Journal: RNA

Article Title: MicroRNA-mediated regulation of extracellular matrix formation modulates somatic cell reprogramming

doi: 10.1261/rna.043745.113

Figure Lengend Snippet: Wisp1 is a key regulator of extracellular matrix genes. ( A ) Wisp1 regulates expression of several ECM genes. Expression of Tgfbi, Igfbp5, Dkk2, Nov, and Ccl20 were dramatically changed upon Wisp1 knockdown. Uninfected and 4F-infected MEFs were transfected with siWisp1 for 2 d and total RNAs were harvested for RT-qPCR analysis of different ECM genes. Error bars represent two independent experiments with duplicate wells. ( B ) Knockdown of Nov, Dkk2, and Tgfbi significantly enhances iPSC generation. MEFs were transduced with 4F at Day 0 and transfected with siRNAs at Day 5 post-infection. GFP + colonies were quantified at around Days 11–13. Error bars represent three independent experiments with triplicate wells. (**) P < 0.01. ( C ) Overexpression of Wisp1-regulated ECM genes compromises reprogramming. The indicated ECM genes were cloned into pMX retroviral vectors. MEFs were transduced with 4F plus the indicated ECM genes and GFP + colonies were quantified at around Days 11–13. Data were normalized to pMX-RFP-transduced cells. Error bars represent three independent experiments with triplicate wells. (**) P < 0.01. ( D ) Addition of recombinant ECM proteins compromises reprogramming. Purified recombinant TGFBI, DKK2, NOV, and CCL20 were added at a final concentration of 100 ng/mL to cultures of 4F-MEFs undergoing reprogramming. GFP + colonies were quantified at Days 11–13. Error bars represent two independent experiments with triplicate wells. (*) P < 0.05.

Article Snippet: Recombinant proteins were obtained from commercial sources as follows: mouse Dkk2 (R&D systems, 2435DK/CF), human NOV/CCN3 (R&D systems, 1640NV), human TGFBI (Prospec, #PRO-568), CCL20 (R&D systems, 760-M3).

Techniques: Expressing, Knockdown, Infection, Transfection, Quantitative RT-PCR, Transduction, Over Expression, Clone Assay, Retroviral, Recombinant, Purification, Concentration Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Increased migration of Langerhans cells in response to HPV16 E6 and E7 oncogene silencing: role of CCL20

doi: 10.1007/s00262-008-0522-5

Figure Lengend Snippet: CCL20 immunostaining in cervical biopsy specimens. Normal exocervical squamous epithelium (a) or high-grade SIL (b) stained with an antibody against CCL20. Semi-quantitative evaluation of CCL20 expression in cervical biopsy specimens (c)

Article Snippet: An anti-human CCL20 antibody (R&D Systems) was used at 10 μg/ml in cell migration neutralization experiments.

Techniques: Immunostaining, Staining, Expressing

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Increased migration of Langerhans cells in response to HPV16 E6 and E7 oncogene silencing: role of CCL20

doi: 10.1007/s00262-008-0522-5

Figure Lengend Snippet: CCL20 secretion in cultures of E6 and E7 siRNA treated SiHa cells measured by ELISA. SiHa cells treated with control siRNA (siC) served as control. Values are expressed as increased CCL20 secretion in supernatants from E6 and E7 siRNA treated cells relative to supernatants from cells treated with siC

Article Snippet: An anti-human CCL20 antibody (R&D Systems) was used at 10 μg/ml in cell migration neutralization experiments.

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Increased migration of Langerhans cells in response to HPV16 E6 and E7 oncogene silencing: role of CCL20

doi: 10.1007/s00262-008-0522-5

Figure Lengend Snippet: a–c Phenotypical analysis of LC generated in vivo. The two-parameter contour plots show CD1a fluorescein isothiocyanate (FITC) on the x-axis and CD14 (a), CCR6 (b) and CD207 (c) phycoerythrin (PE) on the y-axis. The values correspond to the percentages of positive cells in the entire population. d Migration of LC in a Boyden chamber assay with culture supernatants from E6 (siE6), E7 (siE7) or both (siE6/E7) siRNA treated SiHa cells. Culture supernatants from SiHa cells treated with control siRNA (siC) served as controls. Supernatants were treated (greyscale) or not (white) with a CCL20 blocking antibody. Results are expressed as the means ± SD of three experiments. Asterisks indicate statistically significant differences ***P < 0.001

Article Snippet: An anti-human CCL20 antibody (R&D Systems) was used at 10 μg/ml in cell migration neutralization experiments.

Techniques: Generated, In Vivo, Migration, Boyden Chamber Assay, Control, Blocking Assay

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Tauroursodeoxycholic acid increases neural stem cell pool and neuronal conversion by regulating mitochondria-cell cycle retrograde signaling.

doi: 10.4161/15384101.2014.962951

Figure Lengend Snippet: Figure 6. TUDCA mediates neuronal rather than astroglial conversion of NSCs. NSCs were expanded and induced to dif- ferentiate for 24 h in the presence or absence of TUDCA and/or CsA or OligA. Cells were then collected for flow cytome- try and immunocytochemistry, as described in Materials and Methods. (A) Flow cytometry analysis of the ratio between bIII-tubulin- and GFAP-positive cells cultured in optimized neuronal differentiation-inducing medium. (B) Rep- resentative images of immunofluores- cence detection of cells labeled with anti- bIII-tubulin and anti-GFAP antibodies. Nuclei were stained with Hoechst 33258. Scale bar, 10 mm. (C) Flow cytometry anal- ysis of the ratio between bIII-tubulin- and GFAP-positive cells cultured in optimized glial differentiation-inducing medium. Results are expressed as mean § SEM fold-change for at least 3 different experi- ments. *P < 0.01 from non treated cells (control); zP < 0.01 and yP < 0.05 from cells treated with TUDCA alone.

Article Snippet: Analyses of NSC differentiation For detection of Sox2, bIII-tubulin and GFAP expression levels, cells were collected and processed as previously described.15 Subsequently, cells were washed and incubated for 30 min with mouse primary antibodies reactive to Sox2 (MAB2018; R&D Systems Inc.), bIII-tubulin (MMS-435P, TUJ1; Covance) and GFAP (MAB360; Merck Millipore Corp.) at a dilution of 1:100, 1:500 and 1:100, respectively.

Techniques: Immunocytochemistry, Flow Cytometry, Cell Culture, Labeling, Staining, Control