Journal: iScience

Article Title: Epitranscriptomic reader YTHDF2 regulates SEK1( MAP2K4 )-JNK-cJUN inflammatory signaling in astrocytes during neurotoxic stress

doi: 10.1016/j.isci.2024.110619

Figure Lengend Snippet: YTHDF2 is decreased in Mn-gavaged mice, and a conditional knockout of YTHDF2 in the astrocytes of mice leads to increased astrocyte reactivity in substantia nigra pars compacta/reticulata and globus pallidus (A) IHC representation at 40x depicting YTHDF2 decreases and colocalization in GFAP+ cells in the globus pallidus of Mn-gavaged mice (100 μm scale). (B) Immunoblotting of the substantia nigra showing decreases of YTHDF2 in mice gavaged with Mn ( n = 9). (C) Schematic of Y2cKO mice generation. Tamoxifen (tiY2cKO) induces Cre recombination of YTHDF2 exon 4 to produce a band at 748 bp in astrocytes isolated from striatal/hippocampal brain tissue using biotinylated-EAAT1/GLAST-1 antibody. (D) Globus pallidus validation of YTHDF2 protein reduction and increased GFAP immunoreactivity, and also increased m6A staining upon YTHDF2 deletion. YTHDF2 was quantified by parent-child extraction using GFAP as the parent (n = 3–4) (100 μm scale). (E) Globus pallidus representative images of increased C3d immunoreactivity in GFAP-positive cells in the tiY2cKO mice (100 μm scale). (F) Immunoblotting and quantification of substantia nigra- showing tiY2cKO mice with increased GFAP immunoreactivity similar to Mn-gavaged mice, indicating specific loss of YTHDF2 in astrocytes increases their reactivity (n = 4–6). (G) Substantia nigra representative images of increased C3d immunoreactivity in GFAP-positive cells in the tiY2cKO mice (100 μm scale). Data are means ± SEM. Student’s t test or two-way ANOVA with FDR Two-stage step-up method of Benjamini, Krieger, and Yekutieli for multi-group comparison. Q-values < 0.05 considered significant evidence, with q-values between 0.05 and 0.01 considered as weaker evidence taking into consideration variations in data and trends.

Article Snippet: Biotinylated EAAT1/GLAST-1 , Novus Biologicals , Cat# NB100-1869B; RRID: AB_3150018.

Techniques: Knock-Out, Western Blot, Isolation, Staining, Extraction, Comparison

Journal: iScience

Article Title: Epitranscriptomic reader YTHDF2 regulates SEK1( MAP2K4 )-JNK-cJUN inflammatory signaling in astrocytes during neurotoxic stress

doi: 10.1016/j.isci.2024.110619

Figure Lengend Snippet:

Article Snippet: Biotinylated EAAT1/GLAST-1 , Novus Biologicals , Cat# NB100-1869B; RRID: AB_3150018.

Techniques: Recombinant, Extraction, Selection, Negative Control, Software

Journal: Nature Communications

Article Title: Pre-ciliated tubal epithelial cells are prone to initiation of high-grade serous ovarian carcinoma

doi: 10.1038/s41467-024-52984-1

Figure Lengend Snippet: a Distal epithelial cells ( n = 6273) from 62 uterine tubes were identified by their Epcam and Krt8 expression and the subset was represented within the UMAP embedding. b A differentiation trajectory among epithelial cells visualized through the PHATE dimensional reduction technique. c Dot plot reflecting highly expressed, specific markers of each identified epithelial cell cluster. d Monocle3 pseudotime analyses calculated over the PHATE embedding. The expression of Slc1a3 ( e ) and Pax8 ( f ) visualized over the PHATE embedding. Source data are provided as a file.

Article Snippet: Positive selection was then performed on the unbound fraction of cells by incubating with a biotinylated antibody against SLC1A3 (Novus biologicals, NB100-1869B, 0.5 μg per million cells).

Techniques: Expressing

Journal: Nature Communications

Article Title: Pre-ciliated tubal epithelial cells are prone to initiation of high-grade serous ovarian carcinoma

doi: 10.1038/s41467-024-52984-1

Figure Lengend Snippet: a Experimental design. Mice containing Slc1a3-CreERT and Ai9 reporter are injected with tamoxifen to induce expression of modified red fluorescent protein (tdTomato) in cells expressing Slc1a3 . b Top row, tdTomato expression (red) 1, 30, and 360 days post induction (DPI) with tamoxifen. Lower rows, tdTomato+ progeny of Slc1a3 + cells express differentiation markers for stem (SLC1A3, orange, arrows), ciliated (FAM183B, orange, arrows), and secretory (OVGP1, orange, arrows) cells. Counterstaining with DAPI (blue). Scale bar represents 50 μm (tdTomato, FAM1A3/tdTomato, OVGP1/tdTomato rows), and 25 μm (SLC1A3/tdTomato row). c Quantification of cells expressing tdTomato in the distal and proximal regions of the uterine tube 1, 30, and 360 DPI. Quantification of cells expressing tdTomato with stem (SLC1A3, d ), ciliated (FAM183B, e ), or secretory (OVGP1, f ) cells 1, 30, and 360 DPI. c – f Two way ANOVA with the Tukey’s multiple comparison post hoc test for all distal regions. c *** P = 0.0003, **** P < 0.0001 ( d ) **** P < 0.0001 ( e ) **** P < 0.0001 ( f ) * P = 0.0146, ** P = 0.0058, **** P < 0.0001. Data are presented as mean values ± SD. Biological replicates (mice, n ): c Distal 1 DPI n = 6, 30 DPI n = 4, 360 DPI n = 3; Proximal 1 DPI n = 4, 30, and 360 DPI n = 3 each group. d Distal and Proximal n = 3 each group. e Distal 1 DPI n = 6, 30 DPI and 360 DPI n = 4 each group; Proximal 1 DPI n = 4, 30, and 360 DPI n = 3 each group. f Distal 1 DPI n = 6, 30, and 360 DPI n = 4 each group; Proximal n = 3, each group. Source data are provided as a file.

Article Snippet: Positive selection was then performed on the unbound fraction of cells by incubating with a biotinylated antibody against SLC1A3 (Novus biologicals, NB100-1869B, 0.5 μg per million cells).

Techniques: Injection, Expressing, Modification, Comparison

Journal: Nature Communications

Article Title: Pre-ciliated tubal epithelial cells are prone to initiation of high-grade serous ovarian carcinoma

doi: 10.1038/s41467-024-52984-1

Figure Lengend Snippet: a Organoid formation rate of distal tubal epithelial cells isolated for SLC1A3 expression by MACS ( n = 8). b Organoid sections of day 14 SLC1A3+ and SLC1A3- cell derived organoids stained for secretory marker OVGP1 (red, arrows). Counterstaining with DAPI (blue). c Hematoxylin and Eosin (HE) staining of SLC1A3+ and SLC1A3- cell derived organoids after 14 days of culture. Arrow denotes cilia. d Representative images of ciliation (green, acetylated α-Tubulin) between SLC1A3+ and SLC1A3- cell derived organoids. Counterstaining with DAPI (blue). b – d Scale bar all images 200 μm. e Quantification of ciliated cells between SLC1A3+ ( n = 71) and SLC1A3- ( n = 40) cell derived organoids. a , e *** P = 0.0002, **** P = 0.0005, two-tailed unpaired t -tests. Data are presented as mean values ± SD. Source data are provided as a file.

Article Snippet: Positive selection was then performed on the unbound fraction of cells by incubating with a biotinylated antibody against SLC1A3 (Novus biologicals, NB100-1869B, 0.5 μg per million cells).

Techniques: Isolation, Expressing, Derivative Assay, Staining, Marker, Two Tailed Test

Journal: Nature Communications

Article Title: Pre-ciliated tubal epithelial cells are prone to initiation of high-grade serous ovarian carcinoma

doi: 10.1038/s41467-024-52984-1

Figure Lengend Snippet: a tdTomato+ tubal epithelial cells in mice containing Slc1a3-CreERT with floxed Trp53 and Rb1 genes, and an Ai9 reporter 1 and 360 days post induction (DPI) with tamoxifen. Hematoxylin and Eosin (HE) staining (left column) and immunostaining for tdTomato (arrows, brown color), Elite ABC method, hematoxylin counterstaining (right column). Scale bar, all images 200 μm. b PCR analysis of Trp53 and Rb1 gene structure in the same samples of microdissected cells from the tubal epithelium (TE, lanes 5–9) and lung neoplasm (LT, lane 4) of Slc1a3-CreERT Trp53 loxP/loxP Rb1 loxP/loxP Ai9 mice collected 1 year after tamoxifen induction. Samples with known gene structure (wild-type, WT, lane 1, floxed gene, L, lane 2, and recombinant gene, R, lane 3). 316-, 198-, and 163-bp fragments are diagnostic for floxed, excised, and wild-type alleles of the Trp53 gene, respectively. 295-, 269, and 247-bp fragments are diagnostic for floxed, excised, and wild-type alleles of the Rb1 gene, respectively. B, blank control (lane 10), M, DNA marker (Lane 11). Representative of 5 microdissection-PCR experiments. c Apoptotic cells (arrows) in the tubal epithelium 1 day after tamoxifen induction of Cre-mediated inactivation of Trp53 and Rb1 in Slc1a3-CreERT Trp53 loxP/loxP Rb1 loxP/loxP Ai9 mice (MUT TAM) and littermates without Slc1a3-CreERT (WT TAM). Dot-line rectangle indicates respective location of cells shown in the inset in the top image. TUNEL assay, methyl green counterstaining. Scale bar, 50 μm and 21 µm, inset. d Quantification of apoptotic cells 1 day after administration of tamoxifen (TAM+) or vehicle (TAM-) in Slc1a3-CreERT Trp53 loxP/loxP Rb1 loxP/loxP Ai9 mice (DM), Slc1a3-CreERT Trp53 loxP/loxP Ai9 mice (p53) or littermates without Slc1a3-CreERT (WT). d One way ANOVA with the Tukey’s multiple comparison post hoc test (DM TAM+ vs. DM TAM-) * P = 0.0192, (DM TAM+ vs. WT TAM+) * P = 0.0123, (p53 TAM+ vs. DM TAM-) ** P = 0.0035, (p53 TAM+ vs. WT TAM+) ** P = 0.0022. Data are presented as mean values ± SD. Biological replicates (mice, n ): DM TAM+ n = 3, p53M TAM+ n = 5, DM TAM- n = 4, WT TAM+ n = 4. Source data are provided as a file.

Article Snippet: Positive selection was then performed on the unbound fraction of cells by incubating with a biotinylated antibody against SLC1A3 (Novus biologicals, NB100-1869B, 0.5 μg per million cells).

Techniques: Staining, Immunostaining, Recombinant, Diagnostic Assay, Control, Marker, Laser Capture Microdissection, TUNEL Assay, Comparison

Journal: Nature Communications

Article Title: Pre-ciliated tubal epithelial cells are prone to initiation of high-grade serous ovarian carcinoma

doi: 10.1038/s41467-024-52984-1

Figure Lengend Snippet: Early ( a ) and advanced ( b ) neoplastic lesions (arrows) in Pax8-rtTA Tre-Cre Trp53 loxP/loxP Rb1 loxP/loxP Ai9 mice. Hematoxylin and Eosin (HE) staining (left column) and immunostaining for tdTomato (middle column, brown color), and PAX8 (right column, brown color). Elite ABC method, hematoxylin counterstaining. a , b Scale bar, all images 60 µm. Biological replicates n = 6 ( a ) and n = 3 ( b ). c Pseudotime binning along the PHATE embedding to visualize how bins are assigned for 6,273 distal epithelial cells from 62 uterine tubes. d Inferred pseudotime trajectories of secretory and ciliated epithelial cell lineages. The lineages extend from S1 and C1 to S20 and C20 respectively, where S20 and C20 are presumed to be a more differentiated cell state. The percent abundance of each cell type contributing to each pseudotime bin is reflected in black. The average z-scored expression was calculated for each gene to identify genes that best represent smaller transitional states within each lineage. Each pseudotime bin is equally sized and consists of about 150 cells. e Log-normalized expression of Slc1a3, Pax8, Trp53 and Prom1 visualized in a violin plot of epithelial cell clusters. f Dot plot of Krt5 expression among epithelial cell populations. Source data are provided as a file.

Article Snippet: Positive selection was then performed on the unbound fraction of cells by incubating with a biotinylated antibody against SLC1A3 (Novus biologicals, NB100-1869B, 0.5 μg per million cells).

Techniques: Staining, Immunostaining, Expressing

Journal: Nature Communications

Article Title: Pre-ciliated tubal epithelial cells are prone to initiation of high-grade serous ovarian carcinoma

doi: 10.1038/s41467-024-52984-1

Figure Lengend Snippet: The hierarchy begins with Slc1a3 + stem/progenitor cells giving rise to secretory and ciliated cell lineages. Slc1a3 + stem/progenitor cells undergo apoptosis after inactivation of Trp53 alone or together with Rb1 , while inactivation of Trp53 and Rb1 in Krt5 + pre-ciliated transitional cells lead to high-grade serous carcinoma. Colors of epithelial cells resemble the colors used to label cells within UMAP embeddings (Fig. ).

Article Snippet: Positive selection was then performed on the unbound fraction of cells by incubating with a biotinylated antibody against SLC1A3 (Novus biologicals, NB100-1869B, 0.5 μg per million cells).

Techniques: