Journal: Cell Death & Disease

Article Title: Temozolomide promotes glioblastoma stemness expression through senescence-associated reprogramming via HIF1α/HIF2α regulation

doi: 10.1038/s41419-025-07617-w

Figure Lengend Snippet: A Immunofluorescence analysis showed high expression levels of stemness markers CD133, Nestin, and transcription factors Sox2 and Klf4 in aggregation cells. B Elevated expression levels of CD133, Nestin, Sox2, and Klf4 were observed after two weeks of culturing aggregation cells. C Western blot analysis confirmed increased expression of stemness markers CD133, CD15, Nestin, Sox2, and Klf4 after one to two weeks of TMZ treatment, with higher expression levels in aggregation cells. D Aggregation cells (group ②) demonstrated greater invasive capacity compared to controls (group ①). Co-culturing control and aggregation cells showed a higher number of migrating cells when control cells were co-cultured with aggregation cells (group ④) than with control cells (group ③). E RFP-labeled aggregation cells co-cultured with aggregation cells exhibited the highest invasive capacity (group ④) compared to other groups, in the order: group ④ > group ③ (RFP-aggregation cells co-cultured with control cells) > group ② (RFP-control cells co-cultured with aggregation cells) > group ① (RFP-control cells co-cultured with control cells). * P < 0.05 was determined using Student’s t -test.

Article Snippet: These cells were incubated with polyclonal rabbit anti-human CD133 + IgGs (Miltenyi Biotech, Germany) at 4 °C for 15 min, washed with PBS containing 1% BSA, and resuspended in PBSE (108 cells/300 μl).

Techniques: Immunofluorescence, Expressing, Western Blot, Control, Cell Culture, Labeling

Journal: Cell Death & Disease

Article Title: Temozolomide promotes glioblastoma stemness expression through senescence-associated reprogramming via HIF1α/HIF2α regulation

doi: 10.1038/s41419-025-07617-w

Figure Lengend Snippet: A GSEA analysis indicated upregulation of senescence-associated hallmarks and inhibition of growth-promoting pathways. B The heatmap of U87 overlapping differentially expressed genes (DEGs) with SASP at the mRNA level revealed upregulation of most DEGs, including IL6, IL7, CXCL3, CXCL2, ICAM1, CCL2, CCL3, MMP7, and TIMP1. Conversely, senescence-inhibiting genes such as CDC25B, CDC25C, CDC25A, CDKN2D, MSH6, and MSH5 were downregulated. C RT-qPCR analysis demonstrated significant time-dependent increases in the expression of senescence-promoting genes, including IL1a, IL1b, IL6, IL8, CCL2, CDKN1A, CDKN2B, P53, and CXCL3, while the expression of the senescence-inhibiting gene MSH2 decreased significantly. D Mass spectrometry revealed high expression of senescence-promoting SASP proteins, including ITGA4, MMP15, FN1, IGFB3, and FAS, with a concomitant decrease in senescence-suppressing SASP proteins, MSH2 and MSH6. E ELISA confirmed increased expression of IL1a, IL6, and IL8 in a time-dependent manner following TMZ treatment. F SA-β-Gal staining revealed a significant time-dependent increase in β-Gal-positive cells, and the rate of β-Gal positivity was lower in aggregation cells compared to control cells under TMZ treatment. G C 12 FDG expression increased approximately threefold after one week of TMZ treatment in CD133 − CD15 − GBM cells, with lower levels of expression in aggregation cells under equivalent TMZ concentrations. H C 12 FDG-negative and -positive cells were cultured under TMZ for 21 days, showing significantly higher levels of CD133 and CD15 in C 12 FDG-positive cells over time, while CD133 and CD15 expression was not significant difference in C 12 FDG-negative cells. * P < 0.05 and # P > 0.05 were determined using Student’s t -test.

Article Snippet: These cells were incubated with polyclonal rabbit anti-human CD133 + IgGs (Miltenyi Biotech, Germany) at 4 °C for 15 min, washed with PBS containing 1% BSA, and resuspended in PBSE (108 cells/300 μl).

Techniques: Inhibition, Quantitative RT-PCR, Expressing, Mass Spectrometry, Enzyme-linked Immunosorbent Assay, Staining, Control, Cell Culture

Journal: Cell Death & Disease

Article Title: Temozolomide promotes glioblastoma stemness expression through senescence-associated reprogramming via HIF1α/HIF2α regulation

doi: 10.1038/s41419-025-07617-w

Figure Lengend Snippet: A GSEA analysis demonstrated significant upregulation of hypoxia hallmark pathways in newly formed aggregation cells. B HIF1α and HIF2α knockout CD133 − CD15 − cells cultured under TMZ for two months exhibited the lowest expression levels of stemness markers CD133, CD15, Nestin, and transcription factors Sox2 and Klf4. Cells with simultaneous knockout showed the least expression, followed by single knockouts, compared to the control. C HIF1α and HIF2α knockout CD133 − CD15 − cells cultured with TMZ for two months exhibited almost no aggregation formation. Aggregation formation was significantly reduced in single knockouts compared to the control. D Apoptosis and necrosis rates were most significantly increased in cells with simultaneous HIF1α and HIF2α knockouts, followed by single knockouts, compared to the control. E Cell cycle analysis revealed that control cells arrested in the G2/M phase, while HIF1α and HIF2α knockout cells entered the S phase. F The rates of β-Gal-positive and C 12 FDG-positive cells decreased most significantly in simultaneous HIF1α and HIF2α knockout cells, with intermediate decreases in single knockouts compared to the control. G RT-qPCR analysis showed the lowest expression of SASP factors, including IL1a, IL1b, IL6, IL8, CCL2, and others, in simultaneous knockouts, followed by single knockouts, compared to the control in U118 CD133 − CD15 − cells. H GO analysis of miRNA sequences from simultaneous and single HIF1α and HIF2α knockouts revealed activation of terms associated with senescence and stemness, such as stem cell population maintenance, DNA replication, cell cycle arrest, centrosomes, and p53 binding. I KEGG pathway analysis of miRNA sequences from simultaneous and single HIF1α and HIF2α knockouts indicated activation of pathways associated with senescence and stemness, including cell cycle regulation, cellular senescence, Wnt signaling, regulation of pluripotency in stem cells, focal adhesion, and autophagy. * P < 0.05, ** P < 0.01, and # P > 0.05 were determined using Student’s t -test.

Article Snippet: These cells were incubated with polyclonal rabbit anti-human CD133 + IgGs (Miltenyi Biotech, Germany) at 4 °C for 15 min, washed with PBS containing 1% BSA, and resuspended in PBSE (108 cells/300 μl).

Techniques: Knock-Out, Cell Culture, Expressing, Control, Cell Cycle Assay, Quantitative RT-PCR, Activation Assay, Binding Assay

Journal: The Journal of Physiological Sciences : JPS

Article Title: Changes in adrenoceptor expression level contribute to the cellular plasticity of glioblastoma cells

doi: 10.1016/j.jphyss.2025.100016

Figure Lengend Snippet: The antibodies used for Western blotting.

Article Snippet: CD133 rabbit polyclonal , 1:2000 , Proteintech , 18470 −1-AP.

Techniques: Western Blot, Produced

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: iScience

Article Title: CXCL3/TGF-β-mediated crosstalk between CAFs and tumor cells augments RCC progression and sunitinib resistance

doi: 10.1016/j.isci.2024.110224

Figure Lengend Snippet: CAFs-CM and CXCL3 regulated ERK1/2 signaling pathway (A and B) Western blot analysis and quantification of ERK1/2 and phosphorylated-ERK1/2 expression in Renca cells stimulated with CAFs-CM or CXCL3. (C) Expression of ERK1/2 and phosphorylated-ERK1/2 in Renca cells stimulated with conditioned medium from CAFs with CXCL3 interference. (D) Protein expression of ERK1/2 and phosphorylated-ERK1/2 in tumor tissue of tumor-bearing mice. (E and F) Protein expression of N-cadherin and CD133 in Renca cells stimulated with CAFs-CM or CXCL3. (G) Protein expression of CD133 and E-cadherin in tumor tissue of tumor-bearing mice. Data are shown as mean ± SEM, n = 3∼6, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Rabbit anti-CD133 Polyclonal Antibody , Proteintech , Cat# 18470-1-AP; RRID:AB_2172859.

Techniques: Western Blot, Expressing

Journal: iScience

Article Title: CXCL3/TGF-β-mediated crosstalk between CAFs and tumor cells augments RCC progression and sunitinib resistance

doi: 10.1016/j.isci.2024.110224

Figure Lengend Snippet:

Article Snippet: Rabbit anti-CD133 Polyclonal Antibody , Proteintech , Cat# 18470-1-AP; RRID:AB_2172859.

Techniques: Recombinant, Cell Culture, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, RNA Extraction, Sequencing, Over Expression, Plasmid Preparation, Software