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: Cancers

Article Title: Evaluation of the Chemotherapy Drug Response Using Organotypic Cultures of Osteosarcoma Tumours from Mice Models and Canine Patients

doi: 10.3390/cancers13194890

Figure Lengend Snippet: Flow cytometry analysis and fluorescent microscopy of organotypic slices cellular sub-populations at day 0, 4, and 8. ( a ) CD45+ immune cells as a % of total cells. ( b ) Breakdown of mononuclear phagocyte cells as a % of CD45+ population with Ly6C+ low/med macrophages, Ly6C+ high monocytes, and CD11c+ dendritic cells. ( c ) Breakdown of Ly6C+ F4/80+ I-A/I-E low non-activated macrophages and F4/80+ I-A/I-E high activated macrophages (M1, M2). ( d ) sca1+ CD140+ CAFs as a % of total cell population. ( e ) Organotypic slices labelled with DAPI (nuclei, blue), phalloidin (F-actin, red), cleaved caspase-3 (apoptosis, pink), and F4/80 (macrophage marker, green) or sca1 (CAF marker, green). Data is expressed as Mean ± SD of n = 3 samples. p -values; ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: As doxorubicin has an autofluorescence property, another multicolor panel had to be set up to monitor the impact of doxorubicin on stromal cells: CD45 BV421 (BD biosciences) at 1/2000, Ly6A/E FITC (BD Biosciences) at 1/100, TER 119 APC (BD Biosciences) at 1/100, CD140 BUV395 (BD Biosciences) at 1/400 and Nir Zombie as a viability dye.

Techniques: Flow Cytometry, Microscopy, Marker