Journal: Molecular and Cellular Biology

Article Title: Senescent Breast Luminal Cells Promote Carcinogenesis through Interleukin-8-Dependent Activation of Stromal Fibroblasts

doi: 10.1128/MCB.00359-18

Figure Lengend Snippet: List of antibodies utilized in this study

Article Snippet: The utilized antibodies and their sources are listed in . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Antibody Company Anti-Twist1 (10E4E6) Abcam Anti-lamin B1 (119D5-F1) Abcam Anti-lamin A (131C3) Abcam Anti-α-SMA Abcam Anti-IL-6 Abcam Anti-IL-8 (EPR1116(2)) OriGene Anti-Ki-67 Abcam Anti-AUF-1 (hnRNPD/AUF1) Abcam Antivimentin (RV202) Abcam Anti-ZEB1 (4C4) Abnova Corporation Anti-p21 (F-5) Santa Cruz Biotechnology Anti-p53 (DO-1) Santa Cruz Biotechnology Anti-PCNA (PC-10) Santa Cruz Biotechnology Anti-ALDH1/2 (H-85) Santa Cruz Biotechnology Anti-CD24 (C-20) Santa Cruz Biotechnology Anti-GAPDH (14C10) Cell Signaling Technology Anti-p16 BD Biosciences Anti-E-cadherin (24E10) Cell Signaling Technology Anti-N-cadherin (D4R1H) XP Cell Signaling Technology Anti-AKT (C73H10) Cell Signaling Technology Anti-p-AKT (Thr308) Cell Signaling Technology Anti-ERK1/2 (137F5) Cell Signaling Technology Anti-P-ERK1/2 (Thr202/Tyr204)(197G2) Cell Signaling Technology Anti-TGF-β (2Ar2) Abcam Anti-VEGF-A Abcam Anti-JAK2 (D2E12)XP Cell Signaling Technology Anti-P-JAK2 (Tyr1007/1008) Cell Signaling Technology Anti-Nanog (D73G4)XP Cell Signaling Technology Anti-Oct-4 (C30A3) Cell Signaling Technology Anti-KLF4 (D1F2) Cell Signaling Technology Anti-Sox2 (D6D9)XP Cell Signaling Technology Anti-Bmi1 (D20B7)XP Cell Signaling Technology Anti-HIF-1α Cell Signaling Technology Anti-CD34 Abcam Anti-Stat3 (124H6) Cell Signaling Technology Anti-P-Stat3 (Tyr705) (D3A7) Cell Signaling Technology Anti-SDF1 Cell Signaling Technology Anti-EpCAM (D1B3) Cell Signaling Technology Anti-CD44 Sigma-Aldrich Anti-cytokeratin 14 (LL002) Abcam Anti-cytokeratin 19 (EP1580Y) Abcam Anti-cytokeratin 18 (DC 10) Abcam Anti-cytokeratin 5/6 (D5/16 B4) Abcam Anti-c-Kit Abcam FITC–mouse anti-CD90 BD Biosciences Anti-CD326 (EpCAM)–APC Miltenyi Biotec PE-Cy7–mouse anti-human CD10 BD Biosciences APC-Cy7–anti-mouse/human CD44 Biolegend FITC–mouse anti-CD24 BD Biosciences PE–rat anti-CD49 BD Biosciences FITC–mouse anti-MUC-1 BD Biosciences Mouse anti-human CXCL8/IL-8 neutralizing antibody R&D Systems Normal mouse IgG control antibody Millipore Alexa Fluor 594–goat anti-rabbit IgG Life Technologies Alexa Fluor 488–goat anti-mouse IgG Life Technologies Anti-mouse IgG–HRP conjugate Promega Anti-rabbit IgG–HRP conjugate Promega Donkey anti-goat IgG–HRP Promega Open in a separate window List of antibodies utilized in this study

Techniques: Control

Journal: Toxicology reports

Article Title: Silver nanoparticles induces apoptosis of cancer stem cells in head and neck cancer.

doi: 10.1016/j.toxrep.2023.11.008

Figure Lengend Snippet: Fig. 4. CSC isolated from Cal33 characterization by Flow Cytometry. Population 1 and Single cell dot plots show the gating strategy for events of interest and eliminating doublets and triplets. Untreated is a negative control for CD44+PE and CD24+APC staining. Before CSC isolation shows 55% of CD44+ and CD24 low

Article Snippet: FITC Annexin V Apoptosis Detection Kit (Cat no. 640914) was purchased from BD Bioscience, MagCellect Streptavidin Ferrofluid (Cat no. MAG999B), CD24 Biotinylated Antibody (Cat no. FAB52471B), Human CD24 APC-conjugated Antibody (Cat no. FAB5247A) and Human CD44 PE-conjugated Antibody (Cat no. FAB8547P) from R&D Systems Minneapolis, Minnesota, United States.

Techniques: Isolation, Flow Cytometry, Negative Control, Staining

Journal: Breast Cancer Research : BCR

Article Title: Nestin positively regulates the Wnt/β-catenin pathway and the proliferation, survival and invasiveness of breast cancer stem cells

doi: 10.1186/s13058-014-0408-8

Figure Lengend Snippet: Nestin high breast CSC have potent tumorigenicity. The isolated breast CD44 + CD24 − CSC from 12 freshly resected breast cancer tissues were stained with anti-Nestin, and the Nestin + and Nestin − breast CSC were purified as Nestin high or Nestin low CSC by flow cytometry, respectively. The levels of Nestin expression in Nestin high , Nestin low , and unstained control CSC were determined by western blotting, and these CSC were tested for their capacity to form mammospheres in vitro and to induce solid tumors in vivo . Finally, the levels of Nestin expression in the solid tumors induced by Nestin high , Nestin low , or control CSC were determined by western blotting. Data are representative charts and images, and expressed as the mean ± SD of each group of samples (n = 12 per group). (A) Flow cytometric analysis of CSC. (B) Western blot analysis of the levels of Nestin expression. (C) Mammosphere formation. (D) The rates of tumor formation in SCID mice (n = 10 animals per group) from three separate experiments. (E) Western blot analysis of Nestin expression in the formed tumors (n = 2 to 10 per group). CSC, cancer stem cell; SCID, severe combined immunodeficiency; SD, standard deviation.

Article Snippet: The purified CD44 + CD24 − breast CSC from 26 specimens were transfected with human Nestin-specific (sc-36032, a mixture of sc-36032A, sense 5′-CGAGGUCUUUAGAAGAAGAtt-3′ and antisense 5′-UCUUCUUCUAAAGACCUCGtt-3′; sc-36032B, sense 5′-GCCUUUAGAUCUCUAGAAAtt-3′ and antisense 5′- UUUCUAGAGAUCUAAAGGCtt-3; sc-36032C, sense 5′- GGCAAUGAAUCCUCUAGAAtt-3′ and antisense 5′-UUCUAGAGGAUUCAUUGCCtt-3′) or control small interfering RNA (siRNA) (sc-37007, Santa Cruz Biotechnology) using Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturers’ protocols.

Techniques: Isolation, Staining, Purification, Flow Cytometry, Expressing, Control, Western Blot, In Vitro, In Vivo, Standard Deviation

Journal: Breast Cancer Research : BCR

Article Title: Nestin positively regulates the Wnt/β-catenin pathway and the proliferation, survival and invasiveness of breast cancer stem cells

doi: 10.1186/s13058-014-0408-8

Figure Lengend Snippet: Nestin silencing inhibits the mammosphere formation, induces cell cycle arrest at G2/M and promotes apoptosis in breast CSC. The isolated CD44 + CD24 − breast CSC from 26 specimens were transfected with control siRNA, Nestin-specific siRNA, pIRES-Nestin-EGFP, or control pIRES-EGFP to generate Nestin-c, Nestin-si, Nestin + or Nestin-EGFP cells. The Nestin-c, Nestin-si, and Nestin + CSC were tested for the levels of Nestin expression by western blotting and for their ability to form mammospheres in vitro. Their cell cycling progression and spontaneous apoptosis were determined by flow cytometry. Data are representative charts and images, and expressed as the mean ± SD of each group (n = 26 per group). (A) . Flow cytometric analysis of CD44 + CD24 − CSC. (B) Western blot analysis of Nestin expression in breast CSC. Nestin-EGFP and Nestin-c control cells showed similar levels of Nestin expression (data not shown). (C) The mammosphere formation. (D) Cell cycle analysis of Nestin-si and Nestin-c breast CSC; (E) apoptotic Nestin-si and Nestin-c breast CSC. * P <0.05 vs. the control. CSC, cancer stem cell; SD, standard deviation; siRNA, small interfering RNA.

Article Snippet: The purified CD44 + CD24 − breast CSC from 26 specimens were transfected with human Nestin-specific (sc-36032, a mixture of sc-36032A, sense 5′-CGAGGUCUUUAGAAGAAGAtt-3′ and antisense 5′-UCUUCUUCUAAAGACCUCGtt-3′; sc-36032B, sense 5′-GCCUUUAGAUCUCUAGAAAtt-3′ and antisense 5′- UUUCUAGAGAUCUAAAGGCtt-3; sc-36032C, sense 5′- GGCAAUGAAUCCUCUAGAAtt-3′ and antisense 5′-UUCUAGAGGAUUCAUUGCCtt-3′) or control small interfering RNA (siRNA) (sc-37007, Santa Cruz Biotechnology) using Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturers’ protocols.

Techniques: Isolation, Transfection, Control, Expressing, Western Blot, In Vitro, Flow Cytometry, Cell Cycle Assay, Standard Deviation, Small Interfering RNA

Journal: Journal of Cellular and Molecular Medicine

Article Title: Higher expression of SATB2 in hepatocellular carcinoma of African Americans determines more aggressive phenotypes than those of Caucasian Americans

doi: 10.1111/jcmm.14652

Figure Lengend Snippet: Inhibition of SATB2 expression suppresses cell viability in spheroids and stem cell markers and pluripotency maintaining factors in HepG2 and Hep3B cells. A, CSCs derived from primary HCC tissues were transduced with lentiviral particles expressing either SATB2 Crispr/Cas9 or non‐targeting control from a pooled population by targeting four different sites (GeneCopoeia, Rockville, MD). RNA was extracted from cells, and qRT‐PCR analysis was performed to measure the expression of SATB2. Data represent mean ± SD (n = 4). * = significantly different from NTC group ( P < .05). B, CSCs/NTC and CSCs/SATB2 Crispr/Cas9 cells were grown for 6 d, and cell viability in spheroids was measured by XTT assay. Data represent mean ± SD (n = 4). * = significantly different from NTC group ( P < .05). C and D, RNA was isolated from CSCs/NTC and CSCs/SATB2 Crispr/Cas9 cells. The qRT‐PCR analysis was performed to measure the expression of stem cell markers (CD133, CD44 and CD24) and pluripotency maintaining factors (c‐Myc, KLF4, SOX2 and OCT4). GAPDH was used as an internal control. Data represent mean ± SD (n = 4). * = significantly different from CSCs/NTC group, P < .05. Note that the expression of genes in the control group was normalized to 1

Article Snippet: CD24+/CD44+/CD133+ human liver cancer stem cells (CSCs) were isolated from primary HCC (obtained from Celprogen) and grown in well‐defined stem cell culture medium as per the supplier's instructions.

Techniques: Inhibition, Expressing, Derivative Assay, Transduction, CRISPR, Quantitative RT-PCR, XTT Assay, Isolation