Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Temporal Transcript Profiling Identifies a Role for Unfolded Protein Stress in Human Gut Ischemia-Reperfusion Injury

doi: 10.1016/j.jcmgh.2021.11.001

Figure Lengend Snippet: Inhibition of the unfolded protein response decreases pro-apoptotic UPR signaling. ( A ) Experimental set-up. Organoids were exposed to 12 hours of hypoxia without reoxygenation (0R), 30R, and 120R. ISRIB (0, 10, or 100 nmol/L) was added 2 hours before the start of hypoxia. Control samples were not exposed to hypoxia-reoxygenation (HR) (C). ( B ) Bright field images of human intestinal organoids during hypoxia-reoxygenation (images from the same culture are shown), and in control (magnification, 50×). ( C ) Messenger RNA (mRNA) expression of UPR-related genes CHOP, GADD34, ATF4, XBP1s, BiP, and HSPA1A . n = 3 per organoid line (N = 3). Data are presented as fold change compared with C, and shown in Tukey boxplot ( dots indicate outliers). ( D ) Apoptosis measurement using a caspase 3/7 activity assay (Promega). Results are presented as fold change in luminescence compared with C (Tukey boxplot). n = 4 per organoid line (N = 3). Kruskal–Wallis with the Dunn multiple comparisons test was used to compare HR conditions with C (∗indicates significance), and per time point 10 and 100 nmol/L ISRIB were compared with no ISRIB ( # indicates significance). ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, and ∗∗∗∗ P < .0001, # P < .05, ## P < .01, ### P < .001, and #### P < .0001.

Article Snippet: Data are presented as fold change compared with C, and shown in Tukey boxplot ( dots indicate outliers). ( D ) Apoptosis measurement using a caspase 3/7 activity assay (Promega).

Techniques: Inhibition, Control, Expressing, Activity Assay

Journal: Nature Communications

Article Title: A human tissue map of 5-hydroxymethylcytosines exhibits tissue specificity through gene and enhancer modulation

doi: 10.1038/s41467-020-20001-w

Figure Lengend Snippet: a Schematic plot showing all the organ tissues analyzed in this study. Tumor adjacent tissues are marked in red. b Genome Browser view depicting 5hmC genomic distributions at the HOXA gene cluster for one representative donor’s profile for each of the 19 tissue types assayed. The peak outlined with a box shows a highly variable region as an example. c Metagene plot of 5hmC profiles across different tissues showing underrepresentation of 5hmC at TSS regions and over-representation at gene bodies and promoters. The color ranges indicate RPKM values. Upper panel, normal tissues; lower panel, tumor adjacent tissues. TSS, transcription start site. TES, transcription end site. d Boxplots showing genomic enrichment of 5hmC peaks at RefSeq exons, introns, promoters, and intergenic regions. N = 5 biologically independent samples were used ( n = 4 for hypothalamus and n = 6 for sigmoid and transverse colon). For all boxplots, center line represents median, bounds of box represent 25th and 75th percentiles and whiskers are Tukey whiskers. e Enrichment of 5hmC peaks on 15 chromHMM chromatin states from the Roadmap Epigenomics Project for 8 tissue types. Histone modification emissions data are directly from the Roadmap Epigenomics Project. f Enrichment of trans-acting factors binding sites on 5hmC peaks in different tissues. Higher GIGGLE score means higher possibility of enrichment.

Article Snippet: For all boxplots, center line represents median, bounds of box represent 25th and 75th percentiles and whiskers are Tukey whiskers. e Enrichment of 5hmC peaks on 15 chromHMM chromatin states from the Roadmap Epigenomics Project for 8 tissue types.

Techniques: Modification, Binding Assay

Journal: The EMBO Journal

Article Title: Differentiation signals induce APOBEC3A expression via GRHL3 in squamous epithelia and squamous cell carcinoma

doi: 10.1038/s44318-024-00298-9

Figure Lengend Snippet: ( A ) UMAPs heatmap showing gene expression of APOBEC3A and IVL and predicted activity of GRHL3 in scRNA-seq data from four independent tumour cohorts (three HNSCC and one ESCC). ( B ) qRT-PCR-based gene expression measurements of APOBEC3A and GRHL3 in sub-confluent BICR6 (top row) and BICR22 (bottom row) HNSCC cells transfected with control (NC#1) or GRHL3 -specific siRNAs as indicated. Adjusted P values: NC#1 vs GRHL3 siRNA #1 ( APOBEC3A : BICR6 <0.0001; BICR22 = 0.0145; GRHL3 : BICR6 <0.0001; BICR22 <0.0001); NC#1 vs GRHL3 siRNA #2 ( APOBEC3A : BICR6 <0.0001; BICR22 = 0.0035; GRHL3 : BICR6 <0.0001; BICR22 <0.0001). Gene expression in GRHL3 siRNA-transfected cells was compared with control siRNA-transfected cells using one-way ANOVA with Tukey’s multiple comparisons test ( n = 3; error bars represent SEM; **** P < 0.0001; ** P < 0.01 and * P < 0.05). ( C ) matrix showing the relationship between expression of the indicated genes in spatial transcriptomics data from the Southampton HNSCC cohort obtained using the Visium platform (10X Genomics). ( D ) Images displaying expression levels (Visium spot intensities) of selected genes in HN485, an HPV+ve HNSCC case from the Southampton cohort. ( E ) Immunohistochemistry with an antibody specific for APOBEC3A (left) and with an antibody that cross-reacts with APOBEC3A, APOBEC3B and APOBEC3G (right) in sections from the same tissue block from HN485 used for the Visium profiling displayed in part D. ( F ) Boxplot showing expression of APOBEC3A in those cells predicted to be in S-phase in normal tonsil and HNSCC, stratified by binary GRHL3 activity score (on/off). Cells shown in black are outliers relative to the distribution of expression in the cells from healthy tonsils. Inset: UMAP showing the predicted cell cycle phase for each cell in the Southampton HNSCC scRNA-seq dataset. The top, middle and bottom lines of the boxplot represent the upper quartile (Q3), median, and lower quartile (Q1), respectively. The maximum value represented by the top whisker represents the highest observed data point within Q3 + (1.5 × (Q3 − Q1)), and the minimum value represented by the bottom whisker represents the lowest situated point within Q1 − (1.5 × (Q3 − Q1)). Healthy GRHL3 ‘OFF’, n = 19; Healthy GRHL3 ‘ON’, n = 173; Tumour GRHL3 ‘OFF’, n = 98; Tumour GRHL3 ‘ON’, n = 620. .

Article Snippet: Gene expression in GRHL3 siRNA-transfected cells was compared with control siRNA-transfected cells using one-way ANOVA with Tukey’s multiple comparisons test ( n = 3; error bars represent SEM; **** P < 0.0001; ** P < 0.01 and * P < 0.05). ( C ) matrix showing the relationship between expression of the indicated genes in spatial transcriptomics data from the Southampton HNSCC cohort obtained using the Visium platform (10X Genomics). ( D ) Images displaying expression levels (Visium spot intensities) of selected genes in HN485, an HPV+ve HNSCC case from the Southampton cohort. ( E ) Immunohistochemistry with an antibody specific for APOBEC3A (left) and with an antibody that cross-reacts with APOBEC3A, APOBEC3B and APOBEC3G (right) in sections from the same tissue block from HN485 used for the Visium profiling displayed in part D. ( F ) Boxplot showing expression of APOBEC3A in those cells predicted to be in S-phase in normal tonsil and HNSCC, stratified by binary GRHL3 activity score (on/off).

Techniques: Gene Expression, Activity Assay, Quantitative RT-PCR, Transfection, Control, Expressing, Immunohistochemistry, Blocking Assay, Whisker Assay