Journal: International journal of oncology

Article Title: CXCR4 is involved in CD133-induced EMT in non-small cell lung cancer.

doi: 10.3892/ijo.2016.3812

Figure Lengend Snippet: Figure 1. The expression of CD133 in lung cancer and normal lung tissues. Original values are presented as log2 ratios. Spots showing the level of CD133 mRNA in NSCLC and normal tissues. (A) GSE10072. (B) GSE40275. (C) GSE63459; *p<0.05, **p<0.01 and ***p<0.001. (D) Correlation between CD133 and CXCR4 in GSE30219.

Article Snippet: Cells were incubated at 4 ̊C with 100 μl FcR blocking reagent and 100 μl CD133 Micro Beads (130-097-049, Miltenyi Biotec, Germany) for 30 min.

Techniques: Expressing

Journal: International journal of oncology

Article Title: CXCR4 is involved in CD133-induced EMT in non-small cell lung cancer.

doi: 10.3892/ijo.2016.3812

Figure Lengend Snippet: Figure 2. CD133 and CXCR4 are highly expressed in NSCLC patients with metastasis. (A) Representative images of immunohistochemistry staining (mag- nification, x400) of NSCLC patients. (a) CD133 low expression; (b) CD133 high expression; (c) CXCR4 low expression; (d) CXCR4 high expression. Positive staining ratio of CD133 (B)/CXCR4 (C) in metastatic and non-metastatic NSCLC patients. Positive staining score of CD133 (D)/CXCR4 (E) in metastatic and non-metastatic NSCLC patients. (F) Correlation results of CD133 and CXCR4. (G) Correlation analysis between CD133/CXCR4 co-expression and disease- free survival (red and black curves indicate high and low CD133/CXCR4 co-expression groups of patient death, respectively; *p<0.05).

Article Snippet: Cells were incubated at 4 ̊C with 100 μl FcR blocking reagent and 100 μl CD133 Micro Beads (130-097-049, Miltenyi Biotec, Germany) for 30 min.

Techniques: Immunohistochemistry, Staining, Expressing

Journal: International journal of oncology

Article Title: CXCR4 is involved in CD133-induced EMT in non-small cell lung cancer.

doi: 10.3892/ijo.2016.3812

Figure Lengend Snippet: Figure 3. CD133 enhances A549 cells proliferation. (A) Effectiveness of magnetic cell sorting was verified by immunofluorescence assay (magnification, x400). Red and blue indicate CD133 expression and cell nucleus, respectively. (B) Formation of colonies by A549 cell lines after 2-week incubation. (C and D) Cell proliferation capacity was determined in A549 cells, CD133+ A549 cells and CD133-A549 cells by CCK8 assays.

Article Snippet: Cells were incubated at 4 ̊C with 100 μl FcR blocking reagent and 100 μl CD133 Micro Beads (130-097-049, Miltenyi Biotec, Germany) for 30 min.

Techniques: FACS, Immunofluorescence, Expressing, Incubation

Journal: International journal of oncology

Article Title: CXCR4 is involved in CD133-induced EMT in non-small cell lung cancer.

doi: 10.3892/ijo.2016.3812

Figure Lengend Snippet: Figure 4. CXCR4 is upregulated by CD133. Silencing effectiveness of CD133 siRNA was verified by qPCR (A) and western blot analysis (B). (C) Expression of CXCR4 after treatment with CD133 siRNA (50 nM, 48 h) by qPCR analysis. (D) Western blot analysis for the expression of CXCR4 after treatment with CD133 siRNA (50 nM, 72 h). β-actin as a loading control. (E) The statistics of (D). All experiments were performed in triplicate. ***p<0.001.

Article Snippet: Cells were incubated at 4 ̊C with 100 μl FcR blocking reagent and 100 μl CD133 Micro Beads (130-097-049, Miltenyi Biotec, Germany) for 30 min.

Techniques: Western Blot, Expressing, Control

Journal: International journal of oncology

Article Title: CXCR4 is involved in CD133-induced EMT in non-small cell lung cancer.

doi: 10.3892/ijo.2016.3812

Figure Lengend Snippet: Figure 5. CD133+CXCR4+ promotes EMT process in NSCLC cells. (A) Transwell assay for the invasion of CD133-CXCR4+, CD133+CXCR4- and CD133+CXCR4+ cells. (B) Western blot assay for the expression of E-cadherin and Vimentin in CD133-CXCR4+, CD133+CXCR4- and CD133+CXCR4+ cells. (C) qPCR for the expression of E-cadherin, Vimentin, Snail, Slug and Twist in A549/CD133+, A549/CD133+ shRNA-NC, A549/CD133+ shRNA-CXCR4 and A549/CD133+ with amd3100. (D and E) RT-PCR for the expression of E-cadherin, Vimentin, Snail, Slug and Twist in A549/CD133+, A549/CD133+ shRNA-NC and A549/CD133+

Article Snippet: Cells were incubated at 4 ̊C with 100 μl FcR blocking reagent and 100 μl CD133 Micro Beads (130-097-049, Miltenyi Biotec, Germany) for 30 min.

Techniques: Transwell Assay, Western Blot, Expressing, shRNA, Reverse Transcription Polymerase Chain Reaction

Journal: International journal of oncology

Article Title: CXCR4 is involved in CD133-induced EMT in non-small cell lung cancer.

doi: 10.3892/ijo.2016.3812

Figure Lengend Snippet: Figure 6. Vimentin is positively associated with CD133/CXCR4 co-expression. (A) Representative images of immunohistochemistry staining (magnification, x400) of NSCLC patients. (a) E-cadherin low expression; (b) E-cadherin high expression; (c) Vimentin low expression; (d) Vimentin high expression. Positive staining ratio of E-cadherin (B)/Vimentin (C) in metastatic and non-metastatic NSCLC patients. Correlation analysis for E-cadherin (D) or Vimentin (E) with CD133/CXCR4 co-expression in NSCLC patients.

Article Snippet: Cells were incubated at 4 ̊C with 100 μl FcR blocking reagent and 100 μl CD133 Micro Beads (130-097-049, Miltenyi Biotec, Germany) for 30 min.

Techniques: Expressing, Immunohistochemistry, Staining

Journal: PloS one

Article Title: G protein-coupled receptor 87 (GPR87) promotes the growth and metastasis of CD133⁺ cancer stem-like cells in hepatocellular carcinoma.

doi: 10.1371/journal.pone.0061056

Figure Lengend Snippet: Figure 1. CD133+ HCC cells display high invasive and metastatic potential in vitro. (A) Isolation of CD133+ HCC cells sorted by MACS and detection of CD133 expression in SMMC-7721, HCC-LY5, SNU475, and PLC/PRF/5 cells by Western blot. (B) Growth curves of the sorted CD133+ and CD1332 SMMC-7721 and HCC-LY5 cells were obtained by the CCK-8 assay. (C) Transwell migration assay of CD133+ and CD1332 cells sorted from the SMMC-7721 and HCC-LY5 cell lines. (D) Transwell matrigel invasion assay of CD133+ and CD1332 cells sorted from the SMMC-7721 and HCC-LY5 cell lines. doi:10.1371/journal.pone.0061056.g001

Article Snippet: The CD133 cDNA clone (Myc-DDK-tagged ORF clone of Homo sapiens prominin 1 (PROM1), transcript variant 1 as transfectionready DNA NM_006017.1) with full length ORF sequence was purchased from Origene (OriGene Technologies, Inc. Rockville), which was cloned into the pWPXL lentiviral expression vector (Addgene, MA) by replacing the GFP fragment.

Techniques: In Vitro, Isolation, Expressing, Western Blot, CCK-8 Assay, Transwell Migration Assay, Invasion Assay

Journal: PloS one

Article Title: G protein-coupled receptor 87 (GPR87) promotes the growth and metastasis of CD133⁺ cancer stem-like cells in hepatocellular carcinoma.

doi: 10.1371/journal.pone.0061056

Figure Lengend Snippet: Figure 2. CD133+ HCC cells exhibit highly metastatic characteristics in vivo. (A) Representative in vivo bioluminescence imaging of tumor metastases in NOD/SCID mice after orthotopic transplantation with the isolated CD133+ or CD1332 SMMC-7721 cells (picture shown is representative of the group orthotopically transplanted with 10,000 cells) (n = 5 each group). (B) Representative examples of NOD/SCID mice orthotopically transplanted with CD133+ or CD1332 cells isolated from the SMMC-7721 HCC cell line. (C) Numbers of mice manifesting tumorigenicity and liver metastases of CD133+ SMMC-7721 cells in the orthotopic transplant assays are shown in the table. (D) HE staining of the harvested tumors confirmed a primary HCC phenotype. (E) Representative in vivo bioluminescence imaging of tumor metastasis in the NOD/SCID mice in a tumor-homing animal model transplanted with CD133+ or CD1332 cells isolated from the SMMC-7721 cell line (n = 9 each group). (F) Representative examples of the liver tumor formation and metastasis in 2-AAF/PHx animal model transplanted with CD133+ or CD1332 cells isolated from the SMMC-7721 HCC cell line. (G) HE staining of liver tissue sections, a tumor mass from CD133+ HCC cells in 2-AAF/PHx animal model was showed. (H) Numbers of mice manifesting liver metastases of CD133+ or CD1332 SMMC-7721 cells in the tumor-homing animal model are shown in the bar gragh. doi:10.1371/journal.pone.0061056.g002

Article Snippet: The CD133 cDNA clone (Myc-DDK-tagged ORF clone of Homo sapiens prominin 1 (PROM1), transcript variant 1 as transfectionready DNA NM_006017.1) with full length ORF sequence was purchased from Origene (OriGene Technologies, Inc. Rockville), which was cloned into the pWPXL lentiviral expression vector (Addgene, MA) by replacing the GFP fragment.

Techniques: In Vivo, Imaging, Transplantation Assay, Isolation, Staining, Animal Model

Journal: PloS one

Article Title: G protein-coupled receptor 87 (GPR87) promotes the growth and metastasis of CD133⁺ cancer stem-like cells in hepatocellular carcinoma.

doi: 10.1371/journal.pone.0061056

Figure Lengend Snippet: Figure 3. Detection of GPR87 expression in HCC cell lines and human primary cells by qRT-PCR and Western blot analysis. (A) Relative mRNA expression levels of GPR87 and CD133 were determined by quantitative polymerase chain reaction in the CD133+ and CD1332

Article Snippet: The CD133 cDNA clone (Myc-DDK-tagged ORF clone of Homo sapiens prominin 1 (PROM1), transcript variant 1 as transfectionready DNA NM_006017.1) with full length ORF sequence was purchased from Origene (OriGene Technologies, Inc. Rockville), which was cloned into the pWPXL lentiviral expression vector (Addgene, MA) by replacing the GFP fragment.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Real-time Polymerase Chain Reaction

Journal: PloS one

Article Title: G protein-coupled receptor 87 (GPR87) promotes the growth and metastasis of CD133⁺ cancer stem-like cells in hepatocellular carcinoma.

doi: 10.1371/journal.pone.0061056

Figure Lengend Snippet: Figure 4. Overexpression of GPR87 up-regulates CD133 expression and enhances CSC-related properties. (A) Representative examples of proliferation assays examining the effect of GPR87 overexpression in SMMC-7721 and HCC-LY5 cells. (B) The gross features of the tumor-bearing NOD/SCID mice orthotopically transplanted with 26106 SMMC-7721-lenti-GPR87 and SMMC-7721-lenti- pWPXL cells after 6 weeks (n = 6 each group). (C) Transwell migration assay in SMMC-7721 and HCC-LY5 cells overexpressing GPR87. (D) Transwell matrigel invasion assay in SMMC-7721 and HCC- LY5 cells overexpressing GPR87. (E) Immunohistochemical staining of GPR87 and CD133 in HCC tissues with intrahepatic metastasis. (F) The table showed the correlation between CD133 and GPR87 expression in HCC tissues with intrahepatic metastasis. doi:10.1371/journal.pone.0061056.g004

Article Snippet: The CD133 cDNA clone (Myc-DDK-tagged ORF clone of Homo sapiens prominin 1 (PROM1), transcript variant 1 as transfectionready DNA NM_006017.1) with full length ORF sequence was purchased from Origene (OriGene Technologies, Inc. Rockville), which was cloned into the pWPXL lentiviral expression vector (Addgene, MA) by replacing the GFP fragment.

Techniques: Over Expression, Expressing, Transwell Migration Assay, Invasion Assay, Immunohistochemical staining, Staining

Journal: PloS one

Article Title: G protein-coupled receptor 87 (GPR87) promotes the growth and metastasis of CD133⁺ cancer stem-like cells in hepatocellular carcinoma.

doi: 10.1371/journal.pone.0061056

Figure Lengend Snippet: Figure 5. Silencing of GPR87 reduces the levels of CD133 expression. Flow cytometric analysis of the levels of CD133 expression in GPR87 siRNA-treated cells, including the PLC/PRF/5, Hep3B, SNU475 and Huh-7 cell lines. doi:10.1371/journal.pone.0061056.g005

Article Snippet: The CD133 cDNA clone (Myc-DDK-tagged ORF clone of Homo sapiens prominin 1 (PROM1), transcript variant 1 as transfectionready DNA NM_006017.1) with full length ORF sequence was purchased from Origene (OriGene Technologies, Inc. Rockville), which was cloned into the pWPXL lentiviral expression vector (Addgene, MA) by replacing the GFP fragment.

Techniques: Expressing

Journal: PloS one

Article Title: G protein-coupled receptor 87 (GPR87) promotes the growth and metastasis of CD133⁺ cancer stem-like cells in hepatocellular carcinoma.

doi: 10.1371/journal.pone.0061056

Figure Lengend Snippet: Figure 6. GPR87 mediates the expression of CD133 in HCC cell lines. (A) Relative mRNA expression of CD133 in SMMC-7721-lenti- CD133, HCC-LY5-lenti-CD133 and MHCC-97L-lenti-CD133 cells. (B) Relative mRNA expression of GPR87 in SMMC-7721-lenti-CD133, HCC- LY5-lenti-CD133 and MHCC-97L-lenti-CD133 cells. (C) Western blot of GPR87 and CD133 in SMMC-7721-lenti-CD133, HCC-LY5-lenti-CD133 and MHCC-97L-lenti-CD133 cells. doi:10.1371/journal.pone.0061056.g006

Article Snippet: The CD133 cDNA clone (Myc-DDK-tagged ORF clone of Homo sapiens prominin 1 (PROM1), transcript variant 1 as transfectionready DNA NM_006017.1) with full length ORF sequence was purchased from Origene (OriGene Technologies, Inc. Rockville), which was cloned into the pWPXL lentiviral expression vector (Addgene, MA) by replacing the GFP fragment.

Techniques: Expressing, Western Blot

Journal: OncoTargets and therapy

Article Title: NFATC2 is a novel therapeutic target for colorectal cancer stem cells

doi: 10.2147/OTT.S169129

Figure Lengend Snippet: NFATC2 is upregulated in CRC-SCs. ( A ) qRT-PCR analysis of NFATC2 in primary CRC spheres and re-adherent cells relative to adherent cells. Primary CRC cells were isolated from the cancer tissues of CRC patients (No 1, 2, 3, and 4). Spheres were obtained by suspension culture. ( B ) Western blot analysis of NFATC2 in primary CRC spheres and adherent and re-adherent cells. ( C ) qRT-PCR analysis of NFATC2 in sorted CD44 + (left) or CD133 + (right) primary CRC cells relative to negative cells. Primary CRC cells were isolated from the cancer tissues of colorectal cancer patients (No 1, 2, 3, and 4). CD44 + or CD133 + cells were obtained by flow cytometry. ( D ) The correlation between the transcription level of NFATC2 and CD44 (left) and CD133 (right) in pin primary CRC sphere-derived cells. The mRNA level of each gene was determined by qRT-PCR. Data were normalized to GAPDH as ∆CT and analyzed by Spearman’s correlation analysis. Data are represented as mean ± SD; * P <0.05, *** P <0.001; two-tailed Student’s t -test. Abbreviations: CRC, colorectal cancer; NFATC2, nuclear factor of activated T-cells, cytoplasmic 2; CRC-SCs, colorectal cancer stem cells; qRT-PCR, quantitative real-time polymerase chain reaction.

Article Snippet: CD133 , IB , Mouse monoclonal , , Miltenyi Biotec , 130-092-395 , 100.

Techniques: Quantitative RT-PCR, Isolation, Suspension, Western Blot, Flow Cytometry, Derivative Assay, Two Tailed Test, Real-time Polymerase Chain Reaction

Journal: OncoTargets and therapy

Article Title: NFATC2 is a novel therapeutic target for colorectal cancer stem cells

doi: 10.2147/OTT.S169129

Figure Lengend Snippet: Overexpression of NFATC2 promotes the stemness of CRC cells. ( A , B ) Sphere formation assay of NFATC2-overexpressing and control primary CRC cells. Primary CRC cells were isolated from the cancer tissues of CRC patients (No 1 and 2). The 1st passaged spheres were obtained by suspension culture for 15 days and the number and average diameters of the spheres were counted ( A ). The number of 1st, 2nd, and 3rd passaged spheres isolated from the cancer tissues of CRC patients (No 1, 2, 3, and 4) was also counted ( B ). ( C ) qRT-PCR analysis of CD44 (left) and CD133 (right) in NFATC2-overexpressing and control primary CRC cells. Primary CRC cells were isolated from the cancer tissues of CRC patients (No 1 and 2). NFATC2-overexpressing and control cells were generated by lentivirus delivery system. ( D ) Western blot analysis of CD44 and CD133 in NFATC2-overexpressing and control primary CRC cells. Data are represented as mean ± SD; *** P <0.001; two-tailed Student’s t -test. Abbreviations: CRC, colorectal cancer; NFATC2, nuclear factor of activated T-cells, cytoplasmic 2; CRC-SCs, colorectal cancer stem cells; qRT-PCR, quantitative real-time polymerase chain reaction.

Article Snippet: CD133 , IB , Mouse monoclonal , , Miltenyi Biotec , 130-092-395 , 100.

Techniques: Over Expression, Tube Formation Assay, Control, Isolation, Suspension, Quantitative RT-PCR, Generated, Western Blot, Two Tailed Test, Real-time Polymerase Chain Reaction

Journal: OncoTargets and therapy

Article Title: NFATC2 is a novel therapeutic target for colorectal cancer stem cells

doi: 10.2147/OTT.S169129

Figure Lengend Snippet: Knockdown of NFATC2 inhibits the stemness of CRC cells. ( A , B ) Sphere formation assay of NFATC2-knockdown and control primary CRC cells. Primary CRC cells were isolated from the cancer tissues of CRC patients (No 1 and 2). The 1st passaged spheres were obtained by suspension culture for 15 days and the number and average diameters of the spheres were counted ( A ). The number of 1st, 2nd, and 3rd passaged spheres was also counted ( B ). ( C ) qRT-PCR analysis of CD44 (top) and CD133 (bottom) in NFATC2-knockdown and control primary CRC cells. Primary CRC cells were isolated from the cancer tissues of CRC patients (No 1 and 2). NFATC2-knockdown and control cells were generated by lentivirus delivery system. ( D ) Western blot analysis of CD44 and CD133 in NFATC2-knockdown and control primary CRC cells. ( E ) Tumorigenesis of NFATC2-knockdown and control primary CRC cells. Data are represented as mean ± SD; *** P <0.001; two-tailed Student’s t -test. Abbreviations: CRC, colorectal cancer; NFATC2, nuclear factor of activated T-cells, cytoplasmic 2; CRC-SCs, colorectal cancer stem cells; qRT-PCR, quantitative real-time polymerase chain reaction.

Article Snippet: CD133 , IB , Mouse monoclonal , , Miltenyi Biotec , 130-092-395 , 100.

Techniques: Knockdown, Tube Formation Assay, Control, Isolation, Suspension, Quantitative RT-PCR, Generated, Western Blot, Two Tailed Test, Real-time Polymerase Chain Reaction

Journal: OncoTargets and therapy

Article Title: NFATC2 is a novel therapeutic target for colorectal cancer stem cells

doi: 10.2147/OTT.S169129

Figure Lengend Snippet: YAP activity is necessary for NFATC2 for maintaining the stemness in CRC cells. ( A , B ) Sphere formation assay of YAP-knockdown NFATC2-overexpressing primary CRC cells and control cells. Primary CRC cells were isolated from the cancer tissues of CRC patients (No 1 and 2). The 1st passaged spheres were obtained by suspension culture for 15 days and the number of the spheres were counted ( A ). The number of 1st, 2nd, and 3rd passaged spheres was also counted ( B ). ( C ) qRT-PCR analysis of CD44 (top) and CD133 (bottom) in YAP-knockdown NFATC2-overexpressing primary CRC cells and control cells. Primary CRC cells were isolated from the cancer tissues of CRC patients (No 1 and 2). YAP-knockdown NFATC2-overexpressing primary CRC cells and control cells were generated by lentivirus delivery system. ( D ) Western blot analysis of CD44 and CD133 in YAP-knockdown NFATC2-overexpressing primary CRC cells and control cells. ( E ) Tumorigenesis of YAP-knockdown NFATC2-overexpressing primary CRC cells and control cells. Data are represented as mean ± SD; *** P <0.001; two-tailed Student’s t -test. Abbreviations: CRC, colorectal cancer; NFATC2, nuclear factor of activated T-cells, cytoplasmic 2; CRC-SCs, colorectal cancer stem cells; qRT-PCR, quantitative real-time polymerase chain reaction.

Article Snippet: CD133 , IB , Mouse monoclonal , , Miltenyi Biotec , 130-092-395 , 100.

Techniques: Activity Assay, Tube Formation Assay, Knockdown, Control, Isolation, Suspension, Quantitative RT-PCR, Generated, Western Blot, Two Tailed Test, Real-time Polymerase Chain Reaction

Journal: OncoTargets and therapy

Article Title: NFATC2 is a novel therapeutic target for colorectal cancer stem cells

doi: 10.2147/OTT.S169129

Figure Lengend Snippet: Primer sequences used in this study

Article Snippet: CD133 , IB , Mouse monoclonal , , Miltenyi Biotec , 130-092-395 , 100.

Techniques: Reverse Transcription

Journal: OncoTargets and therapy

Article Title: NFATC2 is a novel therapeutic target for colorectal cancer stem cells

doi: 10.2147/OTT.S169129

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: CD133 , IB , Mouse monoclonal , , Miltenyi Biotec , 130-092-395 , 100.

Techniques:

Journal: Genes to Cells

Article Title: Functional Role of COP1 Gene in Hepatocellular Carcinoma Lipid Metabolism and Stemness

doi: 10.1111/gtc.70108

Figure Lengend Snippet: COP1 knockdown reduces motility, and stemness in HCC cells. (a‐b) Migration (a) or invasion (b) assay showing reduced motility in Huh7 and HepG2 cells treated with COP1‐siRNA, as quantified by the relative migration or invaded area. Statistical significance: *, p < 0.05; ** p < 0.01; *** p < 0.001 vs. control. (c) Sphere formation assay images and quantitative data from Huh7 and HepG2 cells, demonstrating a decrease in both the number and size of spheres in COP1‐siRNA‐treated cells. (d) Sphere formation assay images and quantitative data from PLC/PRF/5 CD133+ cells. Compared to the control grpup, both the sphere number and size decreased upon COP1‐siRNA treatment. Scale bar, 100 μm Statistical significance: *, p < 0.05; *** p < 0.001 vs. control.

Article Snippet: The cells were treated with FcR Blocking Reagent and CD133 MicroBeads (130‐100‐857, Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions and incubated for 15 min at 4°C in the dark.

Techniques: Knockdown, Migration, Control, Tube Formation Assay

Journal: PLoS ONE

Article Title: Reduced variability of neural progenitor cells and improved purity of neuronal cultures using magnetic activated cell sorting

doi: 10.1371/journal.pone.0213374

Figure Lengend Snippet: (A) Representative immunofluorescence images from one NPC line demonstrating a reduction in CD271+ cells and an enrichment of CD133+ positive cells following both FACS and MACS compared to unsorted cells. Cell nuclei are labelled with DAPI. Scale bar = 100μm, N = 6. (B) Combined flow cytometry analysis of percent CD271+, CD271- and CD271-/CD133+ cells from 100% live cells (DAPI negative population) comparing unsorted, FACS and MACS conditions. (C) Flow cytometry analysis in B presented as individual cell lines. (D) Reduction in CD271+ cell variability following MACS compared to unsorted cell s . **p<0.01 Variance F-test. (E) Fold change expression of stress-associated genes in cells following FACS compared to MACS. Dotted line denotes equal expression at fold change = 1. (F) Quantification of the percentage surface area covered by live cells imaged in D . (G) Absorbance read at 450nm of LDH assay carried out on media supernatant collected from cells 24 hours following either FACS or MACS sorting. (H) Cell viability assessed by Calcein Violet staining 24 hours following standard passage (unsorted) or following sorting by either FACS or MACS in two representative NPC lines with different survival responses. Scale bar = 100μm, *p<0.05, n.s = not significant. Error bars ± SEM.

Article Snippet: CD271- cells were re-pelleted and re-suspended in fresh MACS Separation Buffer, then labelled using the Indirect CD133 Microbead kit (Miltenyi Biotech, #130-091-895) for 15 minutes at 4°C.

Techniques: Immunofluorescence, Flow Cytometry, Expressing, Lactate Dehydrogenase Assay, Staining

Journal: PLoS ONE

Article Title: Reduced variability of neural progenitor cells and improved purity of neuronal cultures using magnetic activated cell sorting

doi: 10.1371/journal.pone.0213374

Figure Lengend Snippet: (A-B). Clustering of individual cell lines using multidimensional scaling analysis based on gene expression profiles from custom designed Taqman microfluidic cards. (A)Both FACS (red) and MACS (blue) increase gene expression homogeneity between NPC lines (indicated by black circle) compared to unsorted cells (green). (B) MACS (triangles) also improves gene expression homogeneity across different neuron populations (red), as indicated by the red oval, compared to unsorted cells (squares). NPC populations are colored in blue and the homogeneous cluster is indicated by a blue circle, FACS NPCs are circles. Neurons differentiated from the unsorted F12444-3-2 NPC line that failed both FACS and MACS sorting due to low levels of CD271-/CD133+ cells are indicated with a black arrow. (C) Comparison of gene expression of glial and neuronal specific markers in neurons derived from MACS NPCs compared to neurons derived from unsorted NPCs. (D) Representative immunofluorescence images of neurons derived from NPCs demonstrating the enrichment of neuronal markers Tuj1, NeuN and Tau, as well as the depletion of glial markers GFAP and S100β when differentiated from MACS NPCs compared to unsorted NPCs. Cell nuclei are labelled with DAPI. Scale bar = 100μm, N = 5. (E-F) Flow cytometry analysis for neuronal marker TUJ1 (E) and NeuN (F) on neurons derived from MACS NPCs compared to neurons from unsorted NPCs. Relative index for each marker is generated by the multiplication of total number of fluorescent positive cells and its median fluorescence intensity. (G) Relative index of astrocyte marker, S100β by flow cytometry analysis. *p<0.05, **p<0.01, n.s = not significant. Error bars ± SEM.

Article Snippet: CD271- cells were re-pelleted and re-suspended in fresh MACS Separation Buffer, then labelled using the Indirect CD133 Microbead kit (Miltenyi Biotech, #130-091-895) for 15 minutes at 4°C.

Techniques: Gene Expression, Comparison, Derivative Assay, Immunofluorescence, Flow Cytometry, Marker, Generated, Fluorescence

Journal: PLoS ONE

Article Title: Reduced variability of neural progenitor cells and improved purity of neuronal cultures using magnetic activated cell sorting

doi: 10.1371/journal.pone.0213374

Figure Lengend Snippet: (A) Representative immunofluorescence images from a late passage NPC line demonstrating a visible reduction in CD271+ cells and an enrichment of CD133+ positive cells, as well as an enrichment of NPC markers following MACS. Cell nuclei are labelled with DAPI. Scale bar = 100μm. (B-C) Percentage of late passage NPCs positive for SOX2 (B) and NESTIN (C) as measured by flow cytometry analysis. The summary result is shown in unsorted and MACS NPCs compared to the secondary antibody control and CD271+ cells by pooled results of individual cell lines from . (D) Percent NESTIN+ cells by flow cytometry analysis in early (p2-3) and late passage (p10-13) NPCs. (E) Clustering of individual late passage NPC lines using multidimensional scaling analysis based on gene expression profiles from custom designed Taqman microfluidic cards. MACS (triangles) improves homogeneity across late passage (blue) cell lines compared to unsorted cell lines (squares), and exhibits similar gene expression profiles as early passage (red) MACS and FACS (circles) NPCs, indicated by a dotted circle. (F) Representative immunofluorescence images of neurons differentiated from one late passage NPC line demonstrating an enrichment of neuronal specific markers following MACS, but a maintenance of expression of glial-specific markers. Cell nuclei are labelled with DAPI. Scale bar = 100μm. (G) Comparison of gene expression of glial and neuronal specific markers in neurons derived from late passage MACS NPCs compared to neurons derived from early passage NPCs. *p<0.05, **p<0.01, ***p<0.001. Error bars ± SEM.

Article Snippet: CD271- cells were re-pelleted and re-suspended in fresh MACS Separation Buffer, then labelled using the Indirect CD133 Microbead kit (Miltenyi Biotech, #130-091-895) for 15 minutes at 4°C.

Techniques: Immunofluorescence, Flow Cytometry, Control, Gene Expression, Expressing, Comparison, Derivative Assay

Journal: Biotechnology Journal

Article Title: Benchmarking Ultra‐Low Attachment and Photopatterned GelMA 3D Culture Platforms for Modeling Cancer Stemness in High‐Grade Serous Ovarian Cancer

doi: 10.1002/biot.70287

Figure Lengend Snippet: Spheroid formation, maturation, and functional CSC enrichment in HGSOC cell lines cultured under ultra‐low attachment (ULA) conditions. Representative brightfield microscopy images of OVCAR‐3 and OVSAHO cell lines cultured in ULA plates at relevant time points (4× objective). Yellow arrows indicate highly compartmentalized OVCAR‐3 spheroids, whereas red arrows denote overcrowded aggregates observed at extended culture durations (a). Schematic illustration summarizing spheroid morphologies observed under ULA conditions, including loose aggregates, compact spheroids, and compartmentalized spheroids (b). Quantitative analysis of spheroid diameter distribution and representative size comparison of highly compartmentalized OVCAR‐3 spheroids illustrating intra‐well heterogeneity within ULA cultures at Day 48 (10× objective) (c). Representative confocal microscopy images of an OVCAR‐3 spheroid at Day 48 including brightfield image, confocal z‐sections acquired at ∼2 µm intervals, and corresponding z‐stack projection (63× objective). Blue = DAPI (nuclei), green = α‐tubulin. Individual channels are shown separately to facilitate visualization of marker distribution. CD133‐PE monolayer negative controls and uncropped confocal images are provided in Figure S1 (d). Representative confocal microscopy images of additional aggregates and spheroids from the same culture (63X objective). Blue = DAPI, green = α‐tubulin, red = CD133‐PE. Individual channels are shown separately. CD133‐PE monolayer negative controls and uncropped images are provided in Figure (e). Growth inhibition curves and corresponding IC50 bar graphs for carboplatin, niraparib, paclitaxel, olaparib, cisplatin, and doxorubicin comparing OVCAR‐3 monolayer cultures (black bars) and ULA spheroids (gray bars). Viability was assessed using the NCI‐SRB assay (f). Data are presented as mean ± SD from three independent biological replicates. For SRB assays, each biological replicate contained technical triplicates. Statistical significance was determined using unpaired two‐tailed Student's t ‐tests or one‐way ANOVA where appropriate. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: CD133‐PE (Miltenyi Biotec, equal mixing of 130‐110‐962 and 130‐110‐962, 1:50–1:50 = 2:100) antibody was diluted 1:50 in CellO‐IF and incubation was done at 4°C for 20 min.

Techniques: Functional Assay, Cell Culture, Microscopy, Comparison, Confocal Microscopy, Marker, Inhibition, Sulforhodamine B Assay, Two Tailed Test

Journal: Biotechnology Journal

Article Title: Benchmarking Ultra‐Low Attachment and Photopatterned GelMA 3D Culture Platforms for Modeling Cancer Stemness in High‐Grade Serous Ovarian Cancer

doi: 10.1002/biot.70287

Figure Lengend Snippet: Comparative benchmarking of CSC and EMT‐associated traits in HGSOC cells cultured using ULA and photopatterned GelMA platforms. Representative western blot images and corresponding quantitative bar graphs showing expression of CSC‐associated proteins SOX‐2, ALDH1A1, NANOG, and OCT‐4 in OVCAR‐3 and OVSAHO cultures. Protein levels were normalized to Vinculin and Calnexin, with representative loading control bands shown. Different loading controls were selected depending on target abundance, membrane compatibility, and subcellular localization. Quantification was performed using ImageJ (a). Representative flow cytometry pseudo‐dot plots with gates defined using unstained controls, and bar graphs summarizing the percentage of CD133‐PE, CD44‐FITC, CD117‐PE, and CXCR4‐FITC positive populations. Representative gating strategy is provided in Figure (b). Representative western blot images and quantitative bar graphs of EMT‐associated proteins Slug, Snail, TWIST1, vimentin, and fibronectin in OVCAR‐3 cultures (normalized to calnexin). E‐cadherin and N‐cadherin were additionally analyzed (normalized to α‐tubulin), with quantitative N‐cadherin/E‐cadherin ratios shown (c). Representative western blot analysis of stemness‐associated proteins in OVCAR‐3 cells cultured as 2D monolayer, 2D monolayer in spheroid enrichment medium, UV‐treated monolayer, ULA spheroids, and photopatterned GelMA (GelMA‐Pm) spheroids. Due to limited cell yield obtained from GelMA‐Pm cultures, only SOX‐2 and ALDH1A1 expression could be evaluated in these samples. Protein expression was normalized to GAPDH. Corresponding densitometric quantification is shown on the right. Full‐length uncropped blots are provided in Figure . (d). RT‐qPCR analysis of the stemness‐associated genes SOX‐2, NANOG, ALDH1A1, and c‐MYC in OVCAR‐3 cells cultured under 2D monolayer, 2D + sphere medium, 2D + UV, photopatterned GelMA (GelMA‐Pm), and ULA spheroid conditions. Gene expression levels were normalized to β‐actin and are presented relative to 2D monolayer controls (set to 1). Values above 1 indicate increased expression relative to monolayer cultures. Data represent mean ± SD from three independent biological replicates ( n = 3). Statistical significance was determined using multiple t ‐tests; significance symbols are defined as follows: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) (e). Data are presented as mean ± SD from three independent biological replicates. Statistical significance was determined using unpaired two‐tailed Student's t ‐tests or one‐way ANOVA where appropriate. * p < 0.05, ** p < 0.01, *** p < 0.001. Western blot images were cropped for clarity; non‐adjacent lanes from the same membrane are indicated by spaces. Full‐length blots are provided in Figure .

Article Snippet: CD133‐PE (Miltenyi Biotec, equal mixing of 130‐110‐962 and 130‐110‐962, 1:50–1:50 = 2:100) antibody was diluted 1:50 in CellO‐IF and incubation was done at 4°C for 20 min.

Techniques: Cell Culture, Western Blot, Expressing, Control, Membrane, Flow Cytometry, Quantitative RT-PCR, Gene Expression, Two Tailed Test

Journal: Biotechnology Journal

Article Title: Benchmarking Ultra‐Low Attachment and Photopatterned GelMA 3D Culture Platforms for Modeling Cancer Stemness in High‐Grade Serous Ovarian Cancer

doi: 10.1002/biot.70287

Figure Lengend Snippet: Optimization of photopatterned GelMA hydrogels for spheroid formation and CSC enrichment in HGSOC cell lines. Representative brightfield microscopy images of photopatterned GelMA (GelMA‐Pm) cultures of OVCAR‐3 and OVSAHO cells (upper panels: 4× objective; lower panels: 10× objective), showing spheroid formation under optimized conditions (a). Schematic overview of the GelMA‐Pm fabrication workflow and representative image of square photomask‐generated polymerized GelMA structures visible to the naked eye (b). Quantitative comparison of spheroid number and spheroid diameter per mm 2 between ULA and GelMA‐Pm cultures (c). Representative brightfield microscopy images illustrating approximate spheroid diameter distributions within photopatterned GelMA cultures at Day 14, including representative OVCAR‐3 spheroids (∼100 µm) and smaller OVSAHO spheroids (∼50 µm) (d). Representative confocal microscopy images of GelMA hydrogels containing spheroids from OVCAR‐3 and OVSAHO cultures, demonstrating cell viability and spatial distribution (10× objective, with selected regions magnified to 20X as indicated by yellow boxes). Blue = DAPI (nuclei), green = α‐tubulin, red = CD133‐PE. Corresponding brightfield and fluorescence images of identical fields are shown (e). Data are presented as mean ± SD from three independent biological replicates. Statistical significance was determined using unpaired two‐tailed Student's t‐tests or one‐way ANOVA where appropriate. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: CD133‐PE (Miltenyi Biotec, equal mixing of 130‐110‐962 and 130‐110‐962, 1:50–1:50 = 2:100) antibody was diluted 1:50 in CellO‐IF and incubation was done at 4°C for 20 min.

Techniques: Microscopy, Generated, Comparison, Confocal Microscopy, Fluorescence, Two Tailed Test