Journal: Frontiers in Immunology

Article Title: Altered intraperitoneal immune microenvironment in patients with peritoneal metastases from gastric cancer

doi: 10.3389/fimmu.2022.969468

Figure Lengend Snippet: Gating strategy of lymphocytes, myeloid cells and tumor cells in peritoneal fluid. An example of gating method analyzed by Flow Jo software. Among the DAPI(+) peritoneal free cells, dead cells were excluded with FVS780. Tumor cells and leukocytes were defined as CD45(-)CD326 (+) and CD45(+)CD326(-) cells, respectively, and tumor leukocyte ratio (TLR) was calculated as described in Material and Methods. Among the leukocyte populations, lymphocytes and myeloid cells were distinguished as FSC/SCC profile, and expression of antigens was further examined in each cell population.

Article Snippet: APC-conjugated mAb to CD326 (EpCAM) was purchased from Miltenyi Biotec (Auburn, CA).

Techniques: Software, Expressing

Journal: PLoS ONE

Article Title: Histone Deacetylase 1/Sp1/MicroRNA-200b Signaling Accounts for Maintenance of Cancer Stem-Like Cells in Human Lung Adenocarcinoma

doi: 10.1371/journal.pone.0109578

Figure Lengend Snippet: (A 1 , A 2 , A 3 ) Percentage of CD133 + /CD326 + CSCs as analyzed by Flow cytometry at the indicated time after cultivating CD133 + /CD326 + CSCs (acquired by sorting SPC-A1/DTX and H1299/DTX cells, respectively) under differentiation conditions. (B) Mammosphere forming ability of the indicated cells (1000 cells) after 7 days under CSC-cultivating conditions. (C) qRT-PCR detection of relative mRNA levels of the CSC-related markers in the indicated cells. Data was calculated with the 2 −△△Ct method using GAPDH RNA as the reference gene. The expression level was measured relative to the fold change of the parental cells groups that were defined as 1.0. (D 1 , D 2 ) The protein levels of the CSC-related markers in the indicated cells. GAPDH was used as an internal control. (E) Tumor incidence in nude mice that were subcutaneously injected with the indicated cells. Data were presented as mean ± SD of at least three independent experiments. * p <0.05, ** p <0.01.

Article Snippet: Flow cytometric analysis of CD133 + /CD326 + cells was performed with CD133-PE and CD326-FITC antibodies (Miltenyi Biotec) according to the manufacturer's instructions.

Techniques: Flow Cytometry, Quantitative RT-PCR, Expressing, Control, Injection

Journal: PLoS ONE

Article Title: Histone Deacetylase 1/Sp1/MicroRNA-200b Signaling Accounts for Maintenance of Cancer Stem-Like Cells in Human Lung Adenocarcinoma

doi: 10.1371/journal.pone.0109578

Figure Lengend Snippet: ( A ) qRT-PCR detection of miR-200b in CSCs. Data were normalized to U6 rRNA and determined relative to the corresponding parental cell groups. ( B ) qRT-PCR detection of relative mRNA level of miR-200b in docetaxel-resistant LAD cells after transfection with the indicated vectors. Data were normalized to U6 rRNA and determined relative to the corresponding control groups. ( C ) Percentage of CD133 + /CD326 + CSCs as analyzed by flow cytometry after transfection with the indicated vectors. ( D ) Mammosphere forming ability of the indicated cells (1000 cells) after transfection of the indicated vectors. ( E ) Cell viability were measured by Cell Counting Kit-8 (CCK-8) assay. Data were presented as mean ± SD of at least three independent experiments. * p <0.05, ** p <0.01.

Article Snippet: Flow cytometric analysis of CD133 + /CD326 + cells was performed with CD133-PE and CD326-FITC antibodies (Miltenyi Biotec) according to the manufacturer's instructions.

Techniques: Quantitative RT-PCR, Transfection, Control, Flow Cytometry, Cell Counting, CCK-8 Assay

Journal: PLoS ONE

Article Title: Histone Deacetylase 1/Sp1/MicroRNA-200b Signaling Accounts for Maintenance of Cancer Stem-Like Cells in Human Lung Adenocarcinoma

doi: 10.1371/journal.pone.0109578

Figure Lengend Snippet: ( A ) The protein level of Suz-12 in docetaxel-resistant LAD cells after transfection with the indicated sh-RNA vectors (sh-RNA-Suz12#1, sh-RNA-Suz12#2, and sh-RNA-Suz12#3). ( B ) Mammosphere forming ability of docetaxel-resistant LAD cells (1000 cells) after transfection of the indicated vectors. ( C ) Flow cytometry analysis of percentage of CD133 + /CD326 + CSCs as analyzed by in docetaxel-resistant LAD cells after transfection of the indicated vectors. ( D ) The protein level of Suz-12 in CSCs after transfection with the sh-RNA-control or sh-RNA-Suz12#3 vectors. ( E ) CCK-8 analysis of cell viability of the CSCs transfected with the indicated vectors at the indicated time. ( F ) Effect of Suz-12 inhibition on in vivo tumorigenicity of the CSCs from SPC-A1/DTX cells. ( G ) Effect of Suz-12 inhibition on chemoresistance of CSCs. Cell viability was measured by CCK-8 assay. GAPDH was used as an internal control. Data were presented as mean ± SD of at least three independent experiments. * p <0.05, ** p <0.01.

Article Snippet: Flow cytometric analysis of CD133 + /CD326 + cells was performed with CD133-PE and CD326-FITC antibodies (Miltenyi Biotec) according to the manufacturer's instructions.

Techniques: Transfection, Flow Cytometry, Control, CCK-8 Assay, Inhibition, In Vivo

Journal: PLoS ONE

Article Title: Histone Deacetylase 1/Sp1/MicroRNA-200b Signaling Accounts for Maintenance of Cancer Stem-Like Cells in Human Lung Adenocarcinoma

doi: 10.1371/journal.pone.0109578

Figure Lengend Snippet: ( A ) The protein level of Suz-12 in CSCs from SPC-A1/DTX cells transfected with the indicated vectors. GAPDH was used as an internal control. ( B ) Flow cytometry analysis of percentage of CD133 + /CD326 + CSCs in SPC-A1/DTX cells after transfection of the indicated vectors. ( C ) Mammosphere forming ability of SPC-A1/DTX cells (1000 cells) after transfection of the indicated vectors. ( D ) CCK-8 analysis of cell viability of the CSCs (SPC-A1/DTX) transfected with the indicated vectors at the indicated time. ( E ) Suz-12 upregulation partianlly rescued the effects of enforced miR-200b expression on reversing chemoresistance. Cell viability was measured by CCK8 assay. Data were presented as mean ± SD of at least three independent experiments. * p <0.05, ** p <0.01.

Article Snippet: Flow cytometric analysis of CD133 + /CD326 + cells was performed with CD133-PE and CD326-FITC antibodies (Miltenyi Biotec) according to the manufacturer's instructions.

Techniques: Transfection, Control, Flow Cytometry, CCK-8 Assay, Expressing

Journal: PLoS ONE

Article Title: Histone Deacetylase 1/Sp1/MicroRNA-200b Signaling Accounts for Maintenance of Cancer Stem-Like Cells in Human Lung Adenocarcinoma

doi: 10.1371/journal.pone.0109578

Figure Lengend Snippet: ( A ) Western blotting detection of E-cadherin protein expression in the indicated cells transfected with the indicated vectors. ( B ) qRT-PCR detection of E-cadherin mRNA expression as measured by in the CSCs transfected with the indicated vectors. ( C ) Western blotting detection of E-cadherin protein expression in the CSCs transfected with the indicated vectors. ( D ) and ( E ) Suz-12 overexpression rescued the increased level of both mRNA and protein expression of E-cadherin in CSCs (SPC-A1/DTX) mediated by miR-200b upregulation, while silencing of Suz-12 rescued the decreased expression of mRNA and protein of E-cadherin in CSCs (SPC-A1/DTX) induced by miR-200b repression. ( F ) qRT-PCR detection of miR-200b and Suz-12 mRNA expression at the indicated time after plating CD133 + /CD326 + CSCs under differentiation conditions. ( G 1 , G 2 ) The protein levels of Suz-12 and E-cadherin at the indicated time after cultivating CD133 + /CD326 + CSCs under differentiation conditions. ( H ) MiR-200b upregulation decreased the amount of Suz-12 binding to the promoter of E-cadherin in CSCs (SPC-A1/DTX). Data were adjusted with input and determined relative to the control group. ( I ) MiR-200b overexpression reduced trimethylation of histone H3-lysine-27 (H3-K27) at E-cadherin promoter in CSCs (SPC-A1/DTX). Data were adjusted with input and determined relative to the control group. GAPDH was used as an internal control. Data were presented as mean ± SD of at least three independent experiments. * p <0.05, ** p <0.01; # p <0.05, ## p <0.01, compared with corresponding 0 day groups.

Article Snippet: Flow cytometric analysis of CD133 + /CD326 + cells was performed with CD133-PE and CD326-FITC antibodies (Miltenyi Biotec) according to the manufacturer's instructions.

Techniques: Western Blot, Expressing, Transfection, Quantitative RT-PCR, Over Expression, Binding Assay, Control

Journal: PLoS ONE

Article Title: Histone Deacetylase 1/Sp1/MicroRNA-200b Signaling Accounts for Maintenance of Cancer Stem-Like Cells in Human Lung Adenocarcinoma

doi: 10.1371/journal.pone.0109578

Figure Lengend Snippet: (A) Flow cytometry analysis of percentage of CD133 + /CD326 + CSCs in docetaxel-resistant LAD cells after transfection of the indicated vectors. (B) Mammosphere forming ability of docetaxel-resistant LAD cells (1000 cells) after transfection of the indicated vectors. (C) Cell viability as measured by CCK-8 assay of the CSCs (SPC-A1/DTX) transfected with the indicated vectors at the indicated time. (D) MiR-200b inhibition rescued the effects of HDAC1 repression on reversing chemoresistance. Cell viability was determined by CCK8 assay. (E) Repression of HDAC1 downregulated Suz-12 expression and upregulated E-cadherin expression in CSCs from docetaxel-resistant LAD cells partially in a miR-200b-dependent manner. GAPDH was used as an internal control. (F) Suppression of HDAC1 decreased the amount of Suz-12 binding to the promoter of E-cadherin in CSCs (SPC-A1/DTX). Data were adjusted with input and determined relative to the control group. (G) HDAC1 repression reduced trimethylation of H3-K27 at E-cadherin promoter in CSCs (SPC-A1/DTX). Data were adjusted with input and determined relative to the control group. Data were presented as mean ± SD of at least three independent experiments. * p <0.05, ** p <0.01.

Article Snippet: Flow cytometric analysis of CD133 + /CD326 + cells was performed with CD133-PE and CD326-FITC antibodies (Miltenyi Biotec) according to the manufacturer's instructions.

Techniques: Flow Cytometry, Transfection, CCK-8 Assay, Inhibition, Expressing, Control, Binding Assay

Journal: PLoS ONE

Article Title: Histone Deacetylase 1/Sp1/MicroRNA-200b Signaling Accounts for Maintenance of Cancer Stem-Like Cells in Human Lung Adenocarcinoma

doi: 10.1371/journal.pone.0109578

Figure Lengend Snippet: ( A ) Tumorigenicity in nude mice subcutaneously injected with CSCs from SPC-A1/DTX cells (5000 cells/mouse, n = 5) that were stably transfected with the indicated vectors previously. ( B ) Tumor volume in nude mice injected with H1299/DTX cells that were stably transfected with the indicated vectors and were combined with DTX treatment. Data were presented as mean ± SD. ( C ) The protein level of E-cadherin and Suz-12 in tumors of the indicated groups that were provided at 5 weeks after the inoculation. GAPDH was used as an internal control. ( D ) Flow cytometry detection of percentage of CD133 + /CD326 + CSCs in tumors of the indicated groups. ( E ) The mRNA level of miR-200b and Suz-12 in tumors of the indicated groups. Data were normalized to U6 RNA and GAPDH, respectively. Data were presented as mean ± SD of at least three independent experiments. ** p <0.01.

Article Snippet: Flow cytometric analysis of CD133 + /CD326 + cells was performed with CD133-PE and CD326-FITC antibodies (Miltenyi Biotec) according to the manufacturer's instructions.

Techniques: Injection, Stable Transfection, Transfection, Control, Flow Cytometry

Journal: STAR Protocols

Article Title: Protocol for 3D-guided sectioning and deep cell phenotyping via light sheet imaging and 2D spatial multiplexing

doi: 10.1016/j.xpro.2025.104296

Figure Lengend Snippet: 3D light sheet and 2D multi-cyclic imaging data comparison (Human OvCa) (A) Imaris 3D surface rendering of autofluorescence (cyan) and CD326 positive cells (red). (B) Imaris 3D surface rendering of autofluorescence (cyan) with target plane in yellow. (C) Light sheet guided target plane selection representing CD326 positive cell (purple), CD45 positive cells (red), and CD3 positive cells (green). (D) DAPI overview image of selected tissue slice for 2D MACSima™ imaging. (E) Magnified merged six color multiparameter MICS image with CD45 (green), CD326 (cyan), FOLR1 (purple), Collagen III (red), Collagen IV (red), and CD31 (yellow). (F–L) Single staining MICS images (white) of DAPI (F), CD45 (G), CD326 (H), FOLR1 (I), Collagen III (J), Collagen IV (K), and CD31 (L) (gray) (see “Antibodies”). Scale bars: (A–F) 1 mm; (E) 250 μm; (F–L) 500 μm.

Article Snippet: CD326 (EpCam) antibody, anti-human, Vio 780, REAfinity , Miltenyi Biotec B.V. & Co. KG , Cat# 130-136-631 RRID: AB_2904949.

Techniques: Imaging, Comparison, Selection, Staining

Journal: The American Journal of Pathology

Article Title: Profiles of Cancer Stem Cell Subpopulations in Cholangiocarcinomas

doi: 10.1016/j.ajpath.2015.02.010

Figure Lengend Snippet: List of Antibodies Used for IHC and IF

Article Snippet: For magnetic cell sorting, cells were labeled with CD133 (AC133), CD326 (EpCAM), and CD90 MicroBeads, and sorted using the Miltenyi Biotec Cell Isolation Kit, according to the manufacturer’s instruction.

Techniques:

Journal: Nature Medicine

Article Title: Transcriptional mediators of treatment resistance in lethal prostate cancer

doi: 10.1038/s41591-021-01244-6

Figure Lengend Snippet: a , Summary of clinical and select genomics features of patients and biopsies forming the study cohort. Each column represents a single biopsy. Where available, multiple biopsies from the same patient are displayed in adjacent columns. Patients are identified by numerical prefix, while suffixes after a dash, when present, identify biopsies from the same patient. Copy number calls based on whole exome sequencing (WES), but not OncoPanel, are allelic, with calls for the two alleles indicated by two triangles. In cases with whole genome doubling, it is possible for one allele to be amplified and one or both copies of the other allele to be lost. AR and KDM6A are on the X chromosome and so have only a single copy in these patients; they are represented with solid boxes for copy number status. Boxes with diagonal slashes indicate missing data, for example for genes not included in OncoPanel or for low tumor purity samples for which FACETS does not produce a purity estimate. Putative loss of function (LoF) missense mutations were annotated as LoF or likely LoF in OncoKB or mutated the same amino acid as a LoF mutation . b , Study design overview. Dissociated single cells were sorted to enrich for tumor (CD45 - EPCAM + ), immune (CD45 + EPCAM - ), or other populations (CD45 - EPCAM - ). c , Projection of single-cell expression onto the first two dimensions of UMAP space. Each dot represents a single cell, and colors correspond to clusters identified by the Louvain algorithm. Clusters are manually labelled with dominant cell type(s) inferred from cluster-specific expression of marker genes. Cells colored corresponding to ( d ) biopsy of origin or ( e ) metastatic site. Non-malignant cells from different patients jointly cluster by cell type, while cancer cells from different patients largely form distinct clusters.

Article Snippet: Single-cell suspensions in PBS with 2% FBS were stained by incubating for 15 min at room temperature protected from light with anti-human PTPRC (CD45) monoclonal antibody conjugated to FITC (1:200 dilution, VWR ABNOMAB12230), anti-human EPCAM antibody conjugated to PE (1:50 dilution, Miltenyi Biotec, 130-091-253) and either Calcein-AM (1:200 dilution, Thermo Fisher Scientific, C3100MP; biopsies 01115655-TA and 01115665), 7-aminoactinomycin D (7-AAD) (1:200 dilution, Thermo Fisher Scientific, A1310; all other biopsies except sample 01115149-TC) or both (sample 01115149-TC).

Techniques: Sequencing, Amplification, Mutagenesis, Expressing, Marker