Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: ATF2 and ATF7 Are Critical Mediators of Intestinal Epithelial Repair

doi: 10.1016/j.jcmgh.2020.01.005

Figure Lengend Snippet: ATF2 and ATF7 are required to prevent apoptosis and sustain goblet cell differentiation after DSS-induced damage. The experiment was performed as described in Figure 4 A . ( A ) Representative images of immunohistochemistry for BrdU in colons of DSS-treated mutant and control animals. Scale bar : 750 μm. ( B ) Quantification of BrdU-positive cells in intact colonic crypts. ( C ) qRT-PCR of the isolated epithelial fraction for epithelial stem cell markers Cd44 , Lgr5 , and Ascl2 . ( D ) Representative images of colonic RNA scope in situ hybridization for Lgr5 messenger RNA in homeostasis (as described in Figure 1 A ) and on exposure to 2% DSS. ( E ) quantification of Lgr5 messenger RNA particles per colonic Lgr5-positive stem cell ( left panel ) and quantification of Lgr5 -positive cells per colonic crypt ( right panel ). Mice were analyzed for homeostasis: Atf2 wt/wt Atf7 +/+ (n = 8) and Atf2 -/- Atf7 ko/ko (n = 8), and for 2% DSS: Atf2 wt/wt Atf7 +/+ (n = 4) and Atf2 -/- Atf7 ko/ko (n = 4). ( F ) Representative immunohistochemistry image of colonic periodic acid–Schiff (PAS) staining. ( G ) Relative abundance of PAS-positive cells per crypt epithelial cells. ( H ) qRT-PCR for secretory epithelial markers in isolated colonic epithelial fraction. ( I ) TUNEL staining (green) with 4′,6-diamidino-2-phenylindole counterstain (blue). Upper panels : colons from mice not treated with DSS (n = 5 per group). Lower panels : colons from mice treated with DSS for 7 days (n = 6 mice per group). Scale bar : 600 μm. ( J ) Quantification of TUNEL-positive cells per microscopic field (5 random fields per mouse). Mice were analyzed for homeostasis: Atf2 wt/wt Atf7 +/+ (n = 5) and Atf2 -/- Atf7 ko/ko (n = 5), and for 2% DSS: Atf2 wt/wt Atf7 +/+ (n = 6) and Atf2 -/- Atf7 ko/ko (n = 6). ( B , C , G , and H ) n = 12 mice per group. Graph bars show means and SEM. * P < .05, ** P < .01, *** P < .001, Student t test for panels B , C , G , and H , 1-way analysis of variance test for panels E and J followed by the Bonferroni test for selected groups. Rel., relative.

Article Snippet: The single-plex RNAscope probes against mouse Lgr5 (312171; Advanced Cell Diagnostics) were used.

Techniques: Cell Differentiation, Immunohistochemistry, Mutagenesis, Control, Quantitative RT-PCR, Isolation, RNAscope, In Situ Hybridization, Staining, TUNEL Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: ATF2 and ATF7 Are Critical Mediators of Intestinal Epithelial Repair

doi: 10.1016/j.jcmgh.2020.01.005

Figure Lengend Snippet: ATF2 and ATF7 are required to protect epithelial cells against TNF-induced death in organoid culture. Colonic epithelium was isolated from Atf2 wt/wt Atf7 +/+ and Atf2 -/- Atf7 ko/ko mice and cultured as organoids (n = 3, independent organoid cultures). ( A ) qRT-PCR for the floxed region of Atf2 and Atf7 in colonic organoids. ( B ) Representative brightfield images of colonic organoids 72 hours after reseeding. ( C ) Quantification of organoid area and perimeter. ( D ) EdU incorporation assay using flow cytometry 72 hours after reseeding single cells (n = 3 for 2 independent experiments). ( E ) qRT-PCR for stem cell markers Lgr5 and Olfm4 . ( F ) PI viability assay on colon organoids challenged with TNF-α for 12 hours. Scale bar : 250 μm. Red fluorescence indicates cell death. ( G ) Percentage of nonviable organoids quantified per well (n = 2). Graph bars show means and SEM, ∗∗ P < .01, ∗∗∗ P < .001, Student t test.

Article Snippet: The single-plex RNAscope probes against mouse Lgr5 (312171; Advanced Cell Diagnostics) were used.

Techniques: Isolation, Cell Culture, Quantitative RT-PCR, Flow Cytometry, Viability Assay, Fluorescence

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: ATF2 and ATF7 Are Critical Mediators of Intestinal Epithelial Repair

doi: 10.1016/j.jcmgh.2020.01.005

Figure Lengend Snippet: Primers Used for qRT-PCR Experiments

Article Snippet: The single-plex RNAscope probes against mouse Lgr5 (312171; Advanced Cell Diagnostics) were used.

Techniques:

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Glycolytic Regulation of Intestinal Stem Cell Self-Renewal and Differentiation

doi: 10.1016/j.jcmgh.2022.12.012

Figure Lengend Snippet: HK2 is a target gene of Wnt signaling in intestinal cells. ( A ) Expression of HKs was analyzed by scRNA sequencing. “pct.1” is the percentage of HK2 expressing cells in the selected cell type (eg, Goblet). The “pct.2” is the percentage of HK2 expressing cells in the rest of cells (eg, all non-Goblet cells). EI, enterocyte immature; EM, enterocyte mature; EP, enterocyte progenitor. TA, transit amplifying; G1, G1/S cell-cycle phase; G2, G2/M cell-cycle phase. ( B ) RNAscope brown staining of HK2 in intestinal crypts from WT mice. ( C ) HK2 is mainly expressed in LGR5 -positive cells but not in Paneth cells. RNA scope duplex staining of HK2 ( brown ) with LGR5 ( red ) in WT jejunum crypts. ( D ) HK2 is more highly expressed in GFP high cells. Small intestine crypts from the LGR5-EGFP-IRES-creERT2 mice were harvested and digested with Typsin LE for 30 minutes to obtain single cells. GFP high and GFP low cells were isolated by fluorescence-activated cell sorting. Expression of ASCl2 , LGR5 , and HK2 was determined by qPCR. Data represent mean ± SD (n = 4 mice). ( E and F ) Small intestinal crypts from jejunum of WT mice were harvested and seeded in 24-well plates. Organoids were treated with either vehicle control (DMSO) or Wnt agonist CHIR99021 ( D ) or Wnt antagonist XAV939 ( E ) for 3 days. Total RNA was extracted and expression of HK2 and AXIN2 was analyzed by qPCR (n = 5 mice). ( G ) Small intestinal crypts from APC f/f and APC f/f ; Villin-creERT2 mice were harvested and seeded into 24-well plates and cultured for 24 hours followed by treatment with 4-OHT (1 mM) for 24 hours to induce knockout of APC expression. Four days after removing 4-OHT, total RNA was extracted and expression of HK2 and AXIN2 was determined by qPCR (n = 3 mice). ( H ) Western blotting analysis of protein expression in WT and APC f/+ ; Villin-cre mice. Intestinal mucosa from 3-month-old mice was collected for analysis. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .005.

Article Snippet: ISH was carried out on the Ventana Discovery Ultra platform, using RNAScope VS Universal HRP (for single-plex) (Advanced Cell Diagnostics) or RNAScope VS Duplex Reagent (dual stain) (Advanced Cell Diagnostics) with RNAScope probes directed against Ms-HK2 and Ms-LGR5 (Advanced Cell Diagnostics).

Techniques: Expressing, Sequencing, RNAscope, Staining, Isolation, Fluorescence, FACS, Control, Cell Culture, Knock-Out, Western Blot

Journal: Nucleic Acids Research

Article Title: dotdotdot : an automated approach to quantify multiplex single molecule fluorescent in situ hybridization (smFISH) images in complex tissues

doi: 10.1093/nar/gkaa312

Figure Lengend Snippet: Experimental workflows and imaging protocols for smFISH in mouse and human tissues. ( A ) Brains were extracted from wild-type (WT) mice 90 min following electroconvulsive seizures (ECS) or Sham treatment and sectioned on a cryostat. Gene targets were visualized with the RNAscope Multiplex Fluorescence V1 kit. RNAscope technology uses hybridization of two independent probes (double Z probes), referred to as a ‘ZZ pair,’ that must bind to the target sequence in tandem for signal amplification to proceed via the subsequent binding of preamplifiers, amplifiers, and fluorescent detection molecules. Approximately 5–30 ZZ pairs are designed for each target gene. After completion of the RNAscope V1 assay, slides are imaged in x , y and z -dimensions using confocal microscopy. ( B ) Fresh frozen post-mortem human tissue was sectioned on a cryostat and gene targets were visualized using the RNAscope Multiplex Fluorescence V2 kit. The V2 assay uses the same RNAscope technology with added TSA technology for customization of dyes/concentrations and the ability to include a fourth gene target. The V2 assay is also better suited for tissues with autofluorescence, such as post-mortem human brain tissue, which contains an abundance of highly autofluorescent lipofuscin granules. Multispectral imaging and linear unmixing were used to separate individual probe signals and lipofuscin autofluorescence. Lipofuscin signals served as a mask during downstream analysis to exclude pixels confounded by autofluorescence.

Article Snippet: Finally, as chemistry for higher order multiplexing is rapidly coming online, we evaluated the performance of dotdotdot on higher order multiplexed images acquired using the RNAscope Hi-plex assay, which visualizes up to 12 gene targets in a single tissue slice with several rounds of hybridization and stripping ( ). dotdotdot faithfully segments fluorescent signals from 12 positive control probes against different 'house-keeping' genes in the same brain section of mouse tissue (image courtesy of Advanced Cell Diagnostics).

Techniques: Imaging, RNAscope, Multiplex Assay, Fluorescence, Hybridization, Sequencing, Amplification, Binding Assay, Confocal Microscopy

Journal: bioRxiv

Article Title: Benchmark of cellular deconvolution methods using a multi-assay reference dataset from postmortem human prefrontal cortex

doi: 10.1101/2024.02.09.579665

Figure Lengend Snippet: ( A ) Schematic of DLPFC dissections and DLPFC tissue block position depicting order of assays completed (n=10 donors; n=22 tissue blocks, including 7 Anterior, 9 Middle, 6 Posterior). Approximately 50 µm of tissue was trimmed to achieve a flat surface for cryosectioning. Next, several ∼10 µm sections were collected for anatomical validation (RNAScope/IF, H&E) and Visium experiments . Blocks were stored at −80℃ until completion of these assays. At the next cryostat session, blocks were trimmed and ∼1 mm of tissue was collected for snRNA-seq (n=19) , ∼600 µm of tissue was collected for total RNA extraction for bulk-RNAseq, and ∼600 µm of tissue was collected for fractionated RNA extraction for nuclear and cytoplasmic RNA-seq. Finally, four tissue blocks were placed back on the cryostat and trimmed again to obtain a flat surface prior to collecting a ∼10 µm Visium-spatial proteogenomics (SPG) tissue section . ( B ) Tile plot illustrating which assays and configurations (probe/antibody combination for RNAScope/IF and library type/RNA extraction for RNA-seq) were performed on each tissue block and the sample size for that assay before and after quality control (qc) in the format “(n=before:after)”. The tile is blank if an assay configuration was not performed on the tissue block. The tile is blue if the sample passed qc checks and was included in the analysis. Red tiles are not included in the study.

Article Snippet: To quantify six broad cell types across tissue sections (n=16 sections), multiplex single molecule fluorescent in situ hybridization (smFISH) with RNAScope technology (Advanced Cell Diagnostics) was performed in combination with immunofluorescence (IF) using the RNAScope Fluorescent Multiplex Kit v.2, 4-plex Ancillary Kit, and RNA-Protein co-detection ancillary kit (Advanced Cell Diagnostics ACD, Cat No. 323100, 323120, and 323180, Newark, CA.

Techniques: Blocking Assay, RNA Extraction, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Benchmark of cellular deconvolution methods using a multi-assay reference dataset from postmortem human prefrontal cortex

doi: 10.1101/2024.02.09.579665

Figure Lengend Snippet: A. Schematic of experimental design for combined single molecule fluorescent in situ hybridization (smFISH) with immunolabeling using RNAScope/immunofluorescence (IF). The “Star” combination of probes/antibodies marked Excitatory Neurons [Excit], Microglia [Micro], and Oligodendrocyte [Oligo] cell types in 13 tissue blocks. The “Circle” combination marked Astrocytes [Astro], Endothelial cells [Endo], and Inhibitory Neuron [Inhib] cell types in 12 tissue blocks. The total RNA expression gene (TREG) , AKT3 , was also included in each combination to estimate RNA abundance. B. Representative fluorescent image of a single field for the “Star” combination showing expression of SLC17A7 , TMEM119, OLIG2 , AKT3 and the nuclear marker DAPI. C. Representative fluorescent image of a single field for the “Circle” combination showing expression of GFAP, CLDN5, and GAD1 . SLC17A7 , GAD1 , and AKT3 are labeled with RNA probes while TMEM119, OLIG2, GFAP, CLDN5 with antibodies. D. Barplots of estimated cell type proportions from RNAScope/IF data for “Circle” and “Star” experiments. E. Scatter plots of cell type proportions estimated from snRNA-seq data (x-axis) vs. RNAScope/IF, annotated with the Pearson correlation (cor), root mean squared error (rmse), and relative rmse (rrmse) against the mean RNAScope/IF proportions.

Article Snippet: To quantify six broad cell types across tissue sections (n=16 sections), multiplex single molecule fluorescent in situ hybridization (smFISH) with RNAScope technology (Advanced Cell Diagnostics) was performed in combination with immunofluorescence (IF) using the RNAScope Fluorescent Multiplex Kit v.2, 4-plex Ancillary Kit, and RNA-Protein co-detection ancillary kit (Advanced Cell Diagnostics ACD, Cat No. 323100, 323120, and 323180, Newark, CA.

Techniques: In Situ Hybridization, Immunolabeling, Immunofluorescence, Inhibition, RNA Expression, Expressing, Marker, Labeling

Journal: bioRxiv

Article Title: Benchmark of cellular deconvolution methods using a multi-assay reference dataset from postmortem human prefrontal cortex

doi: 10.1101/2024.02.09.579665

Figure Lengend Snippet: A. Scatter plot of cell type proportions estimated by RNAScope/IF (x-axis) vs. the predicted cell type proportions by the deconvolution methods. Points are colored by the cell type and shaped by the combination of bulk RNA-seq RNA extraction method and library type. The annotation lists the overall Pearson’s correlation (cor) and root mean squared error (rmse). B. Correlation (color) between the predicted proportions by deconvolution methods and the estimated RNAScope/IF proportions across RNA extraction method and library type combinations. The 1/rmse values are shown by dot size, where rmse values of 0.25, 0.2, 0.167, and 0.143 respectively correspond to 1/rmse of 4, 5, 6, and 7.

Article Snippet: To quantify six broad cell types across tissue sections (n=16 sections), multiplex single molecule fluorescent in situ hybridization (smFISH) with RNAScope technology (Advanced Cell Diagnostics) was performed in combination with immunofluorescence (IF) using the RNAScope Fluorescent Multiplex Kit v.2, 4-plex Ancillary Kit, and RNA-Protein co-detection ancillary kit (Advanced Cell Diagnostics ACD, Cat No. 323100, 323120, and 323180, Newark, CA.

Techniques: RNA Sequencing Assay, RNA Extraction