Journal: Cells

Article Title: Prominin-1 Knockdown Causes RPE Degeneration in a Mouse Model.

doi: 10.3390/cells13211761

Figure Lengend Snippet: Figure 1. Prom1 mRNA expression in albino mouse RPE in situ. Representative brightfield images showing chromogenic Prom1 RNAscope (red) in albino mouse eyecup sections. The sections were counterstained with hematoxylin (blue). (A,B) The black arrowheads show Prom1 mRNA expression in mouse RPE in situ. The scale bar is 60 µm. (B) The brightfield low-resolution image of the eyecup and the red box showing the area of the eyecup used for imaging. The scale bar is 600 mm. (C) No Prom1 labeling in the negative control for Prom1 RNAscope. The scale bar is 60 mm. (D) Imaging from another mouse eyecup shows Prom1 mRNA expression in mouse RPE in situ (black arrowheads). The scale bar is 60 µm. (E) The brightfield low-resolution image of the eyecup and the red box showing the area of the eyecup used for imaging. The scale bar is 600 µm. (F) Representative fluorescent image showing Prom1 expression in an albino mouse eyecup (white arrows). The section was counterstained with DAPI (blue). The scale bar is 1000 µm. (G) Confocal high-resolution micrograph of Prom1 expression in mouse RPE (white arrows; purple fluorescence). CC = choriocapillaris. The scale bar is 100 µm.

Article Snippet: Samples were incubated with rabbit polyclonal primary antibodies against Prom1 from Origene (Rockville, MD, USA) (catalog number TA354470) and Abcam (Waltham, MA, USA) (catalog number ab19898) at 1:50 dilution for 2 h, followed by the secondary antibody at 1:20 for 1 h. The grids were poststained with 2% uranyl acetate.

Techniques: Expressing, In Situ, RNAscope, Imaging, Labeling, Negative Control, Fluorescence

Journal: Cells

Article Title: Prominin-1 Knockdown Causes RPE Degeneration in a Mouse Model.

doi: 10.3390/cells13211761

Figure Lengend Snippet: Figure 2. Prom1 RNAscope in pigmented C57BL/6J mouse retinal sections. Brightfield 40X slide scanning micrographs of (A) pigmented C57BL/6J retinal sections labeled by chromogenic RNAscope for Prom1 (red puncta). Labeling is present in the photoreceptor inner segments (ISs) and outer nuclear layer (ONL) (black arrowheads) and a cohort of cells in the inner retina (white arrowheads). The scale bar is 60 µm. (B) No labeling was detected in the negative control of chromogenic Prom1 RNAscope. The scale bar is 60 µm. (C) Fluorescent widefield confocal micrographs of Prom1 labeling with Cy5 fluorophore (magenta puncta) and DAPI counterstain in C57BL/6J retinal sections showing Prom1 mRNA expression (magenta puncta) in the mouse RPE (white arrows), photoreceptor inner segments (ISs), ONL, and a cohort of cells in the inner retina (white arrowheads). The scale bar is 200 µm. (D) Negative control of fluorescent Prom1 RNAscope in retinal sections shows no Prom1 labeling. The scale bar is 200 µm. (E) Representative high-resolution widefield confocal microscopy using 100X objective showing Prom1 mRNA expression (magenta puncta) in single RPE cells in situ by a fluorescent RNAscope assay (white arrowheads). Sections were counterstained with DAPI (blue). The scale bar is 200 µm. (F) Representative fluorescent 40× Leica slide scanning images showing the presence of Prom1 mRNA in RPE (white arrows), photoreceptor ISs, and ONL in mouse retinal sections. DAPI was used as a counterstain. The scale bar is 50 µm. CC = choriocapillaris.

Article Snippet: Samples were incubated with rabbit polyclonal primary antibodies against Prom1 from Origene (Rockville, MD, USA) (catalog number TA354470) and Abcam (Waltham, MA, USA) (catalog number ab19898) at 1:50 dilution for 2 h, followed by the secondary antibody at 1:20 for 1 h. The grids were poststained with 2% uranyl acetate.

Techniques: RNAscope, Labeling, Negative Control, Expressing, Confocal Microscopy, In Situ

Journal: Cells

Article Title: Prominin-1 Knockdown Causes RPE Degeneration in a Mouse Model.

doi: 10.3390/cells13211761

Figure Lengend Snippet: Figure 3. Immunogold transmission electron microscopy in C57BL/6J mouse eyecups. Immunogold TEM of Prom1 in C57BL/6J mouse eyecups. (A,B) TEM micrographs showing positive Prom1 labeling in RPE mitochondria (white arrows). The scale bar is 500 nm. (C) Immunogold TEM of mouse RPE in situ showing cytoplasmic localization of Prom1 (white arrow, top left), mitochondria (white arrow, middle left), and in proximity to mitochondria (bottom right). The scale bar is 500 nm.

Article Snippet: Samples were incubated with rabbit polyclonal primary antibodies against Prom1 from Origene (Rockville, MD, USA) (catalog number TA354470) and Abcam (Waltham, MA, USA) (catalog number ab19898) at 1:50 dilution for 2 h, followed by the secondary antibody at 1:20 for 1 h. The grids were poststained with 2% uranyl acetate.

Techniques: Transmission Assay, Electron Microscopy, Labeling, In Situ

Journal: Cells

Article Title: Prominin-1 Knockdown Causes RPE Degeneration in a Mouse Model.

doi: 10.3390/cells13211761

Figure Lengend Snippet: Figure 5. Subretinal injections of AAV2/1.Prom1.gRNA leads to Prom1-KD in mouse RPE in situ. Rep- resentative micrographs of retinal sections of C57BL/6J mouse eyes labeled by fluorescent RNAscope for Prom1 (red puncta). (A,B) Low-magnification fluorescent micrographs with wider views of retinal sections injected with control AAV2/1.CRISPR.scr.gRNA. Scale bars of 60 µm and 100 µm. (C–F) Images of various magnifications showing Prom1 labeling (red puncta) in mouse RPE in control sections (gray arrows). Scale bars ranging from 50 µm to 100 µm. The sections were counterstained with DAPI. (G–I) Fluorescent micrographs with wider views of retinal sections injected with experi- mental AAV2/1.CRISPR.Prom1.gRNA. Scale bars ranging from 80 µm to 90 µm. The white arrow- heads show patchy areas where the Prom1 gene has been knocked down in the RPE in situ. (J–M) Low- and high-magnification micrographs of retinal sections injected with AAV2/1.CRISPR.Prom1.gRNA shows patchy Prom1-KD (white arrowheads) in RPE, with areas showing unaltered Prom1 expression (gray arrows). Scale bars ranging from 50 µm to 100 µm.

Article Snippet: Samples were incubated with rabbit polyclonal primary antibodies against Prom1 from Origene (Rockville, MD, USA) (catalog number TA354470) and Abcam (Waltham, MA, USA) (catalog number ab19898) at 1:50 dilution for 2 h, followed by the secondary antibody at 1:20 for 1 h. The grids were poststained with 2% uranyl acetate.

Techniques: In Situ, Labeling, RNAscope, Injection, Control, CRISPR, Expressing

Journal: Cells

Article Title: Prominin-1 Knockdown Causes RPE Degeneration in a Mouse Model.

doi: 10.3390/cells13211761

Figure Lengend Snippet: Figure 6. Fundus imaging and ERG of mouse eyes with subretinal injection of control or ex- perimental viral vectors. Representative fundus images of mouse eyes injected with (A) control AAV2/1.CRISPR.scr.gRNA or (B,C) experimental AAV2/1.CRISPR.Prom1.gRNA. Images were ob- tained after 11 weeks of injection. Circles with dashed lines show areas of RPE degeneration in mouse eyes injected with Prom1-gRNA. (D) Representative ERG waveforms (a- and b-waves) in mouse eyes injected with scrambled (scr) or Prom1 gRNA. (E) Quantifying scotopic a-wave ERG responses from mouse eyes injected with scr- or Prom1-gRNA. ***, p value 0.0004.

Article Snippet: Samples were incubated with rabbit polyclonal primary antibodies against Prom1 from Origene (Rockville, MD, USA) (catalog number TA354470) and Abcam (Waltham, MA, USA) (catalog number ab19898) at 1:50 dilution for 2 h, followed by the secondary antibody at 1:20 for 1 h. The grids were poststained with 2% uranyl acetate.

Techniques: Imaging, Injection, Control, CRISPR

Journal: Cells

Article Title: Prominin-1 Knockdown Causes RPE Degeneration in a Mouse Model.

doi: 10.3390/cells13211761

Figure Lengend Snippet: Figure 7. Histology of mouse eyes injected with control or experimental viral vectors. Bright- field micrographs of H&E-stained sections of C57BL/6J eyes injected with either (A) control AAV2/1.CRISPR.scr.gRNA or (B–D) experimental AAV2/1.CRISPR.Prom1.gRNA. Prom1 knock- down causes patchy RPE vacuolization and abnormalities with fluid accumulation between the RPE and PRs (back arrowheads)—scale bars for all micrographs, 60 µm.

Article Snippet: Samples were incubated with rabbit polyclonal primary antibodies against Prom1 from Origene (Rockville, MD, USA) (catalog number TA354470) and Abcam (Waltham, MA, USA) (catalog number ab19898) at 1:50 dilution for 2 h, followed by the secondary antibody at 1:20 for 1 h. The grids were poststained with 2% uranyl acetate.

Techniques: Injection, Control, Staining, CRISPR, Knockdown

Journal: Cells

Article Title: Prominin-1 Knockdown Causes RPE Degeneration in a Mouse Model.

doi: 10.3390/cells13211761

Figure Lengend Snippet: Figure 8. Prom1 knockdown in vivo causes RPE apoptosis and degeneration. Retinal sections of C57BL/6J mouse eyes injected with (A,E) control AAV2/1.CRISPR.scr.gRNA or (B,F) experimental AAV2/1.CRISPR.Prom1.gRNA were used to detect active cleaved caspase-3 by immunohistochemistry. White arrows show positive immunolabeling of RPE cells for active caspase-3 in retinal sections obtained from Prom1-gRNA-injected eyes but not in RPE from control eyes. Serial sections from the same mouse eyes were labeled by fluorescent RNAscope for Prom1. (C,G) Prom1 labeling (red puncta) was observed in eyes injected with control AAV2/1.CRISPR.scr.gRNA, but its expression was reduced in eyes injected with (D,H) experimental AAV2/1.CRISPR.Prom1.gRNA (white arrowheads). Scale bars for all micrographs, 50 µm.

Article Snippet: Samples were incubated with rabbit polyclonal primary antibodies against Prom1 from Origene (Rockville, MD, USA) (catalog number TA354470) and Abcam (Waltham, MA, USA) (catalog number ab19898) at 1:50 dilution for 2 h, followed by the secondary antibody at 1:20 for 1 h. The grids were poststained with 2% uranyl acetate.

Techniques: Knockdown, In Vivo, Injection, Control, CRISPR, Immunohistochemistry, Immunolabeling, Labeling, RNAscope, Expressing

Journal: Cells

Article Title: Prominin-1 Knockdown Causes RPE Degeneration in a Mouse Model.

doi: 10.3390/cells13211761

Figure Lengend Snippet: Figure 9. Interrogation of single-cell human RPE datasets using Spectacle shows Prom1 gene expression in human RPE. The single-nucleus ATAC human RPE dataset (PMID: 36775060) [38] was used to analyze

Article Snippet: Samples were incubated with rabbit polyclonal primary antibodies against Prom1 from Origene (Rockville, MD, USA) (catalog number TA354470) and Abcam (Waltham, MA, USA) (catalog number ab19898) at 1:50 dilution for 2 h, followed by the secondary antibody at 1:20 for 1 h. The grids were poststained with 2% uranyl acetate.

Techniques: Gene Expression

Journal: Cells

Article Title: Prominin-1 Knockdown Causes RPE Degeneration in a Mouse Model.

doi: 10.3390/cells13211761

Figure Lengend Snippet: Figure 10. Prom1 gene expression in mouse retinal microglia. Analysis of a mouse single-cell RNA dataset (PMID: 30890724) [45] using Spectacle. (A) Heatmap showing the Prom1 gene is highly expressed in proliferating and activated microglia. (B) Plot showing Prom1 gene expression in a subset of activated microglia in the degenerating mouse retina under dark conditions. A mouse retinal degeneration (induced by light damage) dataset (PMID: 30850344) [46] was used to analyze Prom1 gene expression in adult retinal microglia. (C) The volcano plot shows the levels of Prom1 gene expression in undamaged microglia (MG0), other clusters of microglia (lcMG), and small clusters of microglia (sMG).

Article Snippet: Samples were incubated with rabbit polyclonal primary antibodies against Prom1 from Origene (Rockville, MD, USA) (catalog number TA354470) and Abcam (Waltham, MA, USA) (catalog number ab19898) at 1:50 dilution for 2 h, followed by the secondary antibody at 1:20 for 1 h. The grids were poststained with 2% uranyl acetate.

Techniques: Gene Expression

Journal: Heliyon

Article Title: Identification of TNFAIP6 as a reliable prognostic indicator of low-grade glioma

doi: 10.1016/j.heliyon.2024.e33030

Figure Lengend Snippet: Expression level of TNFAIP6 in Low- and High- CD133 expression groups (A): Representative IHC staining images of CD133 and TNFAIP6 in two stemness group in the Zhujiang in-house cohort. (B): Box plot displaying the pathology score levels of TNFAIP6 and CD133 based between two stemness group in the Zhujiang in-house dataset; (C): The correlation of expression level between TNFAIP6 and CD133.

Article Snippet: The sections were incubated with primary antibodies against CD133 (rabbit, 1:400; YT5192; Immunoway, America), and TNFAIP6 (rabbit, 1:200; AF5492; Affinity, America).

Techniques: Expressing, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Glioblastoma stem cells deliver ABCB4 transcribed by ATF3 via exosomes conferring glioblastoma resistance to temozolomide

doi: 10.1038/s41419-024-06695-6

Figure Lengend Snippet: A Correlation between gene expression of ABCB4 and stem cell markers in CGGA glioma dataset. B Localisation of ABCB4 (green) and CD133 (red) examined by immunofluorescence confocal microscopy. Nuclei stained with DAPI (blue). Scale bar = 50 μm. C Representative images of GSC#1 and GSC#2 cultured in serum-free medium to form floating spheres. Scale bar = 100/200 μm. D Fluorescence staining for CD133 (red) and Nestin (green) in GSCs. Nuclei stained with DAPI (blue). Scale bar = 100/200 μm. E Fluorescence staining for ABCB4 (red) in GSCs. Nuclei stained with DAPI (blue). Scale bar = 100 μm. F Detection of ABCB4, CD133, Nestin, and SOX2 mRNA levels in GSCs and DGCs via qRT-PCR. G Detection of ABCB4, CD133, Nestin, and SOX2 protein levels in GSCs and DGCs via WB. All experiments were repeated independently three times. Data are presented as mean ± standard deviation. * p < 0.05; ** p < 0.01; *** p < 0.001; ns not significant.

Article Snippet: Immunofluorescence staining was performed using primary antibodies against CD133 (CST, USA, #64326), Nestin (CST, USA, #33475), and ABCB4.

Techniques: Gene Expression, Immunofluorescence, Confocal Microscopy, Staining, Cell Culture, Fluorescence, Quantitative RT-PCR, Standard Deviation