Journal: The Journal of Experimental Medicine

Article Title: Dual T cell– and B cell–intrinsic deficiency in humans with biallelic RLTPR mutations

doi: 10.1084/jem.20160576

Figure Lengend Snippet: T cell immunophenotyping of RLTPR-deficient patients. (a) Frequency of naive (CD45RA + CCR7 + ), central memory (CD45RA − CCR7 + ), and effector memory (mem.; CD45RA +/− CCR7 − ) compartments in CD4 + and CD8 + T cells of controls (Ctl) and patients. The horizontal bars represent the median. (Right) Representative FACS plots are depicted. Data show 20 controls and 6 patients. (b) Frequency of T reg cells among CD4 + T cells. Pat, patient. (c–e) Frequencies of MAIT cells (c), γδ T cells (d), and iNKT cells (e) among CD3 + T cells. (b and c) Data show 16 controls and 5 patients. (d and e) Data show 12 controls and 4 patients. (f) Frequency of Th cell subsets in CD4 + T cells. The different markers used to identify Th cell subsets are indicated above the plots. Data show 16 controls and 5 patients. (g) Frequency of CRTH2 + subset in CD4 + T cells. Data show 12 controls and 3 patients. Each symbol corresponds to an individual patient or healthy control. The horizontal bar represents the mean. (a–f) A Mann–Whitney test was used. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Cells were fixed for 10 min at 37°C using Fix Buffer I (BD) and stained for 30 min with mAb against CD3 (BW264/56; Miltenyi Biotec), CD4 (M-T321; Miltenyi Biotec), and CD8 (BW135/80; Miltenyi Biotec).

Techniques: Control, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: Dual T cell– and B cell–intrinsic deficiency in humans with biallelic RLTPR mutations

doi: 10.1084/jem.20160576

Figure Lengend Snippet: Impaired CD28 co-stimulation in patients’ CD4 + T cells. (a) Frequency of TNF + , IFN-γ + , and IL-2 + CD4 + memory T cells in healthy controls and patients after stimulation with the P815 cell line in the presence of 5 µg/ml anti-CD3 and/or 5 µg/ml anti-CD28 mAbs. 40 ng/ml PMA and 10 −5 M ionomycin (Iono) stimulation was used as a positive control. One-way ANOVA and Mann–Whitney tests were used. (b) Frequency of TNF + , IFN-γ + , and CD107a + CD8 + memory T cells in controls and patients after stimulation with the P815 cell line in the presence of 5 µg/ml anti-CD3 and/or 5 µg/ml anti-CD28 mAbs. 40 ng/ml PMA and 10 −5 M ionomycin stimulation was used as a positive control. One-way ANOVA and Mann–Whitney tests were used. (a and b) Data show 20 controls and 6 patients. (c and d) Phospho-P65 (p-P65) detection by flow cytometry in CD4 + (c) and CD8 + (d) PHA blasts after cross-linking of indicated cell surface receptors. Representative FACS plot (left) and recapitulative bar graphs of eight controls (Ctl) and four patients (A3, B1, B2, and C1; right) are shown. The values represent the mean ± SEM. Wilcoxon matched-pairs signed rank test and Mann–Whitney tests were used. *, P < 0.05; **, P < 0.01; ***, P < 0.001. stim, simulated; unstim., unstimulated.

Article Snippet: Cells were fixed for 10 min at 37°C using Fix Buffer I (BD) and stained for 30 min with mAb against CD3 (BW264/56; Miltenyi Biotec), CD4 (M-T321; Miltenyi Biotec), and CD8 (BW135/80; Miltenyi Biotec).

Techniques: Positive Control, MANN-WHITNEY, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Dual T cell– and B cell–intrinsic deficiency in humans with biallelic RLTPR mutations

doi: 10.1084/jem.20160576

Figure Lengend Snippet: Proliferation and differentiation of CD4 + Th cells in vitro. (a) CFSE dilution of naive CD4 + T cells sorted from a representative control (Ctl) and patient (Pat; B2) after 4 d of culture in the presence of CD2/CD3/CD28-coated beads (Th0). (b) Percent IL-2 + naive CD4 + T cells after 4 d of culture under Th0 conditions. Data are mean ± SEM. *, P < 0.05. n = 4–5. (c) CFSE dilution of naive CD4 + T cells sorted from a representative control and patient (B2) after 4 d of culture under Th1 (IL-12), Th2 (IL-4), or Th17 (IL-1β, -6, -21, and -23; TGF-β) cell–polarizing conditions. Similar results were obtained when cells from three additional RLPTR-deficient cells were analyzed under the same conditions. (d) CFSE dilution of memory CD4 + T cells sorted from a healthy control or RLTPR-deficient patient (B2) and then cultured for 4 d in the presence of CD2/CD3/CD28-coated beads (Th0). (e) Percent IL-2 + memory CD4 + T cells after 4 d of culture under Th0 conditions. CFSE profiles are representative of data derived from five independent experiments using cells from different donors and patients. Data are mean ± SEM. (f) Expression of BCL2 , BCL2L1 (Bcl-xL), and BCL2L11 (BIM) by activated CD4 + T cells from five controls and four RLTPR-deficient patients, as determined by qRT-PCR. The values represent the mean ± SEM mRNA levels of the indicated gene expressed by naive (left) and memory (right) CD4 + T cells relative to that expressed by corresponding cells from healthy controls (normalized to 1, indicated by the solid horizontal line; n = 2–3). (g–i) Secretion of Th1 (TNF and IFN-γ) and Th17 (IL-17A, IL-17F, and IL-22) cytokines (g), IL-6 and IL-10 (h), and Th2 (IL-4, IL-5, and IL-13) cytokines (i) by memory CD4 + T cells after 4 d of culture under Th0 conditions. The values represent the mean ± SEM from independent experiments using cells from five different healthy donors or four patients. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. n = 4–5. (j) Secretion of the indicated Th1 (TNF and IFN-γ), Th2 (IL-5 and IL-13), and Th17 (IL-17A, IL-17F, and IL-22) cytokines by naive CD4 + T cells after 4-d culture under Th1, Th2, or Th17 cell–polarizing conditions. The values represent the mean ± SEM and are derived from independent experiments using cells from five different healthy donors or four patients. *, P < 0.05; **, P < 0.01. (k) Expression of TBX21 (Tbet), GATA3 , and RORC (RORγt), as determined by qRT-PCR, by control and RLTPR-deficient naive CD4 + T cells after culture under Th0, Th1, Th2, or Th17 conditions. The values represent the mean ± SEM fold change in expression relative to naive CD4 + T cells from healthy controls cultured under Th0 conditions ( n = 2–3). (l) CD40L frequency and ICOS MFI on sorted naive CD4 + T cells from four patients and three controls after 4 d of culture under Th0 conditions. MFI was normalized to controls’ MFI. Two-way ANOVA was applied for g, I, and j. A Mann–Whitney test was applied for h.

Article Snippet: Cells were fixed for 10 min at 37°C using Fix Buffer I (BD) and stained for 30 min with mAb against CD3 (BW264/56; Miltenyi Biotec), CD4 (M-T321; Miltenyi Biotec), and CD8 (BW135/80; Miltenyi Biotec).

Techniques: In Vitro, Control, Cell Culture, Derivative Assay, Expressing, Quantitative RT-PCR, MANN-WHITNEY

Journal: Cancer research

Article Title: CIP2A modulates cell-cycle progression in human cancer cells by regulating the stability and activity of Plk1.

doi: 10.1158/0008-5472.CAN-13-0888

Figure Lengend Snippet: Figure 1. CIP2A is a cell-cycle regulated protein. A, HeLa cells were synchronized by either a double thymidine block or nocodazole block, released into fresh medium at indicated time points, and then analyzed by immunoblotting with antibodies against the indicated proteins. Synchronization and progression through the cell cycle was confirmed by fluorescence-activated cell sorting analysis. B, HeLa cells were stained with anti-CIP2A antibody (green), anti-pericentrin antibody (red), and DAPI (DNA, blue). C, an enlarged single-cell image from HeLa cells stained with anti-CIP2A antibody (green) and DAPI (blue). D, H1299 cells were transfected with the PTEN-expressing construct or empty vector. E, Hs68 cells were transfected with either control (Ctrl) siRNA or Plk1 siRNA. D and E, after transfection for 48 hours, PTEN-induced G1 arrest or Plk1 depletion–induced G2–M arrest was analyzed by immunoblotting with antibodies against the indicated proteins or by fluorescence-activated cell sorting analysis, respectively. F, Hs68 cells were stained with anti-CIP2A antibody (green) and anti–phospho-H3 antibody (red) or anti-cyclin B1 antibody (red) with DAPI (blue). Data shown represent typical results from at least four independent experiments. Scale bars, 10 mm.

Article Snippet: Briefly, cells were lysed by NP-40 lysis buffer and the lysates were then precipitated with negative control mouse antibody (Santa Cruz Biotechnology, Inc.) or mouse monoclonal antibody against either CIP2A or Plk1 (Santa Cruz Biotechnology, Inc.).

Techniques: Blocking Assay, Western Blot, FACS, Staining, Transfection, Expressing, Construct, Plasmid Preparation, Control

Journal: Cancer research

Article Title: CIP2A modulates cell-cycle progression in human cancer cells by regulating the stability and activity of Plk1.

doi: 10.1158/0008-5472.CAN-13-0888

Figure Lengend Snippet: Figure 2. CIP2A depletion blocks nocodazole-induced mitotic arrest. A, HeLa cells were transfected with the indicated siRNAs (top) or transfected with control (Ctrl) siRNA, CIP2A.1 siRNA, or both CIP2A.1 siRNA and CIP2Arv (bottom) for 48 hours and then analyzed by immunoblotting with anti-CIP2A antibody. B, C, F, and G, HeLa cells were transfected with the indicated siRNAs or Flag-CHFR vector for 36 hours and then incubated with 100 ng/mL nocodazole for 16 hours. MAD2 siRNA or the Flag-CHFR vector was used for a positive control of spindle checkpoint regulation or premitotic regulation, respectively. Cells were analyzed by light microscopy (B, left), fluorescence-activated cell sorting analysis (B, right), or immunoblotted with antibodies against the indicated proteins (C). D and E, HeLa cells were transfected with control (Ctrl) siRNA, CIP2A.1 siRNA, or both CIP2A.1 siRNA and CIP2Arv for 48 hours and then incubated with 100 ng/mL nocodazole for 16 hours. Nocodazole-treated cells were stained with anti-CIP2A antibody (green) and anti–phospho-H3 antibody (red) with DAPI (blue; D). E, the mitotic index was determined by the percentage of phospho-H3–positive cells (bottom) or by FACS (top) and was quantified using CellProfiler software (300 cells for each data point, n ¼ 3; , P < 0.001). F, nocodazole-treated cells were fixed with DAPI (blue) and the percentage of nonmitotic cells with multilobed or interphase nuclei was quantified (300 cells for each data point, n ¼ 3; , P < 0.01). G, representative confocal images of the cells that indicate the different nuclear morphologies, including mitotic arrest, checkpoint bypass, or premitotic arrest. Arrows, multilobed nuclei. Data shown represent typical results from at least three independent experiments. Scale bars, 20 mm. p-H3–positive, phospho-H3–positive.

Article Snippet: Briefly, cells were lysed by NP-40 lysis buffer and the lysates were then precipitated with negative control mouse antibody (Santa Cruz Biotechnology, Inc.) or mouse monoclonal antibody against either CIP2A or Plk1 (Santa Cruz Biotechnology, Inc.).

Techniques: Transfection, Control, Western Blot, Plasmid Preparation, Incubation, Positive Control, Light Microscopy, FACS, Staining, Software

Journal: Cancer research

Article Title: CIP2A modulates cell-cycle progression in human cancer cells by regulating the stability and activity of Plk1.

doi: 10.1158/0008-5472.CAN-13-0888

Figure Lengend Snippet: Figure 3. CIP2A depletion results in delay of mitotic entry and mitotic abnormalities. A, schematic of cell synchronization protocol by double thymidine block and for transfection with siRNA (top). A and B, HeLa cells were transfected with control (Ctrl) siRNA or CIP2A.1 siRNA, synchronized at the G1–S phase by a double thymidine block and released from the secondary thymidine block into medium with or without 100 ng/mL nocodazole (treated 6 hours after release). A, the mitotic index of control- or CIP2A-depleted cells was expressed as the percentage of phospho- H3–positive cells (500 cells at each time point; bottom). B, cells were analyzed by fluorescence-activated cell sorting (FACS) analysis (top) or immunoblotted with the indicated antibodies (bottom). C and D, control (Ctrl) or CIP2A shRNA knockdown cells expressing FUCCI probes were synchronized by double thymidine block and released with fresh medium. C, the time of mitotic entry was determined by observing mitotic cell rounding with signs of DNA condensation and was monitored by time-lapse microscopy (left). Stable knockdown of CIP2A was determined by immunoblotting with anti-CIP2A antibody (right). D, representative time-lapse images of control (Ctrl) or CIP2A shRNA knockdown cells expressing FUCCI probes during cell-cycle progression. E–H, HeLa cells were transfected with either control (Ctrl) siRNA or CIP2A.1 siRNA for 72 hours. E, CIP2Arv was cotransfected with CIP2A.1 siRNA for the rescue of CIP2A. The percentage of normal and aberrant nuclei was quantified using fluorescence microscopy (500 cells for each data point). F, representative images of CIP2A-depleted cells stained with anti-CIP2A antibody (green) and DAPI (blue). Arrows indicate aberrant nuclei. G, CIP2A-depleted cells were scored for abnormal mitosis (200 mitotic cells for each data point, n ¼ 4, error bars, SD). H, representative images of a normal mitotic cells (i) and CIP2A-depleted mitotic cells (ii–vi) stained with anti-CIP2A antibody (green), anti-pericentrin antibody (red), and DAPI (blue). The data shown represent typical results from at least three independent experiments. Scale bars, 10 mm. , P < 0.001. p-H3–positive, phospho-H3–positive; Noc, nocodazole.

Article Snippet: Briefly, cells were lysed by NP-40 lysis buffer and the lysates were then precipitated with negative control mouse antibody (Santa Cruz Biotechnology, Inc.) or mouse monoclonal antibody against either CIP2A or Plk1 (Santa Cruz Biotechnology, Inc.).

Techniques: Blocking Assay, Transfection, Control, FACS, shRNA, Knockdown, Expressing, Time-lapse Microscopy, Western Blot, Microscopy, Staining

Journal: Cancer research

Article Title: CIP2A modulates cell-cycle progression in human cancer cells by regulating the stability and activity of Plk1.

doi: 10.1158/0008-5472.CAN-13-0888

Figure Lengend Snippet: Figure 4. CIP2A binds to Plk1 during mitosis. A and B, HeLa cells were transfected with control (Ctrl) siRNA, CIP2A.1 siRNA, or both CIP2A.1 siRNA and Flag-Plk1 vector for 48 hours and then incubated with 100 ng/mL nocodazole for 16 hours. Cells were analyzed by light microscopy (A) or immunoblotted with antibodies against the indicated proteins (B). C, lysates of HeLa cells were immunoprecipitated with anti-CIP2A antibody, anti-Plk1 antibody, or their respective control immunoglobulin G (IgG) antibodies and immunoblotted with anti-CIP2A or anti-Plk1 antibody. D, lysates of HeLa cells released from thymidine block for the indicated times were immunoprecipitated with anti-CIP2A antibody and immunoblotted with anti-Plk1 or anti-CIP2A antibody. E, lysates of 293T cells expressing Flag, Flag-tagged Plk1 (WT), polo-box–mutant (FAA) Plk1, N-terminal (N) Plk1, and C-terminal (C) Plk1 with Strep-CIP2A were pulled down with Strep-Tactin beads. The Flag-Plk1 protein associated with Strep-CIP2A was detected by immunoblotting with anti-Flag antibody. F–I, HeLa cells were fixed and incubated with mouse anti-CIP2A antibody together with rabbit anti-phospho-Plk1 (Thr210) antibody (top) or rabbit anti-CIP2A antibody together with mouse anti-Plk1 antibody (bottom), followed by in situ PLA analysis. Arrows indicate mitotic cells (F). G and I, dots per cell were counted using CellProfiler (100 cells for each data point; G) or 20 single cells in each respective cell-cycle phase (I); error bars, SD, , P < 0.001). The variation in the number of signal dots between G and I was due to the size difference between multiple- and single-cell images. H, representative confocal images of cells with PLA-positive signals in each respective cell-cycle phase. Data shown represent typical results from at least three independent experiments. Scale bars, 20 mm. p-MPM2, phospho-MPM2.

Article Snippet: Briefly, cells were lysed by NP-40 lysis buffer and the lysates were then precipitated with negative control mouse antibody (Santa Cruz Biotechnology, Inc.) or mouse monoclonal antibody against either CIP2A or Plk1 (Santa Cruz Biotechnology, Inc.).

Techniques: Transfection, Control, Plasmid Preparation, Incubation, Light Microscopy, Immunoprecipitation, Blocking Assay, Expressing, Mutagenesis, Western Blot, In Situ

Journal: Cancer research

Article Title: CIP2A modulates cell-cycle progression in human cancer cells by regulating the stability and activity of Plk1.

doi: 10.1158/0008-5472.CAN-13-0888

Figure Lengend Snippet: Figure 5. CIP2A regulates the stability and activity of Plk1 during mitosis. A, HeLa cells were transfected with the indicated siRNAs, synchronized, and treated with cycloheximide (CHX), which was added 7 hours after release from G1–S. At the indicated times after the addition of cycloheximide, cells were analyzed by immunoblotting with the indicated antibodies. B, the levels of Plk1 were quantified using ImageJ software (n ¼ 3; error bars, SD, , P < 0.01). C, at 7 hours after release from G1–S, cells were treated with MG132 for 3 hours. D, cells cotransfected with vectors for HA-ubiquitin (Ub) and empty or CIP2A-Myc were released from G1–S for 1 hour (interphase) or 7 hours (mitosis) and MG132 was added for 3 hours. Lysates of the cells were immunoprecipitated with anti- Plk1 antibody and immunoblotted with anti-HA antibody. E, HeLa cells transfected with the indicated siRNAs targeting CIP2A, Cdh1, or Cdc20 were synchronized at the G1–S phase, released for 10 hours and then analyzed by immunoblotting with the indicated antibodies. F, HeLa cells transfected with control (Ctrl) siRNA or CIP2A.1 siRNA were synchronized by a double thymidine block and released into fresh medium. Cells were analyzed by immunoblotting with the indicated antibodies. G, purified Strep-CIP2A protein (1mg) incubated with or without recombinant His-Plk1 (0.1 mg) was used as thecontrol to exclude the possibility that purified Strep-CIP2A protein was associated with kinases that can phosphorylate casein (left). Recombinant His-Plk1 (0.1 mg) was incubated with 50 mmol/L BI2536 alone or with serial concentrations of purified Strep-CIP2A proteins (0.25, 0.5, or 1 mg) for 30 minutes (right). Plk1 activity was determined by in vitro kinase assay using casein as a substrate. Data shown represent typical results from at least three independent experiments.

Article Snippet: Briefly, cells were lysed by NP-40 lysis buffer and the lysates were then precipitated with negative control mouse antibody (Santa Cruz Biotechnology, Inc.) or mouse monoclonal antibody against either CIP2A or Plk1 (Santa Cruz Biotechnology, Inc.).

Techniques: Activity Assay, Transfection, Western Blot, Software, Ubiquitin Proteomics, Immunoprecipitation, Control, Blocking Assay, Incubation, Recombinant, In Vitro, Kinase Assay

Journal: Cancer research

Article Title: CIP2A modulates cell-cycle progression in human cancer cells by regulating the stability and activity of Plk1.

doi: 10.1158/0008-5472.CAN-13-0888

Figure Lengend Snippet: Figure 6. Association between CIP2A and Plk1 in lung, gastric, colon, and cervical cancers and their normal tissue counterparts. A, quantification of CIP2A and Plk1 staining intensities in lung, gastric, colon, and cervical cancers and their normal tissue counterparts. B and C, representative microscopic images of lung, gastric, colon, and cervical cancers and their normal tissue counterparts stained with anti-CIP2A antibody (B) or anti-Plk1 antibody (C). Scale bars, 200 mm. D, representative high-magnification images of gastric cancer tissues stained with the indicated antibodies. Scale bars, 100 mm. E and F, tissue sections were incubated with rabbit anti-CIP2A antibody together with mouse anti-Plk1 antibody followed by in situ PLA analysis. Representative confocal images of lung, gastric, colon, and cervical cancers and their normal tissue counterparts (E, left and F). Scale bars, 20 mm. Four different areas were obtained for each sample and 200 to 500 cells were quantified per area (mm2) using CellProfiler (E, right). Nuclei were stained with Hoechst (blue). The green signal represents autofluorescence. N, normal lung tissue. C, cancer tissue. A and E (left), data are presented as box-and-whisker plots. , P < 0.001 compared with their normal tissue counterparts.

Article Snippet: Briefly, cells were lysed by NP-40 lysis buffer and the lysates were then precipitated with negative control mouse antibody (Santa Cruz Biotechnology, Inc.) or mouse monoclonal antibody against either CIP2A or Plk1 (Santa Cruz Biotechnology, Inc.).

Techniques: Staining, Incubation, In Situ, Whisker Assay

Journal: International Journal of Clinical and Experimental Pathology

Article Title: MAPK/ERK signal pathway involved expression of COX-2 and VEGF by IL-1? induced in human endometriosis stromal cells in vitro

doi:

Figure Lengend Snippet: Expression of COX-1 by 4 cytokines in endometriosis ESC by Western Blotting in vitro. Expression of COX-1 was expressed in three ESC, the normal was higher than the eutopic or ectopic, and there was statistical difference (P<0.05). The eutopic and ectopic had no statistical difference (P>0.05). And the expression of COX-1 had no statistical differences after 4 kinds CK (IL-1β, TNF-α, IFN-γ and M-CSF) was added (P>0.05).

Article Snippet: Macrophage colony stimulating factor (MCSF) and vascular endothelial growth factor (VEGF) development ELISA Kit were from PeproTech, Inc (Rocky Hill, NJ, USA), protein assay kit was from Bio-RAD (Hercules, CA, USA), antibody against COX-2 was from Cayman Chemicals (Ann arbor, Mi, USA), antibody against COX-1 was from Santa Cruz Biotechnology (Santa Cruz, CA, USA), the second antibody, a horseradish peroxidase-conjugated anti-mouse antibody and ECL plus western blotting detection system were obtained from Amersham (Piscataway, NJ, USA).

Techniques: Expressing, Western Blot, In Vitro

Journal: Scientific reports

Article Title: Transcription factor profiling identifies Sox9 as regulator of proliferation and differentiation in corneal epithelial stem/progenitor cells.

doi: 10.1038/s41598-018-28596-3

Figure Lengend Snippet: Figure 2. Immunohistochemical localization of SoxE family members in corneoscleral tissue sections. (A) Immunofluorescence microscopy demonstrates nuclear staining for Sox8 (clone 4E4.1) and Sox9 (clone 3C10) in suprabasal epithelial cells at the limbus (left column) and central cornea (right column), whereas Sox10 (clone BC34) is confined to few cells in the basal limbal epithelium. Higher magnification images of basal limbal regions (middle column), as indicated by boxed areas, show differential cytoplasmic (arrows) and nuclear localization of Sox8 and Sox9 in basal and suprabasal limbal epithelial cells. (B) High magnification images of individual channels show cytoplasmic localization of Sox9 in basal stem/progenitor cell clusters and nuclear localization in suprabasal limbal epithelial cells. (C) Double labeling experiments show nuclear co- localization of Sox8 (rabbit IgG) and Sox9 (clone 3C10) (left), distinct localization of Sox9 (clone 3C10) and Sox10 (rabbit IgG) (middle), and localization of Sox10 (clone BC34) to Melan A-positive cells (right). Nuclear counterstaining: DAPI. Individual channels of double labeling experiments are shown in Supplementary Fig. 1.

Article Snippet: Membranes were blocked with SuperBlock T20 Blocking Buffer (Thermo Fisher Scientific) for 1 hour and incubated overnight using monoclonal mouse antibodies against Sox9 (1:5000; clone 3C10; BioRad), Cytokeratin 3/76 (1:5000; clone AE5; Millipore), Cytokeratin 15 (1:1000; clone EPR1614Y; Abcam) and PCNA (1:5000; clone PC10; Abcam).

Techniques: Immunohistochemical staining, Immunofluorescence, Microscopy, Staining, Labeling

Journal: Scientific reports

Article Title: Transcription factor profiling identifies Sox9 as regulator of proliferation and differentiation in corneal epithelial stem/progenitor cells.

doi: 10.1038/s41598-018-28596-3

Figure Lengend Snippet: Figure 3. Co-localisation of Sox9 with markers related to progenitor cell phenotype, differentiation and proliferation in the limbal epithelium. Double-labelling demonstrates co-localisation (arrows) of cytoplasmic Sox9 (red) with the stem/progenitor cell markers (green) N-cadherin, p75 nerve growth factor receptor (NGF-R), p63α, Oct4 and cytokeratin (CK) 15) in basal epithelial cells at the limbus. Suprabasal epithelial cells revealed co-localisation of nuclear Sox9 (red) with differentiation-related markers (green) CK3 and Pax6 as well as proliferation-related marker Ki-67. Sox9 monoclonal mouse antibody (clone 3C10) was used for double labelling experiments with polyclonal antibodies against Oct4, p63α, Pax6 and Ki-67, Sox9 polyclonal rabbit antibody (1) was used for double labelling experiments with monoclonal antibodies against p75 NGF- R, N-cadherin, CK3 and CK15. Nuclear counterstaining: DAPI. Individual channels of all double labelling experiments are shown in Supplementary Fig. 2.

Article Snippet: Membranes were blocked with SuperBlock T20 Blocking Buffer (Thermo Fisher Scientific) for 1 hour and incubated overnight using monoclonal mouse antibodies against Sox9 (1:5000; clone 3C10; BioRad), Cytokeratin 3/76 (1:5000; clone AE5; Millipore), Cytokeratin 15 (1:1000; clone EPR1614Y; Abcam) and PCNA (1:5000; clone PC10; Abcam).

Techniques: Marker, Bioprocessing

Journal: Scientific reports

Article Title: Transcription factor profiling identifies Sox9 as regulator of proliferation and differentiation in corneal epithelial stem/progenitor cells.

doi: 10.1038/s41598-018-28596-3

Figure Lengend Snippet: Figure 4. Expression of Sox9 during limbal epithelial cell expansion and wound healing in vitro. (A) Relative expression levels of Sox9 in cultured limbal epithelial cells expanded as clones on a 3T3 feeder layer or as feeder- free monolayer in passage (P) 0 to P2. Expression levels were determined by quantitative real-time polymerase chain reaction (qRT-PCR) primer assays and normalized against GAPDH. Data are expressed as means (2−∆CT × 1,000) ± SD (n = 3) relative to clonal cultures; *p < 0.01, unpaired t-test. (B) Limbal epithelial cell (LEPC) clones (dashed lines) on 3T3 feeder cells (3T3) stain positively for Sox9 (clone 3C10, red), preferentially towards the proliferating border of the clones; Sox9-expressing cells partly co-localize with Ki-67 (green). Nuclear staining: DAPI. (C) Immunofluorescent staining shows increased levels of nuclear Sox9 (clone 3C10) in basal/suprabasal limbal epithelial cells and central corneal epithelial cells after epithelial debridement and regeneration compared to unwounded control corneas (the background fluorescence seen in the central corneal stroma may be attributed to the epithelial debridement allowing media and serum components to infiltrate the stroma during the wound healing process). Higher magnification images of limbal epithelial progenitor cell clusters show increased nuclear localization of Sox9 in basal epithelial cells in wound healing conditions compared to cytoplasmic retention of Sox9 in control tissues (inserts). Nuclear staining: DAPI. (D) Relative expression of SOX9 in limbal epithelial cells of wounded and unwounded corneas as determined by quantitative real-time polymerase chain reaction (qRT-PCR) primer assays. Normalized data are expressed as means (2−∆CT × 1,000) ± SD (n = 5) relative to unwounded controls; p = 0.08, unpaired t-test.

Article Snippet: Membranes were blocked with SuperBlock T20 Blocking Buffer (Thermo Fisher Scientific) for 1 hour and incubated overnight using monoclonal mouse antibodies against Sox9 (1:5000; clone 3C10; BioRad), Cytokeratin 3/76 (1:5000; clone AE5; Millipore), Cytokeratin 15 (1:1000; clone EPR1614Y; Abcam) and PCNA (1:5000; clone PC10; Abcam).

Techniques: Expressing, In Vitro, Cell Culture, Clone Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Staining, Control, Fluorescence

Journal: Scientific reports

Article Title: Transcription factor profiling identifies Sox9 as regulator of proliferation and differentiation in corneal epithelial stem/progenitor cells.

doi: 10.1038/s41598-018-28596-3

Figure Lengend Snippet: Figure 5. Changes in mRNA expression following knockdown of SOX9 in cultured limbal epithelial cells by RNAi. (A) Results of quantitative real-time polymerase chain reaction (qRT-PCR) showing reduction of SOX9 mRNA transcripts in cultured limbal epithelial cells 24–96 hours after transfection with siRNA to SOX9 relative to control cells transfected with scramble siRNA (Control) (n = 6; Mean ± SD). (B) Transcriptional changes following knockdown of SOX9 in cultured limbal epithelial cells as determined by qRT-PCR. Significant or no relevant changes were seen in the expression levels of stemness-related genes ABCG2 (ATP Binding Cassette Subfamily G Member 2), TP63 (ΔNp63α) and CEBPD (CCAAT/enhancer-binding protein delta); progenitor cell marker genes KRT15 (keratin 15), KRT14 and CDH2 (N-cadherin); differentiation marker genes KRT3, KRT12 and IVL (involucrin); and proliferation-related genes PCNA (proliferating cell nuclear antigen),

Article Snippet: Membranes were blocked with SuperBlock T20 Blocking Buffer (Thermo Fisher Scientific) for 1 hour and incubated overnight using monoclonal mouse antibodies against Sox9 (1:5000; clone 3C10; BioRad), Cytokeratin 3/76 (1:5000; clone AE5; Millipore), Cytokeratin 15 (1:1000; clone EPR1614Y; Abcam) and PCNA (1:5000; clone PC10; Abcam).

Techniques: Expressing, Knockdown, Cell Culture, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Transfection, Control, Binding Assay, Marker

Journal: Scientific reports

Article Title: Transcription factor profiling identifies Sox9 as regulator of proliferation and differentiation in corneal epithelial stem/progenitor cells.

doi: 10.1038/s41598-018-28596-3

Figure Lengend Snippet: Figure 6. Changes in protein expression and proliferation following knockdown of SOX9 in cultured limbal epithelial cells by RNAi. (A) Western blot analysis of limbal epithelial cells transfected with either siRNA that targets SOX9 or non-targeting, scrambled siRNA as a control. Protein expression of Sox9, cytokeratin (CK) 15, CK3 and PCNA was detected with monoclonal antibodies, normalized to the house-keeping gene ß-actin, and expressed as percent of the expression in control cells (Ctrl); (n = 3; mean ± SD); *p < 0.05, **p < 0.005, ***p < 0.0001, unpaired t-test. Uncropped versions of Western blots are shown in Supplementary Fig. 4. (B) BrdU incorporation (i.e., cell proliferation) was determined by measuring absorbance at 450 nm. Statistically significant differences were observed at 72 (*p = 0.005) and 96 hours (**p = 0.009) between cells transfected with siRNA that targets SOX9 (Si-Sox9) and control cells transfected with scramble siRNA (Scr-Crtl) (n = 3; mean ± SD).

Article Snippet: Membranes were blocked with SuperBlock T20 Blocking Buffer (Thermo Fisher Scientific) for 1 hour and incubated overnight using monoclonal mouse antibodies against Sox9 (1:5000; clone 3C10; BioRad), Cytokeratin 3/76 (1:5000; clone AE5; Millipore), Cytokeratin 15 (1:1000; clone EPR1614Y; Abcam) and PCNA (1:5000; clone PC10; Abcam).

Techniques: Expressing, Knockdown, Cell Culture, Western Blot, Transfection, Control, Bioprocessing, BrdU Incorporation Assay

Journal: Scientific reports

Article Title: Transcription factor profiling identifies Sox9 as regulator of proliferation and differentiation in corneal epithelial stem/progenitor cells.

doi: 10.1038/s41598-018-28596-3

Figure Lengend Snippet: Figure 7. Interactions between Sox9 and cell signaling pathways. (A) Changes in mRNA expression of genes centrally involved in Wnt/ß-catenin signaling, i.e., WNT4 (Wnt-4), CTNNB1 (ß-catenin) and GSK3B (glycogen synthase kinase 3 beta), following knockdown of SOX9 in cultured limbal epithelial cells by RNAi relative to mock-transfected control cells. Normalized data are expressed as means (2−∆CT × 1,000) ± SD (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t-test. (B) Changes in SOX9 mRNA expression following exposure of cultured limbal epithelial cells with signaling activators lithium chloride (LiCl), IM- 12, BMP-2, JAG-1 and SAG as well as signaling inhibitors C-59, DMH1, DAPT and Cyclopamine (Cyclo) for 24 hours relative to vehicle-treated control cells (Ctrl). Normalized data are expressed as means (2−∆CT × 1,000) ± SD (n = 3); *p < 0.05, **p < 0.01, unpaired t-test. (C) Changes in Sox9 protein expression following exposure of cultured limbal epithelial cells to Hedgehog signaling activators Sonic hedgehog (SHH), SAG and Purmorphamine (Purmo); BMP-2; Wnt signaling activators Wnt-3a, lithium chloride (LiCl) and IM-12; and Notch signaling ligand JAG-1 for 48 hours relative to vehicle-treated control cells (Ctrl). Sox9 protein expression was detected with the monoclonal antibody (clone 3C10), normalized to the house-keeping gene ß-actin, and expressed as percent of the expression in control cells (Ctrl); (n = 3; mean ± SD). Uncropped versions of Western blots are shown in Supplementary Fig. 1.

Article Snippet: Membranes were blocked with SuperBlock T20 Blocking Buffer (Thermo Fisher Scientific) for 1 hour and incubated overnight using monoclonal mouse antibodies against Sox9 (1:5000; clone 3C10; BioRad), Cytokeratin 3/76 (1:5000; clone AE5; Millipore), Cytokeratin 15 (1:1000; clone EPR1614Y; Abcam) and PCNA (1:5000; clone PC10; Abcam).

Techniques: Protein-Protein interactions, Expressing, Knockdown, Cell Culture, Transfection, Control, Western Blot

Journal: Scientific reports

Article Title: Transcription factor profiling identifies Sox9 as regulator of proliferation and differentiation in corneal epithelial stem/progenitor cells.

doi: 10.1038/s41598-018-28596-3

Figure Lengend Snippet: Figure 8. Model illustrating the mutually repressive interaction of Sox9 and Wnt/ß-catenin signaling as well as potentially involved regulatory signaling pathways in the limbal stem cell niche (mod. after Xu, Z. et al., Elife 4, e10567 (2015) (https://creativecommons.org/licenses/by/4.0).

Article Snippet: Membranes were blocked with SuperBlock T20 Blocking Buffer (Thermo Fisher Scientific) for 1 hour and incubated overnight using monoclonal mouse antibodies against Sox9 (1:5000; clone 3C10; BioRad), Cytokeratin 3/76 (1:5000; clone AE5; Millipore), Cytokeratin 15 (1:1000; clone EPR1614Y; Abcam) and PCNA (1:5000; clone PC10; Abcam).

Techniques: Protein-Protein interactions

Journal: Translational Cancer Research

Article Title: CK1α-targeting inhibits primary and metastatic colorectal cancer in vitro , ex vivo , in cell-line-derived and patient-derived tumor xenograft mice models

doi: 10.21037/tcr.2020.02.13

Figure Lengend Snippet: CRC cells aberrantly express CK1α at mRNA and protein levels. (A) Immunohistochemical staining show enhanced expression of CK1α protein in CRC tissue compared to the normal colon tissue; (B) the relative expression of CK1α mRNA in 8 matched tumor/non-tumor colonic tissues from q-PCR analysis; (C) relative expression of CK1α mRNA in CRC compared to the normal colon tissue in the NCBI GEO dataset; (D) the expression levels of CK1α protein in FHC, HCT116, HT29 and DLD1 cells, as shown by western blot; (E) graph of D. GAPDH served as loading control. *, P<0.05; ****, P<0.0001; data are presented as the mean ± SD of experiments performed three times in triplicates. CRC, colorectal cancer; CK1α, casein kinase 1α; q-PCR, quantitative polymerase chain reaction; NCBI, National Center for Biotechnology Information; GEO, Gene Expression Omnibus; FHC, fetal human normal colonic mucosa; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SD, standard deviation.

Article Snippet: Antibodies against CK1α (sc-74582), Ki-67 (sc-23900) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (sc-47724) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Immunohistochemical staining, Staining, Expressing, Western Blot, Control, Real-time Polymerase Chain Reaction, Gene Expression, Standard Deviation

Journal: Translational Cancer Research

Article Title: CK1α-targeting inhibits primary and metastatic colorectal cancer in vitro , ex vivo , in cell-line-derived and patient-derived tumor xenograft mice models

doi: 10.21037/tcr.2020.02.13

Figure Lengend Snippet: High CK1α expression is implicated in the poor clinical outcome of patients with CRC. Kaplan-Meier curves show the effect of high CK1α or low CK1α on the 5-year (A) OS and (B) DFS in CRC patients. The Mantel-Cox log-rank P value is indicated. CRC, colorectal cancer; CK1α, casein kinase 1α; OS, overall survival; DFS, disease-free survival.

Article Snippet: Antibodies against CK1α (sc-74582), Ki-67 (sc-23900) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (sc-47724) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing

Journal: Translational Cancer Research

Article Title: CK1α-targeting inhibits primary and metastatic colorectal cancer in vitro , ex vivo , in cell-line-derived and patient-derived tumor xenograft mice models

doi: 10.21037/tcr.2020.02.13

Figure Lengend Snippet: Pharmacological inhibition of CK1α using Epiblastin A significantly suppressed the viability of CRC cells without cytotoxicity to normal colonic cells, in vitro . Histograms showing the effect of 0.5–25 μM Epiblastin A on the viability of (A) FHC, (B) HT29, (C) HCT116, and (D) DLD1 cells. *, P<0.05; **, P<0.01; ***, P<0.001; data are presented as the mean ± SD of experiments performed three times in triplicates. CRC, colorectal cancer; CK1α, casein kinase 1α; FHC, fetal human normal colonic mucosa; SD, standard deviation.

Article Snippet: Antibodies against CK1α (sc-74582), Ki-67 (sc-23900) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (sc-47724) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Inhibition, In Vitro, Standard Deviation

Journal: Translational Cancer Research

Article Title: CK1α-targeting inhibits primary and metastatic colorectal cancer in vitro , ex vivo , in cell-line-derived and patient-derived tumor xenograft mice models

doi: 10.21037/tcr.2020.02.13

Figure Lengend Snippet: CK1α expression is enhanced in CRC clinical and PDX tumor samples. (A) Immunohistochemical staining showing the differential expression of CK1α in CRC-001 normal colon, CRC-001 colon tumor, CRC-001 colon tumor/PDX, CRC-002 colon tumor, CRC-002 colon tumor/PDX samples. H&E was used for tissue histology; (B) quantitative representation of A. CRC, colorectal cancer; CK1α, casein kinase 1α; H&E, hematoxylin and eosin; PDX, patient-derived xenograft.

Article Snippet: Antibodies against CK1α (sc-74582), Ki-67 (sc-23900) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (sc-47724) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Immunohistochemical staining, Staining, Quantitative Proteomics, Derivative Assay

Journal: Translational Cancer Research

Article Title: CK1α-targeting inhibits primary and metastatic colorectal cancer in vitro , ex vivo , in cell-line-derived and patient-derived tumor xenograft mice models

doi: 10.21037/tcr.2020.02.13

Figure Lengend Snippet: Epiblastin A effectively inhibits CK1α expression in CRC clinical and PDX tumor samples, ex vivo . (A) Immunohistochemical staining showing the effect of Epiblastin A on the expression of CK1α and Ki-67 in CRC-001 colon tumor/PDX samples compared to the vehicle-treated control PDX tissues. H&E was used for tissue histology. CRC, colorectal cancer; CK1α, casein kinase 1α; H&E, hematoxylin and eosin; PDX, patient-derived xenograft.

Article Snippet: Antibodies against CK1α (sc-74582), Ki-67 (sc-23900) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (sc-47724) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Ex Vivo, Immunohistochemical staining, Staining, Control, Derivative Assay

Journal: Translational Cancer Research

Article Title: CK1α-targeting inhibits primary and metastatic colorectal cancer in vitro , ex vivo , in cell-line-derived and patient-derived tumor xenograft mice models

doi: 10.21037/tcr.2020.02.13

Figure Lengend Snippet: Epiblastin A inhibits tumor growth in DLD1-derived tumor xenograft models by suppressing CK1α expression, in vivo . (A) Graphs showing the effect of 5 mg/kg Epiblastin A on the size of tumors formed (upper) and mice body weights (lower) in DLD1-derived tumor xenograft mice models over the indicated time-course; (B) Immunohistochemical staining of tissue samples from the DLD1-derived tumor xenograft mice models shows the effect of Epiblastin A on the expression of CK1α, compared to the vehicle-treated control tissues. H&E was used for tissue histology. **, P<0.01. CK1α, casein kinase 1α; H&E, hematoxylin and eosin.

Article Snippet: Antibodies against CK1α (sc-74582), Ki-67 (sc-23900) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (sc-47724) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Derivative Assay, Expressing, In Vivo, Immunohistochemical staining, Staining, Control

Journal: Journal of Veterinary Diagnostic Investigation : Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc

Article Title: Activated platelets and platelet-leukocyte aggregates in the equine systemic inflammatory response syndrome

doi: 10.1177/10406387221077969

Figure Lengend Snippet: Percentages of activated platelets (CD62P-positive) and platelet-leukocyte aggregates (PLAs; CD11a/18-positive) in platelet-leukocyte-rich plasma (PLRP) and after activation with collagen (50 μg/mL) measured with Accuri C6 (Becton Dickinson) and the BD Accuri C6 analysis software in 10 healthy adult horses (median, IQR; Prism v.6, GraphPad; Wilcoxon test).

Article Snippet: Activation of platelets was determined with fluorescein isothiocyanate (FITC)-conjugated (LYNX rapid antibody conjugation kit; Bio-Rad) monoclonal mouse anti-human antibodies against CD62P (MCA2419, dilution 1:50 with modified HEPES/Tyrod buffer).

Techniques: Activation Assay, Software

Journal: Journal of Veterinary Diagnostic Investigation : Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc

Article Title: Activated platelets and platelet-leukocyte aggregates in the equine systemic inflammatory response syndrome

doi: 10.1177/10406387221077969

Figure Lengend Snippet: Platelet activation and platelet-leukocyte aggregates (PLAs) in controls ( n = 10) and systemic inflammatory response syndrome (SIRS) group ( n = 17) in native samples (white boxes) and after in vitro activation with collagen (cross-hatched boxes); boxes are median and IQR, whiskers minimum and maximum. A. Percentage of CD62P-positive platelets; a, p = 0.0007; b, p < 0.0001. B. Percentage of PLAs: c, p = 0.048; d, p = 0.0009; e, p = 0.036.

Article Snippet: Activation of platelets was determined with fluorescein isothiocyanate (FITC)-conjugated (LYNX rapid antibody conjugation kit; Bio-Rad) monoclonal mouse anti-human antibodies against CD62P (MCA2419, dilution 1:50 with modified HEPES/Tyrod buffer).

Techniques: Activation Assay, In Vitro

Journal: Journal of Veterinary Diagnostic Investigation : Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc

Article Title: Activated platelets and platelet-leukocyte aggregates in the equine systemic inflammatory response syndrome

doi: 10.1177/10406387221077969

Figure Lengend Snippet: Platelet activation and platelet-leukocyte aggregates (PLAs) in the systemic inflammatory response syndrome (SIRS) group ( n = 13) depending on outcome in native samples (open circles) and after in vitro activation with collagen (black dots); bar = median. A. Individual percentage of CD62P-positive platelets; a, p = 0.03. B. Individual percentage of PLAs.

Article Snippet: Activation of platelets was determined with fluorescein isothiocyanate (FITC)-conjugated (LYNX rapid antibody conjugation kit; Bio-Rad) monoclonal mouse anti-human antibodies against CD62P (MCA2419, dilution 1:50 with modified HEPES/Tyrod buffer).

Techniques: Activation Assay, In Vitro

Journal: PLoS ONE

Article Title: NF-κB-Induced IL-6 Ensures STAT3 Activation and Tumor Aggressiveness in Glioblastoma

doi: 10.1371/journal.pone.0078728

Figure Lengend Snippet: U251-MG cells were grown in the absence or presence of TNF-α (10 ng/ml) for various times, sonicated and soluble chromatin was immunoprecipitated with antibodies specific for p65 or STAT3. Immunoprecipitated DNA was then analyzed by qRT-PCR using primers specific for the IL-6 and SOCS3 promoters. Each sample was normalized to genomic DNA isolated from cells that were cross-linked and processed, yet did not incur the immunoprecipitation step. The results are shown as percentages of input, replicates of three, and error bars represent standard deviation.

Article Snippet: Antibodies against phospho-p65 Ser536 (p-p65 (S536)), phospho-STAT3 Tyr705 (p-STAT3 (Y705)), STAT3, phospho-IKKα/β (S176/180) and IKKα/β were purchased from Cell Signaling Technology (1∶1,000; Beverly, MA).

Techniques: Sonication, Immunoprecipitation, Quantitative RT-PCR, Isolation, Standard Deviation

Journal: PLoS ONE

Article Title: NF-κB-Induced IL-6 Ensures STAT3 Activation and Tumor Aggressiveness in Glioblastoma

doi: 10.1371/journal.pone.0078728

Figure Lengend Snippet: A & B , U251-TR/ sh-p65 cells were incubated with tetracycline (Tet) for 48 h prior to stimulation with TNF-α (10 ng/ml) for the indicated times. Cells were lysed and immunoblotted with the indicated Ab (A) or RNA was isolated, followed by generation of cDNA, and qRT-PCR was performed for the indicated genes (B). Densitometric values of p-STAT3, p-p65 and total p65 were normalized to total STAT3, total p65 and GAPDH, respectively. Data are shown as replicates of three. **, p<0.01.

Article Snippet: Antibodies against phospho-p65 Ser536 (p-p65 (S536)), phospho-STAT3 Tyr705 (p-STAT3 (Y705)), STAT3, phospho-IKKα/β (S176/180) and IKKα/β were purchased from Cell Signaling Technology (1∶1,000; Beverly, MA).

Techniques: Incubation, Isolation, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: NF-κB-Induced IL-6 Ensures STAT3 Activation and Tumor Aggressiveness in Glioblastoma

doi: 10.1371/journal.pone.0078728

Figure Lengend Snippet: A, U251-TR/ sh-p65 cells were injected subcutaneously into nude mice, and tumor growth was measured on the indicated days. At day 53, mice were randomized into 4 groups (n = 4 per group) to begin treatment (Txt) regimens. Treatment groups consisted of vehicle only (vehicle), food supplemented with doxycycline (Dox, a tetracycline analog to induce p65 shRNA expression), AZD1480 (50 mg/kg) in methylcellulose via oral gavage once a day, or both Dox food and AZD1480 (Dox+AZD1480). Data represent mean ± SEM (*, p<0.05, Students t-test at day 76). B, Frozen tumor samples were homogenized and immunoblotted with the indicated Ab. Representative tumor samples from each group are shown. Densitometric values of total p65 and p-STAT3 were normalized to GAPDH and total STAT3, respectively. C, Frozen tumor samples were homogenized, and RNA isolated, followed by generation of cDNA, and qRT-PCR performed for the indicated genes (n = 2 tumors per condition, replicates of three per tumor). *, p<0.05.

Article Snippet: Antibodies against phospho-p65 Ser536 (p-p65 (S536)), phospho-STAT3 Tyr705 (p-STAT3 (Y705)), STAT3, phospho-IKKα/β (S176/180) and IKKα/β were purchased from Cell Signaling Technology (1∶1,000; Beverly, MA).

Techniques: Injection, shRNA, Expressing, Isolation, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: NF-κB-Induced IL-6 Ensures STAT3 Activation and Tumor Aggressiveness in Glioblastoma

doi: 10.1371/journal.pone.0078728

Figure Lengend Snippet: A & B, Xenograft X1046 cells were disaggregated into single cells and briefly propagated as neurospheres in vitro . Cells were pre-treated with AZD1480 (1 µM) and/or WA (5 µM) for 2 h prior to TNF-α (10 ng/ml) for 0.25 or 2 h (A) or 2 h (B). Cells were lysed and immunoblotted with the indicated Ab (A), or RNA was isolated followed by generation of cDNA and qRT-PCR was performed for IL-6 (B). Densitometric values of p-p65, p-IKKα/β and p-STAT3 were normalized to total p65, total IKKα/β and total STAT3, respectively. Data are shown as replicates of three. *, p<0.05. C, Xenograft X1066 cells were treated with the indicated doses of AZD1480 and/or WA for 48 h, and the WST-1 cell viability assay was performed. Data are shown as replicates of three. *p<0.05. D, Nude mice were injected intracranially with Xenograft X1016 cells. Starting at day 3, mice were treated with vehicle (n = 5), AZD1480 (30 mg/kg, twice a day, n = 5), WA (4 mg/kg, alternate days, n = 5) or both AZD+WA (n = 5) for three weeks. Survival was measured, and mice were euthanized at moribund. *, p<0.05 (LogRank).

Article Snippet: Antibodies against phospho-p65 Ser536 (p-p65 (S536)), phospho-STAT3 Tyr705 (p-STAT3 (Y705)), STAT3, phospho-IKKα/β (S176/180) and IKKα/β were purchased from Cell Signaling Technology (1∶1,000; Beverly, MA).

Techniques: In Vitro, Isolation, Quantitative RT-PCR, Viability Assay, Injection

Journal: PLoS ONE

Article Title: NF-κB-Induced IL-6 Ensures STAT3 Activation and Tumor Aggressiveness in Glioblastoma

doi: 10.1371/journal.pone.0078728

Figure Lengend Snippet: NF-κB and STAT3 are competent to ensure activation of themselves and each other, either in an autocrine and/or paracrine manner. Upon stimulation with TNF-α, the NF-κB pathway becomes activated, as shown by the phosphorylation and nuclear translocation of NF-κB p65 and transcription of NF-κB genes, including IL-6 and LIF. Newly synthesized IL-6 is secreted by the cells, and binds in an autocrine or paracrine manner to the IL-6 receptor. This leads to activation of the IL-6R/gp130 complex and the intracellular kinases JAK1/2. STAT3 proteins then become phosphorylated by JAK1/2, dimerize, enter the nucleus and begin the transcription of STAT3 driven genes such as SOCS3 and cIAP2.

Article Snippet: Antibodies against phospho-p65 Ser536 (p-p65 (S536)), phospho-STAT3 Tyr705 (p-STAT3 (Y705)), STAT3, phospho-IKKα/β (S176/180) and IKKα/β were purchased from Cell Signaling Technology (1∶1,000; Beverly, MA).

Techniques: Activation Assay, Phospho-proteomics, Translocation Assay, Synthesized

Journal: Oncotarget

Article Title: The hypoxia-related microRNA miR-199a-3p displays tumor suppressor functions in ovarian carcinoma

doi:

Figure Lengend Snippet: (A) Alignment of potential miR-199a-3p binding site in the 3′-UTR of MET . (B) Luciferase assay. Ovarian cancer cells were transfected with a firefly luciferase reporter vector containing the 3′-UTR of MET or a mutated 3′-UTR. At 24 hours after the transfection, cells were further transfected with pre-miR-199a-3p or negative control miR. Luciferase activity normalized to the activity of Renilla luciferase was measured. Data represent mean ± SEM, n = 5. (C) Immunohistochemistry. Ovarian cancer tissues and normal ovaries from benign diseases were stained with an antibody against c-Met. c-Met is overexpressed in ovarian cancer clinical tissues. Bar represents 100 μm. (D) Western blotting. c-Met were overexpressed in ovarian cancer cell lines than 4 different OSE (ovarian surface epithelium) cells (upper). Densitometric ratio of the expression of c-Met / β-actin (lower). (E) Correlation plots of miR-199a-3p and c-Met by Pearson's product-moment coefficient. Correlation plot from miR-199a-3p and c-Met expression in ovarian cancer specimens (left) and cell line (right) showing that endogenous miR-199a-3p level is inversely correlated with MET protein levels ( R = −0.67, P = 0.013, R = −0.71, P = 0.01, respectively). (F) miRNA RT-qPCR. Cells were transfected with pre-miR-199a-3p or negative control miR. Twenty-four hours after transfection, total RNA was collected and subjected to RT-PCR. 2 −ΔΔCT method was used to calculate the relative abundance of miR-199a-3p with respect to RNU6B expression. Relative fold differences with respect to the control are presented; columns represent the means from 3 independent experiments. (G) Western blotting. Enforced expression of miR-199a-3p inhibits c-Met protein expression in ovarian cancer cell lines. (H) miRNA RT-qPCR. Cells were transfected with anti-miR-199a-3p or negative control miR for 24 hours. (I) Western blotting. Inhibition of miR-199a-3p expression increases c-Met protein expression. **; P < 0.01. n.s.; not significant. Densitometry ratios in each western blotting are shown below each blot.

Article Snippet: Antibodies against β-actin (#4967), phospho-AKT (Ser473; #9271), phospho-ERK 1/2 (p-ERK1/2; Thr202/Tyr204, E10; #9106), and CD44 (156-3C11; #3570) were obtained from Cell Signaling (Danvers, MA, USA).

Techniques: Binding Assay, Luciferase, Transfection, Plasmid Preparation, Negative Control, Activity Assay, Immunohistochemistry, Staining, Western Blot, Expressing, miRNA RT, Reverse Transcription Polymerase Chain Reaction, Control, Inhibition

Journal: Oncotarget

Article Title: The hypoxia-related microRNA miR-199a-3p displays tumor suppressor functions in ovarian carcinoma

doi:

Figure Lengend Snippet: (A) Western blotting. SKOV3ip1 cells were transfected with pre-miR-199a-3p or negative control miR for 24 hours. Cell lysate were collected before and after HGF stimulation (40 ng/mL, 10 minutes). Immunoblotting was performed with antibodies against phosphorylated c-Met (p-c-Met), c-Met, phosphorylated extracellular signal-regulated kinase (p-ERK), phosphorylated Akt (p-Akt), or β–actin. (B) Colony formation assay. Ovarian cancer cells (1 × 10 5 cells; SKOV3ip1 and RMUG-S), cotransfected with miRNA (pre-miR-199a-3p or negative control miRNA) and an pIRES2-EGFP vector (containing MET or the empty control vector), were suspended in 0.33% agarose. After 10 days, colonies with a diameter greater than 100 μm were counted under a microscope at 40× magnification. Representative images are shown. Bar represents 400 μm. Data represent mean ± SEM; n = 10. (C) In vitro adhesion assay. SKOV3ip1 (5 × 10 4 ) cells were cotransfected with miRNA (pre-miR-199a-3p or negative control miR) and the pIRES2-EGFP vector (containing MET or the empty control vector). The cells were plated onto 50 μg/mL fibronectin- or collagen type 1–coated 96-well plates. After being incubated for 75 minutes at 37°C, the plates were washed to discard nonadherent cells, and the number of adherent cells was counted. Representative images are shown. Bar represents 100 μm. Data represent mean ± SEM; n = 6. (D) Matrigel invasion assay. Ovarian cancer cells were cotransfected with miRNA (pre-miR-199a-3p or negative control miR) and an pIRES2-EGFP vector (containing MET or the empty control vector). The cells (5 × 10 4 ) were plated in serum-free medium in modified Boyden chamber system that had been coated with 25 μg Matrigel and allowed to invade the lower chamber, which contained Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, for 24 hours. Noninvading cells were removed with a cotton swab, and invading cells on the underside of the filter were counted. Representative images are shown. Bar represents 200 μm. Data represent mean ± SEM; n = 10. (E) Western blotting. SKOV3ip1 cells were cotransfected with miRNA (pre-miR-199a-3p or negative control miR) and an pIRES2-EGFP vector (containing MET , or the empty control vector) for 4 hours. Cell lysates were then obtained, and immunoblotting was performed with antibodies against c-Met, various integrins (α1, α2, α5, αV, and β1), CD44, matrix metalloproteinase-2 (MMP-2), and β-actin (left). Densitometric ratios of the expression of c-Met, integrin β1, CD44, and MMP-2 (right). * P < 0.05; *** P < 0.001; n.s., not significant. Densitometry ratios in each western blotting are shown below each blot.

Article Snippet: Antibodies against β-actin (#4967), phospho-AKT (Ser473; #9271), phospho-ERK 1/2 (p-ERK1/2; Thr202/Tyr204, E10; #9106), and CD44 (156-3C11; #3570) were obtained from Cell Signaling (Danvers, MA, USA).

Techniques: Western Blot, Transfection, Negative Control, Colony Assay, Plasmid Preparation, Control, Microscopy, In Vitro, Cell Adhesion Assay, Incubation, Invasion Assay, Modification, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Human SMC2 Protein, a Core Subunit of Human Condensin Complex, Is a Novel Transcriptional Target of the WNT Signaling Pathway and a New Therapeutic Target *

doi: 10.1074/jbc.M112.428466

Figure Lengend Snippet: SMC2 is up-regulated in human CRC. A, WB analysis of SMC2 in human CRC. A representative subset of 29 cases studied is shown. Actin was used as loading control. B, quantitative real-time PCR for SMC2 in 16 pairs of colon adenocarcinoma tumors and matched adjacent normal colonic tissues. Data are representative of three independent experiments. The mean values of SMC2 levels were compared using Student's t test (upper boxplot). C and D, SMC2, SMC4, and β-catenin levels were evaluated by WB in both colorectal cancer cell lines (n = 14) and samples from CRC patients (n = 27, a representative subset is shown). Actin was used as loading control. E and F, SMC2, SMC4, and β-catenin protein levels on WB were determined by gel band quantification and normalized to the corresponding actin levels. Values were used to perform correlation studies following Spearman test. G, immunohistochemistry of SMC2 in paraffin-embedded tissue. A representative specimen is shown. Magnified regions of the normal and tumor mucosa are shown on the right. N, normal tissue; T: tumor tissue (adenocarcinoma).

Article Snippet: Blots were probed using antibodies against SMC2 (ab10412, Abcam; and 07-710, Upstate-Millipore, dilution factor of 1:1000), SMC4 (ab17958, Abcam, dilution factor of 1:1000), TCF4 (05-511, Upstate-Millipore, dilution factor, 1:500), NCAPH (HPA003008, Sigma Aldrich, dilution factor, 1:2000), β-catenin (610154, BD Transduction Laboratories, dilution factor, 1:1000) or c-Myc (monoclonal 9E10, sc-40, Santa Cruz Biotechnology, 1:100).

Techniques: Real-time Polymerase Chain Reaction, Immunohistochemistry

Journal: The Journal of Biological Chemistry

Article Title: Human SMC2 Protein, a Core Subunit of Human Condensin Complex, Is a Novel Transcriptional Target of the WNT Signaling Pathway and a New Therapeutic Target *

doi: 10.1074/jbc.M112.428466

Figure Lengend Snippet: SMC2 protein is down-regulated upon WNT signaling inhibition. Ls174T/dnTCF4 (A and B) and Ls174T/pTER-β-catenin (C and D) cell lines were cultured in absence or presence of 5 μg/μl doxycycline (Dox) during the indicated time points. Cells were lysed and analyzed by WB using the indicated antibodies. Representative data from three replicates/independent experiments are shown.

Article Snippet: Blots were probed using antibodies against SMC2 (ab10412, Abcam; and 07-710, Upstate-Millipore, dilution factor of 1:1000), SMC4 (ab17958, Abcam, dilution factor of 1:1000), TCF4 (05-511, Upstate-Millipore, dilution factor, 1:500), NCAPH (HPA003008, Sigma Aldrich, dilution factor, 1:2000), β-catenin (610154, BD Transduction Laboratories, dilution factor, 1:1000) or c-Myc (monoclonal 9E10, sc-40, Santa Cruz Biotechnology, 1:100).

Techniques: Inhibition, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: Human SMC2 Protein, a Core Subunit of Human Condensin Complex, Is a Novel Transcriptional Target of the WNT Signaling Pathway and a New Therapeutic Target *

doi: 10.1074/jbc.M112.428466

Figure Lengend Snippet: Functional study of SMC2 promoter activity. A, schematic representation of human SMC2 promoter. Predicted TCF response elements are also indicated; arrows indicate target sequence for ChIP PCR amplification. B, Ls174T/dnTCF4 (left) and Ls174T/pTER-β-catenin (right) cell lines were transfected with SMC2 promoter-luciferase reporter construct together with control Renilla luciferase reporter pRL-TK for normalization (RLU, relative luciferase units). Where indicated, cells were doxycline (Doxy)-treated to induce the TCF4 dominant-negative form (left) or a siRNA targeting β-catenin (right). TOP-flash vector was used as positive control for WNT signaling activity/repression. A representative result out of at least three different experiments run in triplicates is shown. C, DLD-1 or HCT116 cell lines were co-transfected with SMC2 promoter luciferase construct and pcDNA (empty vector), β-catenin, or VP16-TCF4 expression vectors. D, PCR analyses of DNA pulled down by isotypic antibody (negative control) or anti-TCF-4 monoclonal antibody in ChIP assay. c-myc promoter sequence containing TBE1 element and APC promoter region 1B sequences were amplified as positive and negative controls, respectively. Error bars indicate S.D. (Student's t test; **, p < 0.01).

Article Snippet: Blots were probed using antibodies against SMC2 (ab10412, Abcam; and 07-710, Upstate-Millipore, dilution factor of 1:1000), SMC4 (ab17958, Abcam, dilution factor of 1:1000), TCF4 (05-511, Upstate-Millipore, dilution factor, 1:500), NCAPH (HPA003008, Sigma Aldrich, dilution factor, 1:2000), β-catenin (610154, BD Transduction Laboratories, dilution factor, 1:1000) or c-Myc (monoclonal 9E10, sc-40, Santa Cruz Biotechnology, 1:100).

Techniques: Functional Assay, Activity Assay, Sequencing, Amplification, Transfection, Luciferase, Construct, Dominant Negative Mutation, Plasmid Preparation, Positive Control, Expressing, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Human SMC2 Protein, a Core Subunit of Human Condensin Complex, Is a Novel Transcriptional Target of the WNT Signaling Pathway and a New Therapeutic Target *

doi: 10.1074/jbc.M112.428466

Figure Lengend Snippet: Determination of the minimal regulatory region of SMC2 promoter. A, relative position and sequences of the putative TBEs predicted in silico in the SMC2 promoter and deletion mutants for luciferase (luc) reporters performed. B, determination of fragment 3 as the minimal regulatory region of the SMC2 promoter. Luciferase activity of each deletion mutant was normalized to Renilla luciferase internal control (RLU, relative luciferase units) in DLD1 (left) or HCT116 (right) cell lines; a representative result is shown of at least three independent experiments. *, p < 0.05; **, p < 0.01; Student's t test (promoter activity versus full-length SMC2 promoter (SMC2 FL).

Article Snippet: Blots were probed using antibodies against SMC2 (ab10412, Abcam; and 07-710, Upstate-Millipore, dilution factor of 1:1000), SMC4 (ab17958, Abcam, dilution factor of 1:1000), TCF4 (05-511, Upstate-Millipore, dilution factor, 1:500), NCAPH (HPA003008, Sigma Aldrich, dilution factor, 1:2000), β-catenin (610154, BD Transduction Laboratories, dilution factor, 1:1000) or c-Myc (monoclonal 9E10, sc-40, Santa Cruz Biotechnology, 1:100).

Techniques: In Silico, Luciferase, Activity Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Human SMC2 Protein, a Core Subunit of Human Condensin Complex, Is a Novel Transcriptional Target of the WNT Signaling Pathway and a New Therapeutic Target *

doi: 10.1074/jbc.M112.428466

Figure Lengend Snippet: Elucidation of the TBE responsible for β-catenin·TCF4 transactivation in the SMC2 promoter. A, sequence alignment of SMC2 promoter in different species; Hs, Homo sapiens; Pt, Pan troglodytes; Mmt, Macaca mulatta; Rn, Rattus novergicus; Mms, Mus musculus. Conserved TBEs are highlighted in gray background. B, schematic representation of SMC2 promoter mutant variants. C, DLD1 (left) or HCT116 (right) cell lines were transfected with constructs above. Luciferase activity was normalized to Renilla activity (RLU, relative luciferase units); a representative result is shown out of at least three independent experiments. D, DLD-1 (left) or HCT116 (right) cell lines were co-transfected with Δ3 fragment mutational combinations and expression vectors for β-catenin, TCF4-VP16 (constitutively active form of TCF4), or the empty vector pcDNA3 (pcDNA); a representative result is shown out of at least three independent experiments (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Article Snippet: Blots were probed using antibodies against SMC2 (ab10412, Abcam; and 07-710, Upstate-Millipore, dilution factor of 1:1000), SMC4 (ab17958, Abcam, dilution factor of 1:1000), TCF4 (05-511, Upstate-Millipore, dilution factor, 1:500), NCAPH (HPA003008, Sigma Aldrich, dilution factor, 1:2000), β-catenin (610154, BD Transduction Laboratories, dilution factor, 1:1000) or c-Myc (monoclonal 9E10, sc-40, Santa Cruz Biotechnology, 1:100).

Techniques: Sequencing, Mutagenesis, Transfection, Construct, Luciferase, Activity Assay, Expressing, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Human SMC2 Protein, a Core Subunit of Human Condensin Complex, Is a Novel Transcriptional Target of the WNT Signaling Pathway and a New Therapeutic Target *

doi: 10.1074/jbc.M112.428466

Figure Lengend Snippet: siRNA knockdown of SMC2 impairs tumor growth in a xenograft mouse model. A, schematic representation of the experimental design. DLD1 cells were transiently transfected with an siRNA targeting SMC2 or a scrambled sequence. After 48 h, a second round of transfection was performed. 24 h later, 1.5 × 106 cells were injected subcutaneously in the dorsal flanks of athymic nude mice. B, SMC2 knockdown was assessed by WB using whole cell extracts from in vitro culture until 120 h post-tranfection (sc, scrambled siRNA). C, representative resected tumors from the same animal at day 40 post-injection. Scale bar, 1 cm. D, tumor growth curves. Tumor volume was measured every 2–3 days for 36 days. The graph is representative of two independent experiments. Error bars represent S.E. (n = 11). Differences were evaluated with paired Student's t test (p = 0.0201); (*, p < 0.05, t test in each time point).

Article Snippet: Blots were probed using antibodies against SMC2 (ab10412, Abcam; and 07-710, Upstate-Millipore, dilution factor of 1:1000), SMC4 (ab17958, Abcam, dilution factor of 1:1000), TCF4 (05-511, Upstate-Millipore, dilution factor, 1:500), NCAPH (HPA003008, Sigma Aldrich, dilution factor, 1:2000), β-catenin (610154, BD Transduction Laboratories, dilution factor, 1:1000) or c-Myc (monoclonal 9E10, sc-40, Santa Cruz Biotechnology, 1:100).

Techniques: Transfection, Sequencing, Injection, In Vitro