Journal:

Article Title: A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types

doi: 10.1038/nbt1483

Figure Lengend Snippet: (a) Scheme for infection of puromycin-resistant, Nanog-GFP or Nanog-neo primary MEFs expressing the reverse tetracycline transactivator (M2rtTA) with dox-inducible lentiviruses encoding the four reprogramming factors followed by induction of reprogramming, primary iPS cell colony selection, dox withdrawal, chimera formation and puromycin selection for iPS cell–derived secondary somatic cells. (b) NNeo secondary MEFs isolated from chimeras undergo complete epigenetic reprogramming. Dox-independent cultures express the pluripotency-associated genes alkaline phosphatase, SSEA1 and Nanog. (c,d) MEF-derived NNeo and NGFP2 secondary iPS cells generate cells of all three germ layers in teratomas (c; arrow, mesoderm; asterisk, endoderm; cross, ectoderm) produced in teratoma formation assays and contribute to chimera formation when injected into blastocysts, as indicated by the presence of iPS cell–derived agouti coat color on a black background (d). Scale bars, 250 µm.

Article Snippet: For flow cytometric analysis we used an APC-conjugated anti-mouse SSEA1 (R&D systems) and an alkaline phosphatase substrate kit, Vector Red substrate kit (Vector Laboratories).

Techniques: Infection, Expressing, Selection, Derivative Assay, Isolation, Produced, Injection

Journal:

Article Title: A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types

doi: 10.1038/nbt1483

Figure Lengend Snippet: (a) Secondary MEFs from three ‘primary’ iPS cell lines were treated with dox, and reprogramming was monitored visually. The different MEF populations exhibited morphologic differences 6 d after dox administration, but all formed colonies with ES-cell morphology within 12 d (arrows). Scale bar, 250 µm. (b) Neo-resistant and alkaline phosphate–positive colonies were present in NNeo cultures when the drug was added to the media as early as day 4 after dox induction. (c) Flow cytometric analysis for reactivation of SSEA1 and the Nanog-GFP reporter allele (in NGFP2 and NGFP3 lines) over 18 d of dox culture. (d) Secondary NGFP2 MEFs were plated at densities varying from 0.025–500 cells/mm2 followed by dox addition. GFP+ colonies were counted 4 weeks later. (e) Single secondary MEFs were plated in 96-well plates containing a γ-irradiated MEF feeder layer followed by dox induction. The percentage of single cells able to proliferate sufficiently to form a visible colony on the MEF feeder layer (light gray bars) and the percentage of single cells able to form GFP+ or neo-resistant secondary iPS cell colonies (dark gray bars) were scored 4 weeks later. (f) Comparison of the interexperimental variability in iPS cell colony formation efficiency between direct infection and the secondary system. 3 × 105 Oct4-neo MEFs1 derived from a single embryo were infected with the four factors encoded by Moloney-based retroviral vectors on a 10-cm plate, neo selection was initiated on day 6 and resistant colonies were counted on day 20 (left, direct infection). 3 × 104 secondary NGFP2 MEFs derived from one chimeric embryo were plated in a six-well dish, exposed to dox-containing media and GFP+ colonies were counted 3 weeks later (right, secondary system). The bars represent number of colonies in each of the four independent experiments.

Article Snippet: For flow cytometric analysis we used an APC-conjugated anti-mouse SSEA1 (R&D systems) and an alkaline phosphatase substrate kit, Vector Red substrate kit (Vector Laboratories).

Techniques: Irradiation, Comparison, Infection, Derivative Assay, Retroviral, Selection

Journal: Stem Cells International

Article Title: Small Extracellular Vesicles from Human Fetal Dermal Cells and Their MicroRNA Cargo: KEGG Signaling Pathways Associated with Angiogenesis and Wound Healing

doi: 10.1155/2020/8889379

Figure Lengend Snippet: Physical characterization of EVs by NTA and protein expression. (a) Representative histogram of EVs isolated from secretome of fetal dermal cells showing a peak corresponding to a mode value of 77.5 ± 0.8 nm size and a concentration of 2.59 × 10 12 particles/ml. (b) Representative histogram of EVs isolated from secretome of adult dermal cells showing a peak corresponding to a mode value of 87.2 ± 2.8 nm size and a concentration of 1.12 × 10 11 particles/ml. The results shown are representative of three independent experiments. (c) Representative Western blot analysis of two fetal dermal-derived EV samples showing expression of EV markers Rab5, Alix, and CD63 in total protein extracts of pellet particles. Negative control, calnexin in cell protein extracts, and pellet particles are also shown. NTA: nanoparticle tracking analysis; EVs: extracellular vesicles; cells: total protein extracts of human fetal dermal cells; EV#1: sample 1; EV#2: sample 2.

Article Snippet: The membranes were blocked with 5% nonfat milk in T-TBS (50 mmol/l Tris pH 7.5, 0.9% NaCl, and 0.1% Tween-20) (all from Sigma-Aldrich) overnight at 4°C and incubated 1 hour at room temperature with the following primary antibodies: mouse monoclonal antibody raised against recombinant human Rab5 (F-9, sc-373725, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1 : 500 dilution), mouse monoclonal antibody against Alix (3A9, 2171, Cell Signaling Technology, Danvers, MA, USA; 1 : 1000 dilution), and mouse monoclonal antibody against CD63 (MX-49.129.5, sc-5275, Santa Cruz Biotechnology; 1 : 200 dilution).

Techniques: Expressing, Isolation, Concentration Assay, Western Blot, Derivative Assay, Negative Control

Journal: Cell & bioscience

Article Title: Improving the response to oxaliplatin by targeting chemotherapy-induced CLDN1 in resistant metastatic colorectal cancer cells.

doi: 10.1186/s13578-023-01015-5

Figure Lengend Snippet: Fig. 3 Oxaliplatin-mediated membrane CLDN1 expression is dependent on MAPKp38,/GSK3β/ Wnt-βcat signaling cascade (A) Analysis of MAPK p38 phosphorylation (Pp38) by western botting in SW620 cells after incubation or not (UT) with oxaliplatin for 14 and 24 h (1.2 µM) Top: representative western blotting image; bottom: quantification of MAPK p38 phosphorylation level in the different conditions. (B) CLDN1 expression at the membrane (g-mean quantification) of SW620 cells incubated (+) or not (-) with oxaliplatin and/or LY2228820 (p38 inhibitor) (1 µM) for 72 h p ≤ 0.001 (Student’s t-test). (C) Western blotting showing the expression of GSK3β, GSK3βSer9 and β-catenin in SW620 cells incubated or not with oxaliplatin (5µM for 24 h). (D-E) GSK3βSer9 expression evaluated (D) by flow cytometry and (E) by immunofluorescence using the Celigo™ imaging cytometer. Incubation with H2O2 was used as positive control because it increases GSK3β phosphorylation at Ser9 (20). (F) GSK3β silencing. Left, western blot analysis of GSK3β expression in SW620 cells in which GSK3β was silenced (shGSK3β) or not (WT). Right, effect of GSK3β silencing on CLDN1 membrane expression after incubation with oxaliplatin (1.2 µM for 72 h) evaluated by FACS relative to non-silenced cells ** p = 0.001 (Student’s t-test). (G) Proximity ligation assay (PLA) with oligonucleotide-conjugated antibodies against p38 and GSK3β in SW620 cells incubated or not with oxaliplatin (5µM for 24 h). Left: Fluorescence images, nuclei were counterstained with DAPI (blue). Right: PLA dot counts per cell in the corresponding fluorescence images p ≤ 0.001 (Student’s t-test) (H) Expression and localisation of total and inactive (phosphorylated) βcatenin by immunofluorescence staining in SW620 cells after incubation or not with oxaliplatin (5µM for 72 h). In treated cells, total β-catenin expression increases and inactive β-catenin translocates to the nucleus (arrow) (I) The TOP/FOP Flash luciferase assay shows the transcriptional activation of the Wnt/β-catenin signaling pathway in SW620 cells after oxaliplatin incubation (5µM for 72 h) compared with untreated cells. * p = 0.01 (paired t-test) (J) Oxaliplatin (5µM for 72 h) effect on the mRNA expression of Wnt /β-catenin target genes in SW620 cells compared with untreated cells (Student’s t-test). (K) Oxaliplatin-induced (1.2µM for 72 h) CLDN1 membrane expression increase is reduced when SW620 cells are co-incubated with 7.5 µM XAV939 (small molecule inhibitor of tankyrase) ****p < 0.0001 (one-way ANOVA test)

Article Snippet: The first step was as described above for the immunofluorescence experiments with two primary antibodies against rabbit GSK3β (HPA028017, Sigma-Aldrich) and mouse p38 alpha (F-9; sc-271,120, Santa Cruz), followed by incubation with a pair of oligonucleotide-labeled secondary rabbit and mouse IgG (Duolink® In SituPLA® Probes).

Techniques: Membrane, Expressing, Phospho-proteomics, Western Blot, Incubation, Flow Cytometry, Immunofluorescence, Imaging, Cytometry, Positive Control, Proximity Ligation Assay, Fluorescence, Staining, Luciferase, Activation Assay

Journal: Journal of Biological Chemistry

Article Title: Nuclear Calcium/Calmodulin-dependent Protein Kinase II Signaling Enhances Cardiac Progenitor Cell Survival and Cardiac Lineage Commitment

doi: 10.1074/jbc.m115.657775

Figure Lengend Snippet: FIGURE 4. Nuclear CaMKII overexpression in CPCs promotes increases in cellular size and decreased proliferation. A, CPCeB has decreased proliferative capacity relative to CPCe based on a fluorescent nucleic acid stain measured at 2, 4, and 6 days after seeding equal cell numbers. ** and *** p 0.01 and p 0.0001 CPCeBrelativetoCPCe.B,CPCeBpopulationsshowsanincreaseddoublingtimerelativetoCPCe.C,cellcycleanalysisusingflowcytometryinCPCeandCPCeB.D, fluorescent images of CPCe and E, CPCeB populations in growth media. F, CPCeB shows an increase in relative cell surface area. ***, p 0.0001 CPCeB relative to CPC and CPCe. G, CPCeB shows an increase in length to width ratio relative to non-modified CPCs and CPCe. *, p 0.01 CPCeB relative to CPCe. H, expression of senescence markers p16 and p53 by immunoblot. -Actin was probed as a loading control. I, quantitation of p16 and J, p53 in CPCe and CPCeB populations.

Article Snippet: Next day blots were washed with TBST buffer and incubated in secondary antibodies in milk for 1.5 h. Primary antibodies for Western blot are as follows: rabbit anti-CaMKII (1:15000; UC Davis, Dr. Donald Bers), mouse anti-HDAC4 (1:1,000; Cell Signaling #5392) rabbit anti-phospho-HDAC4 (Ser-632)/HDAC5 (Ser-661)/ HDAC7 (Ser-486) (1:1000; Cell Signaling #3424) goat anti-GFP (1:1000; Rockland 600-101-215), mouse anti-HA tag (1:200; Santa Cruz Biotechnology, Inc. sc-7392), mouse anti-p16 (1:200, Santa Cruz Biotechnology, Inc. F-9 and F-12 clones sc-55600, sc-1661), mouse anti-p53 (1:500, Abcam ab26), rabbit anti- -sarcomeric actinin (1:500; Abcam ab72592), mouse anti- -smooth muscle actin (1:1000; Sigma Aldrich a2547), rabbit anti-Tie2 (1:100; Santa Cruz Biotechnology Inc. sc-9026), mouse anti-mouse -actin (1:500; Santa Cruz Biotechnology Inc sc-81178), rabbit anti-Mef2c (1:500; Cell Signaling #5030), rabbit anti-Cyclin B1 (1:500; Cell Signaling #12231), rabbit anti-Cyclin D1 (1:1000; Cell Signaling #2978), rabbit anti-phospho histone H3 (1:1000, Life Technologies 44 –1190G), mouse anti-GAPDH (1:3000; Millipore AB2302), and rabbit anti-Lamin A (C-terminal) (1:1000; Sigma Aldrich L1293).

Techniques: Over Expression, Staining, Modification, Expressing, Western Blot, Control, Quantitation Assay

Journal: Journal of Biological Chemistry

Article Title: Nuclear Calcium/Calmodulin-dependent Protein Kinase II Signaling Enhances Cardiac Progenitor Cell Survival and Cardiac Lineage Commitment

doi: 10.1074/jbc.m115.657775

Figure Lengend Snippet: FIGURE 9. Knockdown of CaMKIIB in CPCs prevents the up-regulation of pro-apoptotic molecules and promotes properties of cellular senescence. A, CaMKIIB; B, CaMKIIC; and C, Bcl-2 gene expression in CPC sh-Ctrl and CPC sh-B during growth conditions or with the addition of dex. Values are represented as n-fold mRNA relative to CPC sh-Ctrl in GM and after normalization to ribosomal 18S. **, p 0.01. D, proliferation measured using a direct nucleic acid fluorescent dye. Fluorescent values are represented as a fold change relative to day of plating (Day 0). E, cell doubling time in hours. F, cell cycle analysis using propidium iodide to determine percentage of cells in the different stages of the cell cycle analyzed by flow cytometry. G, cell cycle images of CPC sh-Ctrl and H. CPC sh-B. I, fluorescent images of CPC sh-Ctrl; and J, CPC sh-B. K, CPC sh-Ctrl and CPC sh-B cell surface area and L, length to width ratio represented as arbitrary units. M, immunoblot representation of pHDAC4 on the serine 632 and p16. N, quantitation of pHDAC4 and O. Senescence marker p16. Values are represented as a fold change relative to CPC sh-Ctrl and after normalizing to GAPDH.

Article Snippet: Next day blots were washed with TBST buffer and incubated in secondary antibodies in milk for 1.5 h. Primary antibodies for Western blot are as follows: rabbit anti-CaMKII (1:15000; UC Davis, Dr. Donald Bers), mouse anti-HDAC4 (1:1,000; Cell Signaling #5392) rabbit anti-phospho-HDAC4 (Ser-632)/HDAC5 (Ser-661)/ HDAC7 (Ser-486) (1:1000; Cell Signaling #3424) goat anti-GFP (1:1000; Rockland 600-101-215), mouse anti-HA tag (1:200; Santa Cruz Biotechnology, Inc. sc-7392), mouse anti-p16 (1:200, Santa Cruz Biotechnology, Inc. F-9 and F-12 clones sc-55600, sc-1661), mouse anti-p53 (1:500, Abcam ab26), rabbit anti- -sarcomeric actinin (1:500; Abcam ab72592), mouse anti- -smooth muscle actin (1:1000; Sigma Aldrich a2547), rabbit anti-Tie2 (1:100; Santa Cruz Biotechnology Inc. sc-9026), mouse anti-mouse -actin (1:500; Santa Cruz Biotechnology Inc sc-81178), rabbit anti-Mef2c (1:500; Cell Signaling #5030), rabbit anti-Cyclin B1 (1:500; Cell Signaling #12231), rabbit anti-Cyclin D1 (1:1000; Cell Signaling #2978), rabbit anti-phospho histone H3 (1:1000, Life Technologies 44 –1190G), mouse anti-GAPDH (1:3000; Millipore AB2302), and rabbit anti-Lamin A (C-terminal) (1:1000; Sigma Aldrich L1293).

Techniques: Knockdown, Gene Expression, Cell Cycle Assay, Flow Cytometry, Western Blot, Quantitation Assay, Marker