Journal: Scientific Reports

Article Title: Tiliroside as a CAXII inhibitor suppresses liver cancer development and modulates E2Fs/Caspase-3 axis

doi: 10.1038/s41598-021-88133-7

Figure Lengend Snippet: Tiliroside restrained the 3D formation ability and CD133 expression of Hep3B and SNU-449 cells. The representative 3D spheroid models of Hep3B ( A ) and SNU-449 ( B ) cells treated by 40 μM Tiliroside and NC. The relative cross-section area formation efficiency showed significant reduction of area of Hep3B ( C ) and SNU-449 ( D ) in Tiliroside groups. Relative inhibition rates of Hep3B and SNU-449 in response to Tiliroside were calculated by comparing the fluorescence value of Tiliroside to NC, at 24, 48, 72, 96 and 120 h, respectively ( E ). Tiliroside (40 μM) treatment significantly reduced the CD133 relative expression levels in both 3D cultured Hep3B and SNU-449 cells (P < 0.001) ( F ). NC negative control.

Article Snippet: The primers for CD133 were: forward, 5′-GATTCATACTGGTGGCTGGGTGG-3′ and reverse, 5′-GCAGGTGAAGAGTGCCGTAAGT-3′ , and carbonic anhydrase XII qPCR primer pairs(human) were obtain for Sino Biological (CN) (Cat: HP100073).

Techniques: Expressing, Inhibition, Fluorescence, Cell Culture, Negative Control

Journal: Oncology letters

Article Title: In vitro induction of anti‑lung cancer immune response by the A549 lung cancer stem cell lysate‑sensitized dendritic cell vaccine.

doi: 10.3892/ol.2024.14683

Figure Lengend Snippet: Figure 1. Relative expression of CD133, ABCG2 and Sox2 in A549 lung cancer stem cells and flow cytometric identification results of DCs. (A) Relative expres‑ sion of (B) CD133, (C) ABCG2 and Sox2 in A549 lung cancer stem cells. (D) Statistical chart of relative expression levels from the Western blot experiment for Sox2. Expression of IgG (E), CD80 (F), CD83 (G), CD86 (H) and HLA‑DR (I) in DCs. *P<0.05; **P<0.01; ***P<0.001. DC, dendritic cell; ABCG2, ATP‑binding cassette sub‑family G member 2; Sox2, sex determining region Y‑box 2.

Article Snippet: The membranes were then blocked with Protein‐Free Rapid Blocking Buffer (1x) (Shanghai Yamei Biotechnology Co., Ltd.) for 20 min at 4 ̊C, then incubated over‐ night at 4 ̊C with primary monoclonal antibodies against CD133 (1:1,000; cat. no. 48082S; CST Biological Reagents Co., Ltd.), ABCG2 (1:1,000; cat. no. 42078T; CST Biological Reagents Co., Ltd.), Sox2 (1:1,000; cat. no. 5067S; CST Biological Reagents Co., Ltd.) and β‐actin (1:3,000; cat. no. ab8226; Abcam).

Techniques: Expressing, Western Blot

Journal: Scientific Reports

Article Title: Screening of aptamers specific to colorectal cancer cells and stem cells by utilizing On-chip Cell-SELEX

doi: 10.1038/srep10326

Figure Lengend Snippet: ( a ) CR-CSCs form suspension cultured tumorspheres from culture day one to day five; ( b ) anti-CD44 fluorescence immunostaining analysis with CR-CSCs after enrichment suspension culture and colorectal cancer cells (HCT-8); ( c ) anti-CD133 fluorescence immunostaining analysis with CR-CSCs after enrichment suspension culture and HCT-8.

Article Snippet: USA.) was applied for CD44 staining and a CD133 antibody (C24B9, Rabbit, Cell Signaling Tech.

Techniques: Suspension, Cell Culture, Fluorescence, Immunostaining

Journal: British Journal of Cancer

Article Title: CD133/prominin-1 is a potential therapeutic target for antibody-drug conjugates in hepatocellular and gastric cancers

doi: 10.1038/sj.bjc.6604437

Figure Lengend Snippet: Immunohistochemical analysis of CD133 expression demonstrating the scoring intensity and expression pattern. Representative images using anti-CD133 MAb ab5558: ( A ) liver cholangiocarcinoma with multifocal, minimal to mild membranous and cytoplasmic staining, ( B ) pancreatic adenocarcinoma with mild to moderate membranous (apical) staining of luminal structures, ( C ) gastric adenocarcinoma with moderate to strong staining in two distinct cell populations: (1) luminal and apical and (2) cytoplasmic and membranous, ( D ) normal liver with minimal and nonspecific cytoplasmic staining of hepatocytes and apical staining of bile duct, ( E ) normal pancreas with weak to mild, apical membranous staining of acinar epithelium and ductal epithelium, ( F ) normal stomach with minimal to mild apical staining of glandular crypt epithelium. The scale bars represent 50 μ m.

Article Snippet: For mouse xenograft tumours, a rabbit anti-CD133 MAb (Cell Signaling Technology, Danvers, MA, USA) and a rabbit polyclonal anti-CD133 (ab19898, Abcam) were used separately as primary antibodies and the Bond Polymer Alkaline Phosphate (AP) Red Detection kit (Vision BioSystems, Australia) for detection.

Techniques: Immunohistochemical staining, Expressing, Staining

Journal: British Journal of Cancer

Article Title: CD133/prominin-1 is a potential therapeutic target for antibody-drug conjugates in hepatocellular and gastric cancers

doi: 10.1038/sj.bjc.6604437

Figure Lengend Snippet: CD133 expression analysis by immunohistochemistry

Article Snippet: For mouse xenograft tumours, a rabbit anti-CD133 MAb (Cell Signaling Technology, Danvers, MA, USA) and a rabbit polyclonal anti-CD133 (ab19898, Abcam) were used separately as primary antibodies and the Bond Polymer Alkaline Phosphate (AP) Red Detection kit (Vision BioSystems, Australia) for detection.

Techniques: Expressing, Immunohistochemistry

Journal: British Journal of Cancer

Article Title: CD133/prominin-1 is a potential therapeutic target for antibody-drug conjugates in hepatocellular and gastric cancers

doi: 10.1038/sj.bjc.6604437

Figure Lengend Snippet: Tumour and normal cell line expression of CD133 and sensitivity to anti-CD133 antibody-drug conjugate, AC133-vcMMAF

Article Snippet: For mouse xenograft tumours, a rabbit anti-CD133 MAb (Cell Signaling Technology, Danvers, MA, USA) and a rabbit polyclonal anti-CD133 (ab19898, Abcam) were used separately as primary antibodies and the Bond Polymer Alkaline Phosphate (AP) Red Detection kit (Vision BioSystems, Australia) for detection.

Techniques: Expressing, Inhibition

Journal: British Journal of Cancer

Article Title: CD133/prominin-1 is a potential therapeutic target for antibody-drug conjugates in hepatocellular and gastric cancers

doi: 10.1038/sj.bjc.6604437

Figure Lengend Snippet: Activity of anti-CD133 ADC against cancer cell lines. ( A ) ADCs targeting CD133 have potent cytotoxic activity against antigen-positive hepatocellular and gastric carcinoma cell lines. Cytotoxicity was measured by resazurin dye conversion in Hep3B and KATO III cells grown in 96-well plates and exposed to anti-CD133 (AC133-vcMMAF) and control ADCs (IgG-vcMMAF and OKT9-vcMMAF) and crosslinked unconjugated anti-CD133 MAb (AC133) for 96 h. ( B ) Proliferation was measured using [ 3 H]-thymidine uptake in Hep3B and KATO III cells grown in 96-well plates and exposed to anti-CD133 and control ADCs for 96 h. ( C ) Induction of apoptosis in Hep3B cells treated with AC133-vcMMAF. Caspase 3/7 activation, a quantitative measurement of apoptotic cells, was monitored using the Caspase Glo assay at various time points (24–72 h) after addition of ADCs. Caspase 3/7 activation relative to untreated cells was detected by 48 h with optimal measurement after 72 h in Hep3B cells treated with increasing concentrations of AC133-vcMMAF and positive control OKT9-vcMMAF. ( D ) Inhibition of ADC cytotoxicity using internalisation inhibitor, ammonium chloride (NH 4 Cl) in Hep3B cells. Cells were incubated with increasing concentration of NH 4 Cl 30 min before treated with anti-CD133 (AC133-vcMMAF) or control ADCs. Cytotoxicity was measured after 72 h using the rezasurin dye conversion as in ( A ). The percentage inhibition of cytotoxicity relative to control untreated cells at an ADC concentration of 400 ng ml −1 is shown.

Article Snippet: For mouse xenograft tumours, a rabbit anti-CD133 MAb (Cell Signaling Technology, Danvers, MA, USA) and a rabbit polyclonal anti-CD133 (ab19898, Abcam) were used separately as primary antibodies and the Bond Polymer Alkaline Phosphate (AP) Red Detection kit (Vision BioSystems, Australia) for detection.

Techniques: Activity Assay, Control, Activation Assay, Caspase-Glo Assay, Positive Control, Inhibition, Incubation, Concentration Assay

Journal: British Journal of Cancer

Article Title: CD133/prominin-1 is a potential therapeutic target for antibody-drug conjugates in hepatocellular and gastric cancers

doi: 10.1038/sj.bjc.6604437

Figure Lengend Snippet: Subcellular localisation of anti-CD133 ADC, AC133-vcMMAF, in sensitive and resistant cancer cell lines. ( A ) AC133-vcMMAF, partially colocalises (yellow) with the lysosomal marker, CD107a, in Hep3B and KATO III cells. Subcellular localisation of AC133-vcMMAF (red) and CD107a (green) in Hep3B and KATO III cells after 24 h incubation with the ADC. ( B ) Subcellular localisation of AC133-vcMMAF, lysosomal marker, CD107a, and caveolin-1 (Cav-1) in Su.86.86 after 24 h incubation with the ADC. AC133-vcMMAF colocalises with Cav-1 (yellow) and not with CD107a in this resistant cell line. Nuclei were stained blue with DAPI. Images were acquired using a × 63 oil immersion objective with Apotome for optical sectioning.

Article Snippet: For mouse xenograft tumours, a rabbit anti-CD133 MAb (Cell Signaling Technology, Danvers, MA, USA) and a rabbit polyclonal anti-CD133 (ab19898, Abcam) were used separately as primary antibodies and the Bond Polymer Alkaline Phosphate (AP) Red Detection kit (Vision BioSystems, Australia) for detection.

Techniques: Marker, Incubation, Staining

Journal: British Journal of Cancer

Article Title: CD133/prominin-1 is a potential therapeutic target for antibody-drug conjugates in hepatocellular and gastric cancers

doi: 10.1038/sj.bjc.6604437

Figure Lengend Snippet: In vivo efficacy of an anti-CD133 ADC in Hep3B hepatocellular carcinoma model including IHC analysis of CD133 expression following ADC treatment. ( A ) In vivo efficacy of AC133-vcMMAF in Hep3B subcutaneous tumours. SCID mice ( n =7/group) with established (∼100 mm 3 ) Hep3B tumour xenografts were treated by intraperitoneal injection every 4 days for a total of four doses (red arrows) with the anti-CD133 antibody (AC133) or ADC (AC133-vcMMAF) or isotype control mouse IgG1-vcMMAF. An additional group of mice was left untreated as a control. Median tumour volume plots were continued for each group until one or more animals died or were euthanised (see Materials and methods). Tumours were collected when the tumour volume reached 1000 mm 3 . Highly concordant data were in an independent replicate of this experiment. ( B ) CD133 expression in Hep3B xenograft tumours after anti-CD133 drug conjugate treatment. IHC analysis using rabbit anti-CD133 MAb in ( a ) untreated Hep3B xenograft, ( b ) treatment with control IgG-vcMMAF (3.0 mg kg −1 ) and ( c ) treated with AC133-vcMMAF (3.0 mg kg −1 ). ( d ) same tumour as ( c ) stained with control rabbit IgG as primary antibody. Fast Red chromagen was used to detect CD133 expression.

Article Snippet: For mouse xenograft tumours, a rabbit anti-CD133 MAb (Cell Signaling Technology, Danvers, MA, USA) and a rabbit polyclonal anti-CD133 (ab19898, Abcam) were used separately as primary antibodies and the Bond Polymer Alkaline Phosphate (AP) Red Detection kit (Vision BioSystems, Australia) for detection.

Techniques: In Vivo, Expressing, Injection, Control, Staining

Journal: Respiratory Research

Article Title: Chronic intermittent hypoxia promoted lung cancer stem cell-like properties via enhancing Bach1 expression

doi: 10.1186/s12931-021-01655-6

Figure Lengend Snippet: Primers used for real-time PCR

Article Snippet: A549 and SPCA1 were seeded in 12-well plates at a proper concentration and cultured under Nor or CIH condition for 48 h. At the end of CIH cycles, cells were harvested, filtration and centrifugation, and FITC-labeled anti-CD44 (555478) and APC-labeled anti-CD133 (53276) (Cell Signaling Technologies, USA) were used for surface staining.

Techniques:

Journal: Respiratory Research

Article Title: Chronic intermittent hypoxia promoted lung cancer stem cell-like properties via enhancing Bach1 expression

doi: 10.1186/s12931-021-01655-6

Figure Lengend Snippet: CIH promoted CSC-like properties in NSCLC cells. a Microscopic observation of NSCLC cells. A549 and SPCA1 spheroids were obtained and then cultured under Nor or CIH conditions for 24 h. b qRCP analyses in triplicate of CD44, Sox2, Nanog, Oct4, and CD133 in NSCLC cells. Adherent A549 and SPCA1 were treated with 24 h-Nor or CIH exposure. GADPH expression served as an internal control. c Flow cytometry analyses of the percentage of CD44 + CD133 + cells in A549 or SPCA1 populations. Error bars represent the mean ± SEM of at least triplicate experiments. * P < 0.05, ** P < 0.01. CIH chronic intermittent hypoxia, CSC cancer stem cell

Article Snippet: A549 and SPCA1 were seeded in 12-well plates at a proper concentration and cultured under Nor or CIH condition for 48 h. At the end of CIH cycles, cells were harvested, filtration and centrifugation, and FITC-labeled anti-CD44 (555478) and APC-labeled anti-CD133 (53276) (Cell Signaling Technologies, USA) were used for surface staining.

Techniques: Cell Culture, Expressing, Flow Cytometry

Journal: Respiratory Research

Article Title: Chronic intermittent hypoxia promoted lung cancer stem cell-like properties via enhancing Bach1 expression

doi: 10.1186/s12931-021-01655-6

Figure Lengend Snippet: Knockdown of Bach1 decreased the stemness and mitochondrial ROS accumulation in CIH-treated NSCLCs. Bach1 shRNA or parental negative control (NC) was transfected into A549 or SPCA1. Then the cells were cultured under CIH conditions for 48hrs. Flow cytometry analyses of CD44 + CD133 + cells in Nor or CIH-treated A549 ( a ) or SPCA1 ( b ). c The percentage of CD44 + CD133 + cells in Nor or CIH-treated cells was measured and illustrated. Fluorescence microscopy analysis for localization of mtROS production in A549 ( d ) and SPCA1 ( e ). Nuclei was stained with Hoechst (blue), mitochondria ROS were stained with MitoSOX-red. The merged panels showed the MitoSOX-red positive cells in total cells. f The percentage of MitoSOX positive cells was measured and illustrated. All experiments were performed in triplicate, and data are presented as mean ± SEM. * P < 0.05, ** P < 0.01 compared with the control group

Article Snippet: A549 and SPCA1 were seeded in 12-well plates at a proper concentration and cultured under Nor or CIH condition for 48 h. At the end of CIH cycles, cells were harvested, filtration and centrifugation, and FITC-labeled anti-CD44 (555478) and APC-labeled anti-CD133 (53276) (Cell Signaling Technologies, USA) were used for surface staining.

Techniques: shRNA, Negative Control, Transfection, Cell Culture, Flow Cytometry, Fluorescence, Microscopy, Staining

Journal: bioRxiv

Article Title: Synthesis, anticancer properties, and biological profiling of synthetic glycan analogs of proscillaridin A

doi: 10.1101/2025.03.31.646140

Figure Lengend Snippet: Flow cytometry profiling of key markers and cell cycle analysis in human and murine colon cancer cells. A) HCT-116 cells treated with 1 μM of compound 5 exhibit significantly enhanced surface expression of cleaved caspase-3, the hallmark of pre-apoptotic cell death. B) Effect of compounds 1 through 5 on surface CD133 (prominin 1) expression. C) Effect of compounds 1 through 5 on surface CD117 (c-Kit) expression. D) Cell cycle analysis by flow cytometry on HCT-116, HT-29, and CT26 cells treated with 100 nM of compounds 1 through 5

Article Snippet: Cleaved caspase-3 anti-mouse monoclonal antibody (Cat. #Ab156544) and CD117/c-Kit anti-mouse monoclonal antibody (Cat. #Ab096639) were obtained from Aladdin Scientific Corp. Anti-CD133 monoclonal antibody HB#7 was obtained from the DSHB and deposited there by Swaminathan, S.K./Panyam, J./Ohlfest, J.R. Ribonuclease A was obtained from Research Products International (97% purity).

Techniques: Flow Cytometry, Cell Cycle Assay, Expressing