Journal: F1000Research

Article Title: An unexpected effect of TNF-α on F508del-CFTR maturation and function

doi: 10.12688/f1000research.6683.2

Figure Lengend Snippet: Differentiated primary HBE cell cultures grown at an air-liquid interface were incubated with 50ng/ml TNFα for 10–30min, 3–6h and 24h. CFTR immunodetection was performed with 24.1 anti-CFTR antibody and analyzed with confocal microscopy. Green staining represents CFTR (Alexa Fluor 488), red color staining represents ZO-1 protein of tight junctions (Alexa Fluor 594) and blue DAPI staining visualizes nuclei. Independent TNFα treatments and CFTR immunodetection were performed on HBE cells from three different F508del/F508del CF patients. Representative images of one experiment are demonstrated (Scale bars = 20µm).

Article Snippet: Anti-CFTR antibodies (abs): MM13-4 mouse monoclonal ab against N-terminus of CFTR, (Millipore, France, 05-581); 24-1 mouse monoclonal ab against C-terminus of CFTR (R&D Systems, MAB, 25031).

Techniques: Incubation, Immunodetection, Confocal Microscopy, Staining

Journal: bioRxiv

Article Title: Cardiac fibroblasts regulate cardiomyocyte hypertrophy through dynamic regulation of type I collagen

doi: 10.1101/2022.05.25.493406

Figure Lengend Snippet: (A) Whole ventricle mRNA microarray analysis of Col1a2 -/- mouse hearts compared to Col1a2 +/- at 2 months of age, n=3 per genotype. (B) Mass spectrometry analysis of ECM protein changes in Col1a2 -/- mouse hearts compared to Col1a2 +/- hearts at 3 months of age, n=4 per genotype. (C) Representative immunofluorescence images and (D) Western blot analysis of periostin from hearts of Col1a2 +/- and Col1a2 -/- mice at 3 months of age. Scale bar: 25 µm. (E) Flow cytometric gate strategy and (F) analysis of cardiac fibroblasts (MEFSK4 + /CD31 - /CD45 - ) from dissociated hearts of Col1a2 +/- and Col1a2 -/- mice at 3 months of age. (G) Representative immunofluorescence images of platelet-derived growth factor receptor (PDGFR)-α (purple) in Col1a2 +/- and Col1a2 -/- mice at 3 months of age. Wheat germ agglutinin (WGA) staining is green and shows outlines of cardiomyocytes. Scale bar: 100 µm. Relative mRNA expression of Col1a2 (H), Postn (I), Col3a1 (J) and Col5a1 (K) in sorted cardiac fibroblasts (MEFSK4 + /CD31 - /CD45 - ) from Col1a2 +/- and Col1a2 -/- mice at 9 months of age. Student t -test for panels (F), (H), (I), (J) and (K).

Article Snippet: Antibodies against the following proteins were used: periostin (Novus Biologicals NBP1-30042; 1:300 dilution for IF, 1:1000 for Western blot); collagen I (Abcam ab21286; 1:100 for IF); PDGFRα from (R&D Systems AF1062; 1:1000 for IF); collagen 1a2 (Santa Cruz sc-393573; 1:500 for Western blot) Anti-CD31 was from BioLegend (102423; 1:100 for flow cytometry); anti-CD45 was from BD Biosciences (563890; 1:100 for flow cytometry); anti-MEFSK4 was from Miltenyi Biotec (130-120-802; used 1:30 for flow cytometry).

Techniques: Microarray, Mass Spectrometry, Immunofluorescence, Western Blot, Derivative Assay, Staining, Expressing

Journal: Stem cells (Dayton, Ohio)

Article Title: Conversion of human umbilical cord mesenchymal stem cells in Wharton's jelly to dopaminergic neurons in vitro: potential therapeutic application for Parkinsonism.

doi: 10.1634/stemcells.2005-0053

Figure Lengend Snippet: Figure 1. The grouping and scheme of in vitro incubation of HUMSCs. Tyrosine hydroxylase–positive populations were gener- ated from undifferentiated HUMSCs by a three-step in vitro differ- entiation method. Abbreviations: DMEM, Dulbecco’s modified Ea- gle’s medium; FBS, fetal bovine serum; FGF, fibroblast growth factor; HUMSC, human umbilical mesenchymal stem cell; NCM, neuronal-conditioned medium; Shh, sonic hedgehog.

Article Snippet: In stage 3, cells were supplemented with NCM or 10% FBS-DMEM in the presence of the murine N-terminal fragment of sonic hedgehog (SHH) (500 ng/ml, 461-SH, R&D Systems Inc., Minneapolis, http://www.rndsystems.com) and murine FGF8 isoform b (FGF8) (100 ng/ml, 423-F8, R&D Systems Inc.) for 3, 6, 9, or 12 days.

Techniques: In Vitro, Incubation, Modification

Journal: Stem cells (Dayton, Ohio)

Article Title: Conversion of human umbilical cord mesenchymal stem cells in Wharton's jelly to dopaminergic neurons in vitro: potential therapeutic application for Parkinsonism.

doi: 10.1634/stemcells.2005-0053

Figure Lengend Snippet: Figure 2. HUMSC differentiation into dopaminergic, norepinephrine, and GABAergic neurons in vitro. (A): Photomicrographs showing TH immunocytochemistry of cultured HUMSCs. The cells expressed TH after incubation with NCM for 6 days and then SHH and FGF8 in DMEM for 3 days. In addition to TH-positive neurons, DBH-positive (B) and GAD-positive (C) neurons were detected. Human-specific nuclear antigen are in green, and DBH and GAD are in red. Arrows indicate cells stained positively for TH, DBH, or GAD. Scale bar 100 m. (D): Histograms showing the percentage of TH-positive cells after incubation with NCM, SHH, and FGF8. (Results represent the mean standard error from three different experiments. At least 200 cells were counted from 10 randomly selected microscopic fields in each experiment. Statistics consisted of one-way ANOVA followed by the LSD test; *statistical difference at p .05 compared with NCM-only group.) (E): TH expression in cultured cells by Western blotting. The molecular weight of rat and human TH were 60 and 68 kDa, respectively. Rat SN served as positive control. (F): Dopamine concentration in culture medium after HUMSCs were treated with NCM, SHH, and FGF8. (Results represent the mean standard error from three different experiments. Statistics consisted of one-way ANOVA followed by the LSD test; *statistical significance at p .05 compared with DMEM and NCM-only groups.) Abbreviations: ANOVA, analysis of variance; DBH, dopamine--hydroxylase; DMEM, Dulbecco’s modified Eagle’s medium; FGF, fibroblast growth factor; LSD, least-significant difference; HUMSC, human umbilical mesenchymal stem cell; NCM, neuronal-conditioned medium; Shh, sonic hedgehog; SN, substantia nigra; TH, tyrosine hydroxylase.

Article Snippet: In stage 3, cells were supplemented with NCM or 10% FBS-DMEM in the presence of the murine N-terminal fragment of sonic hedgehog (SHH) (500 ng/ml, 461-SH, R&D Systems Inc., Minneapolis, http://www.rndsystems.com) and murine FGF8 isoform b (FGF8) (100 ng/ml, 423-F8, R&D Systems Inc.) for 3, 6, 9, or 12 days.

Techniques: In Vitro, Immunocytochemistry, Cell Culture, Incubation, Staining, Expressing, Western Blot, Molecular Weight, Positive Control, Concentration Assay, Modification

Journal: Stem cells (Dayton, Ohio)

Article Title: Conversion of human umbilical cord mesenchymal stem cells in Wharton's jelly to dopaminergic neurons in vitro: potential therapeutic application for Parkinsonism.

doi: 10.1634/stemcells.2005-0053

Figure Lengend Snippet: Figure 5. Rotation behavior in response to amphetamine tested at 1, 2, 3, 4, and 5 months after lesion. A significant decrease in the number of amphetamine-induced turning was seen in animals with grafted cells treated with NCM SHH FGF8 (, n 6) compared with control (lesion-only) animals (F, n 12) and lesioned animals that received grafted cells treated with NCM (E, n 12). Statistics consisted of two-way ANOVA followed by the LSD test. (* Significant difference at p .05 between NCM SHH FGF8-treated group compared with the control and NCM groups at the same time point. # Significant difference at p 0.05 between the control and NCM groups over 1-month intervals.) Abbreviations: ANOVA, analysis of variance; FGF, fibroblast growth factor; LSD, least-significant difference; NCM, neuronal- conditioned medium; Shh, sonic hedgehog.

Article Snippet: In stage 3, cells were supplemented with NCM or 10% FBS-DMEM in the presence of the murine N-terminal fragment of sonic hedgehog (SHH) (500 ng/ml, 461-SH, R&D Systems Inc., Minneapolis, http://www.rndsystems.com) and murine FGF8 isoform b (FGF8) (100 ng/ml, 423-F8, R&D Systems Inc.) for 3, 6, 9, or 12 days.

Techniques: Control

Journal: iScience

Article Title: Histone H2A ubiquitination resulting from Brap loss of function connects multiple aging hallmarks and accelerates neurodegeneration.

doi: 10.1016/j.isci.2022.104519

Figure Lengend Snippet: Figure 6. Histone H2A ubiquitination accompanied by BRCA1 activation is the hallmark phenotype of BRAP LOF (A) Immunoblotting of histone extracts from NPCs as well as from embryonic, neonatal, adult cerebral cortical tissues, and quantification (Mean G SD) of increases in histone H2Aub (total H2Aub and H2AubK119, respectively) resulted from Brap LOF. n = 3–6 biological replicates. p-values calculated by Student’s t test are indicated. (B) Immunoblotting of Brca1 in various cells and tissues, showing that Brap LOF results in increased Brca1 abundance. (C) Immunoblotting of nuclear vs cytoplasmic fractions of MEFs at P1, showing increased nuclear localization of Brca1 in Brap/ cells. (D) Brca1 (red) and NeuN (green) double immunohistology images of cerebral cortical sections of BrapcKONPC and control mice at four months of age. Representative images are shown. Note the increased intensity and density of Brca1 puncta in the nuclei of BrapcKONPC cortical neurons (NeuN+). (E and F) Immunoblotting analyses of histone extracts from cerebral cortical tissues of three-month-old mice, showing increased ubiquitination of H2A variants targeted by Brca1 (E) along with total histone H2A ubiquitination (F). (G) Double immunohistology staining of cortical sections of 4-month old WT or BrapcKONPC mice with antibodies against Gfap (green) and histone H3 (red), showing reduced nuclear histones in cells surrounded by reactive astrocytes (circles) in BrapcKONPC cortical tissues. Representative images are shown. Nuclear DNA was stained with Hoechst 33342. Bars: 50 um or as indicated. See also Figure S3.

Article Snippet: Phospho-p53 (Ser15) Abcam Cat# ab1431; RRID:AB_301090 Phospho-p53 (Ser15) (D4S1H) Cell Signaling Technology Cat# 12571; RRID:AB_2714036 Phospho-p53 (Ser15) (16G8) Cell Signaling Technology Cat# 9286; RRID:AB_331741 Phospho-ATM(Ser1981) Novus Biologicals Cat# AF1655 Phospho-ATR (Ser 428) Cell Signaling Technology Cat# 2853; RRID:AB_2290281 53 BP1 Novus Biologicals Cat# NB100-304 Brca1 Santa Cruz Biotechnology Cat# sc-642; RRID:AB_630944 Brca1 Novus Biologicals Cat# MAB22101 Brca1 (clone 8F7) Novus Biologicals Cat# NBP1-41186 LC3B (G-9) Santa Cruz Biotechnology Cat# sc-376404; RRID:AB_11150489 LC3B Cell Signaling Technology Cat# 2775; RRID:AB_915950 LC3B Novus Biologicals Cat# NB100-2220 p62SQSTM1 Novus Biologicals Cat# MAB8028 p62SQSTM1 GeneTex Cat# GTX100685; RRID:AB_2038029

Techniques: Ubiquitin Proteomics, Activation Assay, Western Blot, Control, Staining