Ccnd2 (1:1000, CST, #3741), Math1(1:1000, GeneTex, #GTX111,898), NeuN (1:1000, CST, #12,943), Ubiquitin (1:1000, CST, #3936), and actin (1:10,000, CST, #4970).

Techniques: Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Transgenic Assay

Journal: Cell Death and Differentiation

Article Title: Trim32 suppresses cerebellar development and tumorigenesis by degrading Gli1/sonic hedgehog signaling

doi: 10.1038/s41418-019-0415-5

Figure Lengend Snippet: Trim32 knockout enhances GNP proliferation in the postnatal developing cerebellum. a Expression of the Math1-GFP protein (GFP, green) in P7 mouse cerebellar sections from the Math1-GFP/Trim32wt mice and Math1-GFP/Trim32KO mice. Nuclei were counterstained with DAPI (blue). Graph in a representing Math1 positive cells normalized to the length of the EGL edge. The scale bar represents 25 µm. Immunofluorescence staining of NeuN (red, b) and Ki67 (red, c) in P7 mouse cerebellar sections from the Math1-GFP/Trim32wt mice and Math1-GFP/Trim32KO mice. Nuclei were counterstained with DAPI (blue). Graph in b and c respectively representing NeuN or Ki67-positive cells normalized to the length of the EGL edge. The scale bar in b represents 50 µm. The scale bar in c represents 25 µm. oEGL outer external granule layer, iEGL inner external granule layer, ML molecular layer, and IGL internal granule layer. d P7 and P18 cerebellar midsagittal sections were stained for DAPI to show the overall morphology of cerebellum in Trim32wt mice and Trim32ko mice. The scale bar represents 500 µm

Article Snippet: For protein detection, the following antibodies were used: Trim32 (1:1000, GeneTex, #GTX113,937), Gli1 (1:1000, CST, #2643), Ccnd2 (1:1000, CST, #3741), Math1(1:1000, GeneTex, #GTX111,898), NeuN (1:1000, CST, #12,943), Ubiquitin (1:1000, CST, #3936), and actin (1:10,000, CST, #4970).

Techniques: Knock-Out, Expressing, Immunofluorescence, Staining

Journal: Cell Death and Differentiation

Article Title: Trim32 suppresses cerebellar development and tumorigenesis by degrading Gli1/sonic hedgehog signaling

doi: 10.1038/s41418-019-0415-5

Figure Lengend Snippet: Trim32 knockout increases the incidence of MB in Ptch1+/− mice. Quantitative PCR mRNA (a; mean ± SD; n = 9) and protein levels (b; n = 3) of Trim32 in mouse medulloblastomas (MB) from Ptch1+/− mice and normal cerebella. c RNA expression level of Trim32 negatively correlated with Gli1 (n = 51) in human SHH MB samples. Data are analyzed from Oncomine database. d Kaplan–Meier analysis of MB incidence in 63 Ptch1+/−/Trim32wt mice (gray line) versus 17 Ptch1+/−/Trim32KO mice (black line). Ptch1+/−/Trim32wt mice and Ptch1+/−/Trim32KO mice, obtained by interbreeding for at least three generations the progeny of Ptch1 heterozygous and Trim32 knock-out mice, were then monitored for the onset of medulloblastoma; (triple asterisks indicate P < 0.001, Logrank test). e Expression of the Math1-GFP protein (GFP, green) in the Math1-GFP mouse medulloblastomas and adjacent cerebellar cortices sections from Ptch1+/−/Trim32wt mice and Ptch1+/−/Trim32KO mice. Nuclei were counterstained with DAPI (blue). The scale bar represents 200 µm. MB medulloblastoma, IGL internal granule layer. RT-qPCR analysis of SHH target genes (f), Trim32 (g), the granule neuronal progenitor marker Maht1 (h), and the differentiated granule cell markers including Tuj1 and NeuN (i) in adult mouse cerebellums and medulloblastomas from Ptch1+/−/Trim32wt mice and Ptch1+/−/Trim32KO mice. Data are expressed as means ± SD (n = 3). An asterisk indicates P < 0.05, double asterisks indicate P < 0.01, and triple asterisks indicate P < 0.001. j Immunoblotting analysis of Trim32, Gli1, Ccnd2, MycN, and NeuN in MB from Ptch1+/−/Trim32wt mice and Ptch1+/−/Trim32KO mice

Article Snippet: For protein detection, the following antibodies were used: Trim32 (1:1000, GeneTex, #GTX113,937), Gli1 (1:1000, CST, #2643), Ccnd2 (1:1000, CST, #3741), Math1(1:1000, GeneTex, #GTX111,898), NeuN (1:1000, CST, #12,943), Ubiquitin (1:1000, CST, #3936), and actin (1:10,000, CST, #4970).

Techniques: Knock-Out, Real-time Polymerase Chain Reaction, RNA Expression, Expressing, Quantitative RT-PCR, Marker, Western Blot

Journal: Cell Death and Differentiation

Article Title: Trim32 suppresses cerebellar development and tumorigenesis by degrading Gli1/sonic hedgehog signaling

doi: 10.1038/s41418-019-0415-5

Figure Lengend Snippet: mRNA expression analysis

Article Snippet: For protein detection, the following antibodies were used: Trim32 (1:1000, GeneTex, #GTX113,937), Gli1 (1:1000, CST, #2643), Ccnd2 (1:1000, CST, #3741), Math1(1:1000, GeneTex, #GTX111,898), NeuN (1:1000, CST, #12,943), Ubiquitin (1:1000, CST, #3936), and actin (1:10,000, CST, #4970).

Techniques: Expressing